CN105708828A - Application of caffeic acid in preparation of drugs for treating hepatic microcirculation disturbance and hepatic injury - Google Patents

Abstract

The invention relates to an application of caffeic acid in preparation of drugs for treating hepatic microcirculation disturbance and hepatic injury. Through a series of observation of caffeic acid on hepatic ischemia reperfusion rats, caffeic acid is found to improve the hepatic surface blood flow quantity by the action routes: AMPK[alpha] phosphorylation and PKC[delta] membrane translocation are inhibited, NADPH subunit P41 and P47 membrane translocation is inhibited and NADPH-derived peroxides are inhibited; the activity of a hepatic ischemia reperfusion rat mitochondria respiratory chain is improved, and peroxide production caused by mitochondria injury is reduced, so as to increase the production of ATP; hepatocyte apoptosis caused by ischemia reperfusion is inhibited; and hepatic cell internal cytoskeleton injury caused by ischemia reperfusion is attenuated. The new application in preparation of the drugs for treating hepatic microcirculation disturbance and hepatic injury is provided.

Description

Caffeic acid application in preparation treatment hepatic microsome cytochrome P450 and liver injury medicament

Technical field

The present invention relates to a kind of new medicine use, preparing, particularly to caffeic acid, the application treated in hepatic microsome cytochrome P450 and liver injury medicament.

Background technology

Ischemia-reperfusion Injury in Rat: during the serious liver trauma of liver transplantation, liver partial excision operation, process, be required for temporary blocking-up hepatic blood flow, after again recovering blood supply, the ischemical reperfusion injury of liver can be caused.

The oxygen that good blood circulation is histiocyte acquisition abundance supplies and nutrient substance the basic assurance discharging metabolite.Ischemia injury is there is when a variety of causes causes histoorgan hemoperfusion amount to reduce, and after recovering hemoperfusion, cell function metabolism and structural deterioration increase the weight of on the contrary, and this ischemic tissue or organ are recaptured blood perfusion or oxygen, for rear, damaging action produced by tissue and organ be called ischemia reperfusion injury (ischemiareperfusioninjury).During the serious liver trauma of liver transplantation, liver partial excision operation, process, it is required for temporary blocking-up hepatic blood flow, after again recovering blood supply, the ischemical reperfusion injury (hepaticischemiareperfusioninjury, HIRI) of liver can be caused.During law during ischemia/reperfusion, there is the damage of a series of metabolism, 26S Proteasome Structure and Function in liver organization cell, can cause liver microcirculation and hepatic injury, even result in liver failure, is one of principal element affecting disease prognosis, operation success or failure and patient's survival.

One of initial target spot of hepatic ischemia reperfusion is microcirculatory infringement, and endothelial injury destroys microcirculation integrity, causes Oligemia, and this disorder starts again the damage of liver secondary ischemic in turn.In hepatic ischemia reperfusion process, sinusoidal endothelial cell (SEC) and macrophage produce a large amount of potent vasoactive and shrink material, such as ET-1, are combined with the endothelin receptor on stellate cells surface and make the contraction of sinus hepaticus district cause microcirculation disturbance.

Hepatic ischemia reperfusion can also cause the exception of liver cell mitochondria respiratory chain, causes that ATP produces to reduce and produces to increase with peroxide, also can cause the film transposition of nadph oxidase subunit P41 and P47 in cell, activate nadph oxidase, produce peroxide.ATP content reduces can cause hepatocyte inner cell framework collapse, peroxide produces to increase can inducing cell apoptosis, can multi-signal approach in activating cell, such as FN κ B signal pathway, the generation of inducible proinflammatory sex factor and the expression of cell adhesion molecule, the further induced liver injury of generation of proinflammatory cytokines, the expression of cell adhesion molecule can induce leukocyte adhesion in sinus hepaticus, liver central vein, affect the microcirculation of liver, increase the weight of hepatic injury.Liver Microcirculation that ischemia-reperfusion causes and hepatocyte injury abdominal surgery, recurrent hepatic injury after liver surgery, liver transplantation, but lack effective Therapeutic Method at present.

Caffeic acid (Caffeicacid, CA) is one of aqueous soluble active constituent of Radix Salviae Miltiorrhizae, and its molecular structural formula is as follows:

Conventional research confirms that CA can pass through to improve the activation of cell adhesion molecule, relative association of integrins expression and TLR4 signal path and improve fat phylaxin and stimulate sticking of the mononuclear cell caused and endotheliocyte；There is the effect of the oxidation of suppression N-formylmethionyl-leucylphenylalanine (fMLP) human neutrophil stimulated generation ROS and low density lipoprotein, LDL；CA, also by reducing IL-6, IL-1 β, the release of TNF-α and MCP-1 inflammatory factor and mrna expression, raises glutathione oxidase, the expression of superoxide dismutase and catalatic mRNA, the heart tissue of protection diabetes rat；CA can the human endothelial cell apoptosis of cultivation that causes of the low density lipoprotein, LDL of inhibited oxidation；The apoptosis of the human peripheral blood mononuclear cell that CA stress be able to be caused by the machine-processed inhibited oxidation of Bcl-2 non-dependent；CA, by suppressing expression and the activity of Rac1 albumen, reduces the activity of nadph oxidase, light oxidativestress damage, the human arterial smooth muscle cells that protection angiotensin stimulates；CA can also suppress the activity of vitro inhibition PKC；CA can activate Skeletal Muscle AMPK, stimulates the glucose transport of insulin non-dependent；The mice pia mater encephali thin vein leukocyte rolling that homocysteine is induced by CA and be stained with inhibitory action, this effect suppresses the expression of leukocyte adhesion molecule CD11a, the b/CD18 that Src/PI3K/Akt signal path mediates relevant with it.CA can improve the I/R damage of intestines caused；CA can be reduced by delayed ischemic neurological deficits and neuron after improving ischemia, improving brain atrophy, cerebral infarction and astrocyte proliferation, reducing the generation of leukotriene, thus improving focal cerebral ischemia Brain Injury After；Caffeic acid can protect the mitochondrion of rat cerebral tissue, alleviates the brain oxidative damage of intrastriatalquinolinicacid induction.CA can also suppress the duplication of people hepatitis virus B, improves the oxidative damage of the rat liver that nickel causes, and improves acetaminophen and hepatocyte injury that CCL4 causes.

At present, there is not yet Liver Microcirculation after can caffeic acid suppress law during ischemia/reperfusion, alleviate the report of liver tissue injury；The hepatic tissue mitochondrial respiratory chain played a significant role in hepatic microsome cytochrome P450 that particularly ischemia-reperfusion is caused by caffeic acid and course of liver damage, and regulate whether Protein S rt3 has intervention effect；To whether the film transposition of nadph oxidase subunit P41 and P47 in hepatocyte has intervention effect；To dirty microcirculation disturbance, to the hepatocellular apoptosis played an important role in hepatocyte injury, whether the cytoskeleton injury in hepatocyte etc. has intervention effect etc., and there is not been reported.

The present invention is by studying caffeic acid to law during ischemia/reperfusion rat liver microcirculation and the Ultrastructural observation of liver, the mensuration of liver surface blood flow, by flow cytomery Peripheral blood cells adhesion molecule CD11b, the expression of CD18, measure peripheral blood ALT, AST, TNF-α, IL-1 β, MCP-1, measure rat liver cells TNF-α, IL-1 β, MCP-1, MPO, the activity of TACE, measure Caspase8, MDA, 8-OHdG, H2O2/ peroxidase, catalase, SOD, glutathion reductase, glutathione oxidase, Complex I, II, IV, the activity of V, measure many-sided experimentation such as NAD+/NADH and DNA extraction, thus observing caffeic acid is by which kind of approach played to improve the hepatic microsome cytochrome P450 regulating liver-QI damaging action that law during ischemia/reperfusion causes.

Summary of the invention

The present invention relates to caffeinic new opplication.

For this, the present invention provides caffeic acid improve Liver Microcirculation in preparation or alleviate the application of liver injury medicament.

Application of the present invention, it is characterised in that: described Liver Microcirculation or hepatic injury cause due to Ischemia-reperfusion Injury in Rat.

Application of the present invention, it is characterised in that described improve liver microcirculation that law during ischemia/reperfusion causes or hepatic injury refers to and improves ischemia-reperfusion liver surface blood flow.

Application of the present invention, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or hepatic injury refers to and improves rat interlobular veins, the open number of central vein sinus hepaticus after ischemia-reperfusion；Interlobular veins, central vein red cell velocity.

Application of the present invention, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury refer to improve ischemia-reperfusion after rat leukocyte assemble.

Application of the present invention, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury and refer to suppression AMPK α phosphorylation and PKC δ film transposition, it is suppressed that the film transposition of NADPH subunit P41 and P47.

Application of the present invention, it is characterised in that: described improving liver microcirculation that law during ischemia/reperfusion causes or to alleviate hepatic injury be the generation suppressing or reducing peroxide, described peroxide source is in NADPH, mitochondrial injury.

Application of the present invention, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury and refer to the hepatocellular apoptosis suppressing ischemia-reperfusion to cause.

Application of the present invention, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury and refer to the damage alleviating the hepatocyte inner cell skeleton that ischemia-reperfusion causes.

Application of the present invention, it is characterised in that described caffeine is to be administered before ischemia-reperfusion.

Caffeic acid of the present invention is suitable to be administered with the form of pharmaceutical composition.This based composition can mix use with one or more physiologically acceptable carriers or excipient in a usual manner.If likely the caffeic acid of the present invention being administered as crude drug in treatment, it is preferable that active component is directly as pharmaceutical preparation.In the meaning compatible with other compositions and its pill taker is harmless, carrier must be pharmaceutically acceptable.

The pharmaceutical composition of the present invention, can prepare into pharmaceutical dosage forms, in use as required such as oral dosage form, injection form, vagina or supp anal form etc.

The pharmaceutical composition of the present invention, when preparing into pharmaceutical preparation, can add as required and to learn acceptable carrier.

The pharmaceutical composition of the present invention, pharmaceutically acceptable carrier can be contained as required, wherein caffeic acid or its pharmaceutically acceptable salt, solvate and hydrolyzable ester are as active constituents of medicine, its in the formulation shared percentage by weight can be 0.1-99.9%, all the other are pharmaceutically acceptable carrier.The pharmaceutical preparation of the present invention, exists in a unit, and described unit dosage form refers to the unit of preparation, such as every of tablet, and every capsules of capsule, every bottle of oral liquid, granule every bag, every of injection etc..

The pharmaceutical composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms include: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, drop pill, patch.

The pharmaceutical composition of the present invention, the preparation of its oral administration can contain conventional excipient, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, tablet can be carried out coating if desired.

The filler being suitable for includes the filler that cellulose, mannitol, lactose are similar with other.Suitable disintegrating agent includes starch, polyvinylpyrrolidone and starch derivatives, for instance sodium starch glycollate.Suitable lubricant includes, for instance magnesium stearate.Suitable pharmaceutically acceptable wetting agent includes sodium lauryl sulphate.Can passing through to mix, fill, the method that tabletting etc. is commonly used prepares solid oral composition.Carry out mixing repeatedly can make in those compositionss that active substance is distributed in a large amount of filler of whole use.

The form of oral liquid can be such as aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be a kind of available water before use or the composite dry products of other suitable carrier.This liquid preparation can contain conventional additive, such as suspending agent, such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, for instance lecithin, anhydro sorbitol monooleate or arabic gum；Non-aqueous carrier (they can include edible oil), for instance almond oil, the oily ester of ester of fractionated coconut oil, such as glycerol, propylene glycol or ethanol；Preservative, for instance para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if it is required, conventional flavouring agent or coloring agent can be contained.

For injection, the fluid unit dosage form of preparation contains active substance and the sterile carrier of the present invention.According to carrier and concentration, it is possible to this compound is suspended or dissolves.The preparation of solution typically by active substance being dissolved in a kind of carrier, filter-sterilized before being loaded into a kind of suitable bottle or ampoule, then seal.Adjuvant such as a kind of local anesthetic, preservative and buffer agent can also be dissolved in this carrier.In order to improve its stability, after loading bottle, this compositions can be freezed, and under vacuo water be removed.

The pharmaceutical composition of the present invention, applicable pharmaceutically acceptable carrier is optionally added when preparing into medicament, described pharmaceutically acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its derivates, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc..

The pharmaceutical preparation of present invention situation according to patient in use determines usage and dosage, can often taken three times per day, each 1-20 agent, such as: 1-20 bag or grain or sheet, every dose of 1mg-1000mg.

Medicine provided by the invention, for the dosage of adult treatment generally by the scope in 0.02 5000mg every day, it is preferable that 1 1500mg every day.Required dosage can be single dosage or metering repeatedly, by suitable doses at intervals, for instance twice daily, three times, four times or more.Preparation according to the present invention can contain the active component of 0.1 99%, and to tablet and capsule preferably 30 95%, liquid preparation is 3 50% preferably.

Beneficial effects of the present invention is further illustrated below by way of experimental data:

Caffeinic new opplication of the present invention is proved by following experiment:

(1) experimental apparatus

Inverted fluorescence microscope (TE2000-E, Nikon, Tokyo, Japan)；Hypersensitization video camera (USS-301, UNIQvision, SantaClara, CA, the U.S.)；Color video camera (JK-TU53H, Toshiba, Tokyo, Japan)；High-speed CCD photography system (Fastcam-ultimaAPXmodel120k, PHOTRON, the U.S.)；Image synthesizer (SP-460colorquadprocessor, SUNSPO, Japan)；Time video display units (VTG-55B, FOR-A, Japan)；Video distributor (VDA-106, Olympus, Japan)；Monitor (2118A, TCL, China)；DVD burner (DVR-R25, Malata, China)；Laser-Doppler perfusion weighted imaging instrument PeriScanPIM3:PeriScanPIM3System；PERIMED, Sweden；Cold light source (SJ8038HA2, SANJUN, China)；Upright microscope (BH-2, Olympus, Japan)；Colour imagery shot (DigitalsightDS-5M-U1, Nikon, Japan)；Heating blanket (ME04008, FHCBowDOINHAM, the U.S.)；Micro-injection pump (ALC-IP, Ao Lite bio tech ltd, Shanghai, China)；Full-automatic slicing machine (RM2255, LEICA, Germany)；Water bath with thermostatic control (HW SY11-K1, mayor of Beijing bearing instruments and meters company, China)；Centrifuge (centrifuge5471R, EPPENDORF, China)；Analytical balance (SartoriusAG, Germany)；PH meter (Beckman, the U.S.)；High speed centrifugal machine for minim (Heraeus, the U.S.)；High speed centrifuge (Beckman, the U.S.)；Electric drying oven with forced convection (Chongqing experimental facilities factory)；-80 DEG C of cryogenic refrigerators (SANYO, Japan)；Protein electrophorese and transferring film system (Bio-Rad, the U.S.)；Inverted microscope (Olympus, Japan)；Laser scanning co-focusing microscope (TCSSP5, Leica, Mannheim, Germany)；Micro imaging system (cold spring port, the U.S.)；Multi-functional microplate reader (Bio-Rad, the U.S.)；Flow cytometer (FACSCalibur, BD, the U.S.)；Transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan)；Scanning electron microscope (JSM-5600LV, JEOL, Tokyo, Japan)；BiacoreT200Biosensor (Biacore, GEHealthcareLifeSciences, Sweden).

(2) laboratory animal

Body weight is male Spragu-Dawley (SD) rat of 200 soil 20g, is purchased from Department Of Medicine, Peking University's animal center, the quality certification number: SCXK (capital) 2006-0008.Animal feeding is 22 ± 2 DEG C in temperature, and when humidity is 40 ± 5%, free diet, drinking-water, 12 h light/dark is alternately.Animal was in first 12 hours of experiment, and fasting is freely drunk water.Experimental implementation code performs by zooscopy committee of Peking University guide.Experiment draft obtains laboratory animal Ethics Committee of Department Of Medicine, Peking University approval (LA2010-001).

(3) medicine and reagent

Caffeic acid: Sigma, the U.S.；Urethane: Chinese Shanghai Tian Lian Fine Chemical Co., Ltd；Rhodamine 6G (Rhodamine-6G), TEMED, sodium butyrate: Sigma, the U.S.；RIPA lysate, 1.5mol/LTris-Cl (pH8.8), 0.5mol/LTris-Cl (pH6.8), 30% acrylamide-bisacrylamide solution, dodecyl shuttle acid sodium (SDS), 5 × Gel Loading buffer, whole protein extract test kit；Karyon-endochylema-after birth prepares test kit: Pu Lilai, Beijing, China；NE-PER caryoplasm Protein Extraction Reagent kit: ThermoScientific, Fremont, CA, the U.S.；Ammoniumpersulfata (APS): Amresco, the U.S.: BCA protein quantification test kit: Pierce, the U.S.；Bovine serum albumin (BSA): magnificent biology, Beijing, China；Defatted milk powder: Meijer, the U.S.；Pvdf membrane: Zerbrechlich-Fragile, Germany；PBS powder (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；EDTA antigen retrieval buffer (50X) (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；DAB colour reagent box (20 ×) (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；Myeloperoxidase (MPO) (MPO) antibody: ThermoScientific, Fremont, CA, the U.S.；Original position apoptosis detection kit: Roche, Basel, Switzerland；Phalloidin: lifetechnologies, Carlsbad, California, the U.S.；Hoechst33342:Invitrogen, St.Louis, MO, the U.S.；Dylight488-labeledrabbitanti-goatIgGandDylight549-labele dgoatanti-rabbitIgG:KPL, Gaithersburg, MD, the U.S.；25% glutaraldehyde: TedPella, California, the U.S.；Embedding medium SPI-pon812, DDSA, NMA, DMP-30:Westchester, NewYork, the U.S.；ProteinASepharoseCL-4B and PBS-P+buffer:GEHealthcareLifeSciences, London, Britain；ActivehumanSirt3fulllengthprotein:Abcam, Cambridge, MA, the U.S.；Other general chemistry reagent are analytical reagent, purchased from Chemistry In China drug company.

ALT, AST activity analysis test kit: Sigma, the U.S.；TNF-α, IL-1 β, MCP-1:R&DSystems, Minneapolis, Minn, the U.S.；MDA, MPO detection kit: HuanyaBiomedicineTechnology, Beijing, China；QIAampDNA extracts little test kit: QIAGEN, Germany；520TACE (α-Secretase) activity analysis test kit (fluorescence): Fremont, California, the U.S.；OxiSelect^TMDNA oxidative damage ELISA kit (8-OHdG is quantitative), H2O2/ peroxidase assay kit (fluorescence): CellBiolabs, SanDiego, California, the U.S.；Catalase assay kit, SOD assay kit, glutathion reductase assay kit, glutathione oxidase assay kit: CaymanChemicalcompany, AnnArbor, Michigan, the U.S.: Caspase8 assay kit, Complex I, II, IV, V activity detection kit, NAD+/NADH assay kit: Abcam, Cambridge, MA, the U.S..

CD11b, CD18 antibody: BDBiosciences, Pharmingen, SanDiego, CA, the U.S.；ICAM-1, E-selectin, TLR4, p47^phox, p40^phox, p22^phox, NF-κ Bp65, Na+-K+-ATP enzyme antibody, rabbit anti-igg: SantaCruz, SantaCruz, CA, the U.S.；I κ B-α, P-I κ B α, Src, P-src, Histone3, Bax, Bcl-2, CleavedCaspase-3, CleavedCaspase-9, PKC δ, AMPK, P-AMPK, Rac1, Sirt3, SDHA, NDUFA9, MnSOD, Acetylated-Lysine, β-actin:CellSignalingTechnology, Beverly, MA, USA；P67^phox, gp91^phoxAntibody: Abcam, Cambridge, MA, the U.S..

(4) foundation of rat liver I/R model and medication

Rat 2% urethane (2.0g/kg) intramuscular anesthesia, thorax abdomen shaving after anesthesia.Left femoral vein intubates, and with povidone iodine, ethanol is sterilized after thorax abdomen respectively, abdominal transverse incision, clamps muscle with mosquito forceps and reduces hemorrhage, exposes liver.Using bulldog clamp, folder closes hepatic hilar region blood vessel 30min, sets up the full ischemia model of liver；Releasing folder closes, Reperfu-sion 120min.According to table of random number, SD rat is divided into 6 groups (often groups 6):

Caffeic acid is dissolved in normal saline (NS), matching while using, and according to preliminary result, dosage is 15mg/kg/h, and speed is 1.8ml/h, and before sham-operation or ischemia, half an hour gives normal saline or caffeic acid:

1.NS+Sham group: rat threading not ligation, femoral vein instills normal saline (NS) continuously；

2.CA+Sham group: rat threading not ligation, femoral vein instills caffeic acid (CA) continuously；

3.NS+I/R group: rat portal area vascular ligation 30min, releases folder and closes, and Reperfu-sion 120min, femoral vein instills normal saline continuously；

4.CA+I/R group: rat portal area vascular ligation 30min, releases folder and closes, and Reperfu-sion 120min, femoral vein instills caffeic acid continuously.

(5) experimental technique

5.1 Microcirculatory Observations

5.1.1. observing the open number of sinus hepaticus of interlobular veins, central vein, leukocyte adhesion rolls and red cell velocity

(1) anesthesia: 20% urethane, 100g/ml, Rat Right lower limb muscles is anaesthetized, thorax abdomen shaving after anesthesia；

(2) starting device: open water-bath, computer, calorstat, inverted fluorescence microscope, image synthesizer, video distributor, monitor, time video display units, DVD burner, preheating access panel (must clean up), prepares syringe pump (regulating the speed and other parameters)；

(3) left femoral vein intubates: be used for injecting the bright 6G of Luo Da and giving liquid；

(4) liver is exposed: first using iodophor disinfection thorax abdomen, rear alcohol disinfecting thorax abdomen one time, scope is slightly less than iodophor disinfection scope；Skin transverse incision under xiphoid-process, Musclar layer cuts a longitudinal osculum along hunter's line, it is to avoid hemorrhage, cuts off muscle in the middle of mosquito forceps again, then same method cuts off another side muscle, exposes liver after two mosquito forceps laterally clamping each 15s of muscle；

(5) pre-treatment is observed: use fine rule to cut xiphoid-process after tightening xiphoid-process, carefully cut off falciform ligament, it is to avoid puncture diaphragm；Mouse is lain on one's side on access panel, gently liver lobus sinister is routed up with cotton swab, be placed in the middle of plexiglas observation port, it is impossible to use tweezers, it is prevented that hemorrhage, take advantage of a favourable situation to cut off hepatogastric ligament after routing up lobe of the liver；The absorbent cotton of a piece of suitable size is placed on the gastrointestinal side of liver, drips the moistening absorbent cotton of appropriate 37 DEG C normal saline, encase liver gastrointestinal side, gastrointestinal；Another sheet suitable size, it is placed on the diaphragm side of liver with the absorbent cotton that 37 DEG C of appropriate normal saline are moistening, pushes down liver gently, weaken and breathe the liver fluctuation caused；Another sheet suitable size, it is placed on the upside of liver with the absorbent cotton that 37 DEG C of appropriate normal saline are moistening, pushes down front two panels absorbent cotton, fixing liver；

(6) being placed in by access panel on the inverted biological microscope object stage being furnished with 37 DEG C of thermostats together with rat, surface drips 37 DEG C of normal saline continuously；

(7) liver microcirculation is observed dynamically: first observing liver substantially blood flow state, blood flow state is better, can continue to observe without obvious sinus hepaticus edema person；The left femoral vein injection bright 6G0.5ml of Luo Da, open mercury lamp, high-speed camera, leukocyte adhesion is observed after 1min, select without the central vein of leukocyte adhesion, interlobular veins, diameter is about between 20-45 μm, does not have the region that leukocyte adheres on interlobular veins and central vein to observe in 320 μm of visuals field of 400 μ m；Record under fluorescence, at a high speed, central vein, interlobular veins image under colored CCD.

(8) foundation of ischemia-reperfusion injury model: after 10min is observed on basis, liver blood flow is better, can enter group without obvious leukocyte adhesion person, uses bulldog clamp, and folder closes hepatic hilar region blood vessel 30min, sets up the full ischemia model of liver；Releasing folder closes, and is reapposed by lobe of the liver and finds former target under access panel, microscope；Reperfu-sion 30,60,90,120min time observe and record microcirculation, operation completes in-3min 3min, according to fluorescence, at a high speed, colored CCD order record central vein, interlobular veins image.

The mensuration of 5.2 liver surface blood flows

Rat opens breast, is completely exposed by liver, with laser-Doppler perfusion weighted imaging instrument PeriScanPIM3 (PeriScanPIM3System；PERIMED, Sweden) rats'liver lobus sinister blood flow is detected, record is before ischemia (Baseline), and 2cm × 2cm scope liver surface blood flow during Reperfu-sion 120min, each point in time measurement 3 times.Probe is always positioned at being parallel to the position of 18cm above liver surface, and scan depths is 0.5mm.With LDPIwin3.1 (PeriScanPIM3System；PERIMED, Sweden) to image measurement and data assessment, take 3 measurements.It is worth based on liver surface groundwater increment before I/R, calculates the rate of change of leftlobe of liver groundwater increment.

5.3 tectology dyeing

5.3.1. prepared by paraffin section

(1), after Reperfu-sion 120min, irrigating normal saline flushing liver speed with micro-injection pump is 10ml/min, altogether 20ml；

(2) cutting leftlobe of liver lower edge liver, be placed in the fixing 48h of paraformaldehyde solution of newly prepare 4%, note often shaking in fixing process, fixing volumetric ratio is 20-30 times；

(3) liver after fixing is pruned, and with blade by the parallel incision along cross section of bulk liver, makes liver thickness be less than about 5mm；

(4) being stained with dry by the formaldehyde filter paper of liver surface, tri-distilled water cleans, and scavenging period is 12h, and cleaning process often to be shaken, and changes a tri-distilled water at about 6h；

(5) through 70% ethanol, 80% dehydration of alcohol 24h, 95% dehydration of alcohol 12h, 100% dehydration of alcohol 2-3h, constantly to shake in dehydration；

(6) after dehydration terminates, being dried by the ethanol of liver surface with filter paper, entered in dimethylbenzene by tissue, clearing time is 10-30min, will the transparency of tissues observed at any time in clearing process；

(7) the electric drying oven with forced convection temperature of waxdip is decided to be 60 DEG C, and 5-6h opens in advance, makes wax melt.Being dried by the dimethylbenzene on surface with filter paper, action wants fast, it is impossible to making dimethylbenzene volatilization dry, waxdip total time is 2-3h, changes once new wax per hour, and the volumetric ratio of waxdip is 20-30 times；

(8) embedding, is placed on tissue in the mould taking advantage of full paraffin, makes whole tissue all immerse in paraffin, is placed in cold water until paraffin, is removed from the molds by wax stone；

(9) finishing wax stone, is trimmed to trapezoidal by wax stone, and tissue around to reserve enough borders, is positioned in 4 DEG C and preserves；

(10) finally prepare into paraffin mass and be placed in microtome, be cut into the section of 5 μm, be put in drift sheet machine and launch, the wax disk(-sc) jail of flattening is invested on the coated microscope slide of poly-D-lysine；

(11) roasting sheet is wanted in time, must put in an oven before naturally dry, situation about turning white otherwise occurs.The temperature of roasting sheet is 37 DEG C, overnight.

5.3.2. hematoxylin eosin stain

(1) paraffin section dewaxes, and dewax 15min in dimethylbenzene I, and dewax 10min in dimethylbenzene II；

(2) after dewaxing, section 5min in 100% ethanol I, transfers to 3min in 100% ethanol II；

3min in (3) 95% ethanol I, 2min in 95% ethanol II；

2min in (4) 80% ethanol；

1-2min in (5) 70% ethanol；

(6) distillation washing 2-3 time, washing is gently propose section gently up and down, washes 1min every time；

(7) Harris brazilwood extract dyeing 5-10min；

(8) distillation washing, gently carries up and down and washing several times；

(9) break up with 1% hydrochloride alcohol, gently carry several times up and down；

(10) distillation washing, gently carries several times up and down；

(11) 1% ammonia return indigo plant, and Microscopic observation is cut into slices, and control to return the blue time；

(12) distillation washing, gently carries several times up and down；

(13) eosin stains, the time is 3-5s, if desired can Microscopic observation；

(14) distillation washing, gently carries several times up and down；

(15) carrying out breaking up double dehydration with 80% ethanol, differentiation phase Yihong color can shoal, and notes often observing color, checks if desired under mirror；

(16) 95% ethanol carry out dehydration, dewatering time 5min, and the time, oversize meeting was faded, and notice that the moment observes color；

(17) 100% ethanol I dehydration 5min, 100% ethanol II dehydration 3min；

(18) 5min in dimethylbenzene I, 10min in dimethylbenzene II；

(19) use neutral gum mounting, carry out observing and taking pictures with Photobiology microscope (DigitalSightDS-5M-U1, Nikon, Tokyo, Japan).

5.3.3. fluorescent immunohistochemistry

(1) after Reperfu-sion 120min, take off a fritter liver, be placed in the fixing 48h of paraformaldehyde solution of 4%, through dehydration, transparent, waxdip, embedding, prepare wax stone, finally prepare into paraffin section.

(2) paraffin section dewaxes, and dewax 15min in dimethylbenzene I, and dewax 10min in dimethylbenzene II；

(3) after dewaxing, section 5min in 100% ethanol I, transfers to 3min in 100% ethanol II；

3min in (4) 95% ethanol I, 2min in 95% ethanol II；

2min in (5) 80% ethanol；

1-2min in (6) 70% ethanol；

(7) distillation washing 1 time, washing is gently propose section gently up and down, washes 5min；

(8) carrying out antigen retrieval with the sodium citrate of 0.01M, liquor sodii citratis is filled it up with, and specimen is towards one side, and lid is opened, and uses microwave oven to be warmed up to 90 DEG C, then suspends, puts into specimen, regulates firepower to 60, and the time is 10min；

(9) room temperature natural cooling, is generally 2h；

(10) PBS rinses 3 times, and each washing time is 5min；

(11) open incubator in advance, be warmed up to 37 DEG C, PBST is hatched 30min；

(12) PBS rinses 3 times, and each washing time is 5min；

(13) by biological stroke upper border line around tissue, being wiped clean by the water droplet of surrounding with toilet paper, the heart adds several clear working solutions of bleeding in the tissue, is shaken gently for section, makes working solution cover monoblock tissue, reacts 30min；

(14) knocking serum gently, do not rinse, cleaned by the water droplet around boundary line with toilet paper, add the MPO antibody (1:200) prepared, 4 DEG C overnight；

(15) section is moved to room temperature, rewarming 1h from 4 DEG C；

(16) reclaiming primary antibodie, rinse 3 times with PBS, each washing time is 5min；

(17) add the anti-sheep of rabbit two anti-(Zhong Shan company, China), put room temperature 1h；

(18) PBS rinses 3 times, and each washing time is 5min；

(19) add the streptomycin avidin working solution of Radix Cochleariae officinalis enzyme labelling, react 30min；

(20) PBS rinses 3 times, and each washing time is 5min；

(21) DAB colour developing, developing time is as the criterion with the shade seen under mirror；

(22) PBS rinses 5min；

(23) haematoxylin redyes nucleus, distilled water flushing 1min；

(24) with 80% dehydration of alcohol 1min；

(25) 95% ethanol carry out dehydration, dewatering time 5min；

(26) 100% ethanol I dehydration 5min, 100% ethanol II dehydration 5min；

(27) 10min in dimethylbenzene I, 10min in dimethylbenzene II；

(28) use neutral gum mounting, carry out observing and taking pictures with Photobiology microscope (DigitalSightDS-5M-U1, Nikon, Tokyo, Japan).

5.3.4. immunofluorescence dyeing

(1) after Reperfu-sion 120min, take off a fritter liver, be placed in the fixing 48h of paraformaldehyde solution of 4%, through dehydration, transparent, waxdip, embedding, prepare wax stone, finally prepare into paraffin section.

(2) paraffin section dewaxes, and dewax 15min in dimethylbenzene I, and dewax 10min in dimethylbenzene II；

(3) after dewaxing, section 5min in 100% ethanol I, transfers to 3min in 100% ethanol II；

3min in (4) 95% ethanol I, 2min in 95% ethanol II；

2min in (5) 80% ethanol；

1-2min in (6) 70% ethanol；

(7) distillation washing 1 time, washing is gently propose section gently up and down, washes 5min；

(8) carrying out antigen retrieval with the sodium citrate of 0.01M, liquor sodii citratis is filled it up with, and specimen is towards one side, and lid is opened, and uses microwave oven to be warmed up to 90 DEG C, then suspends, puts into specimen, regulates firepower to 60, and the time is 10min；

(9) room temperature natural cooling, is generally 2h；

(10) PBS rinses 3 times, and each washing time is 5min；

(11) open incubator in advance, be warmed up to 37 DEG C, PBST is hatched 30min；

(12) PBS rinses 3 times, and each washing time is 5min；

(13) by biological stroke upper border line around tissue, being wiped clean by the water droplet of surrounding with toilet paper, the heart adds several clear working solutions of bleeding in the tissue, is shaken gently for section, makes working solution cover monoblock tissue, reacts 30min；

(14) knocking serum gently, do not rinse, cleaned by the water droplet around boundary line with toilet paper, add ICAM-1, the E-selectin antibody (1:100) prepared, NF-κ Bp65 antibody (1:200), 4 DEG C overnight；

(15) section is moved to room temperature, rewarming 1h from 4 DEG C；

(16) reclaiming primary antibodie, rinse 3 times with PBS, each washing time is 5min；

(17) incubator is opened in advance, it is warmed up to 37 DEG C, ICAM-1, E-selectin group adds the anti-sheep IgG of Dylight488 labelling, NF-κ Bp65 group adds anti-rabbit IgG, the 1:100 of Dylight549 labelling, dilutes with PBS, the amount of liquid needed on each liver organization is 30 μ l, and in 37 DEG C of climatic chambers, lucifuge hatches 1.5-2h；

(18) PBS rinses 3 times, and each washing time is 5min；

(19) with Hoechst33342 (1:50, MolecularProbes) labeled cell core, Hoechst PBS dilutes, and the amount of liquid of each organization need is 30 μ l, and under room temperature, lucifuge hatches 20min；

(20) PBS rinses 3 times, and each washing time is 5min, wants lucifuge in operating process；

(21) carry out mounting by anti-fluorescent quenching mountant, around scheme upper nial polish after mounting, it is prevented that slide plate；

(22) with laser scanning co-focusing microscope system (Axiovert200M, CarlZeiss, Jena, Germany), carry out observing and taking pictures, wherein nuclei dyeing au bleu, ICAM-1, the E-selectin positive is green, and the NF-κ Bp65 positive is red.

5.3.5. the double; two dyeing of hepatocellular TUNEL and F-actin

(1) after Reperfu-sion 120min, take off a fritter liver, be placed in the fixing 48h of paraformaldehyde solution of 4%, through dehydration, transparent, waxdip, embedding, prepare wax stone, finally prepare into paraffin section.

(2) paraffin section dewaxes, and dewax 15min in dimethylbenzene I, and dewax 10min in dimethylbenzene II；

(3) after dewaxing, section 5min in 100% ethanol I, transfers to 3min in 100% ethanol II；

3min in (4) 95% ethanol I, 2min in 95% ethanol II；

2min in (5) 80% ethanol；

1-2min in (6) 70% ethanol；

(7) distillation washing 1 time, washing is gently propose section gently up and down, washes 5min；

(8) carrying out antigen retrieval with the sodium citrate of 0.01M, liquor sodii citratis is filled it up with, and specimen is towards one side, and lid is opened, and uses microwave oven to be warmed up to 90 DEG C, then suspends, puts into specimen, regulates firepower to 60, and the time is 10min；

(9) room temperature natural cooling, is generally 2h；

(10) PBS rinses 3 times, and each washing time is 5min.

(11) open incubator in advance, be warmed up to 37 DEG C, with phalloidin (1:40, R415, Invitrogen, California, USA) labelling cardiac muscle F-actin, 1:40PBS dilutes, and the amount of liquid that each liver organization needs is 30 μ l, and in 37 DEG C of climatic chambers, lucifuge hatches 1h；

(10) PBS rinses 3 times, and each washing time is 5min；

(11) apoptotic cell is detected with TUNEL (the Nick End labelling method of Deoxydization nucleotide terminal transferase mediation) staining, original position apoptosis detection kit (Roche is selected in experiment, Basel, Switzerland), TUNEL reactant liquor is prepared according to 1:9, the amount of liquid needed in individual heart tissue is 30 μ l, and in 37 DEG C of climatic chambers, lucifuge hatches 1h；

(12) PBS washes 3 times, and each time is 5min, wants lucifuge in operating process；

(13) with Hoechst33342 (1:50, MolecularProbes) labeled cell core, Hoechst PBS dilutes, and the amount of liquid of each organization need is 30 μ l, and under room temperature, lucifuge hatches 20min；

(14) PBS washes 3 times, and each time is 5min, wants lucifuge in operating process；

(15) carry out mounting by anti-fluorescent quenching mountant, around scheme upper nial polish after mounting, it is prevented that slide plate；

(16) with laser scanning co-focusing microscope system (Axiovert200M, CarlZeiss, Jena, Germany), carrying out observing and taking pictures, wherein nuclei dyeing au bleu, F-actin is red, and the nucleus of the TUNEL positive is green.

5.4 electron microscopic observation liver ultrastructures

5.4.1. Electronic Speculum is projected

1. solution preparation

1) phosphate buffer: (0.2MPBS)

Table 1. phosphoric acid buffer liquid making method

Note:

(1) A liquid and B liquid have oneself special bottle, rinse several just passable all over drying in use with the new tri-distilled water steamed；

(2) to configure 200ml just enough every time for A liquid；

(3) when configuring, the bottle of B liquid has the graduation mark of 200ml and 400ml, it is possible to directly add tri-distilled water to groove place, add Na2HPO4.12H2O according to corresponding weight.Prepare the tri-distilled water syringe of the special 50ml inhaling Electronic Speculum tri-distilled water during other volume and inhale tri-distilled water；

(4), when using, A liquid and B liquid are made into the phosphate buffer of 0.2M according to 1:4-1:6 (PH meets 7.2-7.4 and is as the criterion), 4 DEG C of preservations.In formal test, ratio (A:B=80ml:400ml) solution according to A:B=1:5 is placed in the brown bottle holding Electronic Speculum PBS specially.

2) Electronic Speculum perfusate:

Perfusate used by Electronic Speculum is the glutaraldehyde of 2.5%；

Compound method is: take the PBS400ml of 0.2M, adds the glutaraldehyde 100ml of 25%, then with tri-distilled water constant volume to 1L, after filtration, puts into 4 DEG C of preservations.So the pH value pH instrument of the perfusate of preparation is determined as 7.24.The perfusate of 1L probably can 4 rats of perfusion.

3) glutaraldehyde of 2.5%

Table 2. glutaraldehyde compound method

Note:

The amount of (1) 1 25% glutaraldehyde is 10ml, prepares and is placed on 4 DEG C of preservations afterwards；

(2) the glutaraldehyde consumption of the 3% of each tissue is 1.5ml-2ml.

4) osmic acid original solution (2% osmic acid)

Needing during configuration to carry out in fume hood, band double gloves and mask, every gram of osmic acid adds 50mlDDW.

Clean bottle, hard thing puts glove, breaks peace into pieces and cuts open bottle.This solution is configured by the teacher of Electron Microscopy Room.

5) osmic acid solution of 1%:

The osmic acid stock solution of 2% mixes with 0.2MPBS1:1, after preparing, and 4 DEG C of preservations；The amount of the osmic acid of the 1% of each tissue is 1ml, is placed in the EP pipe of 1.5ml, wraps with tinfoil during use.

6) sucrose flushing liquor: (molecular weight is 342.2)

Table 3. sucrose flushing liquor compound method

After preparing, 4 DEG C of preservations；The amount of sucrose flushing liquor is each tissue 3-4ml.Noticing that the sucrose flushing liquor having prepared of a specified duration is easy to become mildewed, in time, notes observing, if become mildewed, and the flushing liquor more renewed immediately.

7) 100% acetone (noting having taken off water)

One day before use, 100% acetone, it should be preceded by water absorbing agent in advance, for instance anhydrous sodium sulfate, anhydrous K CO3, CUSO4 etc..It is most commonly used that anhydrous sodium sulfate at ordinary times.Add the anhydrous sodium sulfate of 1/5 bottle, mixing tens times of turning upside down, it is ensured that fully mix, then stand at least one evening, just can use.Strict moistureproof, it is possible to seal with sealed membrane.

8) embedding medium:

Table 4. embedding medium compound method

Note:

(1) when preparation embedding medium, it should select (humidity is more low more good, the humidity about 10% of four Maies, is best suitable for doing Electronic Speculum) between humidity 30%-40%, before joining 1-2 days, it should dehumidify with dehumidifier；

(2) beaker and the rotor of the 80ml of special configuration embedding medium are taken out, with the new tri-distilled water wash clean steamed, put into dry rotor, first mark scale with DDW, method: add the DDW of 30ml, 12ml, 20ml successively in beaker, mark tri-scales of 30ml, 12ml, 20ml on beaker with marker pen, then water is poured out, dry beaker, standby；

(3) in dry beaker, put into dry rotor, once add Epon81230ml (directly fall, later identical, want slow when falling, very thickness).Add DDSA12ml, be sufficiently stirred for (rotor stirs, not heating) 2min；Then add 20mlNMA, be sufficiently stirred for 2min；Finally it is added dropwise over 40 DMP-30, is sufficiently stirred for 5min.Subpackage deposit in-20 DEG C standby.Note all of stirring operation and be added dropwise over all should being placed on operation in the angle that dehumidifier is formed, open exsiccator；

(4) embedding medium is not placed on room temperature too for a long time, takes out standby in right amount, and remaining embedding medium is as, in the centrifuge tube of large size, sealing with sealed membrane, it is placed in-20 DEG C of refrigerators, is packaged into multiple packaging, on-demand take, avoid multigelation, embedding medium otherwise can be made increasingly harder, cannot normally use.Note indicating the preparation time；

(5) beaker being finished, will use dehydrated alcohol wash clean in time, uses distilled water wash clean afterwards again.

2. experiment flow

1) modeling perfusion and sample treatment:

(1) by rat anesthesia modeling, every rat anesthesia interval time is about 1h；

(2) during modeling, perfusate and normal saline are ready to, changed after liquid every time, pipeline will be tested whether unimpeded, first try perfusate, the speed of perfusate be transferred to 1 second one, try normal saline again, being rinsed by perfusate with normal saline, it is too big that the speed of normal saline is not opened；

(3), after modeling terminates, quickly rat is transferred in the perfusion fume hood of Electronic Speculum；

(4) it is placed in ceramic whiteware dish, opens rapidly breast, insert the needle on the left of the apex of the heart, note necessarily can not inserting too deep, otherwise insert too deeply easily to be inserted through interventricular septum, be inserted in right ventricle and go, notice that the direction of pin is preferably probably parallel with the longitudinal axis of heart, make solution easily flow out.Then having eye scissors to cut off right auricle, open the control valve of conduit bottom, first fill normal saline, the speed of normal saline is not opened too big；

(5) in the process filling normal saline, it is possible to the muscle bone near right auricle and fur are pushed aside, it is impossible to the blood of right auricle flows out to have thing to stop, for the purpose of seeing that blood flow flows out from auricle.In the process of perfusion normal saline, if there is right ventricle excessive bulkiness, turn out pin and insert too deep, be inserted through interventricular septum and reached right ventricle, it should syringe needle is withdrawn；

(6) all expand if there is whole heart, it is possible to problem be right auricle cut off inadequate, it should again wound is opened greatly.Even if noting the situation that whole heart all expands do not occur, also should again being opened greatly by the wound of right auricle in stage of Reperfu-sion normal saline, cutting because only having cut one before, wound is certainly only small；

(7) note observing the liquid flowed out from left ventricle, if it find that liquid no longer has blood, just by where three-way cock reaches perfusate, regulate the speed.Fixing conduit, the time of perfusion is 40-60min；

(8) the successful mark of perfusion is, time perfusion terminates, it is all stiff that whole mouse whole body includes tail, and extremity are all white；

(9) liver is scaled off, carry out repairing block.Liver blade is cut into into the piece of tissue of 1mm × 1mm × 1mm, puts in the EP pipe equipped with 2.5% glutaraldehyde 1ml, standby projection Electronic Speculum.It is put into 4 DEG C of refrigerator overnight.

2) investing tissue is prepared

(1) being taken out from the glutaraldehyde of 2.5% by tissue, rinse 3 times with sucrose, each 15min, 4 DEG C of refrigerators, each EP pipe adds 1ml sucrose solution；

(2) osmic acid is fixed: room temperature 2.5h, by the EP pipe tinfoil lucifuge of 1.5ml, is placed in 4 DEG C；

(3) 0.2MPBS rinses 3 times, each 5min；

(4) acetone serial dehydration: dehydration 10min in 50% acetone, transfer to dehydration 15min in 70% acetone, transfer to dehydration 15min in 90% acetone, transfer to dehydration 10min in 100% acetone I, transfer to dehydration 10min in 100% acetone II, transfer to dehydration 10min in 100% acetone III.Dehydrating operations should be placed in the angle that dehumidifier is formed, and notes that tweezers are with special, rocking frequently in dehydration, at the solution of waiting time preparation preimpregnation.Note: after 100% acetone, want strict drying, moistureproof；

(5) preimpregnation: 100% acetone mixes with embedding medium 1:1, concrete compound method is first add the acetone of 500ml with the EP pipe 500 μ l:500 μ l of 1.5ml, then adds embedding medium to 1ml groove, mixes.Tissue is put in pre-body fluid, is placed in 37 DEG C of baking boxs (Electronic Speculum special oven) overnight；

(6) embedding: first getting out the little scraps of paper printed, write group above clearly, embedding medium toothpick is chosen in embedding plate, add many times, as long as noting not spilling over, adding as far as possible, piece of tissue heading on central authorities above, the scraps of paper are placed on afterbody.First the embedding carried out is room temperature embedding: 37 DEG C of baking box 24h (Electronic Speculum special oven).The embedding again carried out is 60 DEG C of baking boxs (Electronic Speculum special ovens), 48h, it is possible to extend；

(7) embedding medium carries out repairing block, section after solidifying, and slice thickness is 60nm.With acetic acid uranium and lead citrate, ultrathin section is dyeed, observe under transmission electron microscope, make film.

5.4.2. scanning electron microscope

1. solution preparation

1) phosphate buffer: (0.2MPBS)

Table 1. phosphoric acid buffer liquid making method

Note:

(1) A liquid and B liquid have oneself special bottle, rinse several just passable all over drying in use with the new tri-distilled water steamed；

(2) to configure 200ml just enough every time for A liquid；

(3) when configuring, the bottle of B liquid has the graduation mark of 200ml and 400ml, it is possible to directly add tri-distilled water to groove place, add Na2HPO4.12H2O according to corresponding weight.Prepare the tri-distilled water syringe of the special 50ml inhaling Electronic Speculum tri-distilled water during other volume and inhale tri-distilled water；

(4), when using, A liquid and B liquid are made into the phosphate buffer of 0.2M according to 1:4-1:6 (PH meets 7.2-7.4 and is as the criterion), 4 DEG C of preservations.In formal test, ratio (A:B=80ml:400ml) solution according to A:B=1:5 is placed in the brown bottle holding Electronic Speculum PBS specially.

2) Electronic Speculum perfusate:

Perfusate used by Electronic Speculum is the glutaraldehyde of 2.5%；

Compound method is: take the PBS400ml of 0.2M, adds the glutaraldehyde 100ml of 25%, then with tri-distilled water constant volume to 1L, after filtration, puts into 4 DEG C of preservations.So the pH value pH instrument of the perfusate of preparation is determined as 7.24.The perfusate of 1L probably can 4 rats of perfusion.

3) glutaraldehyde of 2.5%

Table 2. glutaraldehyde compound method

Note:

The amount of (1) 1 25% glutaraldehyde is 10ml, prepares and is placed on 4 DEG C of preservations afterwards；

(2) the glutaraldehyde consumption of the 3% of each tissue is 1.5ml-2ml.

4) osmic acid original solution (2% osmic acid)

Needing during configuration to carry out in fume hood, band double gloves and mask, every gram of osmic acid adds 50mlDDW.

Clean bottle, hard thing puts glove, breaks peace into pieces and cuts open bottle.This solution is configured by the teacher of Electron Microscopy Room.

5) osmic acid solution of 1%:

The osmic acid stock solution of 2% mixes with 0.2MPBS1:1, after preparing, and 4 DEG C of preservations；The amount of the osmic acid of the 1% of each tissue is 1ml, is placed in the EP pipe of 1.5ml, wraps with tinfoil during use.

6) sucrose flushing liquor: (molecular weight is 342.2)

Table 3. sucrose flushing liquor compound method

After preparing, 4 DEG C of preservations；The amount of sucrose flushing liquor is each tissue 3-4ml.Noticing that the sucrose flushing liquor having prepared of a specified duration is easy to become mildewed, in time, notes observing, if become mildewed, and the flushing liquor more renewed immediately.7) 100% acetone (noting having taken off water)

One day before use, 100% acetone, it should be preceded by water absorbing agent in advance, for instance anhydrous sodium sulfate, anhydrous K CO3, CUSO4 etc..It is most commonly used that anhydrous sodium sulfate at ordinary times.Add the anhydrous sodium sulfate of 1/5 bottle, mixing tens times of turning upside down, it is ensured that fully mix, then stand at least one evening, just can use.Strict moistureproof, it is possible to seal with sealed membrane.

2. experiment flow

1) modeling perfusion and sample treatment:

(1) by rat anesthesia modeling, every rat anesthesia interval time is about 1h；

(2) during modeling, perfusate and normal saline are ready to, changed after liquid every time, pipeline will be tested whether unimpeded, first try perfusate, the speed of perfusate be transferred to 1 second one, try normal saline again, being rinsed by perfusate with normal saline, it is too big that the speed of normal saline is not opened；

(3), after modeling terminates, quickly rat is transferred in the perfusion fume hood of Electronic Speculum；

(4) it is placed in ceramic whiteware dish, opens rapidly breast, insert the needle on the left of the apex of the heart, note necessarily can not inserting too deep, otherwise insert too deeply easily to be inserted through interventricular septum, be inserted in right ventricle and go, notice that the direction of pin is preferably probably parallel with the longitudinal axis of heart, make solution easily flow out.Then having eye scissors to cut off right auricle, open the control valve of conduit bottom, first fill normal saline, the speed of normal saline is not opened too big；

(5) in the process filling normal saline, it is possible to the muscle bone near right auricle and fur are pushed aside, it is impossible to the blood of right auricle flows out to have thing to stop, for the purpose of seeing that blood flow flows out from auricle.In the process of perfusion normal saline, if there is right ventricle excessive bulkiness, turn out pin and insert too deep, be inserted through interventricular septum and reached right ventricle, it should syringe needle is withdrawn；

(6) all expand if there is whole heart, it is possible to problem be right auricle cut off inadequate, it should again wound is opened greatly.Even if noting the situation that whole heart all expands do not occur, also should again being opened greatly by the wound of right auricle in stage of Reperfu-sion normal saline, cutting because only having cut one before, wound is certainly only small；

(7) note observing the liquid flowed out from left ventricle, if it find that liquid no longer has blood, just by where three-way cock reaches perfusate, regulate the speed.Fixing conduit, the time of perfusion is 40-60min；

(8) the successful mark of perfusion is, time perfusion terminates, it is all stiff that whole mouse whole body includes tail, and extremity are all white；

(9) liver is scaled off, carry out repairing block.Cut the thick lobe of the liver of about 5mm along lobe of the liver longitudinal section, put in the EP pipe equipped with 2.5% glutaraldehyde 10ml, spare scan Electronic Speculum.It is put into 4 DEG C of refrigerator overnight.

2) tissue is prepared

(1) being taken out from the glutaraldehyde of 2.5% by tissue, rinse 3 times with sucrose, each 15min, 4 DEG C of refrigerators, each EP pipe adds 1ml sucrose solution；

(2) osmic acid is fixed: room temperature 2.5h, by the EP pipe tinfoil lucifuge of 1.5ml, is placed in 4 DEG C；

(3) 0.2MPBS rinses 3 times, each 5min；

(4) acetone serial dehydration: dehydration 10min in 50% acetone, transfer to dehydration 15min in 70% acetone, transfer to dehydration 15min in 90% acetone, transfer to dehydration 10min in 100% acetone I, transfer to dehydration 10min in 100% acetone II, transfer to dehydration 10min in 100% acetone III.Dehydrating operations should be placed in the angle that dehumidifier is formed, and notes that tweezers are with special, rocking frequently in dehydration, at the solution of waiting time preparation preimpregnation.Note: after 100% acetone, want strict drying, moistureproof；

(5) tissue is put into CO₂Critical point drying instrument is dry completely to tissue；

(6) tissue is split into fritter, adheres on sample carrier, metal spraying, observe under scanning electron microscope.

The expression of 5.5 flow cytomery Peripheral blood cells adhesion molecule CD11b, CD18

(1), after Reperfu-sion 120min, rat is through abdominal aortic blood, anticoagulant heparin (20unit/ml whole blood).Anticoagulated whole blood is stored in 4 DEG C of refrigerators, for the mensuration of GA molecule within 6h；

(2) traget antibody, takes Falcon pipe, adds anticoagulated whole blood 50 μ l, add GA molecular antibody anti-CD18antibody or anti-CD11bantibody of 1 μ lFITC labelling, be set to positive test pipe；

(3) separately take Falcon pipe, add with experimental group anticoagulated whole blood 50 μ l, add the isotype control Ab that the GA molecular antibody of 1 μ lFITC labelling is corresponding, be set to negative control pipe；

(4), after antibody adds, room temperature lucifuge hatches 20min；

(5) haemolysis, adds 1ml hemolysin, room temperature lucifuge haemolysis 10min in each Falcon pipe.After haemolysis 10min, 1500rpm is centrifuged 5min, abandons supernatant；

(6) washing, adds 1mlPBS (1 ×, pH7.4) in each Falcon pipe, and hands bullet mixes, and 1500rpm is centrifuged 5min, abandons supernatant.Each Falcon pipe adds 300 μ lPBS (1 ×, pH7.4), machine in preparation；

(7) open flow cytometer by boot program, preheat 5～10min；

(8) debugging machine, it is determined that experiment parameter:

From desktop ACQ file, open GA molecule obtain template；

Selecting " ConnecttoCytometer " command option from " Acquire " menu, connect flow cytometer, machine ejects " AcquisitionControl " controller；

From " Cytometer " menu, select " InstrumentSettings " command option, select GA molecular assay basic voltage；

Select " Acquisition&Storage " sets from " Acquire " menu and obtain population as 5000ofG1=R1；

(9) after machine is debugged, upper negative control pipe, selects to obtain at a high speed, adjusts R1 detection door and encloses granulocyte group, from " Cytometer " menu, select " Detectors/Amps " command option, adjust FL-1 voltage and make cell peak between the 100-101 of fluorogram transverse axis；

(10) changing positive test pipe, select to obtain at a high speed, adjust R1 detection door and enclose granulocyte group, collect 5000 granulocytes in door, result represents with cell average fluorescent strength.

(11) data analysis, selects " new " command option from File menu, occurs obtaining masterplate.Rectangular histogram is drawn in obtaining masterplate, dialog box occurs, select Analysis, selecting the data needing to analyze after obtaining in SelectFile, transverse axis selects FL-1, FL-1-count rectangular histogram occurs, negative control result is irised out (near 101) negative district, selecting " QuatStat " order in Stat menu, result is analyzed in output, result is preserved or prints；

(12) flow cytometer is closed by shutdown programm.

5.6 rat peripheral blood ALT, AST, TNF-α, the mensuration of IL-1 β, MCP-1

5.6.1. the mensuration of rat peripheral blood ALT

(1), after Reperfu-sion 120min, rat is through abdominal aortic blood, anticoagulant heparin (20unit/ml whole blood).1000rpm, takes supernatant after centrifugal 5min, and subpackage is saved in-80 DEG C；

(2) take out blood plasma, be put on ice, thaw at room temperature；

(3) take out test kit, ALTAssayBuffer is placed in room temperature, wait melting and recover to room temperature；

(4) preparation standard substance: the 100nmole/ μ lPyruvateStandard of 10 μ l is diluted to the ALTAssayBuffer of 990 μ l；

(5) preparation MasterReactionMix, calculate sample number+standard substance number+positive control+1, ALTAssayBuffer86 μ l is needed according to each sample, FluorescentPeroxidaseSubstrate2 μ l, ALTEnzymeMix2 μ l, the amount preparation of ALTSubstrate10 μ l, fully after mixing, lucifuge is placed；

(6) take 96 clean orifice plates, add the PyruvateStandard of the 1nmole/ μ l of standard substance 0,2,4,6,8,10 μ l in order, then with ALTAssayBuffer polishing to 20 μ l；

(7) positive control of 5 μ l and 15 μ lALTAssayBuffer are added；

(8) good according to schedule order adds sample, every hole 10 μ l, in the sample of every hole, adds 10 μ lALTAssayBuffer with the volley of rifle fire rapidly；

(9) there are sample, standard substance all rapidly with the volley of rifle fire, the hole of positive control add the MasterReactionMix of 100 μ l, shakes 96 orifice plates gently, wrap lucifuge with masking foil rapidly；

(10) opening microplate reader, set absorbance as 570nm, established standards grade is put and the concentration of correspondence, sets all wells；

After (11) 96 orifice plate concussion 2-3min, putting into microplate reader and read first time absorbance and concentration, record saves as T_initial；

(12) 96 orifice plates being put into 37 DEG C of baking boxs, note lucifuge, every 5min reading once, to the maximum absorbance value of sample near or above the absorbance of highest standard product, terminates reading, is designated as T_final.；

(13) evaluation:

ALTActivity=B × SampleDilutionFactor/ (T_final–T_initial)×V

B=T_initialAnd T_finalBetween change concentration

T_initialReading duration=first time

T_final=last the reading duration

V=adds the sample size (mL) in every hole

5.6.2. the mensuration of rat peripheral blood AST

(1), after Reperfu-sion 120min, rat is through abdominal aortic blood, anticoagulant heparin (20unit/ml whole blood).1000rpm, takes supernatant after centrifugal 5min, and subpackage is saved in-80 DEG C；

(2) take out blood plasma, be put on ice, thaw at room temperature；

(3) take out test kit, ASTAssayBuffer is placed in room temperature, wait melting and recover to room temperature；

(4) preparation standard substance: the 0.1MGlutamateStandardsolution of 10 μ l is diluted to the ASTAssayBuffer of 990 μ l, produce 1mM standard substance；

(5) preparation MasterReactionMix, calculate sample number+standard substance number+positive control+1, ASTAssayBuffer80 μ l is needed according to each sample, ASTEnzymeMix2 μ l, ASTDeveloper8 μ l, the amount preparation of ASTSubstrate10 μ l, fully after mixing, lucifuge is placed；

(6) take 96 clean orifice plates, add the standard substance of the 1mM of standard substance 0,2,4,6,8,10 μ l in order, then with ASTAssayBuffer polishing to 50 μ l；

(7) positive control of 5 μ l and 45 μ lASTAssayBuffer are added；

(8) good according to schedule order adds sample, every hole 40 μ l, in the sample of every hole, adds 10 μ lASTAssayBuffer with the volley of rifle fire rapidly；

(9) there are sample, standard substance all rapidly with the volley of rifle fire, the hole of positive control add the MasterReactionMix of 100 μ l, shakes 96 orifice plates gently, wrap lucifuge with masking foil rapidly, 96 orifice plates are put into 37 DEG C of baking boxs；

(10) opening microplate reader, set absorbance as 570nm, established standards grade is put and the concentration of correspondence, sets all wells；

(11) after 2-3min, putting into microplate reader and read first time absorbance and concentration, record saves as T_initial；

(12) 96 orifice plates being put into 37 DEG C of baking boxs, note lucifuge, every 5min reading once, to the maximum absorbance value of sample near or above the absorbance of highest standard product, terminates reading, is designated as T_final.；

(13) evaluation:

ASTActivity=B × SampleDilutionFactor/ (ReactionTime) × V

B=T_initialAnd T_finalBetween change concentration

ReactionTime=T_initialAnd T_finalBetween time

V=adds the sample size (mL) in every hole

5.6.3. the mensuration of rat peripheral blood TNF-α

(1), after Reperfu-sion 120min, rat is through abdominal aortic blood, anticoagulant heparin (20unit/ml whole blood).1000rpm, takes supernatant after centrifugal 5min, and subpackage is saved in-80 DEG C；

(2) take out blood plasma, be put on ice, thaw at room temperature；

(3) various reagent is prepared to specifications, standard substance, positive control and dilute sample 1 times；

(4) calculate sample number+standard substance number+positive control+1, take out 96 appropriate orifice plates, remaining plank is put into packaging bag；

(5) in each hole, add the AssayDiluentRD1-41 of 50 μ l rapidly；

(6) being sequentially added into the standard substance of 50 μ l, positive control, sample, shake plank gently, mix 1min, then wrap plank with sealed membrane, room temperature stands 2h；

(7) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(8) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(9) plank is wrapped with sealed membrane after adding 100 μ lTNF-α Conjugate in every hole, and room temperature stands 2h；

(10) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(11) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(12) every hole adds the SubstrateSolution of 100 μ l, wraps plank with sealed membrane, and room temperature stands 30min, notes lucifuge；

(13) every hole adds the StopSolution of 100 μ l, shakes plank gently, fully mixes liquid；

(14) opening microplate reader, set absorbance as 450nm, established standards grade is put and the concentration of correspondence, sets all wells.Absorbance and concentration is read in 30min；

(15) evaluation: by gained concentration value × 2 (extension rate).

5.6.4. the mensuration of rat peripheral blood IL-1 β

(1), after Reperfu-sion 120min, rat is through abdominal aortic blood, anticoagulant heparin (20unit/ml whole blood).1000rpm, takes supernatant after centrifugal 5min, and subpackage is saved in-80 DEG C；

(2) take out blood plasma, be put on ice, thaw at room temperature；

(3) various reagent is prepared to specifications, standard substance, positive control and dilute sample 1 times；

(4) calculate sample number+standard substance number+positive control+1, take out 96 appropriate orifice plates, remaining plank is put into packaging bag；

(5) in each hole, add the AssayDiluentRD1-21 of 50 μ l rapidly；

(6) being sequentially added into the standard substance of 50 μ l, positive control, sample, shake plank gently, mix 1min, then wrap plank with sealed membrane, room temperature stands 2h；

(7) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(8) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(9) plank is wrapped with sealed membrane after adding 100 μ lIL-1 β Conjugate in every hole, and room temperature stands 2h；

(10) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(11) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(12) every hole adds the SubstrateSolution of 100 μ l, wraps plank with sealed membrane, and room temperature stands 30min, notes lucifuge；

(13) every hole adds the StopSolution of 100 μ l, shakes plank gently, fully mixes liquid；

(14) opening microplate reader, set absorbance as 450nm, established standards grade is put and the concentration of correspondence, sets all wells.Absorbance and concentration is read in 30min；

(15) evaluation: by gained concentration value × 2 (extension rate).

5.6.5. the mensuration of rat peripheral blood MCP-1

(1), after Reperfu-sion 120min, rat is through abdominal aortic blood, anticoagulant heparin (20unit/ml whole blood).1000rpm, takes supernatant after centrifugal 5min, and subpackage is saved in-80 DEG C；

(2) take out blood plasma, be put on ice, thaw at room temperature；

(3) various reagent is prepared to specifications, standard substance, positive control and dilute sample 5 times；

(4) calculate sample number+standard substance number+positive control+1, take out 96 appropriate orifice plates, remaining plank is put into packaging bag；

(5) in each hole, add the AssayDiluentRD1W of 50 μ l rapidly；

(6) being sequentially added into the standard substance of 50 μ l, positive control, sample, shake plank gently, mix 1min, then wrap plank with sealed membrane, room temperature stands 2h；

(7) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(8) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(9) plank is wrapped with sealed membrane after adding 100 μ lMCP-1Conjugate in every hole, and room temperature stands 2h；

(10) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(11) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(12) every hole adds the SubstrateSolution of 100 μ l, wraps plank with sealed membrane, and room temperature stands 30min, notes lucifuge；

(13) every hole adds the StopSolution of 100 μ l, shakes plank gently, fully mixes liquid；

(14) opening microplate reader, set absorbance as 450nm, established standards grade is put and the concentration of correspondence, sets all wells.Absorbance and concentration is read in 30min；

(15) evaluation: by gained concentration value × 5 (extension rate).

5.7 rat liver tissue TNF-α, IL-1 β, MCP-1, MPO, TACE activity, Caspase8, MDA, 8-OHdG, H2O2/ peroxidase, catalase, SOD, glutathion reductase, glutathione oxidase, Complex I, II, IV, V activity, the mensuration of NAD+/NADH and DNA extraction

5.7.1. the extraction of whole protein is organized

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate, 10 × RIPA lysate according to 1:100 add Cocktail protease inhibitor；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 20ml in organizing according to 1g, be put on ice for, fully crack；

(7) weigh the DMSF of 0.0174g, be dissolved in the isopropanol of 1ml, mixing；

(8) add the DMSF of 100 μ l in organizing according to 1g, and carry out ultrasonic；

(9) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(10) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 30min；

(11) supernatant 10 μ l is taken for protein quantification；Take supernatant 100 μ l, many parts-80 DEG C preservations of subpackage；Take supernatant 100 μ l, add 5 × sample-loading buffer 25 μ l, boiling water boils 20min.Put into-80 DEG C of preservations.

5.7.2. the mensuration of protein concentration

(1) weigh BSA to add in tri-distilled water, be completely dissolved into the protein standard substance of 2.0mg/ml, be 1.0mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml with tri-distilled water dilution；

(2) dilute sample, heart tissue generally dilutes 20 times, and final volume is 100 μ l, namely adds the tri-distilled water of 95 μ l in the sample of 5 μ l；

(3) add 1 volume BCA reagent B (50:1) by 50 volume BCA reagent A and prepare appropriate BCA working solution, fully mix；

(4) in the pipe of each sample, all add the protein quantification liquid of 200 μ l, mixing, put into 20min in 37 DEG C of incubators, open microplate reader preheating；

Each hole of (5) 96 orifice plates adds the solution after 200 μ l are hatched；

(6) measure at microplate reader 560nm place.Protein concentration is calculated according to standard curve.

5.7.3. the mensuration of liver tissues of rats TNF-α

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) various reagent is prepared to specifications, standard substance, positive control and dilute sample 1 times；

(3) calculate sample number+standard substance number+positive control+1, take out 96 appropriate orifice plates, remaining plank is put into packaging bag；

(4) in each hole, add the AssayDiluentRD1-41 of 50 μ l rapidly；

(5) being sequentially added into the standard substance of 50 μ l, positive control, sample, shake plank gently, mix 1min, then wrap plank with sealed membrane, room temperature stands 2h；

(6) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(7) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(8) plank is wrapped with sealed membrane after adding 100 μ lTNF-α Conjugate in every hole, and room temperature stands 2h；

(9) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(10) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(11) every hole adds the SubstrateSolution of 100 μ l, wraps plank with sealed membrane, and room temperature stands 30min, notes lucifuge；

(12) every hole adds the StopSolution of 100 μ l, shakes plank gently, fully mixes liquid；

(13) opening microplate reader, set absorbance as 450nm, established standards grade is put and the concentration of correspondence, sets all wells.Absorbance and concentration is read in 30min；

(14) evaluation: by gained concentration value × 2 (extension rate), and divided by protein concentration.

5.7.4. the mensuration of liver tissues of rats IL-1 β

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) various reagent is prepared to specifications, standard substance, positive control and dilute sample 1 times；

(3) calculate sample number+standard substance number+positive control+1, take out 96 appropriate orifice plates, remaining plank is put into packaging bag；

(4) in each hole, add the AssayDiluentRD1-21 of 50 μ l rapidly；

(5) being sequentially added into the standard substance of 50 μ l, positive control, sample, shake plank gently, mix 1min, then wrap plank with sealed membrane, room temperature stands 2h；

(6) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(7) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(8) plank is wrapped with sealed membrane after adding 100 μ lIL-1 β Conjugate in every hole, and room temperature stands 2h；

(9) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(10) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(11) every hole adds the SubstrateSolution of 100 μ l, wraps plank with sealed membrane, and room temperature stands 30min, notes lucifuge；

(12) every hole adds the StopSolution of 100 μ l, shakes plank gently, fully mixes liquid；

(13) opening microplate reader, set absorbance as 450nm, established standards grade is put and the concentration of correspondence, sets all wells.Absorbance and concentration is read in 30min；

(14) evaluation: by gained concentration value × 7 (extension rate), and divided by protein concentration.

5.7.5. the mensuration of liver tissues of rats MCP-1

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) various reagent is prepared to specifications, standard substance, positive control and dilute sample 5 times；

(3) calculate sample number+standard substance number+positive control+1, take out 96 appropriate orifice plates, remaining plank is put into packaging bag；

(4) in each hole, add the AssayDiluentRD1W of 50 μ l rapidly；

(5) being sequentially added into the standard substance of 50 μ l, positive control, sample, shake plank gently, mix 1min, then wrap plank with sealed membrane, room temperature stands 2h；

(6) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(7) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(8) plank is wrapped with sealed membrane after adding 100 μ lMCP-1Conjugate in every hole, and room temperature stands 2h；

(9) the preparation total number+1 of WashBuffer:(sample) × 5 times × 400 μ l；

(10) outwelling the liquid in hole, every hole adds the WashBuffer of 400 μ l, then outwells, quadruplication, after being outwelled by liquid for the last time, knocks all visible water droplets；

(11) every hole adds the SubstrateSolution of 100 μ l, wraps plank with sealed membrane, and room temperature stands 30min, notes lucifuge；

(12) every hole adds the StopSolution of 100 μ l, shakes plank gently, fully mixes liquid；

(13) opening microplate reader, set absorbance as 450nm, established standards grade is put and the concentration of correspondence, sets all wells.Absorbance and concentration is read in 30min；

(14) evaluation: by gained concentration value × 4 (extension rate), and divided by protein concentration.

5.7.6. the mensuration of liver tissues of rats MPO

(1) ELISA Plate is taken out according to Blank (water of 50 μ l), standard substance concentration (50 μ l) from low to high, it is added in the hole of blank, (2) take out-80 DEG C of whole proteins preserved, be put on ice, after thaw at room temperature, be sequentially added into sample；

(3) in each hole, the enzyme labelling liquid of the Enzymeconjugate of 100 μ l redness bottles is added with the volley of rifle fire；

(4) seal with sealed membrane, ELISA Plate is placed in 37 DEG C and hatches 1h；

(5) buffer dilution being become 1 × working solution, ratio is 1:100；

(6) fully ELISA Plate is washed 5 times, every time with the buffer of 300 μ l, for the last time water is all patted dry；

(7) each 50 μ l of A, B two liquid that each hole adds, then first seal with preservative film, then encase with tinfoil, and lucifuge puts into 37 DEG C, take out after 20min；

(8) each hole adds 50 μ l stop buffers, terminates reaction；

(9) 450nm place reads light absorption value, does standard curve, carries out data analysis, and divided by protein concentration.

5.7.7. the mensuration of liver tissues of rats TACE activity

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) dilute sample 50 times, take out the reagent in test kit and recover to room temperature；

(3) calculate sample number+standard substance number+positive control+1, prepare TACEsubstratesolution, prepare standard substance；

(4) every hole adds sample and the standard substance of 50 μ l dilutions；

(5) every hole adds the SubstrateSolution of 50 μ l, fully mixes；

(6) reading is started after 3min, Ex/Em=490nm/520nm, once, 1h, carries out data analysis to every 5min reading altogether afterwards, and divided by protein concentration.

5.7.8. the mensuration of liver tissues of rats Caspase-8 activity

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) upper sample size is 200 μ g, calculates gained volume according to protein concentration, with CellLysisBuffer dilute sample to 50 μ l；

(3) mixed liquor of enough ReactionBuffer and DTT is prepared；

(4) every hole adds the mixed liquor of ReactionBuffer and the DTT of 50 μ l；

(5) every hole adds the 4Mmietd-p-NAsubsrate of 5 μ l, mixes latter 37 DEG C and hatches 1-2h；

(6) 400nm place reads light absorption value, carries out data analysis.

5.7.9. the mensuration of liver tissues of rats MDA

(1) ELISA Plate is taken out according to Blank (water of 50 μ l), standard substance concentration (50 μ l) from low to high, it is added in the hole of blank, (2) take out-80 DEG C of whole proteins preserved, be put on ice, after thaw at room temperature, be sequentially added into sample；

(3) in each hole, the enzyme labelling liquid of the Enzymeconjugate of 100 μ l redness bottles is added with the volley of rifle fire；

(4) seal with sealed membrane, ELISA Plate is placed in 37 DEG C and hatches 1h；

(5) buffer dilution being become 1 × working solution, ratio is 1:100；

(6) fully ELISA Plate is washed 5 times, every time with the buffer of 300 μ l, for the last time water is all patted dry；

(7) each 50 μ l of A, B two liquid that each hole adds, then first seal with preservative film, then encase with tinfoil, and lucifuge puts into 37 DEG C, take out after 20min；

(8) each hole adds 50 μ l stop buffers, terminates reaction；

(9) 450nm place reads light absorption value, does standard curve, carries out data analysis, and divided by protein concentration.

5.7.10. the extraction of liver tissues of rats DNA

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min, now opens water-bath, and design temperature is 56 DEG C；

(4) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh that < 25mg organizes；

(5) be rapidly added 180 μ lALT, 20 μ lProteinaseK, Vortex, mixing, put into 56 DEG C of water-baths, 1-3h, until dissolving, frequently take out Vortex；

(6) simply centrifugal, must the time very short, otherwise have precipitation formed, now water-bath is adjusted to 70 DEG C；

(7) add 200 μ lAL, Vortex15s, put into 70 DEG C of water-baths, 10min；

(8) simply centrifugal, add 200 μ l ethanol, Vortex15s, simply centrifugal；

(9) sample is forwarded Minispincolumn to, cover lid, centrifugal, 8000rpm, 1min, Minispincolumn is put into new collectiontube；

(10) 500 μ lAW1 are added, centrifugal, 8000rpm, 1min, Minispincolumn is put into new collectiontube；

(11) open Minispincolumn, add 500 μ lAW2, centrifugal, 14000rpm, 3min；

(12) Minispincolumn is put into new collectiontube, centrifugal, 14000rpm, 1min；

(13) Minispincolumn being put into new collectiontube, add 200 μ lAE, room temperature waits 1min, is then centrifuged for, 8000rpm, 1min, preserves the liquid in collecting pipe；

(14) Minispincolumn being put into new collectiontube, add 200 μ lAE, room temperature waits 1min, is then centrifuged for, 8000rpm, 3min, preserves the liquid in collecting pipe；

(15) by, after two pipe liquid blendings, taking each group of DNA sample 5 μ l respectively, add 195 μ l tri-distilled waters, fully after mixing, record the absorbance at 260nm/280nm, calculate purity and the concentration of gained sample.

5.7.11. the mensuration of liver tissues of rats 8-OHdG

(1) experiment the previous day: with the appropriate 8-OHdGConjugate1mg/mLto1 μ g/mL of PBS dilution, calculating all of sample and standard substance, take appropriate plank, every hole adds the 8-OHdGConjugate100 μ l of 1 μ g/mL, cover sealed membrane, put into 4 DEG C overnight；

(2) second days, outwell the 8-OHdGConjugate in hole, rinse once with tri-distilled water, and on paper, knock all of visible water droplet；

(3) every hole adds 200 μ lAssayDiluent, covers sealed membrane, and room temperature stands 1h；

(4) plank added with 200 μ lAssayDiluent is put into 4 DEG C standby；

(5) opening water-bath, design temperature is 95 DEG C；

(6) take the DNA sample 60 μ l of extraction, put into 95 DEG C of water-bath 5min, rapidly sample is put into cooled on ice；

(7) take the sample 50 μ l in (6), add the nucleaseP1 of 10 μ l, mixing, 37 DEG C, 2h；

(8) take 10 μ l100mMTris, pH7.5 and add the sample in (7), mix, 37 DEG C, 1h；

(9) centrifugal, 8000rpm, 5min, take supernatant, standby；

(10) preparation standard substance, by (9) gained diluted sample 5 times, by 500 times and the 1000 times dilutions of anti-to anti-8-OHdG antibody and two difference, prepare 1 × WashBuffer；

(11) plank in 4 DEG C is taken out, outwell liquid in hole, in hole, add standard substance and sample, place on vertical shaking table, room temperature 10min；

(12) in hole, add 50 μ lanti-8-OHdG antibody of dilution, place on vertical shaking table, room temperature 1h；

(13) every hole adds 250 μ l1 × WashBuffer, repeats 3 times, knocks all visible water droplets in hole for the last time；

(14) the 50 μ l bis-adding dilution in hole resist, and place on vertical shaking table, room temperature 1h；

(15) every hole adds 250 μ l1 × WashBuffer, repeats 3 times, knocks all visible water droplets in hole for the last time；

(16) every hole adds the recovery SubstrateSolution100 μ l to room temperature, places on vertical shaking table, room temperature, the change of liquid color in viewing hole, if color is deepened rapidly, then terminates reaction；

(17) every hole adds 100 μ LofStopSolution termination reactions, is reading under 450nm in OD value, and records data, when processing data, by gained concentration value × 5 (extension rate).

5.7.12. liver tissues of rats H₂O₂Mensuration

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature, takes out test kit, recovers to room temperature；

(2) to specifications dilution AssayBuffer to 1 ×, prepare ADHP/HRPWorkingSolution, standard substance, positive control and dilute sample 3 times；

(3) every hole adds the sample of 50 μ l dilutions, standard substance and positive control；

(4) every hole adds the ADHP/HRPWorkingSolution of 50 μ l, fully mixes, room temperature lucifuge, reacts 30min；

(5) reading, Ex/Em=530-570nm/590-600nm, result of calculation is by comparing the RFU of sample, and divided by the protein concentration of sample, is multiplied by extension rate.

5.7.12. the mensuration of liver tissues of rats peroxidase (PO)

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature, takes out test kit, recovers to room temperature；

(2) to specifications dilution AssayBuffer to 1 ×, prepare ADHP/H2O2WorkingSolution, standard substance, positive control and dilute sample 3 times；

(3) every hole adds the sample of 50 μ l dilutions, standard substance and positive control；

(4) every hole adds the ADHP/H2O2WorkingSolution of 50 μ l, fully mixes, room temperature lucifuge, reacts 30min；

(5) reading, Ex/Em=530-570nm/590-600nm, result of calculation is by comparing the RFU of sample, and divided by the protein concentration of sample, is multiplied by extension rate.

5.7.13. the mensuration of liver tissues of rats catalase (CAT)

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate: 50mMPotassiumPhosphate, PH7.0,1mMEDTA；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 10ml in organizing according to 1g, be put on ice for, fully crack；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 20min, takes supernatant；

(9) prepare various reagent as requested, prepare standard substance, by diluted sample 2 times；

(10) standard substance of 20 μ l, AssayBuffer, control or sample is added according to the order of design；

(11) every hole adds 100 μ lAssayBuffer, 30 μ lmethanol；

(12) every hole adds 20 μ lHydrogenPeroxide, fully mixes, covers sealed membrane, room temperature shaker 20min；

(13) every hole adds 30 μ lPotassiumHydroxide, and 30 μ lCatalasePurpald fully mix, cover sealed membrane, room temperature shaker 10min；

(14) every hole adds 10 μ lCatalasePotassiumPeriodate, fully mixes, covers sealed membrane, room temperature shaker 5min；

(15) it is reading under 540nm in OD value, and records data；

(16) CATActivity=(μM ofsample/20min) × Sampledilution=nmol/min/ml

(17) each sample is carried out protein quantification, and by (16) acquired results divided by protein concentration.

5.7.14. the mensuration of the super oxygen dismutase (SOD) of liver tissues of rats

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate: 20mMHEPESbuffer, PH7.2,1mMEGTA, 70mMsucrose；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 10ml in organizing according to 1g, be put on ice for, fully crack；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 20min, takes supernatant；

(9) preparing various reagent as requested, standard substance, by diluted sample 2 times；

(10) standard substance or the sample of 10 μ l is added according to the order of design；

(11) every hole adds 200 μ lRadicalDetector；

(12) every hole adds 20 μ lXanthineOxidase, fully mixes, covers sealed membrane, room temperature shaker 20min；

(13) it is reading under 450nm in OD value, and records data；

(14)

SOD (U/ml)=[(sampleLR-y-intercept)/slope] × (0.23ml/0.01ml) × Sampledilution

(15) each sample is carried out protein quantification, and by (14) acquired results divided by protein concentration.

5.7.15. the mensuration of liver tissues of rats glutathion reductase (GPx)

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate: 50mMTris-HCL, PH7.5,5mMEDTA, 1mMDTT；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 10ml in organizing according to 1g, be put on ice for, fully crack；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 20min, takes supernatant；

(9) various reagent is prepared as requested, by diluted sample 2 times；

(10) AssayBuffer, control or the sample of 20 μ l is added according to the order of design；

(11) every hole adds 100 μ lAssayBuffer, 50 μ lCo-SubstrateMixture；

(12) every hole adds 20 μ lCumeneHydroperoxide, fully mixes；

(13) being reading under 340nm in OD value, every 5min reading is once afterwards, reading 6 times altogether, and records data；

(14)

GPxActivity=△ A₃₄₀/min/0.00373μM^-1× (0.19ml/0.02ml) × Sampledilution=nmol/min/ml

(15) each sample is carried out protein quantification, and by (14) acquired results divided by protein concentration.

5.7.16. the mensuration of liver tissues of rats glutathione oxidase (GR)

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate: 50mMPotassiumPhosphate, PH7.5,1mMEDTA；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 10ml in organizing according to 1g, be put on ice for, fully crack；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 20min, takes supernatant；

(9) various reagent is prepared as requested；

(10) AssayBuffer, control or the sample of 20 μ l is added according to the order of design；

(11) every hole adds 100 μ lAssayBuffer, 20 μ lGSSG；

(12) every hole adds 50 μ lNADPH, fully mixes；

(13) being reading under 340nm in OD value, every 5min reading is once afterwards, reading 6 times altogether, and records data；

(14)

GPxActivity=△ A₃₄₀/min/0.00373μM^-1× (0.19ml/0.02ml) × Sampledilution=nmol/min/ml

(15) each sample is carried out protein quantification, and by (14) acquired results divided by protein concentration.

5.7.17. the mensuration of liver tissues of rats Complex I activity

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) according to quantitative result, all samples PBS being diluted to 5.5mg/ml, final volume mixes after being 80 μ l dilutions；

(3) adding the 10 × detergent of 8 μ l in the EP pipe be diluted to 5.5mg/ml, fully mix, now the concentration of sample is 5mg/ml；

(4) sample is placed on hatches 30min on ice；

(5) centrifugal, time centrifugal must trim, rotating speed is 14000rpm, and centrifugation time is 20min, takes supernatant；

(6) by 20 × buffer dilution become 1 × working solution；

(7) the proportions Incubationsolution of Blockingsolution:1 × Buffer=1:9, fully mixes；

(8) fully being mixed by sample 60 μ l and 240 μ lincubationsolution, take 200 μ l i.e. 200 μ g and join in 96 orifice plates, be individually added into 200 μ lincubationsolution and compare, avoid bubble to produce as far as possible, loading speed wants fast；

(9) 3h is at room temperature hatched；

(10) preparation Assaysolution, by 20 × buffer dilution become 1 × buffer working solution, the 1 × buffer of 6.67ml adds the 100 × Dye of 20 × NADH and the 67 μ l of 333 μ l；

(11) 96 orifice plates at room temperature stood are turned, remove whole liquid；

(12) every hole adds 300 μ lbuffer, washes three times, wants the whole liquid of evacuation every time；

(13) each hole adds the Assaysolution of 200 μ l, it is to avoid produce bubble；

(14) program of multi-functional microplate reader is set, as follows:

(15) data, statistical analysis are preserved.

5.7.18. the mensuration of liver tissues of rats Complex II activity

(1) take out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature；

(2) according to quantitative result, all samples PBS being diluted to 5.5mg/ml, final volume mixes after being 80 μ l dilutions；

(3) adding the 10 × detergent of 8 μ l in the EP pipe be diluted to 5.5mg/ml, fully mix, now the concentration of sample is 5mg/ml；

(4) sample is placed on hatches 30min on ice；

(5) centrifugal, time centrifugal must trim, rotating speed is 14000rpm, and centrifugation time is 20min, takes supernatant；

(6) by 20 × buffer dilution become 1 × working solution；

(7) the proportions Incubationsolution of Blockingsolution:1 × Buffer=1:9, fully mixes；

(8) fully being mixed by sample 2.4 μ l and 57.6 μ lincubationsolution, take 50 μ l i.e. 10 μ g and join in 96 orifice plates, be individually added into 50 μ lincubationsolution as comparison, avoid bubble to produce as far as possible, loading speed wants fast；

(9) 2h is at room temperature hatched；

(10) preparation Buffersolution, by 20 × buffer dilution become 1 × buffer working solution, prepare Activitysolution, i.e. Activitybuffer8.3ml, DCPIP83 μ l, Succinate167 μ l and Ubiquinone2 μ l fully mixes；

(11) 96 orifice plates at room temperature stood are turned, remove whole liquid；

(12) every hole adds 300 μ lbuffer, washes three times, wants the whole liquid of evacuation every time；

(13) each hole adds the lipidsolution of 40 μ l, it is to avoid produce bubble, incubated at room temperature 30min；

(14) not outwelling liquid, direct each hole adds the Activitysolution of 200 μ l, it is to avoid produce bubble；

(14) program of multi-functional microplate reader is set, as follows:

(15) data are preserved, statistical analysis:

Rate (mOD/min)=(Absorbance1-Absorbance2)/Time (min)

5.7.19. the mensuration of liver tissues of rats Complex IV activity

(1) taking out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature, 19 times of dilution Tube1 are Solution1；

(2) according to quantitative result, all samples Solution1 being diluted to 5.5mg/ml, final volume mixes after being 80 μ l dilutions；

(3) adding the 10 × detergent of 8 μ l in the EP pipe be diluted to 5.5mg/ml, fully mix, now the concentration of sample is 5mg/ml；

(4) sample is placed on hatches 30min on ice；

(5) centrifugal, time centrifugal must trim, rotating speed is 14000rpm, and centrifugation time is 20min, takes supernatant；

(6) fully being mixed by sample 15 μ l and 285 μ lSolution1, take 200 μ l i.e. 50 μ g and join in 96 orifice plates, be individually added into 200 μ lSolution1 and compare, avoid bubble to produce as far as possible, loading speed wants fast；

(7) 3h is at room temperature hatched；

(8) preparation AssaySolution, namely ReagentC333 μ l, Solution16.67ml fully mix；

(9) 96 orifice plates at room temperature stood are turned, remove whole liquid；

(10) every hole adds 300 μ lSolution1, washes three times, wants the whole liquid of evacuation every time；

(11) each hole adds the AssaySolution of 200 μ l；

(12) program of multi-functional microplate reader is set, as follows:

(15) data are preserved, statistical analysis:

Rate (mOD/min)=(Absorbance1-Absorbance2)/Time (min)

5.7.20. the mensuration of liver tissues of rats Complex V activity

(1) taking out-80 DEG C of whole proteins preserved, be put on ice, thaw at room temperature, 19 times of dilution TUBE1 are SOLUTION1；

(2) according to quantitative result, all samples SOLUTION1PBS being diluted to 5.5mg/ml, final volume mixes after being 80 μ l dilutions；

(3) adding the 10 × detergent of 8 μ l in the EP pipe be diluted to 5.5mg/ml, fully mix, now the concentration of sample is 5mg/ml；

(4) sample is placed on hatches 30min on ice；

(5) centrifugal, time centrifugal must trim, rotating speed is 14000rpm, and centrifugation time is 20min, takes supernatant；

(8) fully being mixed by sample 4.8 μ l and 55.2 μ lincubationsolution, take 50 μ l i.e. 20 μ g and join in 96 orifice plates, be individually added into 50 μ lSOLUTION1 and compare, avoid bubble to produce as far as possible, loading speed wants fast；

(9) 3h is at room temperature hatched；

(10) 96 orifice plates at room temperature stood are turned, remove whole liquid；

(12) every hole adds 300 μ lSOLUTION1, washes three times, wants the whole liquid of evacuation every time；

(13) each hole adds the LIPIDMIX of 40 μ l, it is to avoid produce bubble, incubated at room temperature 45min；

(14) not outwelling liquid, direct each hole adds the REAGENTMIX of 200 μ l, it is to avoid produce bubble；

(14) program of multi-functional microplate reader is set, as follows:

(15) data are preserved, statistical analysis:

Rate (mOD/min)=(Absorbance1-Absorbance2)/Time (min)

5.7.21. the mensuration of liver tissues of rats NAD+/NADH

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh, take about 20 tissues, after washing with PBS, add 400 μ lNADH/NADExtractionBuffer；

(5) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 3 times, each ultrasonic 5s, midfeather 15s；

(6) centrifugal, time centrifugal must trim, rotating speed is 14000rpm, and centrifugation time is 5min, takes supernatant, and this is NADt sample；

(7) open water-bath, be set as 60 DEG C, after reaching temperature, take the sample in 200 μ l (6), 60 DEG C of water-bath 30min；

(8) sample is placed on ice rapidly, and constantly rotate, it is to avoid precipitation produces, and this is NADH sample；

(9) NADH standard substance, NADCyclingEnzymeMix, NADHDeveloper, NADCyclingMix are prepared as requested；

(10) 50 μ l standard substance, NADt sample and NADH sample are added according to the order of design；

(11) every hole adds 100 μ lNADCyclingMix, fully mixes, room temperature reaction 5min, makes NAD be converted into NADH；

(12) every hole adds 10 μ lNADHDeveloper, room temperature reaction 1-4h, is 450nm place reading in OD value, and reading is divided by protein concentration；

(13) [NADt]=(Correctedabsorbance-y-intercept)/Slope

[NADH]=(Correctedabsorbance-y-intercept)/Slope

NAD/NADH=(NADt-NADH)/NADH

5.8 western blot analysis

5.8.1. the extraction of hepatic tissue whole protein

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate, 10 × RIPA lysate according to 1:100 add Cocktail protease inhibitor；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 20ml in organizing according to 1g, be put on ice for, fully crack；

(7) weigh the DMSF of 0.0174g, be dissolved in the isopropanol of 1ml, mixing；

(8) add the DMSF of 100 μ l in organizing according to 1g, and carry out ultrasonic；

(9) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(10) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 30min；

(11) supernatant 10 μ l is taken for protein quantification；Take supernatant 100 μ l, many parts-80 DEG C preservations of subpackage；Take supernatant 100 μ l, add 5 × sample-loading buffer 25 μ l, boiling water boils 20min.Put into-80 DEG C of preservations.

5.8.2. the extraction of hepatic tissue memebrane protein and slurry albumen

(1) prepare test kit packet point with karyon-endochylema-after birth and extract histone；

(2) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(3) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(4) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add 1.6mlCER reagent, add Cocktail protease inhibitor according to 1:100；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) centrifugal, time centrifugal must trim, rotating speed is 800g, and centrifugation time is 5min, takes supernatant, abandon precipitation after centrifugal；

(9) estimating supernatant volume, the MER adding 1/10 accumulated amount immediately mixes with supernatant, ice bath 5min；

(10) 4 DEG C are centrifuged, use maximum (top) speed 13000rpm, centrifugal 30min；

(11) taking out supernatant and transfer in new EP pipe, this is endochylema component；

(12) it is precipitated as membrane fraction, containing the mixture of cell membrane and cell sarcolemma.Brief centrifugation eliminates liquid again；

(13) precipitation adds the resuspended cell membrane of SuspensionBuffer with MER same volume, mixing；

(14) take slurry protein 10 0 μ l, add 5 × sample-loading buffer 25 μ l, add 5 × sample-loading buffer of 1/4 volume according to the amount of memebrane protein, boiling water boils 20min, puts into-80 DEG C of preservations.

(15) take slurry albumen and carry out protein quantification.

5.8.3. the extraction of hepatic tissue nucleoprotein and slurry albumen

(1) prepare test kit packet point with NE-PER caryoplasm albumen and extract histone；

(2) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(3) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(4) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add 2mlCER I reagent, add Cocktail protease inhibitor according to 1:100；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) Vortex mixes 15s, places 10min on ice；

(9) add CER II, CER I/CER II=100 × 2 μ l/5.5 μ l, Vortex and mix 5s, place 1min on ice；

(10) Vortex mixes 5s, and 4 DEG C are centrifuged, 16000g, 5min；

(11) taking supernatant, supernatant is slurry albumen；

(12) precipitation is added NER, CER I: NER=2:1；

(13) Vortex mixes 15s, totally 4 times, every minor tick 10min；

(14) 4 DEG C are centrifuged, 16000g, and 10min takes supernatant, for nucleoprotein；

(15) take slurry protein 10 0 μ l, add 5 × sample-loading buffer 25 μ l, add 5 × sample-loading buffer of 1/4 volume according to the amount of memebrane protein, boiling water boils 20min, puts into-80 DEG C of preservations.

5.8.4. the mensuration of protein concentration

(1) weigh BSA to add in tri-distilled water, be completely dissolved into the protein standard substance of 2.0mg/ml, be 1.0mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml with tri-distilled water dilution；

(2) dilute sample, heart tissue generally dilutes 20 times, and final volume is 100 μ l, namely adds the tri-distilled water of 95 μ l in the sample of 5 μ l；

(3) add 1 volume BCA reagent B (50:1) by 50 volume BCA reagent A and prepare appropriate BCA working solution, fully mix；

(4) in the pipe of each sample, all add the protein quantification liquid of 200 μ l, mixing, put into 20min in 37 DEG C of incubators, open microplate reader preheating；

Each hole of (5) 96 orifice plates adds the solution after 200 μ l are hatched；

(6) measure at microplate reader 560nm place.Protein concentration is calculated according to standard curve.

5.8.3. co-immunoprecipitation

(1) after Reperfu-sion 120min, take the hepatic tissue after perfusion and blot rapidly the water droplet on surface with filter paper, put into cryopreservation tube, after tightening lid, put into liquid nitrogen, from liquid nitrogen, take out cryopreservation tube after 30min, put into-80 DEG C of preservations；

(2) liver organization deposited in-80 DEG C of refrigerators is taken out, be placed on ice, make to be organized in and thaw on ice, general 200mg tissue scissors are shredded, is placed in tinfoil and wraps；

(3) after being wrapped by tissue tinfoil, carrying out group labelling, put in liquid nitrogen, the time can not be too short, about 20min；

(4) in being organized in liquid nitrogen during this period of time in, preparation solution lysate, 10 × RIPA lysate according to 1:100 add Cocktail protease inhibitor, 1:10 adds sodium butyrate；

(5) tissue is taken out from liquid nitrogen, smash, be organized in not melt be previously transferred in centrifuge tube by what pulverize, weigh；

(6) add the lysate of 20ml in organizing according to 1g, be put on ice for, fully crack；

(7) by the power adjustments of Ultrasound Instrument to AMP40%, carry out ultrasonic 5 times, each ultrasonic 8s, midfeather 15s；

(8) centrifugal, time centrifugal must trim, rotating speed is 13000rpm, and centrifugation time is 30min；

(9) supernatant 10 μ l is taken for protein quantification；Take the supernatant result according to protein quantification, subpackage albumen 1mg, and with lysate 10 times dilution before, subpackage dilutes many parts-80 DEG C preservations；Each sample subpackage 250 μ g, subpackage many parts ,-80 DEG C of preservations；

(10) take out four component dress samples and include each group, each 1mg and each 250 μ g: add antibody SDHA in each group of 1mg, NDUFA9, or MnSOD2 μ l, by four sample mix of each 250 μ g, it is subsequently adding 5 μ lIgG, fully puts into 4 DEG C of shaking tables after mixing, and frequently take out sample, put back to shaking table after rapid Vortex；

(11) prepare pearl: take appropriate ProteinA, put in 15ml centrifuge tube, add about 10ml tri-distilled water, put into 4 DEG C after mixing gently, overnight；

(12) ProteinA is washed: be centrifuged by the centrifuge tube in (11) 4 DEG C, 1000rpm, 1min, sucking-off tri-distilled water, adds 1mlPBS, centrifugal, 1000rpm, 1min, sucking-off PBS, add 1mlPBS, in triplicate, after last sucking-off PBS, estimate the volume of ProteinA, add equivalent PBS, put into after mixing 4 DEG C standby；

(13) each ProteinA processed adding 20 μ l or 30 μ l in the sample in (11), fully after mixing, puts into 4 DEG C of shaking table 2h.Note, before taking ProteinA, ProteinA and PBS fully need to be mixed；

(14) joining lysate, formula is with (4)；Join 1 × sample-loading buffer；Open electromagnetic oven to boil water to boiling；

(15) being centrifuged by 4 DEG C of the sample in (11), 1000rpm, 1min, sop up supernatant gently, respectively add the lysate of 750 μ l, 4 DEG C are centrifuged, 1000rpm, and 1min sops up supernatant gently, repeats twice, must inhale clean liquid for the last time；

(16) precipitation adds 50 μ l1 × sample-loading buffer, Vortex1min, boils 5min in boiling water；

(17) 4 DEG C are centrifuged; 12000rpm; standby loading after 15min, notes joining glue with common immunoblot experiment, notes during upper sample: one piece of glue is used for doing destination protein; respectively take 15 μ l and be mixed with the sample of 1 × sample-loading buffer; one piece of glue is used for doing acetylation, respectively takes 35 μ l and is mixed with the sample of 1 × sample-loading buffer, is sequentially added into sample; to suct clear during imbitition, not inhale precipitation.Operation afterwards, with common immunoblot experiment, carries out electrophoresis, transferring film, closes, add primary antibodie, and it is anti-to add two, exposure, son of developing a film, scanning, statistics.

5.8.5. glue and loading

(1) preparation 8%, 10%, 12%SDS-PAGE separation gel, after mixing inject edge strip thickness be 1.5mm two blocks of cleaned glass plates between, notes not having bubble, it adds a thin layer isopropanol, left at room temperature to condense；

(2), after glue to be separated is polymerized completely, blots isopropanol layer, and by tri-distilled water wash clean isopropanol layer, pour into 5% concentration glue, insert application of sample comb, left at room temperature.After glue solidifies completely, extract comb, with electrophoresis wash buffer well for several times；

(3) install electrophoresis tank, irrigate electrophoretic buffer, the sample handled well is added in well in a predetermined sequence.

5.8.6. electrophoresis, transferring film and dyeing

(1) power supply of gel-electrophoretic apparatus, initial voltage 80V are connected；

(2) leading edge of Bromophenol Blue dye improves voltage to 120V after entering separation gel upper limb, and continuation electrophoresis is the lower edge of separation gel until bromophenol blue is swum out；

(3), after electrophoresis terminates, take off PAGE glue and be soaked in transfering buffering liquid；

(4) take and the activation of glue an equal amount of pvdf membrane row methanol, together with filter paper and sponge be soaked in transfering buffering liquid more than 10 minutes stand-by；

(5) successively sponge, three layers filter paper, pvdf membrane, gel, three layers filter paper and sponge are put well in plastic splint with sandwich assay, catch up with the bubble between clean each layer；

(6) plastic splint is put into electricity turn trough, and with gel near negative pole, pvdf membrane, near positive pole, finally covers electricity turn trough, 200mA constant current transferring film 2-3 hour with trash ice；

(7) being soaked in by pvdf membrane in Ponceaux dye liquor, jolting is dyeed 10 minutes；

(8) after protein band occurs, with the Ponceaux background color on deionized water rinsing pvdf membrane；

(9) according to the Position Approximate of albumen Marker estimation purpose albumen, the pvdf membrane of clip corresponding site.

5.8.7. immune detection

(1) pvdf membrane is placed in confining liquid, jog 1 hour under room temperature；

(2) pvdf membrane is enclosed in plastic bag, add the primary antibodie diluted by confining liquid, remove all of bubble, 4 DEG C of shaken over night；

(3) film is washed with TBST, 10 minutes × 3 times；

(4) enclosing in plastic bag by pvdf membrane, add corresponding the two of the alkali phosphatase enzyme mark diluted by confining liquid and resist, remove all of bubble, room temperature is shaken 1-1.5 hour；

(5) film is washed with TBST, 15 minutes × 3 times.

5.8.7. chemiluminescence and exposure

(1) by chemical luminescence reagent kit ECL (Puli's Lay, Beijing, China) in two kinds of reagent respectively take 1ml, mix in the ratio of 1:1, film is dubbed on paper, remove TBST with part, the ECL prepared is dripped to front and the reverse side of film, darkroom is shaken film, makes liquid blending, hatch 3 minutes；

(2) by all bands, put in Puli's Lay lucifuge film magazine and expose, put into 2-3 and open film, compress lid；(3) taking out film, the X-ray with colour developing band is scanned, and uses KODAK exposure system (KODAKMedicalX-RayProcessor) to detect.The relative intensity of each band is normalized by the β-actin of its correspondence, and with using image analysis software Bio-RadQuantityOnesoftware (Bio-Rad, California, USA) semi-quantitative analysis is carried out, statistics is worth based on the value of β-actin, and result used is repeat the result of experiment for three times.

5.9 surface plasma resonances

5.9.1. solution preparation

(1) buffer (1L)

Table 21.SPR buffer

With tri-distilled water constant volume to 1L, titration pH value is to 7.4, and solution prepares afterwards with in the membrane filtration of 0.2 μm to aseptic clean bottle；

(2) sodium acetate

The sodium acetate solution of 10mM is prepared, with the sodium acetate solution of the different pH (4.0,4.5,5.0,5.5,6.0,7.0) of glacial acetic acid preparation with tri-distilled water；

(3) ethanolamine

Preparation 1M ethanolamine solutions, HCl is titrated to pH=8.0.

5.9.2. experimental procedure

(1) CM5 chip is put in surface plasma resonance instrument BIAcoreT200 (GEHealthcare, Buckinghamshire, UnitedKingdom)；

(2) experiment to check whether pipe is blocked before starting, whole passages is all chosen, and there will be a curve risen after Injection, Injection and and the delay that is raised between initial point probably at about 1s, if pipeline is blocked, the time of delay will extend；

(3) test is suitable for the Buffer of pH value, opens and selects RunSensor shortcut after software, isobase stably after, the Buffer of Sirt3 albumen pH4.0 is diluted, 1 μ l is dissolved in the Buffer of 100 μ l.Selecting the general 5 μ l of Injection, flow velocity is 10 μ l/min；

(4) being diluted by the Buffer of Sirt3 albumen pH4.5,1 μ l is dissolved in the Buffer of 100 μ l.Selecting the general 5 μ l of Injection, flow velocity is 10 μ l/min；

(5) being diluted by the Buffer of Sirt3 albumen pH5.0,1 μ l is dissolved in the Buffer of 100 μ l.Selecting the general 5 μ l of Injection, flow velocity is 10 μ l/min.Observe after Injection, it is desirable to:

1) curve gets off again up；

2) value that curve rises is the highest；

3) when rising value is equally high, selection pH value is bigger obtains buffer；Select pH4.5

After (6) Injection, curve has been gone up, it is necessary to waiting until that curve could measure next one buffer after returning to baseline, now passage is not activated, and albumen is connected with passage not by chemical bond, still can rinse and.But the speed declined when curve has is returned very slow, will wait considerable time, the method that the speed that curve declines increases is made to have two kinds

1) increase flow velocity to 10 μ l/min or 20 μ l/min etc., allow mobile phase be swept away by albumen；

2) configuration NaOH solution, Injection volume is 5 μ l, and flow velocity is 10 μ l/min；

(7) suitable pH4.5 sodium acetate buffer is picked out；

(8) prepare chip, join albumen.Change a new chip, carry out a Prime, the mobile phase in machine is all changed once new mobile phase, it is prevented that experimental result is produced interference by the old material in mobile phase.The time period of Prime, the chances are 6 minutes；.

(9) RUNSensor selector channel (single channel) is selected, it is determined that flow velocity 5 μ l/min, selecting paths, name file；

(10) baseline stability is waited；

(11) in the process waited, join activator, N-hydroxy-succinamide: carbodiimide=1:1 mixing, mix with rifle pressure-vaccum, note necessarily can not having bubble, be simply centrifuged on centrifuge after mixing, reduce bubble as far as possible；

(12) QuickInjection, injects activator, and volume is 70 μ l, and flow velocity is 10 μ l/min.Total co-activating 3 times, accumulative RU value rising 301.7；

(13) albumen is connected, it is possible to from low concentration, be gradually increased concentration, connect several times more.If the protein concentration of twice connection is identical during connection albumen, the efficiency that second time connects is just relatively low, so second time to increase concentration.Sirt3 albumen stock solution 10 μ l is dissolved in the sodium acetate solution of pH4.5 of 100 μ l, Injection, injects protein sample, and volume is 30 μ l, and flow velocity is 5 μ l/min；

(14) close with ethanolamine.Flow velocity is transferred to 10 μ l/min, and total amount 70 μ l walks 2-3 time, wait until flushing be over after RU value mean that closing is similar when declining within 10, during closing, for the first time flow velocity is 10 μ l/min, is adjusted to 20 μ l/min afterwards on flow velocity；

(15) it is twice Prime, the mobile phase in machine is all changed once new mobile phase, it is prevented that even experimental result is produced interference by the material in mobile phase during albumen；

(16) experiment is started after stablizing 2-3 hour；

(17) detection CA and Sirt3 is in conjunction with situation, starts experiment, selects the passage that two passages subtract each other；

(18) CA buffer solution, doubling dilution becomes following concentration: 1600 μMs, 800 μMs, 400 μMs, 200 μMs, 100 μMs, 50 μMs, 25 μMs, 12.5 μMs, 6.25 μMs, 3.125 μMs, 1.5625 μMs.Can detection micromolecular compound be combined with Sirt3, with the speed injection 90 μ l of 30 μ l/min.If there being multiple micromolecular compound, then after first compound must be waited to be washed away by mobile phase, waiting until time curve returns to baseline, then entering next little compound；

(19) for calculating affinity constant, therefore have detected the micromolecular compound of 5 concentration, ensure that binding time is 3 minutes simultaneously, Dissociation time 5 minutes；

(20) data are analyzed by BIAT200evaluationsoftwarev2.0 (GEHealthcare, UnitedKingdom), carry out 1:1Langmuir matching, calculate Ka, Kd, KD.

Six, result

6.1 caffeic acids improve I/R rats'liver surface blood flow

Fig. 1 is Doppler and statistical result.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

6.2 caffeic acids improve I/R rat interlobular veins, red blood cell flow speed in central vein

Fig. 2 is interlobular veins, red blood cell flow speed statistical result in central vein.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

6.3 caffeic acids improve I/R rat liver interlobular veins district, the open number of central vein district sinus hepaticus

Fig. 3 is the open number of liver interlobular veins district sinus hepaticus and statistical result.* < 0.05, vs.NS+I/R, arrow indication is the sinus hepaticus closed for p < 0.05, vs.NS+sham, #p.

Fig. 4 is the open number of liver central vein district sinus hepaticus and statistical result.* < 0.05, vs.NS+I/R arrow indication is the sinus hepaticus closed for p < 0.05, vs.NS+sham, #p.

6.4 caffeic acids improve I/R rat liver interlobular veins district, central vein district and sinus hepaticus leukocyte rolling and adhesion

Fig. 5 is liver interlobular veins district's leukocyte rolling and adhesion and statistical result.* p < 0.05, vs.

< 0.05, vs.NS+I/R arrow indication is leukocyte for NS+sham, #p.

Fig. 6 is liver sinus hepaticus leukocyte adhesion and statistical result.* < 0.05, vs.NS+I/R arrow indication is leukocyte for p < 0.05, vs.NS+sham, #p.

Fig. 7 is liver central vein district's leukocyte rolling and adhesion and statistical result.* < 0.05, vs.NS+I/R arrow indication is leukocyte for p < 0.05, vs.NS+sham, #p.

6.5 caffeic acids improve I/R injury of liver

Fig. 8 is HE coloration result and Suzuki appraisal result.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Fig. 9 is transmission electron microscope results

Figure 10 is the ELISA result of plasma A LT, AST.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

6.6 caffeic acids improve I/R liver tissues of rats and express hydrogen peroxide, MDA, 8-OHdG

Figure 11 is hepatic tissue hydrogen peroxide, MDA, the ELISA result of 8-OHdG.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

6.7 caffeic acids improve I/R liver tissues of rats attached molecule and Peripheral blood cells adhesion molecule expression

Figure 12 is the immunofluorescence dyeing of hepatic tissue paraffin section ICAM-1.The ICAM-1 positive Endothelial Cells of arrow instruction liver expression

Figure 13 is WB band and the statistical result of hepatic tissue ICAM-1.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 14 is the immunofluorescence dyeing of hepatic tissue paraffin section E-selectin, the E-selectin positive Endothelial Cells expressed in arrow instruction liver

Figure 15 is the result expressing CD11b and CD18 with flow cytomery Peripheral blood cells.

*p<0.05,vs.NS+sham,#p<0.05,vs.NS+I/R

6.8 caffeic acids improve I/R rat plasma, tissue inflammatory factor expression

Figure 16 is the ELISA result of Plasma TNF-α, IL-1 β, MCP-1.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 17 is the ELISA result of tissue T NF-α, IL-1 β, MCP-1.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

6.9 caffeic acids improve I/R liver tissues of rats leukocyte recruitment

Figure 18 is the ELISA result of the immunohistochemical staining of hepatic tissue MPO and MPO.Arrow indication is the leukocyte that MPO is positive.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 19 is transmission electron microscope results, and arrow indication is leukocyte

6.10 caffeic acids improve I/R liver tissues of rats and express the impact of P-I κ B-α, I κ B-α and NF-κ Bp65 nuclear translocation

Figure 20 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that hepatic tissue expresses P-I κ B-α, I κ B-α and NF-κ Bp65 nuclear translocation

Figure 21 is the immunofluorescence dyeing result of hepatic tissue NF-κ Bp65, and arrow indication is that p65 nuclear translocation is positive.

6.11 caffeic acids improve I/R hepatocyte apoptosis

Figure 22 is the immunofluorescence picture of the double; two dye of TUNEL and F-actin.Arrow indication is that TUNEL is positive.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

6.12 caffeic acids improve I/R liver tissues of rats and express apoptosis-related protein

Figure 23 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that hepatic tissue expresses Bax, Bcl-2, CleavedCaspase-9, CleavedCaspase-3

6.13 caffeic acids improve I/R liver tissues of rats and express MnSOD, superoxide dismutase, catalase, glutathion reductase, glutathione oxidase and peroxidase

Figure 24 is the ELISA result .*p < 0.05, vs. of the WB result of hepatic tissue MnSOD and superoxide dismutase, catalase activity, glutathion reductase, glutathione oxidase and peroxidase

NS+sham,#p<0.05,vs.NS+I/R

6.14 caffeic acids improve the film transposition of each subunit of I/R liver tissues of rats NADPH

Figure 25 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of each subunit expression of NADPH in hepatic tissue slurry albumen

Figure 26 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of each subunit expression of NADPH in hepatic tissue memebrane protein

6.15 caffeic acids improve I/R liver tissues of rats PKC δ film transposition and AMPKa phosphorylation

Figure 27 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that in hepatic tissue slurry albumen and memebrane protein, PKC δ and whole protein AMPKa, P-AMPKa express

6.16 caffeic acids improve I/R liver tissues of rats respiratory chain Complex I, II, IV, V activity

Figure 28 is hepatic tissue Complex I, ELISA result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of II, IV, V

6.17 caffeic acids improve expression and the acetylation of I/R liver tissues of rats SDHA

Figure 29 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that hepatic tissue expresses SDHA expression and Acetylation Level

6.18 caffeic acids improve the acetylation of I/R liver tissues of rats NDUFA9 and MnSOD

Figure 30 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of hepatic tissue NDUFA9 and MnSOD, SDHA Acetylation Level

6.19 caffeic acids improve expression and the NAD+/NADH ratio of I/R liver tissues of rats Sirt3

Figure 31 is hepatic tissue Sirt3 WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R expressed

Figure 32 is result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R detecting NAD+/NADH ratio by ELISA method

6.20 caffeic acids are combined with Sirt3

KD(M)	Rmax(RU)	offset(RU)	Chi2(RU2)
				7.931×10-5	7.61	3.497	0.107

Figure 33 is the affinity curve being combined with Sirt3 with SPR method detection caffeic acid.

Seven, conclusion

The research that the study find that finds that caffeic acid can improve Liver Microcirculation and the hepatic injury that ischemia-reperfusion causes, and the effect that caffeic acid improves the ischemical reperfusion injury of liver is relevant to lower approach.

1, improve I/R rats'liver surface blood flow, improve I/R rat interlobular veins, the open number of central vein sinus hepaticus and I/R rat interlobular veins, central vein red cell velocity；Improve I/R rat leukocyte to assemble.

2, I/R rat AMPK α phosphorylation and PKC δ film transposition are suppressed, it is suppressed that the film transposition of I/R rat NADPH subunit P41 and P47, it is suppressed that the generation of the peroxide in NADPH source.

3, raise I/R rat Sirt3 to express and activity; affect the subunit NDUFA9 of the substrate mitochondrial complex 1 of Sirt3 and the Acetylation Level of the subunit SDHA of complex two; improve the activity of mitochondrial complex 1 and 2; and then affect the generation of ROS; suppress NDUFA9, SDHA acetylation, improve I/R rat mitochondrial respiratory chain activity; reduce the generation of the peroxide that mitochondrial injury causes, increase the generation of ATP.

4, the hepatocellular apoptosis that ischemia-reperfusion causes is suppressed.

5, the damage of the hepatocyte inner cell skeleton that ischemia-reperfusion causes is alleviated.

Accompanying drawing explanation

Fig. 1 is Doppler and statistical result.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Fig. 2 is interlobular veins, red blood cell flow speed statistical result in central vein.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Fig. 3 is the open number of liver interlobular veins district sinus hepaticus and statistical result.* < 0.05, vs.NS+I/R arrow indication is the sinus hepaticus closed for p < 0.05, vs.NS+sham, #p.

Fig. 4 is the open number of liver central vein district sinus hepaticus and statistical result.* < 0.05, vs.NS+I/R arrow indication is the sinus hepaticus closed for p < 0.05, vs.NS+sham, #p.

Fig. 5 is liver interlobular veins district's leukocyte rolling and adhesion and statistical result.* < 0.05, vs.NS+I/R arrow indication is leukocyte for p < 0.05, vs.NS+sham, #p.

Fig. 6 is liver sinus hepaticus leukocyte adhesion and statistical result.* < 0.05, vs.NS+I/R arrow indication is leukocyte for p < 0.05, vs.NS+sham, #p.

Fig. 7 is liver central vein district's leukocyte rolling and adhesion and statistical result.* < 0.05, vs.NS+I/R arrow indication is leukocyte for p < 0.05, vs.NS+sham, #p.

Fig. 8 is HE coloration result and Suzuki appraisal result.* < 0.05, vs.NS+I/R Fig. 9 is transmission electron microscope results for p < 0.05, vs.NS+sham, #p

Figure 10 is the ELISA result of plasma A LT, AST.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 11 is hepatic tissue hydrogen peroxide, MDA, the ELISA result of 8-OHdG.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 12 is the immunofluorescence dyeing of hepatic tissue paraffin section ICAM-1.The ICAM-1 positive Endothelial Cells of arrow instruction liver expression

Figure 13 is WB band and the statistical result of hepatic tissue ICAM-1.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 14 is the immunofluorescence dyeing of hepatic tissue paraffin section E-selectin, the E-selectin positive Endothelial Cells expressed in arrow instruction liver

Figure 15 is the result expressing CD11b and CD18 with flow cytomery Peripheral blood cells.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 16 is the ELISA result of Plasma TNF-α, IL-1 β, MCP-1.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 17 is the ELISA result of tissue T NF-α, IL-1 β, MCP-1.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 18 is the ELISA result of the immunohistochemical staining of hepatic tissue MPO and MPO.Arrow indication is the leukocyte that MPO is positive.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 19 is transmission electron microscope results, and arrow indication is leukocyte

Figure 20 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that hepatic tissue expresses P-I κ B-α, I κ B-α and NF-κ Bp65 nuclear translocation

Figure 21 is the immunofluorescence dyeing result of hepatic tissue NF-κ Bp65, and arrow indication is that p65 nuclear translocation is positive.

Figure 22 is the immunofluorescence picture of the double; two dye of TUNEL and F-actin.Arrow indication is that TUNEL is positive.* p < 0.05, vs.NS+sham, #p < 0.05, vs.NS+I/R

Figure 23 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that hepatic tissue expresses Bax, Bcl-2, CleavedCaspase-9, CleavedCaspase-3

Glutathion reductase, glutathione oxidase and peroxidase

Figure 24 is ELISA result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of the WB result of hepatic tissue MnSOD and superoxide dismutase, catalase activity, glutathion reductase, glutathione oxidase and peroxidase

Figure 25 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of each subunit expression of NADPH in hepatic tissue slurry albumen

Figure 26 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of each subunit expression of NADPH in hepatic tissue memebrane protein

Figure 27 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that in hepatic tissue slurry albumen and memebrane protein, PKC δ and whole protein AMPKa, P-AMPKa express

Figure 28 is hepatic tissue Complex I, ELISA result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of II, IV, V

Figure 29 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R that hepatic tissue expresses SDHA expression and Acetylation Level

Figure 30 is WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R of hepatic tissue NDUFA9 and MnSOD, SDHA Acetylation Level

Figure 31 is hepatic tissue Sirt3 WB result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R expressed

Figure 32 is result .*p < 0.05, vs.NS+sham, the #p < 0.05, vs.NS+I/R detecting NAD+/NADH ratio by ELISA method

Figure 33 is the affinity curve being combined with Sirt3 with SPR method detection caffeic acid.

Detailed description of the invention

By specific examples below, the present invention is further illustrated, but without limitation.

Embodiment 1, tablet

[prescription]

Caffeic acid 100g

Microcrystalline Cellulose 50g

Micropowder silica gel 3g

Magnesium stearate 1.5g

[method for making] takes former, adjuvant mistake 100 mesh sieves respectively；Take caffeic acid, microcrystalline Cellulose, mixing, with 60% appropriate amount of ethanol as binding agent soft material, cross 20 mesh sieve granules, 60 DEG C dry, and take out, and cross 30 mesh sieve granulate, add micropowder silica gel and magnesium stearate, mixing, tabletting, make 1000, to obtain final product.

Embodiment 2, tablet

[prescription]

Caffeic acid 75g

Microcrystalline Cellulose 37g

Micropowder silica gel 2.3g

Magnesium stearate 1.1g

[method for making] takes former, adjuvant mistake 100 mesh sieves respectively；Take caffeic acid, microcrystalline Cellulose, mixing, with 60% appropriate amount of ethanol as binding agent soft material, cross 20 mesh sieve granules, 60 DEG C dry, and take out, and cross 30 mesh sieve granulate, add appropriate micropowder silica gel and magnesium stearate, mixing, and tabletting is made 1000, to be obtained final product.

Embodiment 3, tablet

[prescription]

Caffeic acid 133g

Microcrystalline Cellulose 66g

Micropowder silica gel 4g

Magnesium stearate 2g

[method for making] takes former, adjuvant mistake 100 mesh sieves respectively；Take caffeic acid 1, microcrystalline Cellulose, mixing, with 60% appropriate amount of ethanol as binding agent soft material, cross 20 mesh sieve granules, 60 DEG C dry, and take out, and cross 30 mesh sieve granulate, add appropriate micropowder silica gel and magnesium stearate, mixing, and tabletting is made 1000, to be obtained final product.

Embodiment 4, capsule

The adjuvants such as capsule, takes caffeic acid 100mg, adds appropriate amount of starch, magnesium stearate, granulate, and granulate loads No. 1 capsule, to obtain final product.

Embodiment 5, oral liquid

Oral liquid, takes caffeic acid 100mg, adds suitable amount of sucrose, and preservative, add water 1000ml, is distributed into 10ml mono-, obtains oral liquid.

Embodiment 6, granule

Granule, takes caffeic acid 100mg, adds appropriate dextrin, steviosin, dry granulation, granulate, subpackage, to obtain final product.

Embodiment 7, injection

Injection, caffeic acid 150g adds water

Dissolving, another sodium chloride, ethylparaben add hot water dissolving, mixing, adjust pH value 5-7.Water for injection is diluted to 1000ml, filters with hollow-fibre membrane, fill, sterilizing, to obtain final product.

Claims (10)

1. caffeic acid improves Liver Microcirculation in preparation or alleviates the application of liver injury medicament.

2. apply as claimed in claim 1, it is characterised in that: described Liver Microcirculation or hepatic injury cause due to Ischemia-reperfusion Injury in Rat.

3. apply as claimed in claim 2, it is characterised in that described improve liver microcirculation that law during ischemia/reperfusion causes or hepatic injury refers to and improves ischemia-reperfusion liver surface blood flow.

4. apply as claimed in claim 2, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or hepatic injury refers to and improves rat interlobular veins, the open number of central vein sinus hepaticus after ischemia-reperfusion；Interlobular veins, central vein red cell velocity.

5. apply as claimed in claim 2, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury refer to improve ischemia-reperfusion after rat leukocyte assemble.

6. apply as claimed in claim 2, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury and refer to suppression AMPK α phosphorylation and PKC δ film transposition, it is suppressed that the film transposition of NADPH subunit P41 and P47.

7. apply as claimed in claim 2, it is characterised in that: described improving liver microcirculation that law during ischemia/reperfusion causes or to alleviate hepatic injury be the generation suppressing or reducing peroxide, described peroxide source is in NADPH, mitochondrial injury.

8. apply as claimed in claim 2, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury and refer to the hepatocellular apoptosis suppressing ischemia-reperfusion to cause.

9. apply as claimed in claim 2, it is characterised in that: described improve liver microcirculation that law during ischemia/reperfusion causes or alleviate hepatic injury and refer to the damage alleviating the hepatocyte inner cell skeleton that ischemia-reperfusion causes.

10. apply as claimed in claim 1, it is characterised in that described caffeine is to be administered before ischemia-reperfusion.