CN109394750A - Application of the hydroxyl radical carthamin yellow carthamus A in preparation treatment medication for treating pyemia - Google Patents
Application of the hydroxyl radical carthamin yellow carthamus A in preparation treatment medication for treating pyemia Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
Abstract
The invention belongs to field of medicaments, are related to hydroxyl radical carthamin yellow carthamus A and inhibit pyemic occurrence and development to treat pyemic new application；Pass through the expression of reduction late phase inflammation factor high mobility group protein B 1 (HMGB1) more particularly to hydroxyl radical carthamin yellow carthamus A, reduce the release of tissue factor (TF) and platelet activating factor (PAF), the expression for reducing regulatory T cells (Treg) Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and forked head transcription factor P3 (Foxp3), to treat pyemia.
Description
Technical field:
The invention belongs to field of medicaments, are related to the new therapeutic uses of hydroxyl radical carthamin yellow carthamus A.
Technical background:
Pyemia is the caused mortality organ dysfunction of host response imbalance for infection, and the state of an illness is dangerous, lethal
Rate height is the main reason for patient with severe symptoms is dead.For many years, antibiotic, antiviral drugs, blood vessel boosting drug etc. are used to
Pyemia traditional treatment, but there has been no enough specific drugs for pathogenesis of sepsis mechanism to put into clinical practice.How
Systemic inflammatory reaction, coagulation disorders and immunologic function disorder during timely correction pyemia occurrence and development, as early as possible
Restore proinflammatory-anti-inflammatory dynamic equilibrium of body, is effectively improved patient's prognosis, becomes urgently to be resolved in treatment of sepsis medicament research and development
Important topic.
'Xuebijing ' injection is the dialectical principle according to " three three methods of card ", under the theoretical direction of " bacterium is scorching and controls ", with
Based on Qing Dynasty's Wang Qingren errors in Medicine Corrected contained " xuefu zhuyu decoction ", a kind of intravenous fluid developed, by safflower, red
The modern crafts such as Chinese herbaceous peony, Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, Radix Angelicae Sinensis gomi herbs are extracted, refine, is dry, deploying are prepared, and are suitable for septicopyemia
Disease/because of the systemic inflammatory response syndrome of infection-induced；Can also partner treatment multiple organ disorder syndrome organ function
The impaired phase.Related patents have: 1. Chinese patent 03104977.X disclose " a kind of to treat pyemic Chinese materia medica preparation and its preparation
Method ", the i.e. preparation process of 'Xuebijing ' injection, pharmacodynamics prove that it has the function of anti-endotoxin, can be used for prevent and
Treat pyemia.2. Chinese patent 201611232221.7 discloses " a kind of multicomponent injection ", ingredient is derived from safflower, red
Chinese herbaceous peony, Rhizoma Chuanxiong, Radix Salviae Miltiorrhizae, Radix Angelicae Sinensis gomi herbs, research confirm that it has pyemia cell model and septicopyemia disease mouse model and control
Treatment effect.
Hydroxyl radical carthamin yellow carthamus A (Hydroxysafflor yellow A, HYSA), belongs to flavone compound, is safflower
The highest ingredient of content in uranidin has and increases coronary blood flow, resists myocardial ischemia, and inhibition thrombosis reduces blood
Pharmacological actions, the medical value such as rouge, anti-inflammatory, analgesia, anti-oxidant have been approved.Injection hydroxyl radical carthamin yellow carthamus A is
Carry out the clinic for treating acute arterial atherosclerotic cerebral thrombotic infarction (apoplexy apoplex involving the channels and collaterals blood stasis hinders network card)
Research.Meanwhile hydroxyl radical carthamin yellow carthamus A is one of the main component in 'Xuebijing ' injection, thus, it is further investigated in purulence
Effect in toxication treatment has important practical significance and application value.
Summary of the invention:
The purpose of the present invention is to provide hydroxyl radical carthamin yellow carthamus As to treat the application in pyemic drug in preparation.
Wherein, hydroxyl radical carthamin yellow carthamus A, molecular formula: C27H32O16 molecular weight: 612.53, structural formula is as follows:
Application of the present invention, comprising:
Hydroxyl radical carthamin yellow carthamus A inhibits the application in pyemia in the drug of HMGB1 expression in preparation.
Hydroxyl radical carthamin yellow carthamus A inhibits the application in pyemia in the drug of TF and PAF release in preparation.
Hydroxyl radical carthamin yellow carthamus A inhibits the application in pyemia in the drug of CTLA-4 and Foxp3 expression in preparation.
Hydroxyl radical carthamin yellow carthamus A belongs to existing product, can buy on the market.
It is another object of the present invention to provide a kind of using hydroxyl radical carthamin yellow carthamus A as the pharmaceutical composition of active constituent
The application in pyemic drug is treated in preparation.
Weight percent shared by hydroxyl radical carthamin yellow carthamus A of the present invention can be 0.1-99.9%, remaining is drug
Acceptable carrier.
Pharmaceutical composition of the invention can be prepared into any pharmaceutical dosage form, these dosage forms include: tablet, sugar coated tablet
Agent, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral solution, mouth containing agent, granule, electuary,
Pill, powder, paste, sublimed preparation, suspension, pulvis, solution, injection, suppository, ointment, emplastrum, creme, spray,
Drops, patch.Preparation of the invention, preferably tablet, powder, granule, tincture, pill, capsule, oral solution, atomization
Inhalant, injection.
Pharmaceutical composition of the invention, the preparation of oral administration can contain common excipient, such as adhesive, filling
Agent, diluent, tablet agent, lubricant, disintegrating agent, colorant, flavoring agent and wetting agent when necessary can be coated tablet.
Applicable filler includes cellulose, mannitol, lactose and other similar fillers.Suitable disintegrating agent packet
Include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant includes, such as firmly
Fatty acid magnesium.Suitable pharmaceutically acceptable wetting agent includes lauryl sodium sulfate.
Can be by mixing, filling, commonly method prepares solid oral composition for tabletting etc..Carrying out mixing repeatedly can make to live
Property substance be distributed in entirely using in those of a large amount of fillers composition.
The form of oral liquid for example can be aqueous or oily suspensions, solution, emulsion, syrup or elixir,
Or it can be a kind of dry products that can be compounded with water or other suitable carriers before use.This liquid preparation can contain
Conventional additive, such as suspending agent, such as sorbierite, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxymethyl are fine
Dimension element, aluminium stearate gel or hydrogenated edible fats, emulsifier, such as lecithin, anhydro sorbitol monooleate or Arab
Glue；Non-aqueous carrier (they may include edible oil), for example, apricot kernel oil, fractionated coconut oil, such as glycerol ester oily ester,
Propylene glycol or ethyl alcohol；Preservative, such as para hydroxybenzene methyl esters or propylparaben or sorbic acid, and if desired,
Contain conventional flavouring agent or colorant.
For injection, the fluid unit dosage form of preparation contains active material and sterile carrier of the invention.According to carrier
And concentration, this compound can be suspended or be dissolved.The preparation of solution is usually by the way that active material is dissolved in a kind of load
In body, disinfection is filtered before being loaded into a kind of suitable bottle or ampoule, is then sealed.For example a kind of local anaesthesia of auxiliary material
Agent, preservative and buffer are also soluble in this carrier.It, can be after being packed into bottle by this in order to improve its stability
Kind composition frost, and under vacuum remove water.
Suitable pharmaceutically acceptable carrier is optionally added Chinese materia medica preparation of the invention when being prepared into medicament,
The pharmaceutically acceptable carrier is selected from: mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, sodium thiosulfate, hydrochloric acid
Cysteine, thioacetic acid, methionine, injection Vitamin B_6 DTA disodium, Ethylenediaminetetraacetic Acid Calcium Salt, the carbonate of monovalence alkali metal, acetate,
Phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, malt
Sugar, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its spread out
Biology, alginates, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant,
Polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid material, kaolin, talcum powder, calcium stearate, magnesium stearate etc..
Pharmaceutical preparation of the present invention, preferably injection.
The present invention also provides hydroxyl radical carthamin yellow carthamus As when being used for pyemia, and administration route is intravenous injection or vein drop
Note administration, dosage range is 4.5mg to 1000mg.
Corresponding explanation is made to the English occurred in specification:
Chinese and English (abbreviation) table of comparisons
| Chinese name | English name | English abbreviation |
| Hydroxyl radical carthamin yellow carthamus A | Hydroxysafflor Yellow A | HYSA |
| Endotoxin (lipopolysaccharides) | Lipopolysaccharide | LPS |
| High mobility group protein B 1 | High Mobility Group Protein B1 | HMGB1 |
| Enzyme linked immunosorbent assay | Enzyme-Linked Immunosorbent Assay | ELISA |
| Tissue factor | Tissue Factor | TF |
| Platelet activating factor | Platelet Activating Factor | PAF |
| Regulatory T cells | Regulatory T Cell | Treg |
| Cytotoxic T lymphocyte associated antigen-4 | Cytotoxic T Lymphocyte Associated Antigen-4 | CTLA-4 |
| Forked head transcription factor P3 | Forkhead Box P3 | FoxP3 |
| Phycoerythrin | P-phycoerythrin | PE |
| Fluorescein isothiocynate | Fluorescein Isothiocyanate | FITC |
| Allophycocyanin | Allophycocyanin | APC |
| Cecal ligation and perforation art | CecalLigation and Puncture | CLP |
Specific embodiment
By following embodiment, the present invention is further illustrated, but not as limitation of the invention.
Experiment is all made of hydroxyl radical carthamin yellow carthamus A respectively on different pyemia cell models and animal model below
Pharmacodynamic study.
Acute toxicity test is administered in 1 hydroxyl radical carthamin yellow carthamus A mouse vein of embodiment
1. experimental material
Cleaning grade SD rat (half male and half female), 180-220g, hydroxyl radical carthamin yellow carthamus A, physiological saline (0.9% sodium chloride
Injection).
2. experimental method
40 animals (half male and half female) are screened according to laundering period the weight of animals growth, diet, activity etc. and enter this test, are adopted
It is divided into 2 groups with the weight district's groups method of dividision into groups, every group 20, half male and half female.Experiment uses maximum dosage-feeding method, Sydroxy carthamin
A maximum concentration is 50mg/mL, and rat intravenous injection limitation is 6mL/kg, and to sum up maximum dosage is 300mg/kg.Experimental group
Single-dose, 6mL/kg hydroxyl radical carthamin yellow carthamus A medical fluid (50mg/mL), vein is slowly injected；Control group gives same dose
Normal saline solution.
It is at least observed continuously after administration 2 hours, the 1st day morning and afternoon respectively observed and recorded 1 time after administration, later daily observation note
Record 1 time is observed 14 days altogether.In observation period, the death condition of animal toxicity response situation and groups of animals is observed and recorded, to death
Or the timely dissect of moribund animals.14 days after administration, all surviving animals carry out dissect, and visually observing each main organs, whether there is or not obvious
Anomalous variation；When the change such as volume, color, quality occur in internal organs, it should all record and carry out histopathologic examination.
Drug solution preparation method is as follows: precision balance (1/10000g) weigh 5.0g hydroxyl radical carthamin yellow carthamus A be dissolved in it is suitable
It measures in physiological saline (0.9% sodium-chloride water solution), is settled to 100mL filter filtration sterilization, concentration is 50mg/ at this time
mL。
3. experimental result
The slow intravenous injection 300mg/kg hydroxyl radical carthamin yellow carthamus A of rat single, as the result is shown: (1) after animal administration not
See abnormal symptom；(2) there was no significant difference with control group for administration group the weight of animals and body weight growth rate；(3) substantially dissect has no
It is obvious abnormal.
Embodiment 2, evaluation hydroxyl radical carthamin yellow carthamus A stimulate LPS the influence of lower rat peritoneal macrophages HMGB1 release
1. experimental material
Male cleaning grade SD rat, 180-220g, endotoxin (LPS), hydroxyl radical carthamin yellow carthamus A, RPMI-1640 culture
Base, 24 orifice plates, HMGB1ELISA kit.
2. experimental method
Separating male SD rat peritoneal macrophage, (male SD rat pre-operative anxiety 12h, opens abdominal cavity after anesthesia, to abdomen
Chamber injection pre-cooling PBS liquid 10mL makes liquid in intraperitoneal flowing with the light kneadding stomach wall of finger.Draw the liquid injection in abdominal cavity
In sterile tube, then it is primary with pre-cooling PBS liquid 10mL lavation, operation is same as above.The irrigating solution 250g being collected into, 4 DEG C of centrifugations will be merged
10min abandons supernatant.2mL erythrocyte cracked liquid lysed erythrocyte is added, twice, each 5s is added after standing 5min for slight concussion
4mL D-Hanks solution terminates reaction.It is centrifuged again according to the method described above, abandons supernatant.Precipitating is washed with culture solution, is then resuspended
Cell) 2 × 106/mL cell suspension is made, 24 orifice plates are inoculated in, are placed in 37 DEG C, the cell incubator culture of 5%CO2.Experiment
It is divided into control group, model group, experimental group 1 (0.6 μM), experimental group 2 (1.8 μM), experimental group 3 (5.4 μM), (16.2 μ of experimental group 4
M), experimental group 5 (48.6 μM) and experimental group 6 (145.8 μM), every group of 6 parallel holes.It is collected after cultivating 48h and 72h respectively
Clearly, cytokine content is measured using one step sandwich method enzyme-linked immunosorbent assay (ELISA) of double antibody.
Packet processing method is as follows: model group: LPS (75ng/mL) thorn is added after cell incubator overnight incubation in cell
Swash.Experimental group 1-6: corresponding concentration hydroxyl radical carthamin yellow carthamus A is added and is incubated for 1h, carries out LPS stimulation, various concentration hydroxyl safflower yellow
After pigment A intervenes 48,72h, cell culture supernatant 0.5mL is collected, -20 DEG C of refrigerators save, concentrate and carry out corresponding cell factor
Detection.
Drug solution preparing mode is as follows: weighing 3.0mg hydroxyl carthamin A in precision balance (1/10000g) and is dissolved in 1.96mL
RPMI-1640 culture medium, with filter filtration sterilization, packing is saved.It is at this time 2500 μM of storing liquid.In use, according to each
Experimental group liquor strength needs, and is diluted to required concentration with culture medium (containing 10%FBS).
3. experimental result
The results showed that the hydroxyl radical carthamin yellow carthamus A of each dosage group can significantly reduce 48,72h compared with model group
The emission levels of rat peritoneal macrophages HMGB1 show hydroxyl radical carthamin yellow carthamus A (0.6-145.8 μM) to pyemia cell
Model has presses down scorching effect well.
1. hydroxyl radical carthamin yellow carthamus A of table LPS is stimulated lower rat peritoneal macrophages HMGB1 release influence (n
=6, unit: pg/mL)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
Embodiment 3, evaluation hydroxyl radical carthamin yellow carthamus A stimulate lower rat aorta endothelial cell TF and PAF to discharge LPS
Influence
1. experimental material
Male cleaning grade SD rat, 180-220g, LPS, hydroxyl radical carthamin yellow carthamus A, ECM culture medium, 24 orifice plates, tissue because
Sub (TF) ELISA kit, platelet activating factor (PAF) ELISA kit.
2. experimental method
After putting to death rat with cervical dislocation, it is soaked in 5min in the ethyl alcohol that volume fraction is 75%, successively opens chest, abdomen
Chamber sufficiently exposes chest, abdominal aorta, separates its surrounding tissue, from proximal part separation aorta to arteria iliaca communis bifurcation, is put into
In culture dish containing PBS, the adipose tissue and fibr tissue of sterile removing externa, PBS rinse lumen of vessels.By aorta
It is cut into about 1.5mm × 1.5mm fritter, is placed in the IV Collagenase Type of 6mL 0.25%, 37 DEG C of digestion 15min, every 5min concussion
Once, it carefully siphons away digestive juice, retains tissue block, add the neutral proteinase of 6mL 1.0%, 37 DEG C of digestion 15min, often
5min concussion is primary, siphons away digestive juice, supplements ECM 10mL, blows and beats repeatedly, and 1,000r/min centrifugation 10min discards culture medium
And digestive juice, retain fragment, tissue block is laid in the culture dish bottom of 10cm, put upside down the 2h in 37 DEG C of baking ovens, makes tissue block
It cements, appropriate ECM is added, submerging tissue block is advisable, and puts 37 DEG C, training in the CO2 saturated humidity incubator that volume fraction is 5%
It supports, 3 days one subcultures of replacement.Visible endothelial cell is climbed out of by tissue block edge and is gradually extended outwardly within 7 days or so, in flat
Short shuttle shape or polygonal remove tissue block, at this time with 0.25% trypsin digestion, in 1:3 ratio 25cm2 culture bottle
Middle passage is tested with 3-4 for cell.
According to the cultural method culture cell of rat aorta endothelial cell among the above, cell suspension is prepared, with 1.2 ×
105/mL inoculating cell, by cell suspension inoculation in 24 orifice plates, 37 DEG C are cultivated, and model group and experimental group 1-6 give after about 12h
LPS stimulation, experimental group 1-6 gives various concentration hydroxyl radical carthamin yellow carthamus A intervention respectively after 1h, after stimulation for 24 hours, 48h,
72h collects supernatant.
Packet processing method and drug solution preparing mode are the same as embodiment 2.
3. experimental result
TF is the important procoagulant Factor of rat aorta endothelial cell, and under pyemia state, hypercoagulative state is shown as
A large amount of releases of TF.Under pharmaceutical intervention the reduction of TF facilitate coagulation factor it is anticoagulant/promote solidifying balance, may advantageously facilitate cell/
Body coagulation function restores toward normal condition.
The results showed that the hydroxyl radical carthamin yellow carthamus A of each concentration group can reduce for 24 hours compared with model group, 48h and
The release of 72h rat aorta endothelial cell procoagulant Factor TF, shows hydroxyl radical carthamin yellow carthamus A (0.6-145.8 μM) to purulence
Toxication model has good anticoagulant effect.
2. hydroxyl radical carthamin yellow carthamus A of table LPS is stimulated lower rat aorta endothelial cell TF release influence (n
=6, unit: pg/mL)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
Endothelial cell PAF is the important procoagulant Factor of rat aorta endothelial cell, and the activation of PAF can cause blood coagulation grade
The generation for joining reaction, causes blood coagulation, under pyemia state, body hypercoagulative state shows as a large amount of releases of PAF.It finds effective
Drug intervened, facilitate coagulation factor it is anticoagulant/promote solidifying balance, may advantageously facilitate cell/body coagulation function toward just
Normal state is restored.
The results showed that the hydroxyl radical carthamin yellow carthamus A of each concentration group can significantly reduce for 24 hours compared with model group,
The release of 48h and 72h rat aorta endothelial cell PAF shows hydroxyl radical carthamin yellow carthamus A (0.6-145.8 μM) to septicopyemia
Disease cell model has good anticoagulant effect.
3. hydroxyl radical carthamin yellow carthamus A of table LPS is stimulated lower rat aorta endothelial cell PAF release influence (
N=6, unit: ng/mL)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
Embodiment 4, evaluation hydroxyl radical carthamin yellow carthamus A stimulate lower Rats Spleen regulatory T cells (Treg) function to LPS
It influences
1. experimental material
Male cleaning grade SD rat, 180-220g, LPS, hydroxyl radical carthamin yellow carthamus A, ECM culture medium, 24 orifice plates, algae red egg
White (PE)-anti-rat CD25, fluorescein isothiocynate (FITC) mark anti-rat CD4, allophycocyanin (APC) label film to join egg
White V apoptosis kit；The anti-PE kit of rat, CD4 magnetic bead, MiniMACS Magnetic Isolation instrument and sorting column；PE label
Foxp3 kit, PE labeling CT LA-4 kit, anti-rat CD3 monoclonal antibody, anti-rat CD28 monoclonal antibody.
2. experimental method
The disconnected neck of male SD rat is put to death, and spleen is taken, and asepsis injector piston grinds spleen, and suction pipe draws suspension to centrifugation
Pipe is centrifuged, abandons supernatant for 1,200 turn × 7 minutes, and appropriate MACS Buffer (each spleen of 10mL/) resuspension is added, along tube wall plus
Enter to the upper layer (1:1) of lymphocyte separation medium 3,000 turn × 15 minutes and be centrifuged, draw middle layer liquid with suction pipe, be packed into it is another from
It in heart pipe, is added cleaning solution 1200 turns × 7 minutes and is centrifuged, abandon supernatant, it is spare that MACS Buffer resuspension is added.
The anti-CD25-APC antibody of 1 μ L, 4 DEG C of incubation 15min, MACS Buffer washings are added in every 1 × 107 monocyte.Often
The anti-APC magnetic bead of 20 μ L and 80 μ L MACS Buffer is added in 1 × 107 cell, and 4 DEG C of incubation 15min, MACS Buffer washings add
Appropriate MACS Buffer is resuspended, and LS column magnetic sorting is to get CD25+ cell.
After CD25+ cell count, every 1 × 107 cell is added 20 μ L and dissociates agent, and 4 DEG C of incubation 1min, MACS Buffer are washed
It washs, the anti-CD4 magnetic bead of 20 μ L and 30 μ L terminators are added in every 1 × 107 cell, and 4 DEG C of incubation 15min, MACS Buffer washings add
Enter appropriate MACS Buffer to be resuspended, LS column magnetic sorting is to get CD4+CD25+ cell.
The appropriate culture solution of Treg cell is resuspended, adjustment cell number is 2.5 × 106/mL, and 96 orifice plates, every hole access is carefully
CD3/CD28 (CD3 is that 0.5 μ g/106, CD28 is 1 μ g/106)+LPS (1 μ g/mL) is added in 100 μ L of born of the same parents, model group and experimental group
Stimulation, experiment are divided into control group, model group, experimental group 1 (0.6 μM), experimental group 2 (1.8 μM), experimental group 3 (5.4 μM), experiment
4 (16.2 μM), experimental group 5 (48.6 μM) and experimental group 6 (145.8 μM) are organized, experimental group gives various concentration medical fluid respectively after 1h
Intervened, 37 DEG C of culture 72h of CO2 incubator.
After cell conditioned medium is collected, cell is cleaned with PBS, removes supernatant, CTLA-4-PE fluorescence antibody is added, 4 DEG C are protected from light incubation
30min is added rupture of membranes agent and stays overnight.Second day, rupture of membranes buffer solution for cleaning is added, removes supernatant, it is anti-that Foxp3-PE-Cy7 fluorescence is added
Body, room temperature, which is protected from light, is incubated for 30min, and rupture of membranes buffer solution for cleaning is added, removes supernatant, and 1% paraformaldehyde is fixed, flow cytometer inspection
It surveys.
Packet processing method and drug solution preparing mode are the same as embodiment 2.
3. experimental result
Foxp3+Treg cell inhibits general T cell by generating immunosuppressive factor such as IL-10, IL-35, TGF-β etc.,
And target cell is killed by granzyme B and perforin -1, to play immunosuppressive action.CTLA-4 also can induce DC dendron shape
Cell generates indoleamine 2,3-dioxygenase, and catalysis tryptophan, which is decomposed into cynruin, causes peripheral cell dead, moreover it is possible to induce DC
Cell secretes other amino acid relevant enzymes, thus the proliferation of depression effect T cell, to play immunosuppressive effect.Therefore,
Treg cell is played a very important role in terms of immunity of organism regulation by Foxp3 and CTLA-4, the expression of Foxp3 and tune
Section property T cell (Treg) function is closely related.
Compared with model group, the hydroxyl radical carthamin yellow carthamus A of each dosage group can reduce Rats Spleen regulatory T cells
(Treg) expression of 72hCTLA-4 and Foxp3 shows that hydroxyl radical carthamin yellow carthamus A (0.6-145.8 μM) can be lowered to T
The depression effect of lymphopoiesis and secreting function.
4. hydroxyl radical carthamin yellow carthamus A of table LPS is stimulated lower Rats Spleen regulatory T cells CTLA-4 expression influence (N=6, unit: %)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
5. hydroxyl radical carthamin yellow carthamus A of table stimulates LPS the influence of lower Rats Spleen regulatory T cells Foxsp3 expression Unit: %)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
The influence of embodiment 5, evaluation hydroxyl radical carthamin yellow carthamus A to the CLP rats with sepsis HMGB1 expression induced
1. experimental material
SD male rat, cleaning grade, weight 180-220g, adaptive feeding 1 week.Hydroxyl radical carthamin yellow carthamus A,
HMGB1ELISA detection kit.
2. experimental method
2.1 groupings and intervention
50 rats are randomly divided into control group, model group, experimental group 1 (0.4mg/kg), experimental group 2 (6.0mg/kg), reality
It tests and organizes 3 (90.0mg/kg), every group each 10.12h fasting before testing, weighs and is grouped according to random digits table.Control
Group only opens skin suture after abdomen exposure caecum, subcutaneous injection 10mL physiological saline recovery；Sepsis model group carries out Cecal Ligation
Perforation (CLP) modeling；Experimental group 1- experimental group 3 is after CLP modeling, subcutaneous injection 10mL physiological saline recovery.CLP modeling
Journey is as follows: using ketamine injection+Su Mian Xin II injection 2:1 mixed liquor intramuscular anesthesia rat, prepares septicopyemia using CLP
Disease animal model.Cecal ligation and ileum junction penetrate through 2 formation intestinal fistula of caecum, and 2 drainage strips of indwelling with No. 18 syringe needles
(0.5cm × 2.0cm) prevents pin hole from healing, rear layer-by-layer suture skin, and art finishes subcutaneous injection 10mL physiological saline recovery immediately.Greatly
After mouse row CLP operation, experimental group 1- experimental group 3 2h, 12h after surgery, for 24 hours, 36h, 48h and 60h are through vena dorsalis penis injection point
Not Zhu She the dosage containing above three hydroxyl radical carthamin yellow carthamus A medical fluid；Model group and control group are quiet and secluded through penis in the corresponding time
Arteries and veins injecting normal saline.
The manner of formulation of hydroxyl radical carthamin yellow carthamus A medical fluid and experiment injection volume are as follows: claiming at precision balance (1/10000g)
It takes 2.25g hydroxyl carthamin A to be dissolved in appropriate physiological saline (0.9% sodium-chloride water solution), is settled to 100mL filter
Filtration sterilization, concentration is 22.5mg/mL at this time, is used as high dose group injection and uses, and experiment injection volume is 0.8mL/ times.According to
Each experimental group liquor strength needs, with physiological saline constant gradient (1:15) be diluted to 1.5mg/mL (for middle dose group) and
0.1mg/mL (is used for low dose group), and experiment injection volume is 0.8mL/ times.
2.2 blood samplings and detection
8h after CLP, for 24 hours, 48h and 72h, abdominal aorta sterile blood sampling 3mL after groups of animals is anaesthetized, using ELISA method,
Detect blood plasma HMGB1 content.
3. experimental result
The results showed that containing a small amount of HMGB1 in control group blood plasma；CLP early postoperative period, model group HMGB1 content
It obviously increases, 8h HMGB1 level further increases, and is gradually reduced in for 24 hours, but postoperative 72h is still higher than control group, difference is equal
Statistically significant (p < 0.05).And experimental group 1- experimental group 3, postoperative 8h, for 24 hours, the HMGB1 plasma content of 48h and 72h are bright
It is aobvious to be lower than model group (p < 0.05), close to control group level.Above-mentioned discovery shows compared with model group, the hydroxyl of three dosage groups
Base carthamus tinctorius yellow colour A can significantly reduce the expression of CLP rats with sepsis HMGB1, prompt hydroxyl radical carthamin yellow carthamus A (2.5-
The scorching effect of apparent suppression 90.0mg/kg) is all had to rats with sepsis model, be converted to human dose be about 4.5mg extremely
1000mg.It [is calculated according between humans and animals in " pharmacological experimental methodology " of Xu Shuyun chief editor by body surface area Commutation Law, human body
Dosage=rat dosage/0.018 (in terms of adult weight 70kg, rat body weight 0.2kg)]
The rats with sepsis HMGB1 expression that 6. hydroxyl radical carthamin yellow carthamus A of table induces CLP influence (N=10, it is single
Position: pg/mL)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
The influence of embodiment 6, evaluation hydroxyl radical carthamin yellow carthamus A to CLP rats with sepsis TF and the PAF release induced
1. experimental material
Wistar male rat, cleaning grade, weight 180-220g, adaptive feeding 1 week.Hydroxyl radical carthamin yellow carthamus A,
TFELISA kit, PAFELISA kit.
2. experimental method
2.1 groupings and intervention
50 rats are randomly divided into control group, model group, experimental group 1 (0.4mg/kg), experimental group 2 (6.0mg/kg), reality
It tests and organizes 3 (90.0mg/kg), every group each 10.12h fasting before testing, weighs and is grouped according to random digits table.Control
Group only opens skin suture after abdomen exposure caecum, subcutaneous injection 10mL physiological saline recovery；Sepsis model group carries out Cecal Ligation
Perforation (CLP) modeling, subcutaneous injection 10mL physiological saline recovery.CLP modeling process is as follows: being slept with ketamine injection+speed
New II injection 2:1 mixed liquor intramuscular anesthesia rat prepares pyemia animal model using CLP.Cecal ligation and ileum
Junction penetrates through 2 formation intestinal fistula of caecum with No. 18 syringe needles, and 2 drainage strips (0.5cm × 2.0cm) of indwelling prevent pin hole to be cured
It closes, rear layer-by-layer suture skin, art finishes subcutaneous injection 10mL physiological saline recovery immediately.After rats underwent CLP operation, experimental group 1- is real
Group 3 2h, 12h after surgery are tested, for 24 hours, 36h, 48h and 60h inject the hydroxyl for injecting above three dosage respectively through vena dorsalis penis
Carthamus tinctorius yellow colour A medical fluid；Model group and control group are in the corresponding time through vena dorsalis penis injecting normal saline.
The manner of formulation and experiment injection volume of hydroxyl radical carthamin yellow carthamus A medical fluid are shown in embodiment 5.
2.2 blood samplings and detection
6h, 12h after CLP, for 24 hours, 48h and 72h, abdominal aorta sterile blood sampling 5mL after groups of animals is anaesthetized.Using enzyme-linked
Immunoabsorption (ELISA) detects TF and PAF content.
3. experimental result
The results showed that there is certain expression in control group monocyte TF and PAF；Model group TF (postoperative 12h, for 24 hours,
48h and 72h) and PAF (postoperative 6h, 12h, for 24 hours and 48h) expression be all remarkably higher than control group (p < 0.05), and with Post surgery duration
Extension and gradually increase.Experimental group 1-4 TF (postoperative 12h, for 24 hours, 48h and 72h) and PAF (postoperative 6h, 12h, for 24 hours and
48h) expression occurs significantly reducing (p < 0.05) compared with model group.Above-mentioned discovery prompt, compared with model group, three dosage
The hydroxyl radical carthamin yellow carthamus A of group can significantly reduce the release of CLP rats with sepsis TF and PAF, show hydroxyl radical carthamin yellow carthamus A
(2.5-90.0mg/kg) has the function of significantly inhibiting coagulation factor release, improves hypercoagulative state to rats with sepsis model,
Being converted to human dose is about 4.5mg to 1000mg.[translation method is shown in embodiment 5]
The rats with sepsis TF release that 7. hydroxyl radical carthamin yellow carthamus A of table induces CLP influence (Unit: pg/
mL)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
The rats with sepsis PAF release that 8. hydroxyl radical carthamin yellow carthamus A of table induces CLP influence (Unit:
ng/mL)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group
The influence of embodiment 7, evaluation hydroxyl radical carthamin yellow carthamus A to the CLP pyemia regulatory T cells function of inducing
1. experimental material
SD male rat, cleaning grade, weight 180-220g, adaptive feeding 1 week.Anti- rat CD25, FITC label of PE- is anti-
Rat CD4, APC mark annexin V apoptosis kit；The anti-PE kit of rat, CD4 magnetic bead, MiniMACS Magnetic Isolation instrument
And sorting column；PE flag F oxp3 detection kit, PE labeling CT LA-4 detection kit, anti-rat CD3 monoclonal antibody,
Anti- rat CD28 monoclonal antibody.
2. experimental method
2.1 groupings and intervention
50 rats are randomly divided into control group, model group, experimental group 1 (0.4mg/kg), experimental group 2 (6.0mg/kg), reality
It tests and organizes 3 (90.0mg/kg), every group each 10.12h fasting before testing, weighs and is grouped according to random digits table.Control
Group only opens skin suture after abdomen exposure caecum, subcutaneous injection 10mL physiological saline recovery；Model group and experimental group 1-3 carry out caecum
Ligation and perforation art (CLP) modeling.CLP modeling process is as follows: using ketamine injection+Su Mian Xin II injection 2:1 mixed liquor flesh
Meat injecting anesthetic rat prepares pyemia animal model using CLP.Cecal ligation and ileum junction are penetrated through with No. 18 syringe needles
2 formation intestinal fistula of caecum, and 2 drainage strips (0.5cm × 2.0cm) of indwelling prevent pin hole from healing, rear layer-by-layer suture skin, art finishes
Subcutaneous injection 10mL physiological saline recovery immediately.Experimental group 1- experimental group 3 is injected through vena dorsalis penis respectively after CLP modeling
The hydroxyl radical carthamin yellow carthamus A medical fluid of above three dosage, model group and control group are injected through vena dorsalis penis in the corresponding time and are given birth to
Manage salt water.
The manner of formulation and experiment injection volume of hydroxyl radical carthamin yellow carthamus A medical fluid are shown in embodiment 5.
3.2 isolation and culture of cell
Each group rat is sterile after disconnected neck is put to death respectively to take spleen, and 400 mesh filter screens are crossed after grinding, are added after cell suspension centrifugation
Enter lymphocyte separation medium centrifuging and taking middle layer white cell.The anti-CD25 and PE magnetic bead of PE- is added, positive selection (sun choosing) obtains
CD25+T cell, Solid phase (yin choosing) obtain CD25-T cell.With CD25+T cell dissociation reagent dissociation after, yin select cell with
The anti-CD4 and CD4 magnetic bead sun of FITC- selects up to CD4+CD25+Treg；CD25-T cell is with the anti-CD4 and CD4 magnetic bead sun choosing of FITC-
Up to CD4+CD25-T cell.The purity of flow cytomery double positive cells.With 0.4% trypan blue of mass fraction to CD4+
CD25+Treg is dyed, and cell survival rate is observed.With the RPMll640 culture solution of 20% calf serum containing volume fraction in 48
In well culture plate, cultivated in CO2 incubator.Each group cultivates 12h in separation CD4+CD25+Treg on the 3rd, then with culture solution tune
Whole CD4+CD25+Treg and CD4+CD25-T cell concentration is 1:1 culture, and concanavalin A (Con A, 5mg/L) is stimulated, 37
Centrifuging and taking supernatant after 68h is cultivated in DEG C CO2 incubator, it is to be checked in -70 DEG C of frosts.
3.3 detections and analysis
The detection of Treg apoptosis rate: 12h is cultivated after separation cell, suspension CD4+CD25+Treg (1 × 109/L) uses phosphate
Buffer (PBS) is washed 2 times, and 100 μ L combination buffers are added and APC marks annexin V (20mg/L) 10 μ L, room temperature is protected from light
30min adds 10 μ L of actinomycin D (7-AAD), and after being protected from light 5min, 400 μ L combination buffers, fluidic cell is added
Instrument selects 7-AAD feminine gender annexin V positive cells for apoptotic cell, detects apoptosis rate.
Foxp3 and CTLA-4 detection of expression: the CD4 that 100 μ L are prepared.CD25+Treg (1 × 109/L) plus 1mL are newly prepared
Rupture of membranes liquid, 4 DEG C are protected from light and are incubated for 2h, then are washed with 2mL rupture of membranes buffer；Add the anti-Foxp3 of PE-, after the incubation 30min of 4 DEG C of dark place
It is washed with 2mL rupture of membranes buffer, supernatant is abandoned in centrifugation, and 0.5mL PBS, the mean fluorecence of flow cytomery Foxp3 is added
Intensity.It is resuspended in cell (1 × 109/L) and directly adds PE-CTLA-4,4 DEG C are protected from light incubation 30min, detect CTLA-4 mean fluorecence
Intensity.
3. experimental result
The results showed that model group Treg apoptosis rate is significantly lower than control group (p < 0.05), experimental group 1- experimental group 3 is equal
It is significantly higher than model group (p < 0.05).The expression of model group Foxp3, CTLA-4 is apparently higher than control group (p < 0.05), experimental group 1-
Experimental group 3 is substantially less than model group (p < 0.05).Above-mentioned discovery prompt, compared with model group, the hydroxyl of three dosage groups is red
Anthoxanthin A can promote pyemia Treg apoptosis, lowers the depression effect to T lymphocyte proliferation and secreting function, shows
Hydroxyl radical carthamin yellow carthamus A (2.5-90.0mg/kg) has apparent correction body cell immunosupress to rats with sepsis model
The effect of state, being converted to human dose is about 4.5mg to 1000mg.[translation method is shown in embodiment 5]
9. hydroxyl radical carthamin yellow carthamus A of table expresses rats with sepsis regulatory T cells apoptosis rate and Foxp3, CTLA-4
Influence (N=10, unit: %)
Note:#, indicate compared with the control group, P < 0.05；*, indicate P < 0.05 compared with test group.
Claims (10)
1. hydroxyl radical carthamin yellow carthamus A treats the application in pyemic drug in preparation.
2. application according to claim 1, which is characterized in that hydroxyl radical carthamin yellow carthamus A inhibits in pyemia in preparation
Application in the drug of HMGB1 expression.
3. application according to claim 1, which is characterized in that hydroxyl radical carthamin yellow carthamus A inhibits TF in pyemia in preparation
With the application in the drug of PAF release.
4. application according to claim 1, which is characterized in that hydroxyl radical carthamin yellow carthamus A inhibits in pyemia in preparation
Application in the drug of CTLA-4 and Foxp3 expression.
5. application according to claim 1, which is characterized in that hydroxyl radical carthamin yellow carthamus A is injection.
6. application according to claim 1, which is characterized in that hydroxyl radical carthamin yellow carthamus A dosage range be 4.5mg extremely
1000mg。
7. treating the application in pyemic drug in preparation using hydroxyl radical carthamin yellow carthamus A as the pharmaceutical composition of active constituent.
8. application according to claim 7, which is characterized in that pharmaceutical composition can be prepared into any pharmaceutical dose
Type.
9. application according to claim 7, which is characterized in that contain hydroxyl radical carthamin yellow carthamus A and medicine in pharmaceutical composition
Acceptable carrier on.
10. application according to claim 7, which is characterized in that weight percent shared by hydroxyl radical carthamin yellow carthamus A is 0.1-
99.9%, remaining is pharmaceutically acceptable carrier.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022183539A1 (en) * | 2021-03-01 | 2022-09-09 | 天津红日药业股份有限公司 | Pharmaceutical composition for treating sepsis, and use thereof |
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