CN101120962A - Application of total salvianolic acid in preparing medicine for treating pulmonary fibrosis - Google Patents

Application of total salvianolic acid in preparing medicine for treating pulmonary fibrosis Download PDF

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CN101120962A
CN101120962A CNA2007100240575A CN200710024057A CN101120962A CN 101120962 A CN101120962 A CN 101120962A CN A2007100240575 A CNA2007100240575 A CN A2007100240575A CN 200710024057 A CN200710024057 A CN 200710024057A CN 101120962 A CN101120962 A CN 101120962A
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radix salviae
salviae miltiorrhizae
phenolic acids
total phenolic
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顾振纶
冯一中
林军
周文轩
郭次仪
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SUZHOU INST OF CHINESE MEDICINE
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SUZHOU INST OF CHINESE MEDICINE
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Abstract

The present invention discloses the function of the danshen root phenolic acid in preparation of pulmonary fibrosis medicine. On the basis of mice and rat pulmonary fibrosis model by bleocin replication, the present invention applies three different dosage danshen root phenolic acid in the therapy. The treatment effect, the pathological change and the function mechanism of the danshen root phenolic acid on pulmonary fibrosis caused by the bleocin is proved through anshen root phenolic acid, immunohistochemistry, electron microscope observation and chemical measurement. The experiment result proves that the danshen root phenolic acid has a definite treatment effect in pulmonary fibrosis caused by bleocin. Therefore a new choice for treatment of the pulmonary fibrosis is provided.

Description

The purposes of Radix Salviae Miltiorrhizae total phenolic acids in preparation treatment pulmonary fibrosis medicine
Technical field
The present invention relates to the purposes of a kind of Radix Salviae Miltiorrhizae total phenolic acids in preparation treatment pulmonary fibrosis medicine.
Background technology
(pulmonary fibrosis is a kind of agnogenio PF), with diffusivity alveolitis and alveolar structure disorder, causes alveolar, interstitial pulmonary fibrosis, finally can cause the disease of respiratory failure in pulmonary fibrosis.Sickness rate is about 5,/10 ten thousand, and the trend of increasing is arranged in recent years.It is 2-4 that PF makes a definite diagnosis the average survival period in back, and 5 years survival rates are 50-70%, and existing treatment means does not make patient's survival rate make moderate progress.
The initiating agent that causes pulmonary fibrosis is not clear at present, and the someone thinks may be relevant with genetic predisposition, viral infection, immunologic function.The theory of relatively generally acknowledging relates to cell, cytokine and aspects such as signal transduction mechanism and lipid peroxidation at present.
According to the pathological characteristic of pulmonary fibrosis, its pathological process roughly is divided into three phases, and the phase I is the diffuse lesion of virulence factor to alveolar epithelial cells and vascular endothelial cell, starts the inflammation immunoreation; Second stage is that multiple inflammatory cell participates in, and discharges various cytokines and inflammatory mediator and enlarges tissue injury and cause interstitial proliferation; Phase III is fibroblast, endothelial cell migration, propagation and collagen and other extracellular matrix (Extra cellular matrix, ECM) metabolism disorder increases the weight of inflammatory damage and proliferation response with feedback system, finally causes the normal function tissue to be substituted and to reconstruct.In this process, lung inflammation cell (being mainly mononuclear phagocyte), pulmonary epithelial cells, mastocyte, endotheliocyte and interstitial lung cell (as fibroblast, myofibroblast) are by bioactive substances such as secrete cytokines, inflammatory mediators, and performance is effect directly or indirectly.In fact these three processes exist simultaneously, promote mutually, do not represent the developmental sequence of temporal separation and disease itself.
In the cytokine network that participates in pulmonary fibrosis, transforming growth factor (Transforminggrowth factor β, TGF β) is to study the most deep, most important cytokine.Pulmonary fibrosis animal model and human idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis, IPF) in, TGF β level all raises, TGF β antibody and TGF beta receptor antagonist can both weaken collagen deposition in the lung due to the bleomycin.In the pulmonary fibrosis development, pulmonary macrophage and epithelial cell may be the main sources of TGF β.Transforminggrowthfactor-(Transforming growth factor β 1, TGF β 1) in pulmonary fibrosis generation evolution, plays an important role, it has the various biological effect, fibroblast had chemotaxis, and can stimulate the immaturity growth of fibroblasts, the TGF β 1 that damage location occurs can raise fibroblast and can stimulate immaturity fibroblast proliferation and differentiation from surrounding tissue, promote collagen protein, fibronectin, hyaluronic acid, Dan Baijutang etc. in ECM, to deposit, reduce degradation of extracellular matrix.
Collagen fiber (collagenous fiber) are the important component parts of extracellular matrix, and its biochemical component is collagen protein (collagen is called for short collagen), has found to have at least at present 11 kinds of different collagen protein.I type and III Collagen Type VI are called interstitial collagen, and be widely distributed, also in the lung main between the matter Collagen Type VI.(hydroxyproline is one of the main component of body collagen protein HYP) to hydroxyproline, accounts for 13.4% of its total amino acid content, and other all do not contain HYP except that elastin laminin contains small amount of H YP (about 1%).Therefore HYP content can be used as the metabolic important indicator of collagen tissue in the tissue.
Lipid peroxidation becomes one of research focus of pulmonary fibrosis in recent years.Discover, the workman of contact silicon dioxide and asbestos, its macrophage has lipid peroxidation injury, causes the fibrosis factor thereby be activated to discharge, and causes pulmonary fibrosis.Superoxide dismutase (superoxide dismutase, SOD) oxidation and the antioxidation balance to body plays crucial effects, and this enzyme can be removed ultra-oxygen anion free radical protection cell and avoid damage.Body produces oxygen-derived free radicals by enzyme system and non-enzyme system, and the latter can attack polyunsaturated fatty acid in the biomembrane, and (polyunsaturated fatty acid PUFA), causes lipid peroxidation, and forms lipid peroxide.As: aldehyde radical (malonaldehyde, malondialdehyde, MDA), ketone group, hydroxyl, carbonyl and new free radical etc.Therefore the amount of MDA can reflect the snperoxiaized degree of body inner lipid, reflects the degree of cell injury indirectly.The mensuration of MDA usually cooperatively interacts with the mensuration of SOD, and the height of SOD vigor has reflected that indirectly body removes the ability of oxygen-derived free radicals, and the height of MDA has reflected the order of severity that body cell is attacked by free radical indirectly.
In the treatment of pulmonary fibrosis, generally acknowledge the glucocorticoid of determined curative effect at present, early stage effect is good, can inflammation-inhibiting and immunologic process.Middle and advanced stage is unsatisfactory curative effect then, and the prolonged application side effect is big, has a strong impact on body's immunity, increases the probability of secondary infection and respiratory failure.Clinical data shows only has the patient's hormone therapy below 30% effective.Therefore domestic scholars is many treats pulmonary fibrosis with Chinese medicine, and obtained certain progress, but in the experimentation, compound recipe is limited to a few flavor drug for invigorating blood circulation and eliminating stasis more, and flavour of a drug too much also are unfavorable for the research of pharmacological mechanism, and finding at present has the Chinese medicine research of certain therapeutic effect mainly to concentrate on to pulmonary fibrosis: the damage of (1) free radical resisting; (2) release of inhibition inflammatory factor, performance anti-inflammatory damaging action; (3) influence the collagenic supersession process, thereby reduce fibrinous formation; (4) influence the body immune system function, reduce the intensity of stress.Along with further going deep into of Chinese medicine monomer extraction of active ingredients isolation technics research, from natural product, seek the clear and definite activated monomer of pharmacological action, further illustrate the mechanism of action of different monomers or effective site, stage construction characteristics at the pulmonary fibrosis morbidity are treated, many target position of performance Chinese medicine are treated advantages, have become a developing direction of present treatment pulmonary fibrosis.
Radix Salviae Miltiorrhizae total phenolic acids (salvianolic acids, Sals) be from labiate Radix Salviae Miltiorrhizae (Salviamiltiorrhiza Bunge), to separate the water soluble part that obtains, be a class polyphenol hydroxyl chemical compound, contain salviol acid A, compositions such as salvianolic acid B and rosmarinic acid.Modern experimentation and clinical practice show that all Radix Salviae Miltiorrhizae acts on definitely, effect is remarkable in pulmonary fibrosis resistant, the mechanism of action of its control pulmonary fibrosis is relevant with oxygen radical removing.Discover treatment treated animal lung coefficient, hydroxyproline (HYP), surfactant content, fibroblast growth factor (fibroblast growth factor, FGF) activity all is starkly lower than model group, think the damage of its mechanism of action and non-oxidizability, to keep intracellular Ca2+ stable and to be suppressed to fibrocyte relevant to the conversion of collagen fiber.In the document, about the research of Radix Salviae Miltiorrhizae to pulmonary fibrosis influence, employing be Radix Salviae Miltiorrhizae decocting liquid, Radix Salviae Miltiorrhizae Injection (TANSHINONES) and Radix Salviae Miltiorrhizae liposome.Radix Salviae Miltiorrhizae total phenolic acids has multiple effects such as blood circulation promoting and blood stasis dispelling, antioxidation, blood fat reducing and anti-hepatic fibrosis formation.But whether Radix Salviae Miltiorrhizae total phenolic acids has the effect of pulmonary fibrosis resistant as the effective site of Radix Salviae Miltiorrhizae, is not reported so far.
Bleomycin (bleomycin, BLM) be a kind of clinically tumor chemotherapeutic drug commonly used, polytype tumor all had better therapeutic effect, but most humans can in use produce the pulmonary fibrosis of dose dependent, because BLM is widely used in the preparation of pulmonary fibrosis animal model in scientific research.In the rodent trachea, inject BLM, can cause alveolar epithelium impaired, inflammatory cell oozes out, fibroblast proliferation and collagen deposition, and no matter early stage injury of lung is all similar to human fibrosis pulmonary disease on the histopathology or on the physiology due to the BLM.
Summary of the invention
The present invention seeks to: the purposes of a kind of Radix Salviae Miltiorrhizae total phenolic acids in preparation treatment pulmonary fibrosis medicine is provided.
Technical scheme of the present invention is: the present invention duplicates mice, Pulmonary Fibrosis in Rats animal model by bleomycin, select for use Radix Salviae Miltiorrhizae total phenolic acids as medicine first, the pathological change of research pulmonary fibrosis animal lung tissue, observe the therapeutical effect of Radix Salviae Miltiorrhizae total phenolic acids, provide experimental basis thereby treat pulmonary fibrosis for Radix Salviae Miltiorrhizae total phenolic acids to the pulmonary fibrosis animal model.
Description of drawings
Fig. 1 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mouse lung coefficient (
Figure A20071002405700061
N=10);
Fig. 2 Radix Salviae Miltiorrhizae total phenolic acids to mouse lung organize alveolitis/pulmonary fibrosis degree influence ( N=10);
Fig. 3 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice TGF β 1 positive cell number (
Figure A20071002405700063
N=10);
Fig. 4 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice TGF β 1IOD (
Figure A20071002405700064
N=10);
Fig. 5 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice Collagen I IOD (
Figure A20071002405700065
Figure A20071002405700066
N=10);
Fig. 6 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice CollagenIII IOD (
Figure A20071002405700068
N=10);
Fig. 7 Radix Salviae Miltiorrhizae total phenolic acids to the pulmonary fibrosis mouse lung organize HYP content influence (
Figure A20071002405700069
N=10);
Fig. 8 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung coefficient (
Figure A200710024057000610
N=6);
Fig. 9 Radix Salviae Miltiorrhizae total phenolic acids to the influence of lung tissue of rats alveolitis/pulmonary fibrosis degree (
Figure A200710024057000611
N=6);
Figure 10 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats TGF β 1 positive cell number (
Figure A200710024057000612
N=6);
Figure 11 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats TGF β 1IOD (
Figure A200710024057000613
N=6);
Figure 12 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats Collagen I IOD ( N=6);
Figure 13 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats CollagenIII IOD ( N=6);
Figure 14 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats SOD in serum (
Figure A20071002405700072
N=6);
Figure 15 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats serum MDA ( N=6);
Figure 16 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung tissue SOD (
Figure A20071002405700074
S, n=6);
Figure 17 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung tissue MDA (
Figure A20071002405700075
Figure A20071002405700076
N=6);
Figure 18 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung tissue HYP (
Figure A20071002405700077
N=6);
The 28th day rat lung tissue disease 28 days HE * 100 of reason change-normal control group of Figure 19;
The 28th day rat lung tissue disease 28 days HE * 100 of reason change-model group of Figure 20;
The 28th day rat lung tissue disease of Figure 21 28 days HE * 100 of reason change-prednisolone acetate group;
28 days HE * 100 of the heavy dose of group of the 28th day rat lung tissue disease of Figure 22 reason change-Radix Salviae Miltiorrhizae total phenolic acids;
The 28th day rat lung tissue disease 28 days HE * 100 of reason change-Radix Salviae Miltiorrhizae total phenolic acids small dose group of Figure 23;
The 28th day rat lung tissue ultrastructural change-28 days ultrastructure * 8000 of normal control group of Figure 24;
28 days ultrastructure * 5000 of the 28th day rat lung tissue ultrastructural change-model group of Figure 25;
The 28th day rat lung tissue ultrastructural change-28 days ultrastructure * 10000 of prednisolone acetate group of Figure 26;
28 days ultrastructure * 8000 of the 28th day heavy dose of group of rat lung tissue ultrastructural change-Radix Salviae Miltiorrhizae total phenolic acids of Figure 27;
Expression SABC * 200 of 7 days TGF β 1 of expression-model group of the 7th day rat lung tissue TGF of Figure 28 β 1;
Expression SABC * 200 of the expression of the 7th day rat lung tissue TGF of Figure 29 β 1-7 days TGF β 1 of Radix Salviae Miltiorrhizae total phenolic acids small dose group;
Expression SABC * 200 of 28 days CollagenI of expression-model group of Figure 30 the 28th day rat lung tissue CollagenI, CollagenIII;
Expression SABC * 200 of 28 days Collagen I of the heavy dose of group of the expression of Figure 31 the 28th day rat lung tissue CollagenI, CollagenIII-Radix Salviae Miltiorrhizae total phenolic acids;
Expression SABC * 200 of 28 days CollagenIII of expression-model group of Figure 32 the 28th day rat lung tissue CollagenI, CollagenIII;
Expression SABC * 200 of 28 days CollagenIII of the heavy dose of group of the expression of Figure 33 the 28th day rat lung tissue CollagenI, CollagenIII-Radix Salviae Miltiorrhizae total phenolic acids.
Embodiment:
First's bleomycin causes pulmonary fibrosis mice pathological observation and salvianolic acids administration effect research
Materials and methods
Material
1, laboratory animal
Kunming mouse, 180, male and female half and half, body weight 18-22g, the cleaning level is provided by University Of Suzhou's medical college Experimental Animal Center.Laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.
2, medicine and reagent
2.1 medicine: bleomycin A5 (BLM), Nippon Kayaku K. K, batch number: 640110, import of drugs registration certificate number: H20040205; 4% chloral hydrate; Radix Salviae Miltiorrhizae total phenolic acids, Suzhou Chinese Herbal Medicine Inst plant chamber provides; Prednisone acetate tablets, Xuzhou zero diopter Pharmaceutical Co, batch number: 32022681; Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd, the accurate word H13020657. of traditional Chinese medicines
2.2 reagent: hydroxyproline content is measured reagent, and Nanjing is built up biological preparation company and bought.Rabbit polyclonal antibody TGF β 1, CollagenI, CollagenIII, instant SABC test kit, the DAB developer is produced by Wuhan Boster Biological Technology Co., Ltd..
3, instrument
Optical microscope, OLYMPUS CX31; Spectrophotometer 722 types, Shanghai Precision Scientific Apparatus Co., Ltd produces, factory number: 7070309047; Centrifuge, 80-2 type, Shanghai Surgical Operation Equipment Factory.
One, method
1, grouping and modeling
Kunming mouse, 180, weigh, numbering is divided into: the normal control group at random, model group, prednisolone acetate group (6.67mgkg-1d-1), the heavy dose of group of Radix Salviae Miltiorrhizae total phenolic acids (160mgkg-1d-1), dosage group in the Radix Salviae Miltiorrhizae total phenolic acids (80mgkg-1d-1), Radix Salviae Miltiorrhizae total phenolic acids (40mgkg-1d-1) small dose group, 30 every group.Experiment mice with 4% chloral hydrate (0.01ml/g) intraperitoneal injection of anesthesia after, get the fixed bit of lying on the back, routine disinfection, row neck median incision, passivity is separated the exposure trachea, dosage group and Radix Salviae Miltiorrhizae total phenolic acids small dose group are slowly injected BLM (5mg/kg) in the puncture of tracheal cartilages czermak space in model group, prednisolone acetate group, the heavy dose of group of Radix Salviae Miltiorrhizae total phenolic acids, the Radix Salviae Miltiorrhizae total phenolic acids, and the normal control group is injected the equal-volume normal saline.Skin suture after the injection uprightly rotates 3-5min with mice immediately, and medicinal liquid is uniformly distributed in the lung of both sides, three days continuous intramuscular injection penicillins of operation proxima luce (prox. luc) and postoperative (0.8 ten thousand U-1 day-1) 4 days.Begin to gavage administration next day after each treatment group modeling, and normal control group and model group gavage distilled water.
2, lung coefficient (pulmonary index)
Each treated animal " took off " method old through cervical vertebra on the 7th, 14,28 day respectively at experiment and puts to death 10, isolated two lungs, weigh, according to formula: heavy (the mg)/body weight (g) [18] of lung, calculate the lung coefficient.
3, pathological study
Get right lung, 4% neutral formalin is fixed, routinely the embedding of pathology method, section.Pathological section is carried out HE dyeing, according to [19] methods such as Szaopiel the degree of alveolitis and pulmonary fibrosis is divided into level Four then: 0 grade, do not have obviously and change; 1 grade, MC, extent of disease is less than 20% of full lung; 2 grades, moderate changes, and extent of disease accounts for the 20%-50% of full lung; 3 grades, severe changes, and extent of disease accounts for more than 50% of full lung.
4, immunohistochemistry technology
The main reference reagent of immunohistochemical staining step company test kit description is carried out, and the slice row high temperature high pressure process is replaced trypsinization, promotes antigen-exposed.
4.1 antibody working concentration: TGF β 1 is 1: 100, and Collagen I is 1: 200, and CollagenIII is 1: 100.
4.2 workflow:
1. the thick paraffin section of 3 μ m is attached on the microscope slide that scribbles poly-D-lysine, 60 ℃ of roasting sheet 2hr;
2. the routine of cutting into slices dewaxes to water;
3. 0.05M (PH7.2) PBS washes 3 times, each 2min;
4. antigen retrieval: in pressure cooker, add PH6.0 citrate buffer 1500ml and be heated to boiling, put into buffer with placing the section on the stainless steel frame, continue to be heated to jet back 2min, be cooled to room temperature, take out slide, distilled water flushing 2 times, PBS (PH7.2) flushing 3 times, each 2min;
5. Dropwise 5 %BSA confining liquid, room temperature 20min.Get rid of unnecessary liquid, do not wash;
6. drip one of suitably dilution and resist, about 37 ℃ of 1hr.PBS (PH7.2) washes 3 times, each 2min;
7. it is anti-to drip biotinylation two, 37 ℃ of 20min.PBS (PH7.2) washes 3 times, each 2min;
8. drip SABC reagent (streptavidin-avidin-biotin complex); Hatch 20min for 37 ℃, PBS (PH7.2) washes 3 times, each 2min;
9. drip fresh configuration DAB developer (get the 3ml distilled water, add each 3 of A, B, C reagent, mixing), color development at room temperature, controlling reaction time under the mirror, fully washing; 0
10. haematoxylin is slightly redyed.Dehydration, transparent, mounting.Microscopic examination.
4.3 graphical analysis and processing:
Adopt PAS9000 pathological image analytical system (Ver9.2.1), (Integraloptical density IOD) weighs the content of TGF-β 1 in the lung tissue, Collagen I, III with integral optical density.The density parameter of cell is the photoelectricity transformation principle that utilizes video camera input cell image information, cell is absorbed under same light source or through the numerical value of light as optical density (also claiming absorbance).The measurement target color is dark more, and the light that sees through is few more, and optical density is big more.Therefore, optical density value can be used for weighing the content of certain composition in tissue or the cell.Integral optical density is the stack of every bit optical density in the whole visual field.Be to one of immunohistochemical staining interpretation of result method comparatively accurately.Get the preceding all preheatings of figure more than 20 minutes at every turn, and keep light source, voltage, contrast, sensitivity etc. stable.
1. TGF β 1: 5 high power fields (* 400) are chosen in every section, with the macrophage endochylema dye for brown, pale brown color, brown positive, counting positive cell number/high power field [23].Adopt PAS9000 pathological image analytical system (Ver9.2.1) simultaneously, carry out quantitative analysis, calculate the IOD[24 in each visual field according to the positive painted depth of SABC], average.
2. Collagen I, Collagen III: adopt PAS9000 pathological image analytical system (Ver9.2.1), carry out quantitative analysis, 5 high power fields (* 400) that only contain interstitial lung are chosen in every section, calculate the IOD[24 in each visual field according to the positive painted depth of SABC], average.
5, the lung tissue hydroxyproline (hydroxyproline, HYP) assay:
Measuring the test kit description by hydroxyproline content adopts the sample alkali hydrolysis method to carry out the HYP assay: take by weighing fritter lung tissue (about 30mg) and put in vitro, accurately add hydrolyzed solution 0.1ml, mixing, add a cover back boiling water bath hydrolysis 20min, afterwards each test tube liquid pH value is transferred to 6.0-6.8, add proper amount of active carbon, mixing, the centrifugal 10min of 3500r/min, get supernatant 0.6ml, after adding three kinds of reagent successively, mixing, 60 ℃ of water-bath 15min, the centrifugal 10min of 3500r/min, get supernatant at 550nm, the 0.5cm optical path is measured the absorbance of each pipe down, and formula calculates HYP content in the lung tissue to specifications afterwards.
6, statistical method:
Ranked data are converted into measurement data, adopt SPSS11.0 software to analyze, experimental data with
Figure A20071002405700111
Expression, the comparable group differences carries out the t check.
The result
1, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of mouse lung coefficient:
Each time point model group mouse lung coefficient all is higher than normal control group (p＜0.01) after the modeling, and measured value increased the most remarkable in 28 days; Radix Salviae Miltiorrhizae total phenolic acids and prednisolone acetate group mouse lung coefficient all than the same period model group significantly reduce (P＜0.01, P＜0.05).(see Table 1-1, Fig. 1).
Table 1-1 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mouse lung coefficient (
Figure A20071002405700112
N=10)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 6.67 7.318±0.412 10.266±1.552## 7.705±1.278 ** 6.481±0.562 10.030±1.751## 8.109±1.279 * 5.775±0.473 10.655±5.213## 6.391±0.926 *
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 7.635±1.157 ** 7.328±1.288 ** 8.028±1.165 ** 7.187±1.434 ** 7.630±1.068 ** 8.464±1.425 * 6.351±1.037 * 6.661±1.412 * 6.343±1.148 *
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.
Fig. 1 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mouse lung coefficient ( N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.)
2, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of mouse lung pathomorphism:
2.1 gross examination of skeletal muscle: the two lung pinkiness of normal control group, smooth surface, elasticity is better.The 7th day two lungs of model group mice are kermesinus, and volume is bigger, poor flexibility; 14th, two lungs are pale 28 days the time, volume-diminished, and hardness increases.All the other each treatment groups all are lighter than model group.
2.2 om observation: normal control group lung tissue structure is clear, and alveolar wall is not seen and thickened the alveolar epithelial cells structural integrity.Model group in the time of 7 days alveolitis obvious, alveolar space sees that a large amount of macrophages, neutrophilic granulocyte, plasma cell ooze out, alveolar septum broadening, visible a small amount of fibroblast and substrate hypertrophy thereof; Alveolitis alleviates in the time of 14 days, and alveolar septum sees that fibroblast and substrate increase in a large number; Alveolitis is slight in the time of 28 days, the remarkable broadening of alveolar septum, visible a large amount of fibroblasts and collagen fiber hypertrophy, a small amount of macrophage, lymphocyte and plasmocyte infiltrating.Radix Salviae Miltiorrhizae total phenolic acids and the broadening of prednisolone acetate treatment group mice alveolar septum obviously alleviate than each time point model group, macrophage, lymphocyte and plasmocyte infiltrating are lighter, alveolar structure is normal substantially, and compare fibrosis all has in various degree to alleviate and (see Table 1-2, Fig. 2) with model group.
Table 1-2 Radix Salviae Miltiorrhizae total phenolic acids to mouse lung organize alveolitis/pulmonary fibrosis degree influence (
Figure A20071002405700121
N=10)
Group Dosage (mg/kg) 7 days 14 days 28
Normal control group model group prednisolone acetate group 6.67 0.10±0.32 2.30±0.68## 1.00±0.94 ** 0.10±0.32 2.10±0.74## 1.20±0.63 ** 0.20±0.42 2.70±0.48## 1.20±0.42 **
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 1.50±0.71 * 1.40±0.70 ** 1.50±0.71 * 0.90±0.74 ** 1.40±0.52 * 1.20±0.63 ** 1.70±0.68 ** 2.00±0.82 * 2.00±0.82 *
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.
Fig. 2 Radix Salviae Miltiorrhizae total phenolic acids to mouse lung organize alveolitis/pulmonary fibrosis degree influence (
Figure A20071002405700122
N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
3, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of mouse lung tissue T GF β 1 protein expression:
TGF β 1 is expressed in endochylema, and the cell of expression has bronchial mucosa epithelial cell, pulmonary alveolar macrophage, alveolar epithelial cells, vascular endothelial cell.The endochylema of the minority mucous membrane of bronchiole epithelial cell of normal control group mice, alveolar epithelial cells, vascular endothelial cell has a small amount of positive expression; Model group mice mucous membrane of bronchiole epithelial cell alveolar epithelial cells, vascular endothelial cell TGF β 1 express more obvious and wider; All the other each treatment group mice mucous membrane of bronchiole epithelial cells, alveolar epithelial cells, vascular endothelial cell TGF β 1 expression decreased.
Compare the model group mice with the normal control group and a large amount of TGF β 1 male pulmonary alveolar macrophages (P＜0.01) in 7 days alveolar spaces and a matter, occurs, with single or pulmonary alveolar macrophage appearance in groups, when 14 days and 28 days in an alveolar space and a matter TGF β 1 male pulmonary alveolar macrophage decreased number, compare with the normal control group and to still have significant difference (P＜0.01); 7 days, 14 days, 28 days positive cell numbers of prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group are than the corresponding time point positive cell number minimizing of model group, and significant difference (P＜0.01, P＜0.05), 28 days there was no significant differences were arranged in 7 days and 14 days.(see Table 1-3, Fig. 3)
Each time point IOD of model group is all apparently higher than normal control group (P＜0.01), and prednisolone acetate group and 7 days, 14 days IOD of Radix Salviae Miltiorrhizae total phenolic acids group be than the obvious reduction of model group (P＜0.01, P＜0.05), compares there was no significant difference with the normal control group in 28 days.(see Table 1-4, Fig. 4)
Table 1-3 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin pulmonary fibrosis mice TGF β 1 positive cell number
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 6.67 11.2±2.10 43.2±4.52## 32.2±5.25 ** 12.4±4.60 34.7±5.54## 28.7±4.22 * 10.9±3.07 26.4±4.03## 22.8±3.65
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 35.5±2.01 ** 30.4±4.06 ** 31.7±3.65 ** 26.0±4.00 ** 26.7±4.47 ** 28.0±5.96 * 23.8±4.32 23.0±5.42 21.9±6.17
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.( n＝10)
Fig. 3 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice TGF β 1 positive cell number (
Figure A20071002405700132
N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
Table 1-4 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin pulmonary fibrosis mice TGF β 1 integral optical density (IOD)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 6.67 277.23±22.87 416.94±81.87## 338.34±48.35 * 260.97±10.52 391.34±85.65## 310.34±44.14 * 268.09±23.48 316.18±50.69## 293.99±53.20
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 323.85±42.18 ** 313.94±39.50 ** 313.80±40.70 ** 298.51±47.94 ** 310.24±36.73 * 305.79±40.21 * 304.40±42.49 307.68±21.95 305.17±36.50
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700141
n＝10)
Fig. 4 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice TGF β 1IOD (
Figure A20071002405700142
N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
4, Radix Salviae Miltiorrhizae total phenolic acids is organized the influence of the expression of CollagenI, CollagenIII to mouse lung:
CollagenI, CollagenIII present brown, pale brown color or sepia behind immunohistochemical staining.Normal control group mouse lung organizes medium vessels wall, bronchus, bronchioles wall that the expression of CollagenI, CollagenIII is arranged, and the expression of being dispersed in property is arranged in the interstitial lung; Visible CollagenI, CollagenIII are focal, patch shape, streak distribution in the model group mice interstitial lung.Both IOD all began to increase in the 7th day, and progressively increased.The prednisolone acetate group is compared with model group with each time point IOD of Radix Salviae Miltiorrhizae total phenolic acids group, all has significance to reduce (P＜0.01, P＜0.05).(see Table 1-5, Fig. 5, table 1-6, Fig. 6)
Table 1-5 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin pulmonary fibrosis mice CollagenI integral optical density (IOD)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 6.67 189.03±8.01 302.71±21.90## 280.43±23.13 * 190.13±16.84 326.30±31.06## 287.88±28.56 * 181.47±17.95 361.42±47.99## 311.65±28.32 *
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 274.92±24.32 * 281.37±22.40 * 272.88±34.16 * 294.05±20.23 * 287.47±19.98 ** 296.18±14.81 * 314.27±35.38 * 302.44±24.59 ** 312.47±32.19 *
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700143
n＝10)
Fig. 5 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice CollagenI IOD (
Figure A20071002405700144
Figure A20071002405700145
N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
Table 1-6 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin pulmonary fibrosis mice CollagenIII integral optical density (IOD)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 6.67 288.75±22.78 388.89±23.11## 355.79±36.61 * 278.28±21.12 409.32±31.71## 379.17±20.39 * 290.43±6.65 457.42±40.11## 416.89±44.62 *
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 365.30±15.57 * 359.02±12.73 ** 359.87±26.67 * 378.14±19.06 * 370.10±11.23 ** 373.07±15.38 ** 406.25±24.49 ** 401.18±24.22 ** 417.37±25.83 *
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700151
n＝10)
Fig. 6 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin pulmonary fibrosis mice CollagenIII IOD (
Figure A20071002405700152
Figure A20071002405700153
N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
5, Radix Salviae Miltiorrhizae total phenolic acids is organized the influence of HYP content to mouse lung:
Model group mouse lung HYP content and normal control group began than 14 days to raise, and 28 days the most remarkable; Each time point HYP content of the heavy dose of treatment of Radix Salviae Miltiorrhizae total phenolic acids back is all than the obvious reduction of model group (P＜0.05, P＜0.01); In, low dose of mice treats back 7 days, 28 days lung HYP content and significantly reduces (P＜0.01).Administration in the time of 14 days mouse lung HYP content downward trend is arranged; Though the visible HYP content in prednisolone acetate treatment back reduces than model group, does not have significant difference.(see Table 1-7, Fig. 7)
Table 1-7 Radix Salviae Miltiorrhizae total phenolic acids is organized the influence of HYP content (μ g/mg) to the pulmonary fibrosis mouse lung
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 6.67 0.40±0.06 0.37±0.05 0.34±0.04 0.43±0.19 0.61±0.14# 0.56±0.10 0.44±0.09 0.66±0.07## 0.60±0.14
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 160.0 80.0 40.0 0.30±0.04 0.29±0.04 0.25±0.04 0.46±0.07 * 0.57±0.10 0.56±0.16 0.52±0.14 * 0.52±0.10 ** 0.45±0.09 **
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700161
n＝10)
Fig. 7 Radix Salviae Miltiorrhizae total phenolic acids to the pulmonary fibrosis mouse lung organize HYP content influence ( N=10)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
Brief summary
1, the Radix Salviae Miltiorrhizae total phenolic acids administration all can obviously reduce the lung coefficient of pulmonary fibrosis mice due to the bleomycin after 7,14,28 days;
2, Radix Salviae Miltiorrhizae total phenolic acids can alleviate the inflammatory reaction that bleomycin causes the pulmonary fibrosis mouse lung in early days, can suppress lung tissue collagen deposition and fibrosis late period and form;
3, Radix Salviae Miltiorrhizae total phenolic acids can reduce the expression of pulmonary fibrosis mouse lung tissue T GF β 1 due to the bleomycin in early days;
4, Radix Salviae Miltiorrhizae total phenolic acids can reduce due to the bleomycin deposition of I type, III Collagen Type VI in the pulmonary fibrosis mice interstitial lung;
5, Radix Salviae Miltiorrhizae total phenolic acids can reduce the content of the lung hydroxyproline of pulmonary fibrosis mice due to the bleomycin.
The second portion bleomycin causes lung fibrosis in rats pathological observation and salvianolic acids administration effect research
Materials and methods
One, material
1, laboratory animal
The SD rat, 108, male and female half and half, body weight 180-220g, the cleaning level is provided by University Of Suzhou's medical college Experimental Animal Center.Laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.
2, medicine and reagent
2.1 medicine: same first.
2.2 reagent: SOD vitality test reagent, MDA assay reagent, Nanjing build up biological preparation company and buy.The same first of other reagent.
3, instrument
Interior cut tissue refiner, ZS88-1 type, West Zhejiang Province machinery plant; Desk-top refrigerated centrifuge, 5180R type, U.S. Effendorf company; The H-600 of Hitachi transmission electron microscope; The same first of Other Instruments.
Two, method
1, grouping and modeling [16,17]
The SD rat, 108, weigh, numbering is divided into: the normal control group at random, model group, prednisolone acetate group (3.33mgkg-1d-1), the heavy dose of group of Radix Salviae Miltiorrhizae total phenolic acids (80mgkg-1d-1), dosage group in the Radix Salviae Miltiorrhizae total phenolic acids (40mgkg-1d-1), Radix Salviae Miltiorrhizae total phenolic acids (20mgkg-1d-1) small dose group, 18 every group.Experimental rat with 4% chloral hydrate (1ml/g) intraperitoneal injection of anesthesia after, get the fixed bit of lying on the back, routine disinfection, row neck median incision, passivity is separated the exposure trachea, dosage group and Radix Salviae Miltiorrhizae total phenolic acids small dose group are slowly injected BLM (5mg/kg) in the puncture of tracheal cartilages czermak space in model group, prednisolone acetate group, the heavy dose of group of Radix Salviae Miltiorrhizae total phenolic acids, the Radix Salviae Miltiorrhizae total phenolic acids, and the normal control group is injected the equal-volume normal saline.Skin suture after the injection uprightly rotates 3-5min with rat immediately, and medicinal liquid is uniformly distributed in the lung of both sides, three days continuous intramuscular injection penicillins of operation proxima luce (prox. luc) and postoperative (80,000 U-1 day-1) 4 days.Begin to gavage administration next day after each treatment group modeling, and normal control group and model group gavage distilled water.
2, lung coefficient
Each treated animal is put to death 6 through cervical vertebra from disconnected method respectively at experiment the 7th, 14,28 day, isolates two lungs, weighs, according to formula: heavy (the mg)/body weight (g) of lung, calculating lung coefficient.
3, pathological study
The same first of method.
4, transmission electron microscope observing
Get 28 days rat lung tissues after 4% glutaraldehyde fixative is fixing, fixing behind the reuse osmic acid, the dehydration of acetone gradient, 618 epoxy resin embeddings, the section of LKB ultramicrotome, the H-600 of Hitachi transmission electron microscope observing.
5, immunohistochemistry technology
5.1 antibody working concentration: TGF β 1 is 1: 150, and CollagenI is 1: 200, and CollagenIII is 1: 100.
5.2 workflow: same first.
5.3 graphical analysis and processing: same first.
6, the mensuration of SOD in serum, MDA
Getting serum 5 μ l adopts xanthine oxidase to carry out the SOD vitality test by SOD mensuration test kit description; Getting serum 0.1ml adopts thiobarbituricacid (TBA) method to carry out the MDA assay by MDA mensuration test kit description.
7, lung tissue SOD, MDA index determining
7.1 the preparation of tissue homogenate: take by weighing lung tissue piece (about 150mg), remove blood, wipe away driedly with filter paper, add cold saline, volume is 9 times of lung tissue piece weight, makes 10% lung tissue homogenate with interior cut tissue refiner.
7.2SOD, MDA measures: get 10% lung tissue homogenate with the centrifugal 4min of desk-top refrigerated centrifuge 10000r/min, stay supernatant, get 0.1ml and measure test kit description employing thiobarbituricacid (TBA) method by MDA and carry out the MDA assay; Other gets 0.1ml and adds the 0.9ml normal saline and make 1% lung tissue homogenate, gets 30 μ l and measures the test kit description by SOD and adopt xanthine oxidase to carry out the SOD vitality test.
8, lung tissue HYP assay
The same first of method.
9, statistical method
Ranked data are converted into measurement data, adopt SPSS11.0 software to analyze, experimental data with
Figure A20071002405700181
Expression, the comparable group differences carries out the t check.
The result
1, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of induced lung coefficient:
Each time point model group induced lung coefficient all is significantly higher than normal control group (p＜0.01) after the modeling; Radix Salviae Miltiorrhizae total phenolic acids and prednisolone acetate group induced lung coefficient all than the same period model group significantly reduce (P＜0.01, P＜0.05).(see Table 2-1, Fig. 8).
Table 2-1 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung coefficient (
Figure A20071002405700182
N=6)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 5.08±0.07 13.17±0.73## 9.88±2.49 ** 5.45±0.92 13.22±1.43## 11.17±1.20 * 5.88±1.16 13.31±1.68## 10.38±2.37 *
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 9.93±2.21 ** 11.03±2.08 * 9.78±2.99 * 10.46±1.95 * 11.06±0.61 * 10.69±1.99 * 10.33±2.44 * 7.67±1.21 ** 11.54±0.74 *
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.
Fig. 8 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung coefficient (
Figure A20071002405700191
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
2, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of lung tissue of rats pathomorphism:
2.1 gross examination of skeletal muscle: the two lung pinkiness of normal control group, smooth surface, elasticity is better.The 7th day two lungs of model group rat are kermesinus, and volume is bigger, poor flexibility; 14th, two lungs are pale 28 days the time, volume-diminished, and hardness increases.All the other each treatment groups all are lighter than model group.
2.2 om observation: normal control group lung tissue structure is clear, and alveolar wall is not seen and thickened the alveolar epithelial cells structural integrity.Model group in the time of 7 days alveolitis obvious, alveolar space sees that a large amount of macrophages, neutrophilic granulocyte, plasma cell ooze out, alveolar septum broadening, visible a small amount of fibroblast and substrate hypertrophy thereof; Alveolitis alleviates in the time of 14 days, and alveolar septum sees that fibroblast and substrate increase in a large number, alveolitis is slight 28 days the time, the remarkable broadening of alveolar septum, visible a large amount of fibroblasts and collagen fiber hypertrophy, a small amount of macrophage, lymphocyte and plasmocyte infiltrating.Radix Salviae Miltiorrhizae total phenolic acids and the broadening of prednisolone acetate treatment group mice alveolar septum obviously alleviate than each time point model group, macrophage, lymphocyte and plasmocyte infiltrating are lighter, alveolar structure is normal substantially, and compare fibrosis all has in various degree to alleviate and (see Table 2-2, Fig. 9) with model group.(accompanying drawing 19,20,21,22,23 is seen in the reason change of the 28th day rat lung tissue disease)
Table 2-2 Radix Salviae Miltiorrhizae total phenolic acids to the influence of lung tissue of rats alveolitis/pulmonary fibrosis degree ( N=6)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 0.83±0.41 2.83±0.41## 1.67±0.52 ** 0.67±0.52 2.50±0.55## 1.33±0.52 ** 0.83±0.41 2.67±0.82## 1.17±0.75 **
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 1.50±1.05 * 1.83±0.41 ** 1.67±0.52 ** 1.33±0.82 * 1.50±0.55 * 1.50±0.55 * 1.50±0.55 * 1.33±0.52 ** 1.17±0.41 **
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.
Fig. 9 Radix Salviae Miltiorrhizae total phenolic acids to the influence of lung tissue of rats alveolitis/pulmonary fibrosis degree (
Figure A20071002405700201
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
2.3 Ultrastructural observation: in the time of 28 days, normal control group I, II type alveolar epithelial cells structural integrity, iuntercellular connects normal, and vascular endothelial cell and basement membrane are intact.Model group part I, II type alveolar epithelial cells endoplasmic reticulum and mitochondrial swelling, its ridge is sparse, the karyon pyknosis, visible downright bad self-dissolving changes in the kytoplasm; II type alveolar epithelial cells quantity reduces, and lamellar body increases in the kytoplasm, and the emptying phenomenon is arranged; The distortion of vascular endothelial cell nuclear, basal membrane thickening.The alveolar space atrophy, wherein visible downright bad free II type epithelial cell; Alveolar septum broadening is particularly evident, and visible fibroblast is increased, and collagen fiber deposition irregular in a large number, arrangement disorder is arranged therebetween, a light weight degree edema, and the mononuclear cell neutrophil infiltration is arranged.The still visible endoplasmic reticulum of prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group I, II type alveolar epithelial cells, the accidental a small amount of cell infiltration of mitochondrion mild swelling; Between in the matter deposition of collagen fiber than model group alleviating in various degree arranged.(rat lung tissue ultrastructural change was seen accompanying drawing 24,25,26,27 in the 28th day)
3, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of lung tissue of rats TGF β 1 protein expression:
TGF β 1 is positioned endochylema, and bronchial mucosa epithelial cell, pulmonary alveolar macrophage, alveolar epithelial cells, vascular endothelial cell all can be expressed.The endochylema of the minority mucous membrane of bronchiole epithelial cell of rats in normal control group, alveolar epithelial cells, vascular endothelial cell has a small amount of positive expression; Model group rat mucous membrane of bronchiole epithelial cell, alveolar epithelial cells, vascular endothelial cell then TGF β 1 are expressed more obvious and wider; All the other each treatment group rat mucous membrane of bronchiole epithelial cells, alveolar epithelial cells, vascular endothelial cell TGF β 1 expression decreased.
Compare the model group rat with the normal control group and a large amount of TGF β 1 male pulmonary alveolar macrophages (P＜0.01) in 7 days alveolar spaces and a matter, occurs, with single or pulmonary alveolar macrophage appearance in groups, when 14 days and 28 days in an alveolar space and a matter TGF β 1 male pulmonary alveolar macrophage decreased number, compare with the normal control group and to still have significant difference (P＜0.01); 7 days, 14 days, 28 days positive cell numbers of prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group are than the corresponding time point positive cell number minimizing of model group, and significant difference (P＜0.01, P＜0.05), 28 days there was no significant differences were arranged in 7 days and 14 days.(see Table 2-3, Figure 10)
Table 2-3 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin lung fibrosis in rats TGF β 1 positive cell number
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 7.33±2.07 26.67±3.01## 20.50±4.14 * 7.67±4.13 23.17±2.40## 18.33±1.21 ** 8.17±2.14 13.50±2.07## 11.50±2.74
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 19.50±4.68 * 20.33±3.39 ** 20.50±5.68 * 19.50±2.07 * 19.00±2.61 * 19.17±2.79 * 12.83±2.48 13.00±2.97 11.67±4.18
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700211
n＝6)
Figure 10 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats TGF β 1 positive cell number (
Figure A20071002405700212
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
Each time point IOD of model group is all apparently higher than normal control group (P＜0.01), and prednisolone acetate group and 7 days, 14 days IOD of Radix Salviae Miltiorrhizae total phenolic acids group be than the obvious reduction of model group (P＜0.01, P＜0.05), compares there was no significant difference with the normal control group in 28 days.(see Table 2-4, Figure 11), (accompanying drawing 28,29 is seen in the expression of the 7th day rat lung tissue TGF β 1)
Table 2-4 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin lung fibrosis in rats TGF β 1 integral optical density (IOD)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 211.90±20.34 312.81±21.79## 269.11±20.85 ** 221.37±36.14 299.88±23.55## 268.19±21.68 * 226.82±27.29 276.25±5.12## 255.37±21.20
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 276.50±35.22 * 273.25±16.17 ** 275.48±19.15 * 263.95±12.16 ** 266.61±15.08 * 268.85±14.95 * 260.12±13.47 264.11±24.69 263.53±12.37
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700221
n＝6)
Figure 11 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats TGF β 1 IOD (
Figure A20071002405700222
Figure A20071002405700223
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
4, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of the expression of lung tissue of rats Collagen I, CollagenIII:
CollagenI, CollagenIII present brown, pale brown color or brown behind immunohistochemical staining.Rats in normal control group lung tissue medium vessels wall, bronchus, bronchioles wall have the expression of Collagen I, CollagenIII, and the expression of being dispersed in property is arranged in the interstitial lung; Visible CollagenI, CollagenIII are focal, patch shape, streak distribution in the model group rat interstitial lung.Both IOD all began to increase in the 7th day, and progressively increased.The prednisolone acetate group is compared with model group with each time point IOD of Radix Salviae Miltiorrhizae total phenolic acids group, all has significance to reduce (P＜0.01, P＜0.05).(see Table 2-5, Figure 12, table 2-6, Figure 13), (accompanying drawing 30,31,32,33 is seen in the expression of the 28th day rat lung tissue Collagen I, CollagenIII)
Table 2-5 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin lung fibrosis in rats CollagenI integral optical density (IOD)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 238.13±31.73 329.30±17.93## 300.84±10.03 ** 242.39±15.08 352.82±9.51## 328.49±35.15 ** 248.53±23.17 394.27±17.05## 340.72±16.50 **
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 300.78±9.96 ** 299.95±13.39 * 302.23±11.13 * 311.42±28.21 ** 323.55±22.72 * 323.86±21.25 * 330.35±14.48 ** 332.71±23.73 ** 335.61±13.78 **
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700224
n＝6)
Figure 12 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats CollagenI IOD (
Figure A20071002405700231
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
Table 2-6 Radix Salviae Miltiorrhizae total phenolic acids is to the influence of bleomycin lung fibrosis in rats CollagenIII integral optical density (IOD)
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 241.13±20.53 332.73±13.55## 290.77±6.56 ** 251.65±12.71 371.95±12.41## 348.65±15.37 * 253.09±19.86 380.64±11.74## 347.80±9.73 **
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 305.81±9.71 ** 308.30±8.19 ** 312.51±8.37 * 349.30±5.73 ** 350.13±16.11 * 348.54±21.93 * 354.34±5.65 ** 363.52±9.65 * 358.07±9.49 **
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700232
n＝6)
Figure 13 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats CollagenIII IOD (
Figure A20071002405700233
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
5, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of rat blood serum SOD, MDA:
5.1SOD variation: model group SOD numerical value increases gradually, compares with the normal control group, and the 7th, 14 day SOD vigor has significance to reduce (P＜0.01, P＜0.05), and the 28th day is near with the normal control winding, no significant difference.Prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group all are higher than model group at the 7th, 14 day SOD vigor, are lower than the normal control group, recover normal gradually on the 28th day.Compare with model group, prednisolone acetate group and the large and small dosage group of Radix Salviae Miltiorrhizae total phenolic acids had significant difference (P＜0.05) at the 7th day; The dosage group all had significant difference (P＜0.01, P＜0.05) in the 7th, 14 days in the Radix Salviae Miltiorrhizae total phenolic acids.(see Table 2-7, Figure 14)
Table 2-7 Radix Salviae Miltiorrhizae total phenolic acids is to the influence (U/mgprot) of bleomycin lung fibrosis in rats SOD in serum
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 279.95±20.83 236.04±21.21## 264.86±16.15 * 273.77±20.91 249.73±13.58# 266.47±19.91 278.90±9.69 268.17±9.45 280.94±10.91
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 272.27±21.51 * 261.19±10.13 * 268.79±16.25 * 266.97±15.03 271.89±9.35 ** 262.53±20.93 277.37±25.92 277.88±16.86 272.77±14.76
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700241
n＝6)
Figure 14 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats SOD in serum (
Figure A20071002405700242
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
5.2MDA variation: model group MDA numerical value reduces gradually, compares with the normal control group, and the 7th, 14 day MDA content all has significance to raise (P＜0.01) the 28th day there was no significant difference.Prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group are lower than model group at the 7th, 14 day MDA content, are higher than the normal control group, recover normal gradually on the 28th day.Compare with model group, prednisolone acetate group and the large and small dosage group of Radix Salviae Miltiorrhizae total phenolic acids had significance to reduce (P＜0.01, P＜0.05) in the 7th, 14 days, and the dosage group had significance to reduce (P＜0.01) on the 7th day in the Radix Salviae Miltiorrhizae total phenolic acids.(see Table 2-8, Figure 15)
Table 2-8 Radix Salviae Miltiorrhizae total phenolic acids is to the influence (nmol/mgprot) of bleomycin lung fibrosis in rats serum MDA
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 1.52±0.48 3.24±0.55## 2.43±0.62 * 1.62±0.41 2.45±0.23## 2.06±0.24 * 1.49±0.38 1.79±0.22 1.52±0.27
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 2.40±0.43 * 1.96±0.28 ** 2.45±0.56 * 1.85±0.29 ** 2.06±0.40 2.01±0.21 ** 1.58±0.09 1.64±0.22 1.52±0.27
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700243
n＝6)
Figure 15 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats serum MDA (
Figure A20071002405700244
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
6, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of lung tissue of rats SOD, MDA:
6.1SOD variation: model group SOD numerical value increases gradually, compares with the normal control group, and the 7th, 14 day SOD vigor has significance to reduce (P＜0.05), and the 28th day is near with the normal control winding, no significant difference.Prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group all are higher than model group at the 7th, 14 day SOD vigor, are lower than the normal control group, recover normal gradually on the 28th day.Compare with model group, prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids small dose group had significant difference (P＜0.05) at the 7th day; The big or middle dosage group of Radix Salviae Miltiorrhizae total phenolic acids all had significant difference (P＜0.01, P＜0.05) in the 7th, 14 days.(see Table 2-9, Figure 16)
Table 2-9 Radix Salviae Miltiorrhizae total phenolic acids is to the influence (U/mgprot) of bleomycin lung fibrosis in rats lung tissue SOD
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 33.46±3.42 27.87±1.51# 30.76±2.40 * 34.60±5.70 28.50±1.62# 27.32±6.69 32.93±2.66 30.44±1.94 27.08±4.10
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 31.06±2.49 * 29.56±0.88 * 30.27±1.29 * 34.01±2.05 ** 32.91±2.58 ** 32.22±3.76 33.77±6.32 33.51±4.06 30.09±3.03
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700251
n＝6)
Figure 16 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung tissue SOD (
Figure A20071002405700253
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
6.2MDA variation: model group MDA numerical value reduces gradually, compares with the normal control group, and the 7th, 14 day MDA content all has significance to raise (P＜0.01) the 28th day there was no significant difference.Prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group are lower than model group at the 7th, 14 day MDA content, are higher than the normal control group, recover normal gradually on the 28th day.Compare with model group, prednisolone acetate group and the large and small dosage group of Radix Salviae Miltiorrhizae total phenolic acids had significance to reduce (P＜0.01, P＜0.05) in the 7th, 14 days, and the dosage group had significance to reduce (P＜0.01) on the 7th day in the Radix Salviae Miltiorrhizae total phenolic acids.(see Table 2-10, Figure 17)
Table 2-10 Radix Salviae Miltiorrhizae total phenolic acids is to the influence (nmol/mgprot) of bleomycin lung fibrosis in rats lung tissue MDA
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 3.556±1.215 6.094±2.126## 4.441±0.956 * 3.528±1.041 6.268±0.880## 5.129±0.483 * 3.447±0.925 5.026±1.041 4.219±0.889
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 4.610±0.472 * 4.453±0.699 * 4.374±0.392 * 5.113±0.389 * 4.667±2.323 3.732±0.753 ** 4.380±1.989 4.677±0.551 4.866±1.956
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.(
Figure A20071002405700261
n＝6)
Figure 17 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung tissue MDA (
Figure A20071002405700262
Figure A20071002405700263
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
7, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of lung tissue of rats HYP content:
Model group induced lung HYP content and normal control group began than 14 days to raise 28 days (P＜0.01) the most remarkable; Though prednisolone acetate is organized each time point and compares HYP with model group and descend to some extent, does not all have significant difference.Each time point HYP content of Radix Salviae Miltiorrhizae total phenolic acids treatment back all reduces than model group, and there were significant differences in the 28th day (P＜0.05, P＜0.01).(see Table 2-11, Figure 18)
Table 2-11 Radix Salviae Miltiorrhizae total phenolic acids is to the influence (μ g/mg) of bleomycin lung fibrosis in rats lung tissue HYP
Group Dosage (mg/kg) 7 days 14 days 28 days
Normal control group model group prednisolone acetate group 3.33 0.63±0.053 0.62±0.107 0.55±0.057 0.71±0.052 0.81±0.131 0.80±0.122 0.87±0.096 1.35±0.104## 1.26±0.222
Radix Salviae Miltiorrhizae total phenolic acids Dosage group small dose group in heavy dose of group 80.0 40.0 20.0 0.46±0.068 0.46±0.076 0.43±0.019 0.77±0.069 0.8±0.070 0.79±0.119 1.18±0.134 * 1.01±0.139 ** 0.96±0.249 **
Annotate: #P＜0.05, ##p＜0.01 is compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group.( n＝6)
Figure 18 Radix Salviae Miltiorrhizae total phenolic acids to the influence of bleomycin lung fibrosis in rats lung tissue HYP (
Figure A20071002405700265
N=6)
(#P＜0.05, ##p＜0.01 are compared with the normal control group; *P＜0.05, *P＜0.01 is compared with model group)
Brief summary
1, the Radix Salviae Miltiorrhizae total phenolic acids administration can obviously reduce the lung coefficient of lung fibrosis in rats due to the bleomycin after 7,14,28 days;
2, the Radix Salviae Miltiorrhizae total phenolic acids administration can alleviate bleomycin and causes the lung fibrosis in rats alveolitis after 7 days, and administration can alleviate the pulmonary fibrosis degree after 14,28 days;
3, the Radix Salviae Miltiorrhizae total phenolic acids administration can reduce the expression of lung fibrosis in rats lung tissue TGF β 1 due to the bleomycin in 7,14 days;
4, Radix Salviae Miltiorrhizae total phenolic acids can obviously strengthen the early stage lung tissue of lung fibrosis in rats due to the bleomycin, serum activity of SOD, obviously reduces MDA content;
5, Radix Salviae Miltiorrhizae total phenolic acids can reduce due to the bleomycin deposition of I type, III Collagen Type VI in the lung fibrosis in rats interstitial lung.
6, the Radix Salviae Miltiorrhizae total phenolic acids administration can reduce the lung tissue hydroxyproline content of lung fibrosis in rats due to the bleomycin in 28 days.
Discuss
Under the invention process condition, modeling is in the time of the 7th day, model group animal alveolitis is (P＜0.01) obviously, hydroxyproline content obviously increases (P＜0.01) light microscopic, SABC and Electron microscope showed lung fiber degree than normal group and obviously raises in the 28th day lung tissue, the a large amount of hypertrophy of fibroblast and collagen fiber, fibrosis has become major lesions, and pulmonary fibrosis animal model replication success due to the BLM is described.Preparing with BLM on the basis of pulmonary fibrosis animal model, this paper experimental result shows, give the Radix Salviae Miltiorrhizae total phenolic acids treatment after, the animal pulmonary fibrosis obviously alleviates.
Now bleomycin is caused the evaluation of pulmonary fibrosis animal model and Radix Salviae Miltiorrhizae total phenolic acids bleomycin is caused the curative effect mechanism of pulmonary fibrosis animal model, be discussed below:
One, bleomycin causes the evaluation of pulmonary fibrosis animal model
The cause of disease of pulmonary fibrosis it be not immediately clear that its pathogenesis is not also illustrated fully.It is generally acknowledged that dust sucks, infects, sucks some gas and uses some drugs all can cause pulmonary fibrosis.For preliminary therapeutical effect and the mechanism of inquiring into Radix Salviae Miltiorrhizae total phenolic acids to pulmonary fibrosis, at first must duplicate stable pulmonary fibrosis animal model; And that experimental animal model is selected is correct, duplicate stable is the basic assurance that this experiment is carried out smoothly.Consulting on the basis of pertinent literature, this project selection bleomycin cause the experimental animal model of pulmonary fibrosis.
Quartz commonly used both at home and abroad at present, bleomycin cause the pulmonary fibrosis animal model and study reason and the mechanism that pulmonary fibrosis forms.Bleomycin (BLM) be from streptomyces one produce the one group of glycopeptide class that extracts in the antibiotic bacterial strain, be a kind of cancer chemotherapy preparation.Pulmonary fibrosis is the BLM common complication, is usually used in making the animal pulmonary fibrosis model.It is still indeterminate that BLM causes fibrosis mechanism.Relatively the viewpoint of Gong Rening is that BLM energy and ferrous ion form the Blemycin-Fe++ complex, and in the presence of O2, this complex can provide electronics for oxygen molecule, forms midbody products such as peroxide and free hydroxyl group, makes the deoxyribose of DNA produce free radical.A large amount of radical pair lung tissues produces lipid peroxidation injury, promotes hypertrophy, propagation such as fibroblast, makes matter generation fibrosis between alveolar.Because the BLM molecular weight is big, stable in properties did not reduce at room temperature and tires in 2 years, entered the tissue back and was slowly absorbed by cell, so BLM has persistence to the damage of organizing.
Inject bleomycin through trachea and cause the animal pulmonary fibrosis, its pathological process is similar to human idiopathic pulmonary interstitial fibrosis, is widely used as the classical animal model of pulmonary fibrosis.
This experimental applications bleomycin duplicates mice, Pulmonary Fibrosis in Rats model, observe and find that 7 days alveolitises of model group pathological change are obvious, formed fibrosis in 14 days, 28 days, major lesions is all consistent with bibliographical information with index, the Ultrastructural change of lung tissue simultaneously shows that rat not only has the pathological changes of interstitial lung, also have tangible pulmonary parenchyma to comprise the damage of alveolar epithelial cells, vascular endothelial cell and basement membrane, consistent with bibliographical information, the animal model replication success is described.
Two, Radix Salviae Miltiorrhizae total phenolic acids suppresses injury of lung and inflammatory reaction
The pulmonary fibrosis process originates in alveolar, and it may be the early stage incident that pulmonary fibrosis takes place that alveolar epithelium damage, alveolitis and macrophage quantity increase.In this experiment, can make alveolar epithelial cells and vascular endothelial cell impaired by injecting bleomycin in the trachea, the chemical chemotactic substance that impaired lung tissue produces can make a large amount of inflammatory cell migration at first in interstitial lung and alveolar space, inflammatory cell can further discharge the various kinds of cell factor and inflammatory mediator afterwards, for example IL-1, TGF-β, TNF-α, IFN-γ, PDGF, MMPs etc., these factor pair pulmonary fibrosiss form all has important function.In addition, injury of lung can make alveolar epithelial cells and vascular endothelial cell permeability increase due to the BLM, and plasma protein extravasation finally causes ECM to be deposited in alveolar space and the interstitial lung.This shows that damaged lung tissue, inflammatory cell are assembled can promote fibrosis formation, protection alveolar epithelial cells, the reaction that reduces inflammation, minimizing inflammatory cell ooze out then and can improve pulmonary function.Traditional treatment medicine such as glucocorticoid at pulmonary fibrosis is exactly the inflammation-inhibiting reaction with its starting point of other immunosuppressive drugs at present, thereby alleviates the degree of pulmonary fibrosis.In this experiment, after the Radix Salviae Miltiorrhizae total phenolic acids treatment, can significantly alleviate the inductive mice of BLM, the early stage acute stage inflammatory reaction of rat pulmonary, reduce the lung coefficient, pathomorphology inspection and Electron microscope showed pneumonia cell ooze out obvious minimizing, the alveolar epithelial cells structure is normal substantially, are one of main mechanism of treatment pulmonary fibrosis so infer inhibition injury of lung and inflammatory reaction.
Three, Radix Salviae Miltiorrhizae total phenolic acids suppresses the pulmonary fibrosis model oxygen free radical injury
Radical damage plays an important role in pulmonary fibrosis generation evolution, is one of present known important mechanisms.Report oxygen-derived free radicals such as Murrel can stimulate fibroblast proliferation.Fibroblast is the main cell of secretion collagen protein, and its enhancing or fibroblastic a large amount of propagation synthetic and secretion collagen ability all may cause the increase of collagen total amount.This experiment shows: in 1-7 days, pathological changes is based on alveolitis behind the injection bleomycin, and alveolitis reaches the peak in the time of the 7th day, and begin to occur alveolar septum and thicken, fibroblast and blood capillary proliferation form stable fibrosis in the time of the 28th day, consistent with bibliographical information.Therefore bleomycin causes the pulmonary fibrosis pathological process and divided for two phases: promptly be alveolitis in early days, be pulmonary fibrosis late period.In the alveolitis stage, bleomycin stimulates a large amount of inflammatory cells generation oxygen-derived free radicals, cytokine and enzyme to participate in injury of lung, and oxygen-derived free radicals stimulates the fibroblasts to secrete collagen protein to form pulmonary fibrosis subsequently.
Under physiological condition, the tissue injury that oxygen-derived free radicals causes can be suppressed by the endogenous antioxidant.Yet, be oxidation antioxidation system when unbalance when these polyphenoils are not enough to resist a large amount of oxides, irreversible damage will take place in tissue.Bleomycin can produce oxygen-derived free radicals in the presence of oxygen molecule and Fe2+, oxygen-derived free radicals can destroy biomacromolecule such as DNA, protein and lipid, damage dna and signal transducer, final trigger cell membrane lipid peroxidization, the biomembrane afunction, thereby make alveolar epithelium suffer irreversible destruction, cause injury of lung; On the other hand, the alveolar epithelial cells damage can cause that a large amount of inflammatory cell infiltrations are in a matter or alveolar space, the gathering of inflammatory cell and activation also can produce excessive oxygen-derived free radicals, except the damage that further increases the weight of lipid peroxidation and lung tissue, also can cause the fibrosis production of cytokines, promote the formation of pulmonary fibrosis indirectly by activating NF-κ B rise.Therefore, play an important role in the inductive inflammation of the excessive BLM of being created in of peroxide and radical reaction intermediate product and the fibrosis.SOD is the enzyme of removing oxygen-derived free radicals important in the body, and its activity increase can stop the chain reaction of oxygen-derived free radicals, removes oxygen-derived free radicals and oxidation product thereof, and protection lung tissue cell alleviates injury of lung.The height of its vigor has reflected the ability of body removing oxygen-derived free radicals indirectly.MDA is the product of lipid peroxidation, and its height has reflected the order of severity that body cell is attacked by free radical indirectly.Therefore both measure simultaneously and have better judge value.
The chemical constituent of Radix Salviae Miltiorrhizae mainly is divided into water soluble ingredient and liposoluble constituent two large divisions.TANSHINONES is the main fat-soluble effective ingredient of Radix Salviae Miltiorrhizae, and the TANSHINONES that studies show that in the past has cardiovascular protection effects such as atherosclerosis, reduction myocardial oxygen consumption, inhibition thrombosis.The water-soluble fluidity effective ingredient of Radix Salviae Miltiorrhizae mainly is a phenolic acid compound, has result of study to show that Radix Salviae Miltiorrhizae total phenolic acids plays a significant role for preventing and treating atherosclerosis.Other studies show that Radix Salviae Miltiorrhizae total phenolic acids has significant protective effect to the vascular smooth muscle of H2O2 oxidative damage.
This experimental result shows that lung fibrosis in rats is after Radix Salviae Miltiorrhizae total phenolic acids and prednisolone acetate treatment, and early stage lung tissue, serum activity of SOD are apparently higher than model group; MDA content is starkly lower than model group.Illustrate that pulmonary fibrosis is early stage, Radix Salviae Miltiorrhizae total phenolic acids has the effect of good antioxidant radical.
Four, Radix Salviae Miltiorrhizae total phenolic acids inhibition TGF-β secretes in a large number
TGF-β is distributed widely in the body, participates in the various cells physiological function, as migration, differentiation and the propagation of cell.It is in that to cause aspect the fibrosis effect performance particularly outstanding, and the fibrosis sexually transmitted disease (STD) that relates to due to a plurality of organs and the multiple reason becomes.In the cytokine network that participates in pulmonary fibrosis, TGF-β studies the most deep, most important cytokine.TGF β has three kinds of isomers in mammal, promptly TGF-β 1, TGF-β 2, and TGF-β 3, wherein TGF-β 1 distribution is the widest, studies also at most.Aubert report TGF-β 1 is distributed widely in the normal rat lung, comprises pulmonary alveolar macrophage, matter between lung is interior, and bronchial epithelial cell is in air flue and the circumvascular connective tissue.Wherein pulmonary alveolar macrophage is considered to secrete the main cell of TGF-β 1.
TGF-β 1 is a kind of cytokine with various biological effect, fibroblast had chemotaxis, and can stimulate the immaturity growth of fibroblasts, the TGF-β 1 that damage location occurs can raise fibroblast and can stimulate immaturity fibroblast proliferation and differentiation from surrounding tissue.In vitro study shows that TGF-β 1 can stimulate lung fibroblast to synthesize a large amount of collagens.
Former studies shows that Radix Salviae Miltiorrhizae can suppress the expression of pulmonary fibrosis mouse lung tissue T GF-β, the relevant report of not seeing Radix Salviae Miltiorrhizae total phenolic acids.
This experimental result shows, the model group mice, lung tissue of rats TGF-β 1 integral optical density value is obviously more than the normal control group, the generation of TGF-β 1 in the stimulated lung tissue of bleomycin is described, thereby cause the synthetic and deposition of various extracellular matrixs, and prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group mice, the early stage lung tissue TGF-β 1 integral optical density value of rat is starkly lower than model group, find that simultaneously Radix Salviae Miltiorrhizae total phenolic acids can reduce the expression of TGF-β 1 at macrophage, illustrate that pulmonary fibrosis is early stage, Radix Salviae Miltiorrhizae total phenolic acids and prednisolone acetate can suppress TGF-β 1 secretion, reduce its expression in lung tissue.
Five, Radix Salviae Miltiorrhizae total phenolic acids is to the influence of pulmonary fibrosis model collagen and HYP
Collagen is the main component of extracellular matrix, and it is a key character of interstitial pulmonary fibrosis that collagen gathers.Its synthetic speed is greater than the speed of degraded, and collagen fiber significantly increase in the lung, finally cause the accumulation gradually of collagen.Experiment also confirms, in the lung tissue of rat and people's interstitial pulmonary fibrosis the collagenic supersession disorder arranged, and shows as the I type and the III Collagen Type VI is out of proportion, and collagen gathers in interstitial lung.HYP is one of main component of body collagen protein, accounts for 13.4% of its total amino acid content, and other all do not contain HYP except that elastin laminin contains small amount of H YP (about 1%).Therefore HYP content can be used as the metabolic important indicator of its collagen tissue [9] in the tissue.The synthetic significant process of collagen is that proline changes into hydroxyproline through the hydroxylase effect, will have nearly 100 hydroxyproline residues just can make the triple-helix structure of collagen stable at least in every α peptide chain in each tropocollagen molecule.This stable proportionate relationship of α peptide chain in hydroxyproline and the tropocollagen molecule, the mensuration that makes hydroxyproline content become the sensitive indicator of indirect reflection collagen content and are widely used in the research of pulmonary fibrosis.And the content by hydroxyproline measuring and calculating collagen protein can reflect the situation of collagenic supersession on the one hand intuitively, judges Fibrotic degree, can provide foundation for the medicine of screening prevention and treatment simultaneously.
This experiment Collagen I, CollagenIII Showed by immune group result mice, rat model group I type and III Collagen Type VI integral optical density all began at 7 days to increase, and until 28 days, still be continuous increase trend, alveolar structure destroyed this moment, fibrosis clearly as seen, fibrous tissue is bar rope sample, and the patch shape distributes.And prednisolone acetate group and Radix Salviae Miltiorrhizae total phenolic acids group mice, rat I, the 7th, 14,28 days integral optical density values of III Collagen Type VI are starkly lower than model group.Pointed out Radix Salviae Miltiorrhizae total phenolic acids can suppress I, III Collagen Type VI deposition at interstitial lung.
This experiment lung tissue HYP result show, 14,28 days the time, compares with normal group after modeling, and model group mice, lung tissue of rats HYP content obviously raise; Radix Salviae Miltiorrhizae total phenolic acids group mice, lung tissue of rats HYP content were compared with model group in 14 days, all have in various degree to descend, but there was no significant difference; 28 days Radix Salviae Miltiorrhizae total phenolic acids group mices, lung tissue of rats HYP content is compared with model group that remarkable decline is all arranged, and has reflected that indirectly Radix Salviae Miltiorrhizae total phenolic acids can reduce the content of lung tissue collagen, plays the effect that alleviates pulmonary fibrosis.Prednisolone acetate group mice, lung tissue of rats HYP content were compared with model group in 14 days, 28 days, all have in various degree to descend, but there was no significant difference.Prompting prednisolone acetate therapeutic effect at a specified future date is not good.
In sum, PNS has clear and definite therapeutical effect to the inductive animal pulmonary fibrosis model of BLM, for the Drug therapy of pulmonary fibrosis provides a new selection.The mechanism of PNS anti-experimental character pulmonary fibrosis is: 1. anti-damage and anti-inflammatory effect: PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM shows that PNS can protect alveolar epithelial cells, significantly alleviates the degree and the scope of alveolitis.2. antioxidation: PNS can significantly improve SOD vigor in lung tissue of rats homogenate and the serum in this experiment, reduces MDA content, and prompting PNS can play certain inhibitory action to injury of lung and fibrosis by suppressing lipid peroxidation.3. regulate cytokine network: SABC shows that PNS can significantly reduce the positive expression that causes fibrosis factor TGF-β in the lung tissue, thereby ECM deposition such as collagen is reduced.4. be suppressed to fibroblast proliferation, activation: find that through pathomorphology and electron microscopic observation PNS can suppress the collagen deposition that caused by a large amount of hypertrophy of fibroblast.
Conclusion
1, this experiment result of implementation shows: by injecting BLM in the trachea, successfully duplicated the pulmonary fibrosis model of mice, rat;
2, Radix Salviae Miltiorrhizae total phenolic acids can obviously reduce the lung coefficient that bleomycin causes pulmonary fibrosis mice, rat;
3, pathological observation shows: Radix Salviae Miltiorrhizae total phenolic acids can alleviate bleomycin in early days and cause pulmonary fibrosis mice, rat alveolitis, alleviates fibrosis late period;
4, Electron microscope showed: Radix Salviae Miltiorrhizae total phenolic acids can alleviate the damage of lung fibrosis in rats I, II type epithelial cell degree and between the deposition of collagen in the matter;
5, can obviously the raise early stage lung tissue of lung fibrosis in rats due to the bleomycin, serum activity of SOD of Radix Salviae Miltiorrhizae total phenolic acids obviously reduces MDA content;
6, Radix Salviae Miltiorrhizae total phenolic acids can reduce the high expressed of crossing that bleomycin causes pulmonary fibrosis mice, lung tissue of rats TGF β 1 in early days;
7, Radix Salviae Miltiorrhizae total phenolic acids can reduce the expression that bleomycin causes pulmonary fibrosis mice, rat interstitial lung CollagenI, CollagenIII; Significantly reduce the content of lung tissue HYP.
Above result shows: Radix Salviae Miltiorrhizae total phenolic acids has clear and definite therapeutical effect to animal pulmonary fibrosis due to the bleomycin, this effect suppresses and its anti-damage, anti-inflammatory response, the antioxidant radical damage, suppress a large amount of secretions of TGF-β and fibroblast proliferation, treatment is relevant.

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1. a Radix Salviae Miltiorrhizae total phenolic acids is preparing the purposes for the treatment of in the pulmonary fibrosis medicine.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104095839A (en) * 2013-04-05 2014-10-15 滨州医学院 Application of rosmarinic acid in drug for treating or preventing pulmonary fibrosis
CN104784676A (en) * 2015-05-06 2015-07-22 中国科学院合肥物质科学研究院 Method for establishing rat single lung pulmonary fibrosis model

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104095839A (en) * 2013-04-05 2014-10-15 滨州医学院 Application of rosmarinic acid in drug for treating or preventing pulmonary fibrosis
CN104784676A (en) * 2015-05-06 2015-07-22 中国科学院合肥物质科学研究院 Method for establishing rat single lung pulmonary fibrosis model

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