Study Of Anti-fibrosis Effect Of Sodium Tanshinone ⅡA Injection

Hepatic Fibrosis is a important reversible pathological stage of chronic liver disease when liver cells meet necrose or inflammation ,also is material foundation for hepatic cirrhosis. Hepatic stellate cell is a kind of interstitial cell in Space of Disse.when the Injury of liver at later stage, HSCs' phenotype changes and propagates to produce various kinds of extracellular matrix(ECM) ,and can secrete matrix metalloproteinase to refrain degradation of ECM, so it have important effect on the development of Hepatic Fibrosis. Clinically, liver biopsy is the most reliable method to diagnose hepatic fibrosis . In vivo tests ,the four indexes and pathology detection are used usually.In our country ,disease incidence of liver disease is very high .According to a conservative estimation,there are over a hundred million patients who carry hepatitis virus.So the Medicine intervention study for the reversion of hepatic fibrosis at reversible stage has significant meaning of theory and value ofapplication.OBJECTIVEsodium tanshinone IIA sulphonate injection is Clinically used to treatangiocardiopathy(CD) at present,because of it's indeed therapeutic effect, social needs for which will increase gradually.now ,the manufacture technology of sodium tanshinone IIA sulphonate injection is very mature and the implantation of danshen root is standardized,this will provid much convenience and promising perspective for treatment of hepatic fibrosis in the future.The effect of promoting HSC apoptosis of tanshinone IIA had been reported. For danshen root,the effect of anti-hepatic-fibrosis has been reported,But which of sodium tanshinone IIA sulphonate injection has not been reported.The objective of my research is to provide theoretical evidence for clinical application by the study of the effect and mechanism of anti-fibrosis in vivo and vitro.so the study and exploitation for new pharmacological action of sodium tanshinone IIA sulphonate injection must bring great social effectsand economic returns.METHODSSTUDY IN VITRO:1. cell culture in vitro: Activated HSCs-T₆ were incubated with DMEM (high glucose) medium containing 10% fetal calf serum in 37°C, 5%CO₂.when HSCs become monolayer adherence and occupy 80%-90% of areas of bottle, HSCs were passaged.2. trypan blue staining detect cell survival rate of HSCs-T₆3. MTT colorimetric method detect effects on cell proliferation by different concentrations of sodium tanshinone IIA sulphonate injection .4. The detection of activity of lactic add dehydrogenase(LDH).5. Radio-immunity methods detect effects on type IV collage by different concentrations of sodium tanshinone IIA sulphonate injection .6. ELISA methods detect expressed albumen of TGF-β₁ excreted by HSCs cultivated by different concentrations of sodium tanshinone IIA sulphonate injection.7. Flow Cytometry detect apoptosis rate of HSCs.8. Cell appearances were observed continuously by inverted microscope in control group and medicine group in culture flask.9. Fluorescence staining detect normal, apoptosis and dead HSCs.STUDY IN VIVO1. preparation for experimental animal and model :108 rats of sanitation gradewere bought from the center of experimental animal of Henan province,which intraperitoneal injection were given for two times per week and 0.5ml every rat once for 12 weeks ,except for normal control. Hepatic fibrosis emerged after 6 weeks.2. pathospecimens were made: liver left lobe of identica position were obtained,which were fixed routinly , then have paraffin imbedding, slicing, HE staining for observation of Cell degeneration of necrosis degree.3. Radio-immunity methods detect content change of HA,LN,PC III, IV-C of blood serum.RESULTS IN VITRO1. With the elongation of time and increasing progressively of drug concentration,inhibitted effect by medicine of HSCs became more and more obvious,so the inhibition for proliferation between HSCs and drugs have a quantity- potency and time- potency relationship. Contrasting control group with drug group, inhibition ratio has a significant difference.2. According to cell morphology observed by inverted microscope, with the increasing of drug concentration, division growth of HSCs tend to become slower , cell shape also change from irregula star, fusiform, and so on to ellipse spherical and some cells broke to pieces.3. Depressant effect of HSCs by sodium tanshinone IIA sulphonate was not caused by non-specificity cytotoxicity.4. Content of type IV collagen became lower and lower with the increasing of drug concentration. Difference between control group and drug group has a significant difference.5. protein level of TGF-β₁ became lower and lower with the increasing of drug concentration. Difference between control group and drug groups has a significant difference.6. Apoptosis rate of HSCs increased gradually with the increasing of drugconcentration,as show a dosedependent relationship.IN VIVO1. animal condition: normal control group developed well and activity werenormal. Condition of model group is general .Increasing of body weight is slow and have no significant difference, drug group was better than model group.2. patho- observation:In blank group , lobules of liver demarcation were not obvious,and had no necrosis or phlegmasia infiltration or fibroplasia(picture l).In model group , hepatic tissue pathological lesion was serious, hepatic cord spreaded in a state of chaos. Structure of lobules of liver was destroyed.Around hepatic lobules necrosis obviously appeared and fake lobules come into being.The high , middle, low dose precaution groups or therapy groups which had been treated by sodium tanshinone IIA had not fake lobules,and fiber proliferated lessly which was confined to header area. Structure of lobules of liver gently disordered(picture3, picture 4).In low-dose group , curative effect was comparatively little and fibrous tissue spreaded from header area to peripherad.3.The effects on HA,CIV,PCIII,LN by sodium tanshinone IIAsulphonateContrasting model group with normal control group, Content of HA,CIV,PCIII,LN in rat serum increased significantly, but which strikingly depressed in all precaution groups and therapy groups of different drug concentration Contrasting with model group. Furthermore, high dose groupdepressed the most significantly.CONCLUSIONS IN VITRO1. Sodium tanshinone IIA sulphonate can inhibit proliferation of HSCs in vitro, and have a good quantity-poten -cy,time- potency relationship.2. Sodium tanshinone IIA sulphonate can inhibit synthesis of type IV collagen and proteinum expression of TGF-β₁ ,can raised apoptosis rate ofHSCs.IN VIVO1. according to pathology , Sodium tanshinone IIA sulphonate can make rathepatic fibrosis induced by immune hepatic injury with hog blood serum ameliorate. 2. Sodium tanshinone IIA sulphonate can make content of HA,CIV,PCIII,LN decrease in hepatic fibrosis rats induced by immune Hepatic injury with hog blood serum.