Protective Effect Of Salvianolate Lyophilized Injection On Liver Fibrosis Rats And Its Mechanism Research

ObjectiveLiver fibrosis is a reversible fibrous scar caused by a variety of chronic liver diseases.It is mainly manifested by excessive proliferation and deposition of extracellular matrix(ECM)in liver tissue,which causes abnormal changes in liver tissue structure.Salvianolate lyophilized injection(SLI)is a salt compound with magnesium lithospermate B(MLB)as the main component extracted from the single-flavored Chinese medicine Salvia miltiorrhiza by a three-level fingerprint.It has anti-inflammatory properties,anti-oxidant,pharmacological effects of liver protection.At present,the protective effect and mechanism of SLI in liver fibrosis are rare.This experimental study aims to explore the protective effect of SLI on liver fibrosis in rats and to explore related molecular mechanisms on human liver stellate cells LX-2.Methods1.Model construction and drug intervention72 SD rats were divided into normal group,model group,colchicine group(0.12 mg/kg),SLI high-dose group(50 mg/kg),SLI medium-dose group(25 mg/kg),and SLI low-dose group(12.5 mg/kg)by random interval grouping according to body weight,12 in each group,half male and half female.On the first day of modeling,all the groups except the normal group were subcutaneously injected with a 20%CC14(V/V)peanut oil mixed solution at a rate of 5 ml/kg,and then subcutaneously injected at a rate of 3 ml/kg at a 3-day interval.Rats were intraperitoneally injected with pig serum at a rate of 3 ml/kg on the second day of modeling,and then injected intraperitoneally at a rate of 1 ml/kg at intervals of 3 days.Beginning at the 3rd week of the model,20%CC14 peanut oil mixed solution and pig serum were injected once a week until the 12th week.During the experiment,the normal group was fed with ordinary diet,and the rest of the groups were fed with 10%ethanol and fed with high-fat corn flour(795 g/kg corn flour+200 g/kg lard+5 g/kg cholesterol).From the 5th week,the experimental groups were administered with drug intervention.The normal group and the model group were orally administered with the same volume of distilled water.The colchicine group was orally administered once at 0.12 mg/kg,the SLI high-medium-low-dose group was injected intraperitoneally once daily at 50 mg/kg,25 mg/kg and 12.5 mg/kg for 8 weeks.2.Detection indexAfter the 12th week of the experiment,serum was collected via abdominal aorta and liver was extracted.The liver pathological changes were observed by HE staining,collagen deposition was observed by Masson staining,and the optical density value(0D value)of fibrosis products in each group was analyzed by Image Pro Plus 6.0;α-SMA in liver tissue of each group was measured by immunohistochemical staining method Content;Elisa method was used to measure serum ALT,AST,four indexes of liver fibrosis,TGF-β1,MMP-2,TIMP-2 levels,and Elisa method was used to detect cytokines TNF-α,IL-1β,IL-6 and IFN-γ content in liver tissue;qRT-PCR was used to detect the relative expression of Col-Ⅰ,Ⅲ mRNA in liver tissue.3.Preliminary exploration of the mechanism of action of SLI in vitroCCK-8 method was used to determine the effect of SLI on the proliferation of human hepatic stellate cells LX-2,qRT-PCR was used to detect the changes of Bax,Bcl-2,Caspase-3,and Caspase-9 mRNA and Western Blot was used to detect the corresponding protein changes to explore some biological mechanisms of SLI on LX-2 cells in vitro.Results1.Liver pathological morphology showed that liver fibrosis model was successfully established.SLI can reduce the degree of liver fibrosis caused by CC14 combined with pig serum.2.SLI can reduce the increase of ALT and AST caused by CC14(P<0.05),reduce the degree of inflammation and necrosis of liver tissue,and have good liver function recovery and liver injury protection;it can reduce the four indexes of liver fibrosis(P<0.01、P<0.05),improve the degree of fibrosis;can increase the expression of MMP-2(P>0.05),reduce the expression of TIMP-2 and TGF-β1(P<0.01、P<0.05),by regulating MMP-2/TIMP-2 balance to regulates the process of matrix degradation;can reduce the expression of pro-inflammatory factors TNF-α,IL-6,IL-1β(P<0.01、P<0.05),and increase the expression of anti-inflammatory factor IFN-y(P<0.05);It can reduce the expression of Col-Ⅰ and Col-Ⅲ mRNA in liver tissues,and can reduce collagen deposition.3.SLI can inhibit the proliferation of LX-2 in a dose-and time-dependent manner.SLI can promote the expression of Caspase-9 mRNA,Caspase-3 mRNA,and Bax mRNA,while suppressing the expression of Bcl-2 mRNA to promote the apoptosis of LX-2 cells,and the WB results show the same trend.ConclusionSLI can alleviate the degree of liver fibrosis induced by CC14 combined with pig serum by inhibiting the production of liver inflammatory factors,regulating the MMP-2/TIMP-2 balance,and reducing collagen synthesis.In addition,SLI can inhibit the proliferation activity of LX-2 cells in vitro,and the mechanism may be related to increasing the activities of Bax,Caspase-9,and Caspase-3 by inhibiting Bcl-2.