Antifibrotic Effects Of Liuweiwuling Tablets On Liver Fibrosis And Its Mechanisms

Liver fibrosis is a kind of non-self-limited chronic liver disease to the progress of cirrhosis of the intermediate pathological process,is a common disease that threatens human health,and is also one of the most risky factors of primary liver cancer.Professor Hans Popper,the founder of the world’s modern liver disease scholar,said,“Who can stop or delay the onset of liver fibrosis,who will cure most chronic liver disease.” Modern studies have found that liver fibrosis can be prevented,intervened,or even reversed.However,there has not appeared such safe and effective,liver-specific targeting and well-tolerated chemical drugs which can be approved for the treatment of human liver fibrosis by the Food and Drug Administration(FDA)yet.In China,the National Food and Drug Administration(CFDA)has approved drug like Fuzheng Huayu capsule(tablets)and compound turtle soft liver tablets for treating liver fibrosis as its indications of traditional Chinese medicine.Liuweiwuling tablets(LWWL)is a fine six drugs to protect the liver.Clinical study found that LWWL combined with nucleoside(acid)drugs in the treatment of chronic hepatitis B liver fibrosis has a significant effect.This study a comprehensive search of its randomized controlled study(randomized controlled study,RCT)for meta-analysis.On the basis of revealing the potential therapeutic effect of LWWL on chronic hepatitis B-related liver fibrosis,the objective evaluation of LWWL anti-hepatic fibrosis was further evaluated objectively.To explore the pharmacological effects of LWWL on hepatic fibrosis,and to explore its potential role in the activation,a bile duct ligation(BDL)and carbon tetrachloride(carbon tetrachloride,CCl4)caused rat liver fibrosis model has been used,and LWWLshows the inhibition of the formation of collagen fibers,and inhibiting of activating hepatic stellate cells.,reduce the formation of extracellular matrix(ECM)and promote the degradation of ECM.Through the clinical research we stimulate the basic research and then return to clinical research ideas,with a view to LWWL secondary development to expand its clinical indications to provide a reference,and to provide some new methods and ideas.The specific research contents of this subject are as follows:Part 1 The systematic analysis of LWWL for the treatment of liver fibrosis resulting from chronic hepatitis BObjective:To make a systematic evaluation on the efficacy and safety of LWWL combined with nucleotide drugs for the treatment of liver fibrosis resulting from chronic hepatitis B.Methods:Major databases(such as Pubmed,Embase,Cochrane Library(Issue 9,2015),Chinese Biomedical Database(CBM),China National Knowledge Infrastructure(CNKI),VIP,WanFang Data were searched from their inception to September 9,2015 to collect comprehensively RCT of LWWL combined with nucleotide drugs on the treatment of liver fibrosis resulting from chronic hepatitis B.After two reviewers separately screened literature according to the inclusion and exclusion criteria,extracted data and assessed the methodological quality of the included studies,data were analyzed with RevMan 5.3 software.Results:The total 7 RCTs were included.Among all 1021 patients involved in,529 were in the treatment group,while the other 492 were in the control group.The findings showed that compared with using nucleotide drugs alone for antiviral treatment,LWWL combined with nucleotide drugs were more efficiently in down-regulating serum liver fibrosis index HA [ SMD=-1.26,95%CI(-1.92,-0.59),P=0.0002],LN[SMD=-1.55,95%CI(-2.27,-0.83),P<0.0001],PC-Ⅲ[SMD=-1.39,95%CI(-2.21,-0.58),P=0.0008],Ⅳ-C[SMD=-1.49,95%CI(-2.25,-0.72),P=0.0001],improved the liver function ALT[SMD=-1.17,95%CI(-1.41,-0.94),P<0.00001],AST[SMD=-1.25,95%CI(-1.54,-0.96),P<0.00001],TBIL[SMD=-0.87,95%CI(-1.21,-0.53),P<0.00001],increased total clinical effective rate[OR=4.59,95%CI(2.05,10.25),P=0.0002].While no advantage was found in reducing the incidences of HBVDNA overcast[OR=1.51,95%CI(0.96,2.39),P=0.08],No significant adverse reactions were reported.Conclusion:LWWL combined with nucleotide drugs can were effectively in improving liver function,controlling liver fibrosis and increasing total clinical effective rate while compared with using the nucleotide drugs alone,LWWL has less adverse reaction and better tolerability.In clinical LWWL may have the potential effect to inhibit liver fibrosis.Part 2 To study the pharmacological effects of LWWL on BDL and CCl4-induced hepatic fibrosis in model rats and to explore its possible mechanism.Objective:To study the therapeutic effect and mechanism of LWWL on hepatic fibrosis induced by BDL and CCl4 in rats.Method:(1)BDL induced rat liver fibrosis model.Wistar rats were randomly divided into sham operation group(control group),BDL model group,colchicine group(BDL+Colchicine 0.0002 g/kg),LWWL of low,medium and high dose group(BDL+LWWL 0.4 g/kg,1.6 g/kg,6.4 g/kg).In addition to the sham operation group,the common bile duct was sutured after removal of the common bile duct,and all the other groups were sutured with the open common bile duct after ligation,and fourth days after operation,the rats were sacrificed for the first twenty-first days.Using the method of HE and Sirius red staining to observe the pathological change of liver tissue,serum alanine than color method alanine transaminase(ALT)and aspartate aminotransferase(AST)and hydroxyproline(HyP).Using immunohistochemical to detecte alpha-smooth muscle actin(α-SMA)and collagen type I(Collagen I)and nuclear factor kappa B(NF-κB,p65)expression.Using ELISA for detecting serum transforming growth factor(TGF-β1)and tumor necrosis factor alpha(TNF-α)level,α-SMA(Acta2),TGF-β1,collagen I,Platelet derived growth factor(PDGF),vascular endothelial growth factor(VEGF),matrix metalloproteinase 2(MMP2),matrix metalloproteinase 13(MMP13),matrix metalloproteinase inhibitor 1(TIMP1),matrix metalloproteinase inhibitor 2(TIMP2),Smad3,TNF-α,interleukin-6(IL-6)and interleukin-1 beta(IL-1β)mRNA expression of liver tissue was detected with real time quantitative PCR(RT-PCR).The expression of α-SMA,Bambi,Smad3,Smad2/3,Smad7,p-Smad3,IκBα,p-IκBα and NF-κB p65 in liver tissue were detected with Western blot.(2)CCl4 induced rat liver fibrosis model.SD rats were randomly divided into solvent control group,CCl4 model group,colchicine group(CCl4+Colchicine 0.0002 g/kg),LWWL of low medium and high dose group(CCl4+LWWL 0.4 g/kg,1.6 g/kg,6.4 g/kg),all animals were treated by intraperitoneal injection molding of CCl4 with olive oil(1:1),two times a week,8 weeks after administration.Animals were sacrificed at twelfth weeks,blood and liver were took from all animals to be detected like above.Results:(1)LWWL could effectively improve rat liver function and inhibit the formation of collagen fibers in rat liver.LWWL could reduced the proliferation of small hepatic ducts in rats,reduced the infiltration of inflammatory cells,reduced the generation of fibrous septa,significantly reduced the serum ALT,AST levels and liver tissue HyP content,in addition to improve the BDL of the rat liver weight increase in a dose dependent manner.(2)LWWL could inhibit the activation,proliferation and chemotaxis of HSC.LWWL significantly reduced the expression of HSC-activated phenotype α-SMA and CollagenI,and inhibited the expression of PDGF and VEGF,which promoted the proliferation and chemotaxis of HSC.(3)LWWL by regulating MMPs and their specific inhibitors TIMPs,inhibition of pathological matrix degradation,accelerated abnormal proliferation of ECM degradation.The expression of MMP2,TIMP1 and TIMP2,which were positively correlated with ECM production,were down-regulated by LWWL.(4)LWWL could inhibit the transcriptional regulation of TGF-β/Smad signaling pathway.The expression of TGF-β1,Smad3,Smad2/3 and P-Smad3 was down-regulated by LWWL,and up-regulated the protein expression of Bambi and Smad7.(5)LWWL could inhibit gene transcription of NF-κB signaling pathway.LWWL can reduce the phosphorylation of IκB-α protein,reduce the degradation of NF-κB p65 and reduce the expression of NF-κB p65 protein in a dose-dependent manner.In addition,LWWL dose-dependently reduced NF-κB-regulated expression of inflammatory cytokines TFN-α,IL-6 and IL-1β.Conclusion:LWWL can effectively block and inhibit the progression of BML and CCl4-induced rat liver fibrosis,and its mechanism may be through negative regulation of TGF-β/Smad and NF-κB signaling pathway conduction and transcriptional activation,thereby reducing the liver Inflammatory response,inhibition of HSC activation,proliferation and sustained activation,promote ECM degradation,and ultimately delay,block or even reverse the development of liver fibrosis.LWWL’s multi-link,multi-target anti-fibrosis characteristics can be considered for the secondary development of LWWL to expand its clinical indications for the treatment of liver fibrosis provides a reference.