Evaluation Of Hepatoprotective Effect Of Schisandrae Sphenantherae Fructus And Schisandrae Chinensis Fructus And Study On Anti-fibrosis Effect On Liver Of Their Main Active Constituent:Schisandrol B

The liver is an important substantive organ of the human body,which regulates and controls the physiological processes of metabolism,detoxification and excretion.As the liver is the key site for metabolism and elimination of toxic substances in the human body,it is most vulnerable to pathological damage,causing liver damage and even developing into liver fibrosis,liver cirrhosis and liver cancer.China is a large country with a high incidence of liver diseases,and preventing and reversing the process of liver fibrosis has become an important strategy for the clinical treatment of chronic liver diseases.At present,there is still a lack of first-line drugs for anti-fibrosis treatment with western medicine,while traditional Chinese medicine has a good preventive effect and therapeutic effect on liver fibrosis,which is irreplaceable in the treatment of liver injury.The three northeastern provinces mainly produce the dry and ripe fruit of Schisandra Chinensis（Turcz.）Baill,which is known as"Schisandrae Chinensis Fructus（Sch）";The dry and ripe fruit Tof Schisandra Sphenanchera Rehd.et Wils.in central China,mainly produced in southwest and southern provinces,is known as"Schisandrae Sphenantherae Fructus（S.sph）".In the 1960s,professor Chen jumei discovered that Schisandrae Chinensis Fructus had a good effect of protecting liver and lowering transaminase,and developed a series of preparations of Fufang Biejia Ruangan Pian,which achieved better clinical efficacy.The Chinese pharmacopoeia listed both Sch and S.sph as authentic products,and began to list them separately in the 2000 edition.They have established their own quality standards and continuously improved them until now.However,no distinction has been made on their efficacy.The common phenomenon of mixed use of Sch and S.sph exist in traditional Chinese medicine prescription and modern clinical medicine,but due to the producing area of Sch and S.sph is different,its chemical composition is bound to have certain differences and their did not have enough evidence that the effects of protect liver is consistent,so the comparative study in this paper is to explore whether Sch and S.sph play a role in the protection of liver injury and whether the effect is different,and probe its main efficacy components protect liver.It is expected to provide scientific basis for the clinical application and development of Sch and S.sph.Explore the pharmacodynamic substances that can effectively inhibit the development of liver fibrosis,and provide support for the development of traditional Chinese medicine for anti-fibrosis components.The specific contents of this research are as follows:Part one:Study on the protective effect and pharmacodynamic difference of Schisandrae Chinensis Fructus and Schisandrae Sphenantherae Fructus on liver.Objective:To establish an in vitro and in vivo model of hepatic cell injury caused by acetaminophen（APAP）,and to investigate the hepatoprotective effects and pharmacodynamic differences of Sch and S.sph.Methods:1.Ethanol reflux extraction and freeze-drying were used to prepare the ethanol extracts of the effective ingredient of Sch and S.sph.2.CCK-8 method used IC₅₀ as evaluation index to construct APAP-induced LO2 hepatocyte injury model.To investigate the effects of Sch extracts and S.sph extracts on the survival rate of LO2 cells.3.The survival rate of cells in the experimental groups were determined by CCK-8 and aspartate aminotransferase（AST）and alanine aminotransferase（ALT）levels were detected by colorimetry.Through the above two test methods,the protective effects of extracts on APAP-induced LO2 cell injury were evaluated.4.APAP-induced acute drug-induced liver injury in mice was used to verify whether the liver protection effects of different batches of Sch and S.sph from different regions were consistent.Serum ALT and AST levels were detected by colorimetry.Superoxide dismutase（SOD）,reduced glutathione assay kit（GSH）,cellular glutathione peroxidase assay kit（GSH-PX）and malondialdehyde assay kit（MDA）were detected in liver tissue homogenates.The histopathological changes of liver were observed by HE staining and TUNEL staining.Results:1.When the concentration of APAP was 22mmol/L,after 24h,the cell survival rate was about 50%.The concentration of 1mg/ml of Sch extracts and S.sph extracts had no effect on cell viability.2.Sch and S.sph can significantly improve APAP-induced cell damage.All groups of Sch and S.sph ethanol extracts could improve the cell survival rate and reduce the increase of cell supernatant transaminase.Remove the effects of wild batches,the protective effects of various batches of Sch ethanol extracts were relatively consistent,and the ethanol extracts of S.sph from different producing areas or different batches in the same producing area have different protective effects on APAP-induced LO2cell injury..3.The alcohol extracts of Sch and S.sph both have protective effects on APAP-induced acute drug-induced liver injury.They can inhibit the elevation of transaminase,relieve oxidative stress reaction,reduce the apoptosis of liver cells and improve liver tissue lesions.The effect of the alcohol extracts of Sch were the same,and the difference was not statistically significant.The effect of the alcohol extract groups of S.sph were different,which was consistent with the results of cytology.Conclusion:Sch and S.sph showed significant protective effects on cell models and animal models of acute liver injury induced by paracetamol.Based on the above experimental model.Set aside the effects of wild batches,the protective effects of different batches of Sch on liver injury were relatively consistent,the liver protection effect of the alcohol extract of S.sph from the same origin is quite different,believe that with the decrease of unstable factors causing the difference of liver protective effect,strengthen the quality control of medicinal materials,the difference in liver-protection efficacy will be gradually reduced,and the drug effect will be more stable.The clear difference in efficacy is to provide guidance for the safe and rational use of Sch and S.sph in clinical hepatoprotective treatment.Part two:Screening of hepatoprotective constituents of Schisandrae Chinensis Fructus and Schisandrae Sphenantherae Fructus and its pharmacological action and mechanism of anti-BDL and CCL₄ induced liver fibrosis in mice.Objective:To study the most effective constituent of Sch and S.sph in protecting liver and its pharmacological effect of anti-BDL and CCl₄ induced liver fibrosis in mice,to explore the mechanism of anti-liver fibrosis.Methods:1.APAP-induced LO2 hepatocyte injury model and CCK-8 method were used to screen the chemical components of Sch and S.sph.The most effective ingredients were found to investigate the cytotoxicity and the minimum onset concentration was explored.At the same time,the colorimetric method was used to detect the content of lactate dehydrogenase（LDH）and flow cytometry（FCM）to investigate the effectiveness of the protective effect.2.Chemical liver injury model was used to induce liver fibrosis in mice with CCL₄.C57BL/6 mice were randomly grouped,divided into control group（Control）,CCL₄ model group（Model-CCL₄）,positive drug group（Colchicine,0.2mg/kg,Col）,and test groups（10mg/kg,20mg/kg,40mg/kg）.5%CCL₄ was prepared with olive oil and injected intraperitoneally with 2mL/kg once a day for 2 weeks.After 2 weeks,different doses of medication were given,and 5%CCL₄ was intraperitoneally injected every 2 days.At 6 week,the mice were treated after the injection.The eyeballs were removed and blood was taken.Observe fresh samples,HE staining sections to investigate the influence of different doses of drugs on liver histopathology;The expression of alpha-smooth muscle actin（alpha-SMA）,Sirius red and Masson was detected by immunohistochemistry.AST,ALT,direct bilirubin（DBIL）and total bilirubin（TBIL）were detected in serum by colorimetry.Serum hydroxyproline（Hyp）was detected by ELISA.Western blot method analysis of collagen I,p-Smad3,Smad2/3 in the liver tissue;RT-PCR method in detection of a-SMA（Acta2）,Collagen I,Smad2,Smad3,matrix metalloproteinases（MMPs）,tissue inhibitor of metalloproteinases（TIMPs）and related protein mRNA expression.3.Liver fibrosis model in mice was induced by BDL.C57BL/6 mice were randomly grouped,divided into sham group（Control）,BDL model group（Model-BDL）,positive drug group（Colchicine,Col,0.2mg/kg）,test groups（10mg/kg,20mg/kg,40mg/kg）.The sham operation group was freed from the common bile duct and was not ligated.The remaining groups underwent bile duct ligation according to the surgical procedure.The mice that were successfully modeled were given the corresponding drugs on the 7th day after the operation,and were administered by intragastric administration until the 21st day.Pharmacological and mechanistic research indicators are the same as above.Results:1.Multiple chemical components in Schisandrae Chinensis Fructus have a protective effect on APAP-induced LO2 cell damage.Schisandrol B（Sch B）has the strongest protective effect at a low concentration.2.Sch B significantly inhibited the pathological changes of liver tissue induced by BDL and CCL₄ in mice,and reduced the liver index.Hyp expression in liver was significantly reduced.The improvement of liver function in mice inhibited the increase of hepatic transaminase and bilirubin in a dose-dependent manner.3.Sch B can inhibit the sustained activation of BDL and CCL₄induced stellate cells.According to the WB and PT-PCR,according to the results of SchB can significantly reduce the HSC activation markers a-SMA expression,at the same time reduce the expression of growth factors promoting the fibrosis;4.According to WB and RT-PCR results,Sch B down-regulated the protein expression in a dose-dependent manner of Collagen I,p-smad3,Smad2,Smad3 by affecting the TGF-β/Smad signaling pathway.It regulates the expression of MMPs and its natural tissue inhibitor TIMPs,affects the degradation of ECM,prevents excessive deposition of ECM in the liver,and down-regulates the protein expression of MMP2,MMP9,TIMP1 and TIMP2.The protein expression of MMP13 was up-regulated.Conclusion:Sch B is the component with the strongest hepatoprotective effect among the lignans.The model of liver fibrosis induced by CCl₄ and BDL in mice confirmed that Sch B could effectively alleviate liver fibrosis,improve the pathological characteristics of liver fibrosis in mice,enhance liver function and reduce the expression of pro-fibrosis factors.The mechanism study showed that Sch B could play an anti-fibrosis role by influencing the TGF-β/Smad signaling pathway,thus delaying the development of liver fibrosis and even reversing liver fibrosis.To lay the foundation for the research and development of Chinese herbal medicine against liver fibrosis based on Sch and S.sph.