The Inhibition Of Liver Cancer And The Protection Against Acute Liver Failure Through Coptisine From Coptis Chinensis Franch

Rhizoma Coptidis,one of famous and precious traditional Chinese medicinal herb,is the dry rhizoma of Coptis chinensis Franch,Coptis teeta Wall and Coptis dletoidea C.Y.Cheng et Hsiao.It tastes bitter,quality of cold,with heat dampness,purging fire detoxification effects,with characteristics of long history,extensive source and medicinal value.In recent years,with the development of traditional Chinese medicine and medical technology,more and more pharmacological effects of Rhizoma Coptidis have been displayed.RC displays the roles in anti-bacterial,anti-inflammatory,anti-viral,anti-cancer,anti-oxidation,lowering blood pressure,blood fats and blood sugar,anti-platelet aggregation,improvement of cardiac cerebral vessel.RC contains many kinds of isoquinoline alkaloids,mainly composed of berberine,coptisine,palmatine,epiberberine,columbamine and jatrorrhizine,the richest content is berberine.Now,because berberine is widely studied,many pharmacological effects have been discovered,but other alkaloids need further study.Coptisine?COP?,which has a similar parent structure to berberine,and is little structural differences between them.Hepatocellular carcinoma?HCC?is one of the most common and aggressive malignant tumors worldwide,while the molecular mechanisms underlying HCC tumorigenesis are not completely clear.Many studies proved that microRNA-122?miR-122?is a liver special miRNA,which accounts for 70%of the total liver miRNA.Also regard as a tumor suppressor gene and is usually down-regulated in liver cancer.So,this research is based on BALB/c liver cancer mode by implanting subcutaneously with HepG2cells to preliminarily study the anti-tumor function of coptisine and it's possible mechanism.Also study the liver protective effect of coptisine on acute liver failure.The following is the main contents and results of the study:?We investigated the safety of the COP.The HepG2,Huh7 and L02 cells with a good status in logarithmic phase were used to investigate the cyto toxicity of COP in different concentrations by MTT assay.The results indicated that COP selectively induced cell apoptosis in HepG2 and Huh7 cells while has no cytotoxicity in normal liver cells.When the concentration of COP reached at 25?g/mL,both of the survival rates of HepG2 and Huh7 cells were less than80%,but there were no changes to L02 cells,but when the concentration of COP reached at 300?g/mL,the survival rate of L02 cells was less than 80%.Therefore,COP is a kind of compound with low toxic and can protect liver.The value of IC50 of HepG2,Huh7 and L02 cells were47.056?g/mL,61.831?g/mL and 329.265?g/mL respectively.?The anti-liver cancer activity of COP in vitro:We tested the influence of COP on cell proliferation,migration,apoptosis and miR-122 in HepG2 and Huh7 cells which were evaluated by MTT assay and colony formation assay,wound-healing assay,hoechst 33258 staining and flow cytometry,RT-qPCR,respectively.The results of experiments showed that the value of IC₅₀in HepG2 cells was 47.056?g/mL,the value of IC₅₀0 in Huh7 cells was 61.831?g/mL,which indicated HepG2 was more sensitive to COP.Moreover,25?g/mL of COP completely inhibited proliferation and induced death of HepG2 cells after 7d treatment of COP.Also,25?g/mL of COP could effectively inhibit the migration of HepG2,Huh7 cells and HepG2 cells transfected with miR-122 mimics.COP also induced apoptosis in HepG2 and Huh7 cells.?The anti-liver cancer activity of COP in vivo:HepG2 in logarithmic phase with 95%viability were implanted subcutaneously into the right flanks of male BALB/c nude mice at the age about 4-week-old.After injection,mice were randomLy allocated to four groups as follows:the normal control group,the tumor control group,the Sorafenib group?120.13mg/kg?,and the COP group?150mg/kg?,each group contained eight mice.Tumor growth was measured by calipers every 5 days for 30 days.COP and Sorafenib were dissolved in ultrapure water and administrated by gavage one times a day after tumor formation.The mice in tumor control group administrated by gavage with saline.The histopathology of liver and tumor were valued by H&E staining,Western blot and RT-qPCR were used to analysis the proteins of liver and tumor,and the expression of miR-122,and also detected the changes of the tumor volume,the body weight,and the ratio of the liver to body weight,the ratio of the kidney to body weight,and the ratio of the spleen to body weight.The results of study demonstrated that comparing to tumor control group,both of the Sorafenib group and COP group effectively inhibited the growth of tumor?P<0.01?,but there was no significant difference between them.COP improved the level of miR-122 in mice liver.No matter observed in macro by eyes or in micro by H&E staining,COP displayed a protective role in liver.The ratios of organ to body weight further showed the low toxicity of COP.?The liver protection effect of COP in vitro:MTT assay was used to screen out the suitable concentration of LPS,COP in subsequent study.The viability of HepG2 and L02 after treatment of LPS and/or COP for 72 h was detected by MTT.The expression of miR-122 was evaluated by RT-qPCR assay.The cell apoptosis of HepG2,L02 cells and transfected HepG2,L02 cells were detect by flow cytometry.The expressions of related apoptotic proteins in HepG2 and L02 cells were detected by western blot.The results indicated that 10?g/mL of LPS could establish acute liver failure mode,COP showed a selective role in liver protection,high toxicity to HepG2 cells,low toxicity to L02 cells.The expression of miR-122 down-regulated in COP treatment HepG2cells,but up-regulated in COP treatment L02 cells.COP promoted cells apoptosis in HepG2 cells,in the contrary,COP inhibited cells apoptosis to protect L02 cells.?The liver protection effect of COP in vivo:Male Kunming mice?4 weeks of age?were randomLy divided into:normal control group?NC?;model group?MC?;37.5 mg/kg body weight of COP?COPL?and 150 mg/kg body weight of COP?COPH?,each group had 10 mice.The COPL and COPH groups were pretreated intragastrically for 7 days with COP before injected with LPS?500??g/kg?/D-GalN?400?mg/kg?by intraperitoneally?i.p.?to induce acute hepatic injury.After animals sacrificed,the activities of AST and ALT in serum,and those of SOD and GSH in liver were tested by corresponding kits.RT-qPCR was used to analysis the expression of miR-122 in liver and cells.The morphological changes of the liver were analyzed by H&E staining,and the changes of related apoptotic proteins of liver were analyzed by immunohistochemistry.The data showed that LPS/D-GalN administration resulted the apoptosis of liver,but COP revised the level of GSH,SOD,miR-122,and reduced the level of AST,ALT,COP had a pre-protective effect in acute liver failure study.In conclusion,COP were found to inhibit the proliferation,migration and to promote apoptosis of HCC cells,and inhibit the tumor growth of nude mice and protect liver in acute liver failure by up-regulating the expression of miR-122.