| ObjectiveTo study the effect of HaoBieYangYinRuanJian Prescription(HBYYRJP)on alcoholic liver disease in rats,and explore the possible mechanisms by upgrading the Nrf2 signaling pathway in liver,to provide experimental basis of hepatic fibrosis for clinical use of HBYYRJP.A large number of cell signaling pathways participate in the activation of hepatic stellate cells,so understanding the mechanisms of these cell signaling pathways is very important.As the etiology of alcohol liver disease,acetaldehyde have been used widely as an activator of hepatic stellate cells in experiment.In experiment,the role of Nrf2 signaling pathway and the effect of HBYYRJP on Nrf2 pathway in the activation of rat hepatic stellate cells was studied by detecting protein expression levels of downstream molecules' in Nrf2 signaling pathway.Methods1.78 healthy Wistar male rats,weighted 200~240g,were taken from Academy of Military Science,and female rats and male rats each account for half.The model of alcohol liver disease in rats was established by feeding alcohol and fat diet.Rats were randomly divided into 8 groups: low dose of total water extraction group(2.59 g.kg-1.d-1),high dose of total water extraction group(8.2g.kg-1.d-1).low dose of water followed by 95% ethyl alcohol extraction group(2.59 g.kg-1.d-1),high dose of water followed by 95% ethyl alcohol extraction group(8.2g.kg-1.d-1).Low dose of water extraction and alcohol precipitation group(2.59 g.kg-1.d-1),high dose of water extraction and alcohol precipitation group(8.2g.kg-1.d-1).Powder group(0.09 mg.kg-1.d-1)and Compound Biejiaruangan Troche group(0.09 mg.kg-1.d-1).model group(30% ethanol is filled with stomach,once a day),normal group(distilled water: 1.2ml.g-1.d-1).The therapeutic groups: alcohol was given in the morning and drugs were given in the afternoon;the model group: alcohol was given in the morning and distilled water was given in the afternoon;the normal group: distilled water was given in the morning and in the afternoon.After 10 weeks,rats were put to death,and samples were taken.Pathological changes of hepatic tissue under light microscope were observed by immunohistochemical method;hyp of hepatic tissue was measured by acid hydrolysis.Nrf2 and ?-GCS protein expresstion from Nrf2/ARE pathway were detected by Western blot method.2.In this experiment,the LX-2 cell lines cultured in vitro were treated with two different extraction schemes,with a certain concentration and a certain concentration of acetaldehyde.In addition,model group and normal group were set as the control group according to the grouping situation.Besides,the LX-2 cell lines cultured in vitro were treated with two different extraction schemes and Nrf2 pathway inhibitor ATRA and observe the proliferation condition of the activated LX-2 cells.The GSH content of cultured cells was detected by using glutathione(GSH)test kits.Results1.Liver tissue immunohistochemical results: Nrf2 and ?-GCS are most expressed in cytoplasm and nucleus with Model group and treatment group,which have also been expressed near portal area but protein expresstion were less than cytoplasm and nucleus.In addition,as for administered group,Nrf2 and ?-GCS protein expresstion increase with the increase of the dose.Namely,high dose group of downstream protein expression quantity is higher than the low dose group,and downstream protein expression quantity dosage group was obviously higher than that of model group(P<0.01).2.Liver tissue collagen content: Powder group and the 95% dose of alcohol after water group differences in high dose group had no statistical significance(P>0.05);But both low and High dose group of full of water therapy and water extract-alcohol precipitation therapy were significant differences between Powder group and the low water after alcohol group(P<0.05);Liver tissue collagen content of each extract therapy within low and high doses of is also different,but there is no statistically significant differences(P>0.05).3.Protein expression results of Nrf2 and ?-GCS: We found that protein expression of Nrf2 and ?-GCS were enhanced in model group compared with normal group(P<0.01).The protein expression of Nrf2 and ?-GCS were upgraded obviously in powder group and water followed by 95% ethyl alcohol extraction group(P<0.01).The protein expression of Nrf2 and ?-GCS were also different but there is no statistically significant differences(P>0.05).4.Early screening of extracting therapy results determined by MTT method: The difference of inhibition rate with Powder and water after alcohol low-dose therapy was not statistically significant(P>0.05);But the difference of inhibition rate with low-dose of Powder,full water extraction of water and water extract-alcohol precipitation therapy has significantly difference(P<0.05).5.Later results determined by MTT method: Acetaldehyde stimulus alone can make the LX-2 cells proliferated with the rapid rate,different extraction therapy of HBYYRJ prescription can obviously decrease the growth and proliferation of LX-2 after the stimulus of acetaldehyde.To determine the optimal concentration and action time of the 95% dose of alcohol after water therapy,powder group,full water extraction of water therapy,water extract-alcohol precipitation therapy,positive drug sulforaphane(SFN)and the specific inhibitor of Nrf2 pathway ATRA.The LX-2 cell lines cultured in vitro were treated with two different extraction schemes and Nrf2 pathway inhibitor ATRA,The inhibition rate of activated LX-2 cell lines decreased sharply(P<0.05).6.The results of GSH content from activated LX-2 cell lines: Water followed by 95% ethyl alcohol extraction group or powder group combined use with ATRA can decrease GSH content of activated LX-2 cell lines(P<0.05).After BSO inhibits GSH synthesis in cells,water followed by 95% ethyl alcohol extraction group or powder group combined use with ATRA can decrease GSH content of activated LX-2 cell lines sharply(P<0.01).Conclusions1.The model of alcohol-injured hepatic fibrosis in rats and in LX-2 cell lines were successfully established.2.Water followed by 95% ethyl alcohol extraction group and powder group both have therapeutical effects on alcohol-injured hepatic fibrosis in rats and in LX-2 cell lines.3.The opening Nrf2/?-GCS signaling pathway may be involved in the effects with HaoBieYangYinRuanJian Prescription on inhibition of alcoholic liver fibrosis.The intervention of two extraction groups of HaoBieYangYinRuanJian Prescription can inhibit alcoholic liver fibrosis by effectively upgrading the Nrf2/?-GCS signaling pathway,improving the activity of ?-GCS protein in liver tissues.4.The opening Nrf2/?-GCS signaling pathway may be involved in the effects with HaoBieYangYinRuanJian Prescription on activation of hepatic stellate cells stimulated by acetaldehyde.The intervention of two extraction groups of HaoBieYangYinRuanJian Prescription can reduce the proliferation of hepatic stellate cell through effectively upgrading the Nrf2/?-GCS signaling pathway,improving the activity of ?-GCS protein and increase the content of GSH in activated cells. |
PDF Full Text Request