Protective Effect And Mechanism Of Hugan Tablets On Liver Injury Caused By Rifampicin

ObjectiveTaking the endoplasmic reticulum stress signal pathway as the research target,the therapeutic effect of Hugan Tablets against tuberculosis drugs in patients with liver damage caused by liver and gallbladder damp-heat syndrome and drug-induced liver injury and schizandrin B which is the active ingredient contained in Schisandra chinensis in Hugan Tablets against study on the protective effects of tuberculosis drugs on normal hepatocyte damage.To explore the mechanism of Hugan Tablets regulating endoplasmic reticulum stress signal pathway in the treatment of drug-induced liver injury.Methods1.Clinical ResearchAccording to the different treatment methods,68 patients with liver injury caused by liver,gallbladder damp-heat syndrome and drug-induced liver injury were divided into treatment group and control group.Both groups of patients were treated with "2HRZE/4HR" for antituberculosis treatment,while the control group was given oral polyene phosphatidylcholine capsules for oral treatment.On the basis of treatment in the control group,the treatment group was treated with Hugan Tablets orally.The course of treatment is 14 days.During the treatment of the two groups,observe the two groups of patients:?improvement of TCM syndrome indicators;? liver function-related laboratory indicators:alanine aminotransferase,total bilirubin,alkaline phosphatase,and international standard time ratio of coagulation;?Detection indicators of human serum endoplasmic reticulum stress pathway related proteins:changes in GRP78,IRE1,PERK,ATF6 and CHOP protein content.2.In vitro cell test?Treat human normal liver L02 cells with different concentrations of schizandrin B(0,25?M,50 ?M,100 ?M,200 ?M,400 ?M)and rifampicin(0,25 ?M,50 ?M,100 ?M,200 ?M,400?M)for 48 h.Determined by MTT experiment schizandrin B's protective concentration and rifampicin's modelling concentration.50 ?M schizandrin B was selected as the cytoprotective concentration,and 200 ?M rifampicin was used as the cell modeling concentration.?Experimental grouping:control group:L02 cells were left untreated;rifampicin group:L02 cells were given 200 ?M rifampicin;rifampicin+schizandrin B group:L02 cells were given 50?M schizandrin B and 200 ?M rifampicin.After 0-72h treatment,the survival rate of L02 cells was detected by MTT assay.?L02 cells were treated with 200 ?M rifampicin for 12 h,24 h,and 48 h,and the expression of proteins and genes related to the endoplasmic reticulum stress pathway were observed.?Experimental grouping:control group:L02 cells were left untreated;rifampicin group:200?M rifampicin was used to treat L02 cells for 48 h;rifampicin+schizandrin B group:200 pM rifampicin and different concentrations of schizandrin B(25 ?M,50 ?M,100?M)L02 cells were treated for 48h,and WB,qRT-PCR,and flow cytometry were used to detect the endoplasmic reticulum stress pathway-related proteins,gene expression and apoptosis of L02 cells in each group.?Experimental grouping:control group:L02 cells were left untreated;rifampicin group:L02 cells were given 200 ?M rifampicin;rifampicin+schizandrin B group:L02 cells were given 100 ?M schizandrin B and 200 ?M rifampicin.After 48 hours of treatment,the expression of ER stress pathway-related proteins in L02 cells of each group was detected by immunofluorescence staining.Results1.Clinical Research?Compared with the control group,the therapeutic effect of TCM syndromes was significantly better than that of the control group,and the difference was statistically significant(P<0.05).? Compared with the control group,the improvement of TCM symptom scores before and after treatment was significantly better than that of the control group.? After 1 week of treatment in the treatment group,ALT and ALP decreased significantly compared with before treatment,and the differences were statistically significant compared with before treatment.The differences in TBIL and INR were not statistically significant compared with before treatment.Results Compared with the control group after 1 week of treatment,only ALT improved more significantly,and the difference was statistically significant.There was no significant difference in ALP improvement.After 2 weeks of treatment in the treatment group,except for ALT and ALP,TBIL levels began to decrease compared with before treatment,and the difference was statistically significant compared with before treatment.Compared with the control group,2 weeks after treatment,TBIL improved more significantly.There was no significant difference in the improvement of INR compared with before treatment and 2 weeks after treatment in the control group.? The serum levels of endoplasmic reticulum stress pathway related proteins GRP78,IRE1,PERK,ATF6 and CHOP in the two groups of patients increased significantly before treatment.After 1 week of treatment in the treatment group,the expressions of GRP78,IRE1,PERK,ATF6,and CHOP in patients' serum decreased only slightly,and the differences were not statistically significant(P>0.05).After 2 weeks of treatment in the treatment group,the expressions of GRP78,IRE1,PERK,ATF6 and CHOP in the serum of the patients were significantly lower than those before treatment.However,the expressions of GRP78,IREI,PERK,ATF6,and CHOP in the control group patients did not decrease significantly after 1 or 2 weeks of treatment.After 2 weeks of treatment,the treatment group compared with the control group.The serum GRP78,IRE1,PERK,ATF6 and CHOP expressions in the patients decreased,and the differences were statistically significant.2.In vitro cell test?Rifampicin+ schizandrin B group,compared with rifampicin group,the survival rate of L02 cells significantly increased,indicating that schizandrin B can reduce the damage of rifampicin to L02 cells.? Rifampicin can activate the endoplasmic reticulum stress pathway and promote protein expression of GRP78.PERK,ATF4,CHOP,ATF6,ARMET,p-IRE1,and XBP-1.At the same time,rifampicin promotes the genes GRP78,PERK,ATF4 and the expression level of CHOP mRNA was time-dependent.? WB and qRT-PCR methods suggest that schizandrin B can effectively inhibit rifampicin-activated endoplasmic reticulum stress pathway,down-regulate the expression levels of GRP78,PERK,ATF4,CHOP,ATF6,ARMET and XBP-1 proteins,and inhibition was positively correlated with schizandrin B concentration.?Flow cytometry and WB methods suggest that schizandrin B can effectively inhibit rifampicin-induced apoptosis of L02 cells and reduce the expression of apoptotic proteins PARP and Cleaved caspase3,and this inhibition is positively correlated with schizandrin B concentration.?The immunofluorescence staining method suggested that schizandrin B could effectively inhibit the ER stress-activated endoplasmic reticulum stress pathway and down-regulate the expression levels of GRP78,PERK,ATF6,p-IRE1 and XBP-1 proteins in the nucleus of L02 cells.Conclusion?Hugan Tablets can effectively improve the TCM syndromes of patients with liver injury caused by drug-induced liver injury of liver-biliary damp-heat syndrome.?Hugan Tablets can effectively improve liver function related indexes of patients with liver injury of liver gallbladder dampness and heat syndrome:alanine aminotransferase,total bilirubin,alkaline phosphatase.?Hugan Tablets can effectively inhibit the expression of ER stress-related proteins,and play a role in treating drug-induced liver injury caused by antituberculosis drugs.?Schizandrin B can effectively improve the survival rate of rifampicin-injured liver cells and reduce the apoptosis rate,and at the same time can down-regulate the expression levels of GRP78,PERK,ATF4,CHOP,ATF6,ARMET and XBP-1,and inhibit activated endoplasm Net stress pathway,and the inhibitory effect is positively correlated with its concentration.It is suggested that the liver protection mechanism may be achieved by inhibiting the endoplasmic reticulum stress pathway.