The Study Of Rifampicin-induced Liver Injury In Mice And The Construction Of ABCC2 Gene Overexpressing Cell Line

Background and ObjectiveRecent years,the incidence of tuberculosis has increased significantly compared with previous years,the mortality rate has also been significantly improved,which is ranking the first place in all types of infectious diseases.China is one of the largest tuberculosis countries from all over the world [1-2].Among the treatments of anti-tuberculosis,these anti-TB drugs can cause liver injury and the incidence is about 2%-28% [3-5].Among the adverse reactions of anti-TB drugs,liver injury accounted for the first,and the severe liver injury incidence is about 1%-2% [6-7],but the mechanisms are still unclear.Therefore,we need to find the mechanisms of liver injury caused by anti-tuberculosis drugs.To minimize the liver injury caused by anti-TB drugs or to find new anti-TB drugs,which are hot clinical problems need to be solved.Rifampicin(RIF)is one of semi-synthetic antibiotics,which belongs to rifamycin class.It can inhibit the synthesis of bacterial RNA and it can be used for the treatment of tuberculosis,intestinal tuberculosis,tuberculous peritonitis,joint tuberculosis,Cocci and other infections.It is one of the most commonly used anti-TB drugs in clinical practice,so that it has become a common cause of acute liver injury in China.Rifampicin has many side effects,liver toxicity is the most common adverse reaction,which seriously affected its clinical application [8-11].The mechanisms of liver injury caused by rifampicin is still unclear.There is no evidence that during the process of the treatment,rifampicin will be toxic in the liver metabolism [12].In this study,mice were given different doses of rifampicin to observe whether rifampicin can induce liver injury and the effects of different doses of rifampicin on liver injury in mice.It makes a foundation to further explore the mechanisms of rifampicin-induced liver injury.We constructed the ABCC2 gene overexpressing cell line to lay the foundation for the study of the relationship between ABCC2 gene,MRP2 protein expression,and rifampicin-induced cholestasis.Methods1.Rifampicin-induced liver injury in mice research methods:24 healthy female ICR mice were randomly divided into 4 groups,6 rats in each group: control group,low-dosage group,middle-dosage group and high-dosage group.They were fed once at every 8:30 am for 2 weeks.The mice of low-dosage group were given rifampicin for 100 mg/kg per day;these of middle-dosage group were given rifampicin for 200 mg/kg per day;these of high-dosage group were given rifampicin for 400 mg/kg per day;the control group were given the equal volume of aqueous solution of sodium carboxymethyl cellulose.After the last administration,the rats were sacrificed for 6 h,and the blood samples were collected and serological tests were carried out.The liver of the mice was collected,and to observe the pathological changes of the mice.2.ABCC2 gene overexpression of cell lines:(1)ABCC2-e GFP site-structured integration vector construction,(2)electric transfer HEK-293 cells,screening positive clones.Results1.There was no significant difference in all liver biochemical indexes between the four groups.2.Significant pathological changes of liver in mice were observed among the three experimental groups.3.HE staining: in the experimental group: liver cell disorder,part of the liver cell swelling,and some vacuolar cells can be observed,which was considered as different degrees of steatosis cells.And in the portal area,a large number of inflammatory cell infiltration and cholestasis can be observed.It was most significant in the high-dosage group.4.Electron microscopy: in different experimental groups,liver capsule was filled with a lot of fat droplets,some of the capillary bile ducts were slightly expanded,and cholestasis was found.These damage were increased along with the increase of rifampicin concentration.5.To Constructed ABCC2 gene overexpressing cell line.ConclusionDifferent doses of rifampicin can cause liver injury in mice for 2 weeks,which mainly manifested as hepatic steatosis,inflammatory cell infiltration,partial mitochondrial swelling,endoplasmic reticulum rupture and scattered cholestasis.Furthermore,rifampicin-caused liver injury was concentration-dependent,but the mechanisms are still unclear which need to be further studied.We successfully constructed the ABCC2 gene overexpressing cell line.Objective To evaluate the influence of SLCO1B1 T521 C genetic polymorphisms on statins pharmacokinetics.Methods Pub Med,Cochrane Library,EMBASE,CBM,CNKI,WANFANG,CQVIP data bank were searched by calculator for studying the impact of SLCO1B1 T521 C genomic polymorphisms on statins pharmacokinetics.The pharmacokinetic parameters including AUC0-6h,AUC0-12 h,AUC0-24 h,AUC0-inf,Cmax and CL/F were extracted.The standardized mean differences(SMD)and 95% confidence intervals(95% CI)were calculated to compare the TT genotype and mutant genotypes.Subgroup analyses were based on different ethnic populations and statin types.Sensitivity analysis was used to perform heterogeneity tests.The Meta analysis was performed by Rev Man5.3 software.Results A total of 25 articles were analyzed in this study,which include 23 litertures in Engish and 2 literatures in Chinese.Altogether 903 people were involved.The results of Meta analysis showed:the curve AUC0-inf of TT-TC,TT-CC,TT-(TC+CC)was(SWD=0.51,95% CI(0.29,0.72))),(SWD=1.80,95% CI(1.31,2.29))and(SWD=0.78,95% CI(0.57,0.99))respectively.Overall,the stand ard mean difference of AUC0-inf was higher in mutant genotype than that in wild genotype.The same result was obtained in subgroup analysis of Caucasians and Asians.The curve Cmax of TT-TC,TT-CC,TT-(TC+CC)was(SWD=0.48,95% CI(0.18,0.77)),(SWD=1.24,95% CI(0.77,1.70))and(SWD=0.65,95% CI(0.43,0.87))respectively.Also,the standard mean difference in Cmax was higher in the mutant genotype.The same result was obtained in subgroup analysis of Caucasians and Asians.The curve CL/F of TT-TC,TT-CC,TT-(TC+CC)was(SWD=-1.01,95% CI(-1.91,-0.11)),(SWD=-1.51,95% CI(-2.08,-0.94))and(SWD=-1.07,95% CI(-1.55,-0.59))respectively.CL / F standard mean difference in mutant genotype was lower than that inwild genotype.Conclusion The SLCO1B1 T521 C allele may have an important effect on the pharmacokinetics of statins.