The Role Of PXR-MRP2 Pathway In Cyclosporine-Induced Drug-Induced Liver Injury

Objective:At present,China's organ transplantation ranks second in the world.Cyclosporine A(Cs A)is one of the most commonly used immunosuppressive agents in the field of organ transplantation.How to improve the survival rate of patients and grafts,reduce the incidence of liver injury,improve the safety of clinical application of the drug,and avoid harmful interactions has now become a hot issue in global research.Therefore,studying the risk factors of liver injury caused by Cs A after transplantation is of great significance for clinical use.Based on the previous research basis of this research group,the mechanism of PXR-MRP2 pathway in drug-induced liver injury induced by cyclosporine was explored.This project uses RNA interference(RNAi)technology to construct MRP2 gene silenced cell lines;The Cs A-induced liver injury model of Hep G2 cells established by the research group in the early stage combined with the construction of a successful silent cell line,different concentrations of Cs A were given to investigate the effects of Cs A on relevant biochemical indicators of Hep G2 normal cells,MRP2 silenced cells,and PXR silenced cells,and their MRP2,PXR protein expression and m RNA.To investigate whether Cs A regulates the expression of MRP2 through the PXR signaling pathway at the cellular level and to clarify the role of MRP2 in Cs A-induced liver injury.,providing theoretical and experimental basis for clinical medicationMETHOD:1.Using the p HBLV plasmid as a vector,artificially synthesize the MRP2-sh RNA sequence.The MRP2-sh RNA sequence was ligated with digested p LK0.1 plasmid,introduced into competent cells.It was then screened and sequenced.;2.Hep G2 cells were pre-experimented to select the appropriate concentration of puromycin.A three-plasmid packaging system was used to transfect 293 T cells.Hep G2 cells were infected with lentivirus carrying the sh RNA fragment of interest,and the recombinant plasmid was integrated into the genome of Hep G2 cells.The p HBLV plasmid is puromycin-resistant,and MRP2 silenced cell lines that have been successfully transfected are selected by puromycin,and the silencing efficiency is detected by western blotting;3.The Cs A-induced Hep G2 cell liver injury model established by the research group in the early stage and the PXR silenced cell line previously constructed by the research group,combined with the successful MRP2 silenced cell line,different concentrations of Cs A were given to investigate the effects of Cs A on relevant biochemical indicators of Hep G2 normal cells,MRP2 silenced cells,and PXR silenced cells,and their MRP2,PXR protein expression and m RNA.To investigate whether Cs A regulates MRP2 expression through the PXR signaling pathway at the cellular level and clarify the mechanism of Cs A affecting MRP2 expressionResult:1.Sequencing the MRP2-sh RNA recombinant plasmid,no nucleotide mutations were found in the sequencing results,indicating that the p HBLV-MRP2-sh RNA recombinant plasmid was successfully constructed.2.Comparing the MRP2 silencing group with the Hep G2 group,the MRP2 protein expression in the MRP2 silencing group was significantly reduced(P<0.05),which was statistically significant.MRP2 silencing cell lines were successfully constructed.Western blotting results showed that the MRP2 silencing efficiency of MRP2 silencing cell lines was about 50%,and the PXR silencing efficiency of PXR silencing cell lines was about 55%.3.MRP2 and PXR are genes related to liver injury caused by Cs A in Hep G2 cells.Decreasing the expression of these two genes will lead to a significant increase in liver function indicators AST and LDH values,which exacerbates liver injury caused by Cs A in Hep G2 cells.When the MRP2 gene is underexpressed,low concentrations of Cs A cause elevated AST and LDH values;4.In Hep G2 cells and PXR silenced cells,the expression of MRP2 protein in the two groups of cells decreased with increasing Cs A concentration.Compared with Hep G2 group,the expression of MRP2 protein in PXR silenced group was lower than that in Hep G2group(P<0.05,P<0.01)5.In Hep G2 cells and PXR silenced cells,MRP2 m RNA expression of the two groups of cells decreased with increasing Cs A concentration.Compared with Hep G2 group,MRP2 m RNA expression in PXR silenced group was lower than Hep G2 group(P<0.05)Conclusion:Successfully constructed the p HBLV-MRP2-sh RNA lentiviral expression plasmid and MRP2 silenced cell line;MRP2 and PXR are genes related to liver injury of Hep G2 cells caused by Cs A,which aggravates liver injury of Hep G2 cells caused by Cs A;comparing the effects of Cs A on Hep G2 cells and PXR silenced cells,it was found that Cs A affects MRP2 m RNA and The protein expression has inhibitory effect,and the inhibitory effect becomes more obvious with the increase of Cs A concentration.At the same time,it was found that the inhibitory effect of Cs A on MRP2 m RNA and protein expression of PXR silenced cells was significantly lower than that of Hep G2 cells,indicating that the silence of PXR gene can significantly reduce the inhibitory effect of Cs A on MRP2.This confirmed that Cs A can regulate the expression of MRP2 m RNA and protein levels through PXR nuclear receptors.