Bidirectional regulation of triptolide on Nrf2/P65 and related toxic/therapeutic effects
Objective To determine the dual role of triptolide(TP),in regulating both the toxicity and protection through the Nrf2/P65 pathway and related mechanism.Methods Molecular docking simulations were performed to analyze the potential interaction between TP and Nrf2/P65.In the cell experiments,different concentrations of TP were used to intervene human normal liver cells(L02)and mouse Kupffer cells(KC).The cell viability was assessed with the MTT assay,and the protein expression of Nrf2,HO1,P65,and IL-6 was examined by Western blot.In the animal experiments,TP was administered for model rats with diabetic nephropathy.The kidney pathology was evaluated with HE staining,the serum BUN level was measured by indophenol blue colorimetric method,and changes in the serum levels of inflammatory cytokines TNF-α,IL-6,and IL-1β were determined by ELISA.The mRNA levels of P65,podocin,and Kim-1 in the kidney tissue were measured with RT-qPCR,and protein expression of P65 in the kidney tissue with Western blot.Immunohistochemistry was performed to observe the protein expression of P65 and Nrf2 in the kidney tissue.Results The molecular docking showed good binding between TP with Nrf2 and P65.In vitro experiments demonstrated that TP reduced the cell viability in L02 cells,decreased Nrf2 protein expression in a dose-and time-dependent manner,reduced the cell viability in KC cells,inhibited the expression of Nrf2 and its downstream target HO-1,and upregulated the expression of P65 and IL-6.The animal experiments showed that TP had obvious protective effect on DN rats,namely reducing the mRNA and protein expression of P65 in the kidney tissue,upregulating Nrf2 protein expression,and downregulating the levels of inflammatory cytokines TNF-α,IL-6,and IL-1β in the serum.Conclusion TP can induce toxic damage to liver cells by downregulating Nrf2 protein expression and upregulating P65 protein expression.Meanwhile,it can also exert a protective effect on DN rats by upregulating Nrf2 protein expression and downregulating the expression of P65 protein and mRNA.Therefore,TP can exert dual regulation on the Nrf2/P65 pathway,displaying either toxicity or therapeutic effects.
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