Study Of Potential Role And Mechanism On Nrf2 In Sulforaphane Alleviating Renal Injury In Ln

Objective:SLE is a chronic and recurrent autoimmune disease,and LN is the most important complication of SLE.The pathogenesis of LN is mainly related to the deposition of IC in the kidney and the imbalance of oxidation/antioxidation balance.The aim of this study was to explore the role of Nrf2 in alleviating kidney injury in LN by sulforaphane（SFN）and its related mechanism.Methods:The study was divided into two parts:（1）Animal experiments:（i）20 male MRL/lpr mice were randomly divided into SFN-treated group and model control group;10 male C57BL/6J mice were selected as normal control group.（ii）Mice in the three groups were intraperitoneally injected with SFN or equal volume of PBS from 24weeks of age to 28 weeks of age,general condition of mice was monitored,blood creatinine,blood urea nitrogen and urine protein concentrations were detected,serum anti-ds DNA antibody,IL-6,IL-10and TNF-αlevels were determined by enzyme-linked immunosorbent assay,mouse kidney tissue samples were obtained for HE staining,Masson staining and PAS staining,and immunohistochemistry was performed to detect the expressions of Nrf2,HO-1,GPX4,SLC7A11 and Transferrin in kidney.The expression levels of PAI-1,Nrf2,HO-1,GPX4,SLC7A11 and Transferrin in renal tissues were detected by western blot.（2）Cell experiments:（i）Human glomerular mesangial cells（HMC cells）were selected to construct oxidative stress model（H₂O₂treatment）and ferroptosis model（RSL3 treatment）.The effects of SFN on HMC cells under oxidative stress and ferroptosis were observed:cell viability was examined with CCK-8,cell proliferation was detected by EDU-555,reactive oxygen species（ROS）levels were tested by DCFH-DA fluorescence staining,IL-6,IL-10 and TNF-αlevels in cell supernatant were determined by Enzyme-linked immunosorbent assay.The expression levels of Nrf2,HO-1,GPX4,SLC7A11 and Transferrin were detected by western blot.（ii）The Nrf2 gene of HMC cells was silenced by RNA interference,and the effects of SFN on the protein expression levels of Nrf2,HO-1 and GPX4 in HMC cells after Nrf2 silencing were observed under oxidative stress.Results:（1）The results of animal experiments showed that:（i）The levels of urine protein concentrations,serum creatinine and blood urea nitrogen,serum anti-ds DNA antibody,IL-6,IL-10 and TNF-αin the SFN-treated MRL/lpr mice were lower than those in the model control group and higher than those in the normal control group.（ii）Compared with the model control group,the renal pathological damage of MRL/lpr mice in SFN-treated group was significantly alleviated.（iii）The protein expression levels of PAI-1,Nrf2 and HO-1 in SFN-treated group were lower than those in model control group,while the protein expression levels of GPX4,SLC7A11 and Transferrin were significantly higher than those in the model control group.（2）The results of cell experiments showed that:（i）In oxidative stress cell model,the proliferation and activity of HMC cells stimulated by H₂O₂were significantly decreased,the production of intracellular ROS was significantly increased,the levels of inflammatory cytokines IL-6,IL-10 and TNF-αwere significantly increased,and the expressions of ferroptosis regulatory proteins GPX4,SLC7A11 and Transferrin were significantly decreased.The molecular expression of Nrf2/HO-1 signaling pathway was significantly up-regulated.And the results of ferroptosis cell model showed that the expression of GPX4,SLC7A11 and Transferrin in HMC cells stimulated by RSL3 was significantly decreased,and the expression of Nrf2/HO-1signaling pathway was significantly up-regulated;but SFN can significantly inhibit the above effects of H2O2 or RSL3 stimulation.（ii）After the expression of gene Nrf2 was silenced by si RNA-Nrf2 in HMC cells,SFN up-regulated Nrf2 and HO-1 expression in HMC cells and HMC cells stimulated by H₂O₂were significantly inhibited,and SFN also inhibited the up-regulation of GPX4 expression.Conclusions:（1）SFN can inhibit renal damage and mesangial cell damage caused by oxidative stress and cell ferroptosis in LN.（2）The protective effect of SFN on kidney and mesangial cells of MRL/lpr mice was mediated by Nrf2 signaling pathway.