Micheliolide Attenuates Renal Tubular Epithelial Cells Inflammation By Modulating The MROS/NF-κB/NLRP3 Axis

Background:Chronic kidney disease(CKD)is a disease with high morbidity and mortality.Currently,there is no effective treatment.Studies have shown mitochondrial reactive oxygen species,nuclear factor kappa B(NF-κB)pathway and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome activation play important roles in renal inflammation,and the three are closely related,but the specific mechanism is unclear.Micheliolide(Micheliolide,MCL)is a novel compound derived from parthenolide with antioxidant and anti-inflammatory effects.Micheliolide(Micheliolide,MCL)is a novel compound derived from parthenolide with antioxidant and anti-inflammatory effects.In this study,we first established a mouse model of unilateral ureteral obstruction in vivo,and then tested the effect of MCL on the NLRP3 inflammasome activation pathway and related inflammatory factors in the state of renal inflammation induced by unilateral ureteral obstruction.In vitro experiments,we established lipopolysaccharide(LPS)-induced rat renal proximal tubular cell line(NRK-52E)inflammation model,and then studied the effect of MCL on the NF-κB pathway and related inflammatory factors in the inflammatory state of renal tubular epithelial cells induced by lipopolysaccharide.And studied the effect of MCL on NLRP3 inflammasome activation pathway and related factors.Finally,we studied the mechanism of regulation effect of mitochondrial reactive oxygen species on the activation of NLRP3 inflammasome in renal tubular epithelial cells and the role of MCL in it.Methods:In vivo experiments were conducted to establish a mouse model of unilateral ureteral obstruction.Immunohistochemistry and Western blotting were used to detect the effect of MCL on the NLRP3 inflammasome activation pathway and related inflammatory factors in the state of renal inflammation induced by unilateral ureteral obstruction.In vitro experiments,we established a lipopolysaccharide or rotenone-induced rat kidney proximal tubule cell line(NRK-52E)inflammation model and intervened with MCL or Mitoq,an inhibitor of mitochondrial reactive oxygen species.The effect of MCL on cell viability was detected by MTT experiment.The expressions of NLRP3,capase-1,MCP-1,IL-1β,IL-18 and TNF-α were detected by western blotting experiment,quantitative Real-time PCR and enzyme-linked immunosorbent assay experiment.The fluorescent probe DCFH-DA and flow cytometer were used to measure the release of reactive oxygen species in renal tubular epithelial cells,and the mitochondrial membrane potential(MMP)of the cells was measured with the JC-1 kit and fluorescence microscope.Results:(1)MCL intervention significantly down-regulated the expression of NLRP3,caspase-1,IL-1β and TNF-α in the kidney tissue of mice with unilateral ureteral obstruction.(2)Low concentration(1-10μM)MCL has no significant effect on the viability of NRK-52E cells after 48 hours of incubation.The high concentration of 20μM MCL will significantly reduce the viability of NRK-52E cells.(3)MCL intervention significantly down-regulated the expression of MCP-1,TNF-α and nuclear NFκBp65 in NRK-52E cells induced by LPS.(4)MCL intervention significantly down-regulated the expression of NLRP3,caspase-1 p10,IL-1β and IL-18 in NRK-52E cells induced by LPS.(5)MCL intervention inhibits the decrease of mitochondrial membrane potential of NRK-52E cells and reduces the release of mitochondrial reactive oxygen species induced by lipopolysaccharide.(6)The intervention of MCL and Mitoq,an inhibitor of mitochondrial reactive oxygen species,inhibited the expression of NLRP3,caspase-1 p10,IL-1β and IL-18 in NRK-52E cells induced by lipopolysaccharide or rotenone.Conclusion:In vivo and in vitro experimental results show that MCL intervention can improve renal tubular epithelial cell inflammation by inhibiting the NF-κB pathway and the NLRP3 inflammasome activation pathway.MCL intervention can improve renal tubular cell inflammation by inhibiting the NF-κB pathway and the NLRP3 inflammasome activation pathway.Further experiments showed that MCL inhibited lipopolysaccharide-induced decline in the mitochondrial membrane potential of renal tubular epithelial cells,and reduced the release of mitochondrial reactive oxygen species.Mitoq,an inhibitor of mitochondrial reactive oxygen species,inhibits lipopolysaccharide-induced NLRP3 inflammasome activation pathway.Both Mitoq and MCL can inhibit NLRP3 inflammasome activation induced by rotenone,a drug that promotes the release of mitochondrial reactive oxygen species.,This indicates that MCL inhibits the activation of NLRP3 inflammasomes by inhibiting the release of mitochondrial reactive oxygen species,thereby improving renal tubular inflammation.Therefore,we speculate that MCL inhibits the inflammatory response of renal tubular cells by inhibiting the mROS/NF-κB/NLRP3 axis.