| Objective: Establish a renal tubular epithelial cell injury model induced by high uric acid and a hyperuricemia mouse model,and to investigate the mechanism of Shizhifang regulating ROS inhibiting NEK7-NLRP3 signaling pathway to improve uric acidinduced renal injury.Methods:NRK-52 E cells were cultured in vitro and stimulated with 50 and 100ug/ml uric acid for 24 hours.Flow cytometry and TUNEL staining were used to detect apoptosis,ROS was detected by flow cytometry.Western blotting was used to detect Cle-caspase-3,Bax,Bcl-xl,NEK7,NLRP3,LC3 B and P62 protein,and m RNA of Caspase-3,Bax and Bcl-xl were detected by q PCR.Subsequently,the cultured cells were divided into normal group,model group(100ug/ml),ROS inhibitor group(NAC) and Shizhifang group.ROS,apoptosis and related proteins were detected as in the first part of the experiment,and the expression of IL-1β and IL-18 protein were also detected.In vivo,50 SPF BALB/c male mice aged 9 weeks were selected,including 10 mice in the normal group,model group,NAC group,allopurinol group and Shizhifang group.The hyperuricemia model of mice was induced by intraperitoneal injection of potassium oxonate solution(250mg/kg).After 2 weeks,blood biochemical tests were performed.Renal pathology was observed by HE and Masson.Uric acid crystal was detected by silver hexamine staining,ROS was detected by DHE,and NEK7/NLRP3 pathway,inflammatory factors,apoptosis and autophagy protein expression were detected by Western blotting and immunohistochemistry.Tunel assay was used to detect renal tubular epithelial cell apoptosis.Results: In vitro results showed that,compared with normal cells,NRK-52 E apoptosis cells were increased after uric acid stimulation,the Cle-caspase-3,Bax,NEK7/NLRP3 pathway and LC3 B Ⅱ/I protein were up-regulated,the protein levels of Bcl-xl and P62 were down-regulated,and ROS production was increased.Among them,Bcl-xl and P62 were negatively correlated with uric acid concentration,while the other indicators were positively correlated with uric acid concentration.Compared with the model group(100ug/ml),the apoptosis of NRK-52 E and ROS levels were decreased in NAC and Shizhifang groups,and the protein levels of Cle-caspase-3,Bax,NEK7/NLRP3 pathway,LC3B Ⅱ/I,IL-1β,IL-18 and TNF-α were down-regulated,while the protein levels of Bcl-xl and P62 were up-regulated.In vivo experiments showed that compared with normal mice,renal function of HUA model mice was decreased,the renal tubule inflammatory injury,the renal tissue ROS increased,the expressions of Cle-caspase-3,Bax,NEK7/NLRP3 pathway proteins,LC3 B Ⅱ/I,IL-1β,IL-18 and TNF-α were upregulated,and the protein levels of Bcl-xl and P62 were down-regulated.Apoptosis and autophagy of renal tubular epithelial cells were increased.Compared with model group,NAC,allopurinol and Shizhifang group renal function was improved,renal ROS,renal cell apoptosis and autophagy were decreased,Cle-caspase-3,Bax,NEK7/NLRP3 pathway,LC3B Ⅱ/I,IL-1β and IL-18 were down-regulated,and Bcl-xl and P62 protein levels were up-regulated,and reduced renal injury.Conclusion: Uric acid activates the NEK7/NLRP3 signaling pathway through ROS,causing apoptosis,autophagy and inflammation of renal tubule epithelial cells.Shizhifang inhibits ROS,and then inhibits activation of NEK7/NLRP3 signaling pathway,improving renal function of hyperuricemia mice,alleviating renal injury,reducing the release of inflammatory factors,renal tubule epithelial cell apoptosis and autophagy,and protect kidney.ROS is the key target of Shizhifang in inhibiting NEK7/NLRP3 signaling pathway and alleviating renal injury in hyperuricemia mice. |
