Effect And Mechanism Of Resveratrol On Kidney Inflammation Of Exhausted Exercise Rats Via Modulating SIRT1/NF-?B/NLRP3 Inflammsome Pathway

Objective: Resveratrol is a naturally-occurring small molecule agonist of sirtuin1(SIRT1),As one of the most extensively studied polyphenols,its effectiveness,safety and pharmacokinetics have been verified in several clinical trials,this study aims to explore the effects of resveratrol supplementation on the regulation of SIRT1/NF-?B/NLRP3 inflammasome pathway and effects on renal inflammation of exhausted exercise rats.Methods: In vitro experiments,human proximal renal tubular epithelial cells HK-2 cells were used,MTT assay was applied to detect the effects of albumin and resveratrol on the activity of HK-2 cells,and inflammatory damage model of renal tubular epithelial cells was established by albumin simulated vivo proteinuria environment.Resveratrol,SIRT1 inhibitor and NF-?B inhibitor were used in the model.SIRT1,NF-?B p65,NLRP3,ASC and caspase-1 expression were detected by Western blot,the co-localization of NLRP3 and ASC was detected by immunofluorescence double staining,Hoechst33342/PI staining was used to observe pyroptosis,and ELISA was used to detect the levels of inflammatory factors IL-1? and IL-18.In vivo experiments,the effect of resveratrol supplementation on renal inflammation in rats with exercise-induced actue kidney injury was explored.36 male SD rats were randomly divided into 4 groups,control group(Con,n=8),resveratrol group(Rsv,n=8),exhausted exercise group(Ex,n=10),and exhausted + resveratrol group(Ex+Rsv,n=10).Rats in Ex and Ex+Rsv groups performed exhausted treadmill exercise for 4 weeks,with a slope of 10 °,speed starting from 5 m/min,increasing 5 m/min every 5 minutes,and the maximum speed was 35 m/min.Exhausted time was recorded,and random urine was taken once a week to detect proteinuria.In Rsv and Ex+Rsv groups,resveratrol was orally administered daily at a dose of 50 mg/kg BW/d.Anesthesia treatment was taken 24 hours after the last training.Blood and kidney tissues were collected for detection,Scr,BUN,urine protein,urine albumin,and urinary creatinine levels were measured by an automatic analyzer;urinary NGAL and KIM-1,TNF-?,IL-6,IL-1? and IL-18 levels in renal were detected by ELISA;HE staining was used to observe the changes of renal tissue morphology and structure.Western blot was used to detect SIRT1,NF-?B p65,Ac-NF-?B p65,NLRP3,ASC,caspase-1,IL-1? and IL-18 expression at the protein level,immunofluorescence double staining was applied to observe the colocalization of NLRP3 and ASC in renal tubules.Results:(1)MTT results showed that bovine serum albumin(BSA)(20 mg/ml,40 mg/ml)reduced the proliferation activity of HK-2 cells obviously;with a certain concentration of albumin(20 mg/ml),resveratrol(50 ?M,100 ?M)can significantly increase the proliferative activity of HK-2 cells;(2)BSA induced a significant increase in the levels of inflammatory factors IL-1?,IL-18 in HK-2 cells,NLRP3,ASC and caspase-1 expression of protein were increased,as was the same with pyroptosis;(3)Resveratrol up-regulated the expression and deacetylation activity of SIRT1 in HK-2 cells,reduced the levels of inflammatory factors,down-regulated NF-?B and NLRP3 inflammasome expression and also alleviated pyroptosis.After inhibiting SIRT1,BSAinduced inflammatory damage in renal tubular epithelial cells increased;further suppressing NF-?B,the levels of inflammatory factors IL-1? and IL-18 decreased,the protein expression level of NLRP3 inflammasome was decreased;(4)In vivo animal experiments,the levels of Scr and BUN in serum and KIM-1 and NGAL in urine were increased in exhausted exercise rats,and HE light microscopy of renal tissue showed renal tubular injury;(5)The protein expressions of NF-?B,NLRP3,ASC and caspase-1 in kidney of the exhausted exercise group were increased,and the same with the content of IL-1? and IL-18;(6)Resveratrol supplementation increased the expression of SIRT1 in kidney and reduced the expression of Ac-NF-?B,then further down-regulated the protein expression of NF-?B and NLRP3 inflammasome,the morphological changes of kidney caused by exhaustive exercise in rats was alleviated,the levels of inflammatory factors were reduced.Conclusions:(1)Stimulation of albumin at a certain concentration can activate NLRP3 inflammasome in renal tubular epithelial cells and cause pyroptosis,which promotes the development of inflammation;(2)Resveratrol acts on SIRT1/NF-?B/NLRP3 inflammasome pathway that resulted in the alleviation of albumin-induced inflammatory response;(3)NLRP3 inflammasome is activated and participates in kidney inflammation in rat caused by exhaustive exercise,resveratrol can play an antiinflammatory effect in exhausted exercise rats;(4)Resveratrol can effectively inhibit the excessive activation of inflammasome in kidney during exhaustive exercise,which is one of the candidates for relative health products.