| Objective:To investigate the expression of NLRP3 inflammasome and its related inflammatory factors and fibrosis factors in renal tubular epithelial cells of rats and diabetic rats cultured in high glucose environment.NLRP3 siRNA silencing technique and angiotensin receptor antagonist(ARB class)were used to intervene diabetic nephropathy rats and rats renal tubular epithelial cells under high glucose environment,and detect the changes in the expression of these indicators.To explore the potential mechanism of NLRP3 inflammasome in diabetic kidney disease,to preliminarily explore whether irbesartan can block the action of NLRP3 inflammasome.We designed different time points to investigate the changes of NLRP3 inflammasome and related molecules in the pathway under high glucose.Methods:NLRP3 siRNA was transfected into cells and rats using liposomes.The positive transfection rate of NRK-52 E cells was observed under fluorescence microscope,and the transfection efficiency was verified by fluorescence quantitative PCR.The experiment was divided into two parts:Cell Experiment Part:Divide into 5 groups at multiple time points : Low glucose group(Control group),high glucose group(HG group),high glucose + no-load group(HG+blank group),high glucose +NLRP3 siRNA group(HG+ NLRP3 siRNA group),high glucose + ARB group(HG+ ARB group).The m RNA expressions of NLRP3,Caspase-1 and Vimentin at each time point were detected by Q-PCR.The protein expressions of NLRP3,HSP47,NF-k B p-p65,Collagen IV and Vimentin were detected by Western Blot.ELISA was used to detect the expression levels of IL-1βand TNF-alpha in cell supernatants.Animal experiments:Divided into 5 groups and multiple time points(4W,8W,12W): Normal control group(Control group),high glucose model group(Model group),high glucose model+ no-load group(Model +blank group),high glucose model +NLRP3 siRNA group(Model + NLRP3 siRNA group),high glucose model + ARB group(Model + ARB group).The changes of P-P65 in renal tissue were observed by immunohistochemistry.The changes of Vimentin and NLRP3 in renal tissue were observed by immunofluorescence.Results:Cell Experiment Part:1.After 48 hours of cell intervention,compared with Control group,the m RNA expressions of NLRP3 and Vimentin in HG group and HG+blank group were significantly increased(P<0.01),and the m RNA expression of Caspase-1 was significantly increased(P<0.05).The protein expression levels of NLRP3,HSP47,p-p65,Vimentin Collagen IV were significantly increased(P<0.01).Compared with HG group,the m RNA expression of NLRP3 in HG+NLRP3 siRNA group and HG+ARB group was significantly down-regulated(P<0.01),the m RNA expression of Caspase-1 and Vimentin were significantly down-regulated(P<0.05),and the protein expressions of NLRP3,HSP47,p-p65,Vimenti Collagen IV were significantly down-regulated(P<0.01).There was no statistical significance in the above indexes between HG group and HG+blank group.2.After 48 hours of cell intervention,compared with Control group,the m RNA expressions of NLRP3,Caspase-1 and Vimentin in HG group and HG+blank group were significantly increased(P<0.01),and the protein expressions of NLRP3,HSP47,p-p65,Vimentin Collagen IV were significantly increased(P<0.01).Compared with HG group,the m RNA expression of NLRP3 and Vimentin in HG+NLRP3 siRNA group and HG+ ARB group were significantly down-regulated(P<0.05),the m RNA expression of Caspase-1 was significantly down-regulated(P<0.01),and the protein expressions of NLRP3,HSP47,p-p65,Vimenti Collagen IV were significantly down-regulated(P<0.01).There was no statistical significance in the above indexes between HG group and HG+blank group.3.After 48 hours of cell intervention,compared with Control group,the protein expression levels of NLRP3,HSP47,p-p65,Vimentin Collagen IV in HG group and HG+blank group were significantly increased(P<0.01).Compared with HG group,the protein expressions of NLRP3,HSP47,p-p65 and Vimenti in HG+NLRP3 siRNA group and HG+ ARB group were significantly down-regulated(P<0.01),and the protein expressions of Collagen IV were significantly down-regulated(P<0.05).There was no statistical significance in the above indexes between HG group and HG+blank group.4.At each time point(24h,48 h,72h),the expression levels of IL-1β and TNF-αin HG group and HG+blank group were higher than those in Control group.Compared with HG group,the expression levels of IL-1β and TNF-α in HG+NLRP3siRNA group and HG+ ARB group were lower.The expression of IL-1β and TNF-αin HG group increased with time from 24 h to 72 h.The expression of IL-1β,TNF-α in HG+NLRP3 siRNA group and HG+ ARB group also increased,but the expression was less than that in HG group.Animal experiments:1.After 4 weeks of intervention,compared with Control group,the m RNA expressions of NF-KB,NLRP3,HSP47,TRAF6 and Collagen IV in the Model group and the Model+Blank group were significantly increased(P<0.01),and the protein expressions of NLRP3,HSP47,α-SMA,Vimentin and p-p65 were increased(P<0.01).Compared with Model group,m RNA expressions of NF-KB,NLRP3,HSP47,TRAF6 and Collagen IV in Model+ARB group were significantly down-regulated(P<0.05),protein expressions of NLRP3 and HSP47 in Model+ARB group were significantly down-regulated(P<0.05),and α-SMA,Vimentin and p-p65 were significantly down-regulated(P< 0.01).The protein expressions of α-SMA,Vimentin and p-p65 in Model+NLRP3 siRNA group were significantly down-regulated(P <0.01),and the protein expressions of NLRP3 and HSP47 were significantly down-regulated(P < 0.05).There was no statistical significance in the above indexes between Model group and Model+blank group.2.After 8 weeks of intervention,compared with the Control group,the m RNA expressions of NF-KB,NLRP3,HSP47,TRAF6 and Collagen IV in the Model group and the Model+Blank group were significantly increased(P<0.01),and the protein expressions of NLRP3,HSP47,α-SMA,Vimentin and p-p65 were increased(P<0.01).Compared with Model group,m RNA expressions of NF-k B,NLRP3,HSP47,TRAF6 and Collagen IV in Model+ARB group were significantly down-regulated(P<0.05),protein expressions of NLRP3,α-SMA,Vimentin and p-p65 in Model+ARB group were down-regulated(P< 0.01),and HSP47 was significantly down-regulated(P<0.05).The protein expressions of NLRP3,Vimentin and p-p65 in Model+NLRP3-siRNA group were significantly down-regulated(P < 0.01),while those of SP47 and α-SMA were significantly down-regulated(P < 0.05).There was no statistical significance in the above indexes between Model group and Model+blank group.3.After 12 weeks of intervention,compared with the Control group,the m RNA expressions of NF-KB,NLRP3,HSP47,TRAF6 and Collagen IV in the Model group and the Model+Blank group were significantly increased(P<0.01),and the protein expressions of NLRP3,HSP47,α-SMA,Vimentin and p-p65 were increased(P<0.01).Compared with Model group,the expressions of NF-k B and NLRP3 in Model+NLRP3-siRNA and Model+ARB group were significantly down-regulated(P<0.01),the m RNA expressions of HSP47,TRAF6 and Collagen IV were significantly down-regulated(P<0.05),and the protein expressions of NLRP3,HSP47,α-SMA,Vimentin and p-p65 were significantly down-regulated(P<0.05).The protein expressions of NLRP3,HSP47,α-SMA and p-p65 in Model+NLRP3-siRNA group were significantly down-regulated(P < 0.05).There was no statistical significance in the above indexes between Model group and Model+blank group.Conclusion:High glucose can activate the expression of NLRP3 inflammasome,leading to the up-regulation of the expressions of inflammatory factors NF-k B,TRAF6,IL-1β,TNF-α and fibrosis factors HSP47,α-SMA and Collagen IV,inducing phenotypic transformation of renal tubular epithelial cells in rats with glucose and fibrosis of renal tissue in diabetic nephropathy rats.Specific NLRP3 siRNA gene silencing and the angiotensin receptor blocker(irbesartan)can block the activation of NLRP3 inflammators induced by high glucose,down-regulate the release of inflammatory factors and the expression of fibroproteins,and thus delay the progression of nephropathy. |
