| Objective:To investigate the expression of NLRP3 in cultured human renal tubular epithelial cells(HK-2)under high glucose stimulation at different concentrations and time points,and to detect the interstitial fibrosis m RNA and protein expression of NLRP3 and as well as its signaling pathway after intervention with the traditional angiotensin antagonist ARB and new small molecule inhibitor MCC950.To primarily explore the possible immune mechanism of Diabetic Kidney Disease and provide new ideas for the treatment of Diabetic Kidney Disease.Methods:1.To explore the differential expression of NLRP3 under high glucose or hypertonic stimulation.Hk-2 cells were divided into three groups: a low glucose control group(Con,5.6mmol/L glucose),a mannitol group(Ma,5.6 mmol/L glucose +34.4 mmol/L mannitol)and a high glucose group(HG,40 mmol/L glucose).The effects of high glucose or high osmotic concentration on NLRP3 expression were primarily investigated after48 hours of culture.2.To explore the differential expression of NLRP3 under different glucose concentrations.Hk-2 cells were divided into three groups: a low-glucose control group(Con,5.6mmol/L glucose),a group with 20mmol/L glucose and a group with 40mmol/L glucose.The effects of different concentrations of glucose stimulation on NLRP3 expression were primarily investigated after 48 hours of culture.3.To explore the mechanism of traditional angiotensin antagonist ARB on NLRP3 and its signaling pathway as well as indexes related to interstitial fibrosis.Hk-2 cells were divided into three groups: Con group,HG group,HG+irbesartan group(HG+ARB group),and cultured for 24 h,48h and 72 h.NLRP3,Caspase-1,NF-κB P65,Vimentin,N-cadherin were detected by q PCR and WB in the Con group,HG group and HG+ Irbesartan group(HG+ARB group).The expression of Vimentin was detected by the way of immunofluorescence.4.To explore the mechanism of MCC950,the inhibitor of NLRP3,on NLRP3 and its signaling pathway as well as indicators interstitial fibrosis related.Hk-2 cells were divided into three groups: Con group,HG group,HG+irbesartan group(HG+ARB group),and cultured for 24 h,48h and 72 h,q PCR and WB were used to detect NLRP3,Caspase-1,NF-κB P65,Vimentin,N-cadherin in the Con group,the HG group,and the HG+MCC950 group(MCC950 was added before the intervention and was not removed during the intervention period of high glucose).Immunofluorescence was used to detect Vimentin’s expression.Results:1.In HK-2 cells,after high glucose treatment for 48 h,compared with the Con group,the NLRP3 m RNA and protein expressions in the HG group were increased,and the difference was statistically significant(P<0.05).2.In HK-2 cells,treated with different glucose concentrations for 48 h,compared with the Con group,the m RNA and protein expressions of NLRP3 with a glucose concentration of 40mmol/l in the HG group increased,and the difference was statistically significant(P<0.01),while the group with glucose concentration of20mmol/l showed no statistical difference.3.Stimulate HK-2 cells with high glucose and intervene with ARB for 24 h,Compared with the Con group,the m RNA expressions of NLRP3,Caspase-1,P65 and a-SMA in the HG group were increased,and the protein expressions of NLRP3,P65,Vimentin and N-cadherin in the HG group were increased,and the differences were statistically significant(P<0.05),in the HG+ARB group,compared with the HG group,the m RNA expressions of NLRP3,Caspase-1,P65,and a-SMA were significantly decreased,and the protein expressions of NLRP3,P65,Vimentin,and N-cadherin were significantly decreased,the difference was statistically significant(P<0.05);Stimulate HK-2 cells with high glucose and intervene with ARB for 48 h,Compared with the Con group,the m RNA expressions of NLRP3,Caspase-1,P65 and a-SMA in the HG group were increased,and the protein expressions of NLRP3,P65,N-cadherin in the HG group were increased,and the differences were statistically significant(P<0.05),in the HG+ARB group,compared with the HG group,the m RNA expressions of NLRP3,Caspase-1,P65,and a-SMA were significantly decreased,and the protein expressions of NLRP3,P65,N-cadherin were significantly decreased,the difference was statistically significant(P<0.05);Stimulate HK-2 cells with high glucose and intervene with ARB for 72 h,Compared with the Con group,the m RNA expressions of NLRP3,Caspase-1,P65 and a-SMA in the HG group were increased,and the protein expressions of NLRP3,P65,N-cadherin in the high glucose group were increased,and the differences were statistically significant(P<0.05),in the HG+ARB group,compared with the HG group,the m RNA expressions of NLRP3,Caspase-1,P65,and a-SMA were significantly decreased,and the protein expressions of NLRP3,P65,N-cadherin were significantly decreased,the difference was statistically significant(P<0.05).4.Stimulate HK-2 cells with high glucose and intervene with MCC950 for 24 h,Compared with the Con group,the m RNA expressions of NLRP3,Caspase-1,P65 and a-SMA in the HG group were increased,and the protein expressions of NLRP3,P65,Vimentin and N-cadherin in the HG group were increased,and the differences were statistically significant(P<0.05),in the HG+MCC950 group,compared with the HG group,the m RNA expressions of NLRP3,Caspase-1,P65,and a-SMA were significantly decreased,and the protein expressions of NLRP3,P65,Vimentin,and N-cadherin were significantly decreased,the difference was statistically significant(P<0.05);Stimulate HK-2 cells with high glucose and intervene with MCC950 for48 h,Compared with the Con group,the m RNA expressions of NLRP3,Caspase-1,P65 and a-SMA in the HG group were increased,and the protein expressions of NLRP3,P65,Vimentin in the HG group were increased,and the differences were statistically significant(P<0.05),in the HG+MCC950 group,compared with the HG group,the m RNA expressions of NLRP3,Caspase-1,P65,and a-SMA were significantly decreased,and the protein expressions of NLRP3,P65,Vimentin were significantly decreased,the difference was statistically significant(P<0.05);Stimulate HK-2 cells with high glucose and intervene with MCC950 for 72 h,Compared with the Con group,the m RNA expressions of NLRP3,Caspase-1,P65 and a-SMA in the HG group were increased,and the protein expressions of NLRP3,P65,N-cadherin in the HG group were increased,and the differences were statistically significant(P<0.05),in the HG+MCC950 group,compared with the HG group,the m RNA expressions of NLRP3,Caspase-1,P65,and a-SMA were significantly decreased,and the protein expressions of NLRP3,P65,N-cadherin were significantly decreased,the difference was statistically significant(P<0.05).5.Under the high glucose stimulation,the fluorescence of Vimentin in the HG group was stronger than that in the Con group at different time periods and it was mostly expressed in the cytoplasm.The fluorescence of the HG+ARB group and HG+MCC950 group was weaker than it in the HG group,which could inhibit the expression of Vimentin.Conclusion:1.High glucose can stimulate the activation of NLRP3 inflammatory bodies and related pathways in human renal tubular epithelial cells,leading to epithelial-interstitial transformation in epithelial cells and participating in the immune inflammatory response of DKD renal interstitial fibrosis.2.The activation of NLRP3 inflammasome was correlated with glucose concentration up to a certain extent.3.Irbesartan,a traditional angiotensin inhibitor,can significantly down-regulate NLRP3-Caspase-1 and NF-κB pathways,reduce the immune effect of renal epithelial cells induced by high glucose,reverse the phenotypic transformation of epithelial interstitial cells.4.MCC950,the inhibitor of NLRP3,can significantly down-regulate NLRP3-Caspase-1 and NF-κB pathways,reduce the immune effect of renal epithelial cells induced by high glucose,reverse the phenotypic transformation of epithelial interstitial cells. |
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