Mechanism Of Innate Immune Response Of Rat Mesangial Cells Mediated By NLRP3 Inflammasome Under High Glucose

ObjectiveThe purpose of this study was to study the expression of NLRP3 inflammasome and the expression of related inflammatory and fibrotic factors in rat Mesangial cells(HBZY-1)cultured in high glucose environment.NLRP3 si RNA interference technique and angiotensin receptor antagonist(ARB Drugs)were used to interfere with HBZY-1 stimulated by high glucose,and the expression of these factors were detected to explore the potential mechanism of NLRP3 inflammasome in diabetic kidney disease.a preliminary study on whether irbesartan can block the effect of NLRP3 inflammasome Different time points were designed to verify the effects of high glucose stimulation on the expression of NLRP3 inflammasome and pathway-related molecules.MethodsThe NLRP3 si RNA was packaged with lentivirus,and GFP-Bsd with green fluorescence was used as a negative control.The concentration gradient of MOI value was designed,and the positive rate of cell transfection was observed by negative virus infection HBZY-1,under a fluorescence microscope,and the expression of NLRP3 m RNA was detected by Q-PCR to explore the most suitable MOI value for follow-up experiments.The experiment was divided into three parts:Part 1:the cells were divided into three groups: low glucose control group(LG,5.6 mmol/L glucose),mannitol group(M,5.6 mmol/L glucose + 24.4 mmol/L mannitol)and high glucose group(HG,30 mmol/L glucose).The effects of high glucose and hyperosmotic on the expression of NLRP3 were preliminarily verified.Part 2: after lentivirus infection,the silenced NLRP3 plasmid was transfected into HBZY-1 cells.The cells were divided into three groups: low glucose group(LG group),low glucose + NLRP3 si RNA group(LG+NLRP3-sh group)and low glucose+ no load group(LG+sh group).The efficiency of NLRP3 si RNA interference was verified.Part 3: the experiment was divided into 5 groups at two-time points(24 h and48 h): LG group,HG group,HG+sh group,HG+NLRP3-sh group,HG+ irbesartan group(HG+ARB group).Q-PCR was used to detect the expression of NLRP3,caspase-1 and nuclear factor-kB(NF-kB)m RNA at 24 h and 48 h,and Western Blot was used to detect the protein expression of NLRP3,heat shock protein 47(HSP47),NF-k B and type IV collagen(Coll V),actin-SMA(ɑ-SMA)at 24 h and 48 h.The expression levels of interleukin-1β(IL-1β)and tumor necrosis factor-1(TNF-ɑ)in the supernatant of the cells were detected by ELISA at 24 h and 48 h.Results1.Compared with the low glucose group,the NLRP3 m RNA expression increased in the high glucose group(P<0.05),the difference was statistically significant,and the mannitol group NLRP3 m RNA expression was not statistically significant(P>0.05).2.After 24 hours of cell intervention treatment in the five groups,compared with the low glucose group,the expression of NLRP3 m RNA in the high glucose group and the high glucose + sh group increased significantly(P<0.01),and the expression of Caspase-1 and NF-kB m RNA increased significantly(P<0.05),the expressions of NLRP3,HSP47,NF-kB,ɑ-SMA,and COl IV protein were significantly up-regulated(P<0.05),and the expression of IL-1β and TNF-ɑ in the cell supernatant increased significantly(P<0.01);and Compared with the high glucose group,the expressions of NLRP3 and Caspase-1 m RNA in the high glucose + NLRP3-sh and high glucose +ARB groups were significantly down-regulated(P<0.01).The expression of NF-kB m RNA was significantly down-regulated(P<0.05).The expression was significantly down-regulated(P<0.01),and the expressions of IL-1β and TNF-ɑ were significantly down-regulated(P<0.01).There was no statistically significant difference between the high glucose group and the high glucose + sh group.3.After 48 hours of cell intervention treatment in the five groups,compared with the low glucose group,the expression of NLRP3 m RNA in the high glucose group and the high glucose + sh group increased significantly(P<0.01),and the expression of Caspase-1 and NF-kB m RNA increased significantly(P<0.05),the expressions of NLRP3,HSP47,NF-kB,ɑ-SMA,and COl IV protein were significantly up-regulated(P<0.05),and the expression of IL-1β and TNF-ɑ in the cell supernatant increased significantly(P<0.01);and Compared with the high glucose group,the expressions of NLRP3 and Caspase-1 m RNA in the high glucose + NLRP3-sh and high glucose +ARB groups were significantly down-regulated(P<0.01).The expression of NF-kB m RNA was significantly down-regulated(P<0.05).The expression was significantly down-regulated(P<0.01),and the expressions of IL-1β and TNF-ɑ were significantly reduced(P<0.01).There was no statistically significant difference between the high glucose group and the high glucose + sh group.4.The cells were treated with high glucose intervention for 24 h and 48 h.Compared with the 24 h high glucose group,the expression of NLRP3 in the 48 h high glucose group was significantly up-regulated(P<0.01),and the expression of Caspase-1 and NF-k B m RNA was significantly down-regulated(P<0.05),The expressions of NLRP3,HSP47,NF-k B,ɑ-SMA,COl IV protein increased significantly(P<0.01),the expressions of IL-1β and TNF-ɑ increased significantly(P<0.05),and the expression of TNF-ɑ increased significantly(P<0.01).1.ConclusionHigh glucose can activate the expression of NLRP3 inflammasome and Induce glomerular Mesangial cells to secrete inflammatory factors and up-regulate the expression of fibrosis factors.Irbesartan and specific gene silencing can block the activation of NLRP3 inflammasome induced by high glucose and down-regulate the release of inflammatory factors and the expression of fibrosis proteins.And through this experiment,it was found that the activation expression of NLRP3 inflammasome has a certain correlation with time.