Study On The Role And Mechanism Of Macrophage Pyroptosis In The Progression Of Lupus Nephritis

Objective:Pyroptosis has been known to be characterized by programmed cellular inflammatory death.Some studies have found that pyroptosis affects the occurrence and development of lupus nephritis(LN).There is infiltration of macrophages and other inflammatory cells in LN,and macrophage pyroptosis has been shown to play a role in other immune-related diseases.Thus we speculate that macrophage pyroptosis may participate in the occurrence and development of LN.Combined with the important role of NF-κB and NLRP3 in LN and pyroptosis,this study intends to start with clinical and in vitro experiments.The purpose of this study was to explore whether there is macrophage pyroptosis in LN and to clarify the correlation between the level of macrophage pyroptosis and the clinical data with LN.To further explore the relationship between the occurrence of macrophage pyroptosis and NF-κB/ NLRP3 pathway.Methods:1.56 patients diagnosed with lupus nephritis by renal pathology and 20 patients diagnosed with minimal changes disease were collected.Renal tissues and clinical data of the patients were collected.Macrophage pyroptosis was detected by double immunofluorescence,and IL-1β and IL-18 levels in blood were detected by ELISA.Further analysis of their correlation with clinical indicators.2.THP-1 cells were cultured in vitro,and THP-1 was induced into macrophages by PMA.The experiments were divided into Control group,LPS+Nigericin(pyroptosis inducer)group,MCC950(NLRP3 inhibitor)group and BAY 11-7082(NF-κB inhibitor)group.Macrophage pyroptosis model was established by LPS+Nigericin.Cell status was detected by CCK-8cell viability assay.The expressions of IL-1β and IL-18 in cell supernatant were detected by ELISA,and the expressions of NLRP3,caspase-1 p20 and GSDMD were detected by western blot.The expressions of NF-κB p65,NLRP3,caspase-1 and GSDMD m RNA were detected by PCR,and the nuclear shift of NF-κB p65 was detected by cellular immunofluorescence.Detection of macrophage pyroptosis status by Hoechst 33342/PI assay.Results:1.Clinical trial:(1)compared with MCD group,macrophage pyroptosis occurred in glomeruli and renal interstitium of LN patients.(2)Analysis of correlation between macrophage pyroptosis and clinical data: The degree of macrophage pyroptosis in the glomerulus was negatively correlated with C3,C4,hemoglobin,platelet count,and positively correlated with ds DNA,urine protein,uric acid(P<0.05).The degree of macrophage pyroptosis in the renal interstitium was positively correlated with BUN,SCr,CRP,ESR,and BMI,and negatively correlated with vitamin D and e GFR(P<0.05).In addition,the degree ofmacrophage pyroptosis in renal interstitial was positively correlated with pathological inactivity index(CI).2.Cell experiment:(1)Compared with Control group,the cell viability of LPS+Nigericin group was significantly decreased(P<0.0001),and the expression of IL-1βand IL-18 in cell supernatant was significantly increased(P<0.0001).(2)WB assay showed that the protein expression of NLRP3,caspase-1and GSDMD in LPS+Nigericin group was higher than that in Control group(P<0.05),and MCC950 inhibited the increase(P<0.05).(3)Compared with Control,the expression of NF-κB p65,NLRP3,caspase-1 and GSDMD m RNA in LPS+Nigericin group was higher(P <0.01).(4)In NF-κB p65 nuclear shift assay,NF-κB p65 transferred from cytoplasm to nucleus in LPS+Nigericin group,but there was no obvious nuclear transfer in cells pretreated with BAY 11-7082.(5)Hoechst 33342/PI staining showed that the number of pyroptosis cells(strong blue light and strong red light)increased in LPS+Nigericin group,and decreased significantly after pretreatment with MCC950 and BAY11-7082.Conclusions:1.Macrophage pyroptosis exists in glomeruli and renal interstitium in LN;2.The degree of macrophage pyroptosis is related to lupus activity in LN;3.THP-1 cell experiments shows that the progression of macrophage pyrogenesis can be blocked by inhibiting NF-κB/NLRP3 pathway.