| Objective:Hepatitis B virus associated glomerulonephritis(HBV-GN)is one of the main extrahepatic organ diseases infected by hepatitis B virus(HBV).Podocytes are the last layer of glomerular filtration barrier.The phenotype and quantity of podocytes change,including the reduction or disappearance of effective podocytes,and finally the podocytes separated from the basement membrane,resulting in serious damage to the structure and physiological function of the kidney.Nod-like receptor protein 3(NLRP3)inflammasome is involved in the pathogenesis of many kidney diseases,but no study has proved that NLRP3 inflammasome is involved in HBV-GN and finally mediates podocyte pyroptosis.Therefore,the purpose of this study was to investigate the action mechanism of NLRP3 inflammasome in the signal transduction pathway of podocyte pyroptosis in HBV-GN.Hepatitis B virus associated glomerulonephritis(HBV-GN)is one of the main extrahepatic organ diseases infected by hepatitis B virus(HBV).The phenotypic and quantitative changes of podocytes will lead to serious damage to renal structure and physiological function.Nucleotide binding oligomerization domain like receptor protein 3(NLRP3)inflammasome is involved in the pathogenesis of many kidney diseases,but the mechanism of NLRP3 inflammasome in HBV-GN is not clear.Therefore,the purpose of this study was to explore the mechanism of NLRP3 inflammasome in podocyte pyroptosis in hepatitis B virus associated glomerulonephritis.Methods:(1)PcDNA3.1/myc HBx plasmid and NLRP3 siRNA interference plasmid was constructed and transfected into human podocyte strain.Podocytes were divided into blank control group,empty transfection group,HBx transfection group and HBx + NLRP3 siRNA transfection group.(2)Rt-pct was used to detect the expression of HBx to explore whether HBx induced podocyte pyroptosis.(3)Flow cytometry: the pyroptosis rate of podocytes in each group was detected by flow cytometry.(4)Hoechst 33342 staining was used to observe the morphological changes of podocyte nucleus.(5)HBx,NLRP3,apoptosis associated speek-like protein containing CARD(ASC),caspase-1 and expression of IL-18 and IL-1βwere detected by qRT-PCR and Western blot.(6)Caspase-1 activity detection kit,lactate dehydrogenase(LDH),ELISA kit,human interleukin-1 β ELISA kit and human interleukin-18 ELISA kit were used to detect activity of caspase-1,level of LDH,IL-1β And IL-18 in podocyte supernatant respectively.(7)Immunofluorescence staining was used to detect the expression of Nephrin protein and Desmin protein in podocytes of each group.Results:(1)HBx was successfully transfected into renal podocytes(P < 0.001).(2)HBx overexpression significantly increased the podocyte pyroptosis rate(P < 0.001)(3)The expression of NLRP3 inflammasome related proteins(P < 0.05),including NLRP3,ASC and caspase-1,was up-regulated.(4)NLRP3 inflammasome mediates caspase-1 activation after activation,and then activates inflammatory factor IL-1β And IL-18(P < 0.01).(5)HBx mediates abnormal expression of podocyte damage-associated proteins,including the enhancement of Desmin(P < 0.01),and the down-regulation of podocyte structural protein Nephrin(P < 0.01).(6)NLRP3 siRNA transfection slowed down the process of podocyte pyroptosis(P < 0.05)inhibited the increase of pyroptosis related protein expression during HBx overexpression(P < 0.05),and increased the expression of Nephrin(P< 0.01).(7)HBx can mediate the increase of LDH expression(P < 0.01),and NLRP3 siRNA transcription reduces LDH production(P<0.001).Conclusion:(1)HBx activates caspase-1 and then activates IL-1β and IL-18 by activating NLRP3 inflammasome,finally induced podocyte pyroptosis.(2)Interfering with the formation of NLRP3 inflammasome can reduce the production of cytokines and the degree of podocyte pyroptosis.This discovery can be cited in the control of the disease and even the prevention of HBV-GN,and provides a new thought for clinical treatment. |
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