Mechanism Of Hepatitis B Virus X Protein Mediated Podocyte Pyroptosis In Hepatitis B Virus-associated Glomerulonephritis

Objective: Hepatitis B associated glomerulonephritis(HBV-GN)is one of the main secondary renal diseases in China.The expression of hepatitis B virus X protein(HBx)may play a crucial role in the pathogenesis of HBV-GN,but the specific mechanism was not clear.Podocyte pyroptosis is one of the important pathological changes of HBV-GN,and mitochondrial damage is closely related to it;micro RNA(mi RNA)has been shown to be associated with a variety of physiological and pathological processes in the body,but its role in HBV-GN podocyte pyroptosis was not clear.Therefore,the aim of this study was to investigate the potential function and related mechanisms of oxidative stress and mi RNAs in HBx protein-induced pyroptosis in HBV-GN podocytes.Methods: Overexpression of HBx gene in mouse renal podocytes and human renal podocytes was used to mimic the pathogenesis of HBV-GN,respectively.Mouse renal podocytes were divided into 5 groups: blank control group(no special treatment),negative control group(transfected control lentivirus),HBx overexpression group(transfected HBx overexpression lentivirus),HBx overexpression + NLRP3 si RNA group(co-transfected HBx overexpression lentivirus and NLRP3 si RNA),HBx overexpression + ROS inhibitor group(transfected HBx overexpression lentivirus and added ROS inhibitor);Human renal podocytes were divided into the following nine groups: control group(no special treatment),empty plasmid group(transfected empty plasmid),HBx overexpression group(transfected HBx overexpression lentivirus),HBx overexpression + mi RNA 223 mimic group(cotransfected HBx overexpression lentivirus and mi RNA 223 mimic),HBx overexpression +mi RNA 223 inhibitor group(co-transfected HBx overexpression lentivirus and mi RNA 223inhibitor),HBx overexpression + mi RNA 223 + NLRP3 group(co-transfected HBx overexpression lentivirus,mi RNA 223 mimic and NLRP3 overexpression plasmid),HBx overexpression + mi RNA 223 mimic + NLRP3 si RNA group(co-transfected HBx overexpression lentivirus,mi RNA 223 mimic and NLRP3 si RNA),HBx overexpression +mi RNA 223 inhibitor + NLRP3 group(co-transfected HBx overexpression lentivirus,mi RNA 223 inhibitor and NLRP3 overexpression plasmid),HBx overexpression + mi RNA223 inhibitor + NLRP3 si RNA group(co-transfected HBx overexpression lentivirus,mi RNA 223 inhibitor and NLRP3 si RNA).The morphological changes of podocytes were observed under electron microscope;The generation of ROS was detected by dichlorodihydrofluorescein diacetate assay(DCFH-DA);Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei;Enzyme-linked immunosorbent assay was used to detect caspase-1 activity,and the levels of lactate dehydrogenase,interleukin(IL)-1β and IL-18;TUNEL staining and flow cytometer were used to detect the number of pyroptosis cells;Dual-luciferase reporter gene assay was used to verify the downstream targets of mi RNA 223;Immunofluorescence staining was used to detect the expression levels of desmin and nephrin;Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression levels of m RNA and protein of pyroptosis-related protein,such as NLRP3,apoptosis-associated speck-like protein containing card(ASC),caspase-1,IL-1β and IL-18.Results:(1)Role of the ROS/NLRP3 in HBV-GN:(1)After successful infection of podocytes with HBx-overexpressing lentivirus,pyroptosis-related morphological changes in the cells were observed under electron microscope.(2)The level of ROS in the HBx overexpression group was significantly higher compared to the negative control group(P<0.05).(3)Hoechst 33342 staining revealed condensed nuclei in the HBx overexpression group.(4)TUNEL staining and flow cytometer demonstrated that podocytes underwent increased pyroptosis in the HBx overexpression group.(5)The m RNA and protein expression levels of pyroptosis-related proteins such as NLRP3,ASC,caspase-1,IL-1β and IL-18 were up-regulated upon HBx overexpression(all P<0.05).(6)Caspase-1 enzyme activity,lactate dehydrogenase and desmin expression levels were enhanced after HBx overexpression(all P < 0.05).(7)NLRP3 knockdown or addition of ROS inhibitors attenuated the pyroptosis and increased expression levels of pyroptosis-related proteins caused by HBx overexpression(all P<0.05).(2)Regulatory effect of mi R-223 on NLRP3 in HBV-GN:(1)mi RNA‐223 was down‐regulated in HBx overexpression group compared with the control group(P < 0.05).(2)TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression(P < 0.05).(3)Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of mi RNA‐223.(4)Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of mi RNA‐223 in podocyte injury(P < 0.05).(5)The addition of mi RNA‐223 mimic and NLRP3 si RNA decreased the expression of NLRP3 inflammasome and cytokines,and reduced the number of pyroptosis cells induced by HBx overexpression(all P < 0.05);The addition of mi RNA‐223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome,cytokines,and caspase‐1 activity,and the number of pyroptosis cells(all P < 0.05).Conclusion: ROS/NLRP3 pathway plays an important role in HBx-induced podocyte pyroptosis.In addition,HBx may promote podocyte pyroptosis via downregulating mi RNA‐223 of HBV-GN targeting NLRP3 inflammasome,suggesting that mi RNA ‐ 223 is expected to be a potential target for the treatment of HBV‐GN.