Hepatitis B virus X protein mediates podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis through reactive oxygen species/NLRP3 signaling pathway / 中华肾脏病杂志

ABSTRACT

Objective:To investigate whether hepatitis B virus X protein (HBx) mediates the podocyte injury through reactive oxygen species (ROS) /nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway.Methods:HBx-overexpressing lentivirus was transfected into renal podocytes of mouse to mimic the pathogenesis of hepatitis B virus-associated glomerulonephritis. Podocytes were divided into the following five groups blank control group (no special treatment), negative control group (transfected with control lentivirus), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+NLRP3 siRNA group (transfected with HBx overexpression lentivirus and NLRP3 siRNA), and HBx overexpression+ROS inhibitor group (transfected with HBx overexpression lentivirus and adding ROS inhibitor). The morphological changes of podocytes were observed with 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. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression levels of mRNA and protein of pyroptosis-related protein, such as NLRP3, apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β and IL-18. TUNEL staining and flow cytometer were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression levels of desmin and nephrin.Results:After successful infection of podocytes with HBx-overexpressing lentivirus, pyroptosis-related morphological changes in the cells were observed under electron microscope. The level of ROS in the HBx overexpression group was significantly higher compared to the negative control group ( P<0.05). Hoechst 33342 staining revealed condensed nuclei in the HBx overexpression group. TUNEL staining and flow cytometer demonstrated that podocytes underwent increased pyroptosis in the HBx overexpression group. The mRNA 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). Caspase-1 enzyme activity, lactate dehydrogenase and desmin expression levels were enhanced after HBx overexpression (all P<0.05). However, 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). Conclusion:ROS/NLRP3 pathway plays an important role in HBx-induced podocyte pyroptosis.