乙型肝炎病毒X蛋白通过调控活性氧/NLRP3信号通路介导乙肝相关性肾小球肾炎足细胞焦亡

Hepatitis B virus X protein mediates podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis through reactive oxygen species/NLRP3 signaling pathway

目的探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)是否通过调控活性氧(reactive oxygen species,ROS)/核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)信号通路引起足细胞焦亡。方法采用小鼠肾足细胞过表达HBx基因来模拟乙型肝炎相关性肾炎的发病机制。将足细胞分为以下5组:空白对照组(不予特殊处理)、阴性对照组(转染对照慢病毒)、HBx过表达组(转染HBx过表达慢病毒)、HBx过表达+NLRP3 siRNA组(共转染HBx过表达慢病毒和NLRP3 siRNA)、HBx过表达+ROS抑制剂组(转染HBx过表达慢病毒和添加ROS抑制剂)。电镜下观察足细胞的形态学改变;二氯二氢荧光素二乙酸酯(dichlorodihydrofluorescein diacetate assay,DCFH-DA)法检测ROS的生成;Hoechst 33342染色观察足细胞细胞核的形态和数量变化;酶联免疫吸附测定试验分别检测胱天蛋白酶1(Caspase-1)酶活性、乳酸脱氢酶、白细胞介素(IL)-1β和IL-18水平;实时荧光定量PCR和Western印迹法分别检测细胞焦亡相关蛋白如NLRP3、凋亡相关斑点样蛋白(ASC)、Caspase-1、IL-1β和IL-18 mRNA和蛋白水平的表达;TUNEL染色和流式细胞术检测焦亡细胞数量;免疫荧光染色检测Desmin和Nephrin的表达。结果HBx过表达慢病毒成功感染足细胞后,电镜下观察到细胞发生焦亡相关形态学改变;与阴性对照组相比,HBx过表达组ROS水平显著升高(P<0.05);Hoechst 33342染色发现HBx过表达后细胞核浓缩;TUNEL染色和流式细胞术证明HBx过表达后足细胞发生了焦亡;焦亡相关蛋白NLRP3、ASC、Caspase-1、IL-1β和IL-18 mRNA和蛋白表达显著上调(均P<0.05);Caspase-1酶活性、乳酸脱氢酶和Desmin表达水平升高(均P<0.05)。而NLRP3敲低或ROS抑制减弱了HBx过表达引起的足细胞焦亡以及相关蛋白表达增加(均P<0.05)。结论ROS/NLRP3通路在HBx过表达引起的足细胞焦亡中起着重要作用。

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)-1βand 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-1βand 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-1βand 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.