Department of Nephrology, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China.
Int J Mol Med. 2013 May;31(5):1017-29. doi: 10.3892/ijmm.2013.1295. Epub 2013 Mar 7.
Hepatitis B virus X protein (HBx) is a multifunctional protein, and it activates multiple signal transduction pathways in multiple types of cells and regulates the process of cell apoptosis. In the present study, we mainly investigated the correlation between HBx and renal tubular epithelial cell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. Cell apoptosis in nephridial tissues of patients with HBVGN were determined by the TUNEL method. HBx, p-STAT3 and STAT3 levels in nephridial tissues were determined by immunohistochemical assay, and a correlation analysis between HBx expression levels and apoptosis index in nephridial tissues was conducted. The activation of the JAK2/STAT3 signaling pathway in HK-2 cells and the expression of the apoptosis-related proteins Bax and Bcl-2 were determined by western blot analysis following transfection with the HBx eukaryotic expression vector. Cellular proliferation activity was determined by the CCK‑8 method, and cell apoptosis was determined with HO33342 staining using transmission electron microscopy and Annexin V/PI double staining flow cytometry. The results revealed that the apoptosis index in nephridial tissues of patients with HBVGN was significantly higher when compared to that of the control group, and p-STAT3 expression levels in HBVGN nephridial tissues were significantly increased. In the control group, no HBx expression was observed in the nephridial tissues, whereas HBx expression was found in the nephridial tissues of 86% of the patients with HBVGN. The HBx expression levels had a linear correlation with the apoptosis index in the nephridial tissues. After target gene HBx infection, expression levels of both p-JAK2 and p-STAT3 in human proximal HK-2 cells were significantly increased, and the Bax/Bcl-2 ratio was also significantly increased. At the same time, cellular proliferation of HK-2 cells was significantly inhibited, and the rate of apoptosis was increased. After incubation with AG490, the JAK2/STAT3 signaling pathway was partially blocked, which caused a decrease in the Bax/Bcl-2 ratio and reduced cell apoptosis caused by HBx. In conclusion, HBx upregulates the Bax/Bcl-2 ratio by activating the JAK2/STAT3 signaling pathway to cause renal tubular epithelial cell apoptosis, and it is possibly involved in the pathogenic mechanism of nephridial tissue damage caused by HBV.
乙型肝炎病毒 X 蛋白 (HBx) 是一种多功能蛋白,它可在多种类型的细胞中激活多种信号转导通路,并调节细胞凋亡过程。本研究主要探讨乙型肝炎病毒相关性肾小球肾炎(HBVGN)中 HBx 与肾小管上皮细胞凋亡的相关性及其可能的信号机制。采用 TUNEL 法检测 HBVGN 患者肾组织细胞凋亡情况,免疫组化法检测肾组织中 HBx、p-STAT3 和 STAT3 水平,并进行 HBx 表达水平与肾组织凋亡指数的相关性分析。用 HBx 真核表达载体转染 HK-2 细胞,采用 Western blot 法检测 JAK2/STAT3 信号通路的激活及凋亡相关蛋白 Bax、Bcl-2 的表达,CCK-8 法检测细胞增殖活性,透射电镜 HO33342 染色观察细胞凋亡,Annexin V/PI 双染流式细胞术检测细胞凋亡。结果显示,HBVGN 患者肾组织的凋亡指数明显高于对照组,且 HBVGN 肾组织中 p-STAT3 的表达水平明显升高。对照组肾组织未见 HBx 表达,而 86%的 HBVGN 患者肾组织中存在 HBx 表达。HBx 表达水平与肾组织凋亡指数呈线性相关。HBx 基因转染后,人近端 HK-2 细胞中 p-JAK2 和 p-STAT3 的表达水平均明显升高,Bax/Bcl-2 比值也明显升高。同时,HK-2 细胞的增殖受到明显抑制,细胞凋亡率增加。用 AG490 孵育阻断 JAK2/STAT3 信号通路后,Bax/Bcl-2 比值降低,HBx 引起的细胞凋亡减少。综上所述,HBx 通过激活 JAK2/STAT3 信号通路上调 Bax/Bcl-2 比值,导致肾小管上皮细胞凋亡,可能参与了乙型肝炎病毒引起的肾组织损伤的发病机制。