Department of Physiology, Basic Medical College, Nanchang University, Nanchang, 330006, People's Republic of China.
The Second Clinical Medical College, Nanchang University, Nanchang, 330006, People's Republic of China.
Inflammation. 2016 Oct;39(5):1814-26. doi: 10.1007/s10753-016-0417-0.
This study was aimed at exploring the effects of P2X7 receptor on BV2 microglia cell injury induced by glycoprotein gp120 (gp120) and its underlying mechanisms. We used the MTS method to study the influence of different gp120 concentrations on BV2 microglia cells, and to test the degree of cell injury in each gp120 treatment group; quantitative real-time PCR (qPCR) and Western blot were used to detect the P2X7 mRNA and receptor protein expressions. Immunocytochemistry and Western blot were used to detect the P2X7 receptor expression and P65 NF-κB, respectively. We also measured the content of TNFα, IL-1β, nitric oxide (NO) and reactive oxygen species (ROS). We found that the cell survival rate generally decreased as gp120 concentration increased, and the cell survival rate of the gp120 + Brilliant Blue G (BBG) group was higher than that of the gp120 group. Western blot and qPCR results showed that the expressions of P2X7 receptor protein and mRNA were positively dose-dependent with gp120 concentration; the results of immunocytochemistry and Western blot showed that the expressions of P2X7 receptor and P65 NF-κB in the gp120 group increased significantly compared to those of the control (Ctrl) group, but those in the gp120+BBG group decreased. Taken together, these results confirmed that the P2X7 receptor is involved in gp120-induced BV2 microglial cell injury and that the underlying mechanism may be associated with the over-activation of microglia caused by P2X7 receptor up-regulation, which leads to abundant release of inflammatory factors which exert toxic effects on the cells.
本研究旨在探讨 P2X7 受体在糖蛋白 gp120(gp120)诱导的 BV2 小胶质细胞损伤中的作用及其机制。我们使用 MTS 法研究不同 gp120 浓度对 BV2 小胶质细胞的影响,并检测各 gp120 处理组细胞损伤程度;采用实时定量 PCR(qPCR)和 Western blot 检测 P2X7 mRNA 和受体蛋白表达。免疫细胞化学和 Western blot 分别检测 P2X7 受体表达和 P65 NF-κB。同时还检测了 TNFα、IL-1β、一氧化氮(NO)和活性氧(ROS)的含量。结果发现,随着 gp120 浓度的增加,细胞存活率普遍降低,gp120+BBG 组的细胞存活率高于 gp120 组。Western blot 和 qPCR 结果表明,P2X7 受体蛋白和 mRNA 的表达与 gp120 浓度呈正相关;免疫细胞化学和 Western blot 结果显示,gp120 组 P2X7 受体和 P65 NF-κB 的表达明显高于对照组(Ctrl),而 gp120+BBG 组则降低。综上所述,这些结果证实 P2X7 受体参与 gp120 诱导的 BV2 小胶质细胞损伤,其机制可能与 P2X7 受体上调引起的小胶质细胞过度激活有关,导致大量炎症因子释放,对细胞产生毒性作用。