雌激素受体GPR30通过TXNIP/NLRP3信号通路减弱氧糖剥夺/复氧诱导的BV-2细胞氧化应激损伤和炎症反应

Estrogen receptor GPR30 attenuates oxygen-glucose deprivation and reperfusion-induced oxidative stress injury and inflammation in BV-2 cells through TXNIP/NLRP3 signaling pathway

摘要:

目的 通过研究雌激素受体G蛋白偶联受体30（G protein-coupled receptor 30，GPR30）对氧糖剥夺/再灌注（oxygen-glucose deprivation and reperfusion，OGD/R）BV-2小胶质细胞的氧化应激损伤和炎症反应的调控作用，探讨其对OGD/R BV-2小胶质细胞的保护作用及其相关机制。方法 取BV-2小胶质细胞ODG 4 h后再灌注2、4、6或12 h。Western blot检测细胞中GPR30的蛋白表达水平。将pcDNA3.1、pcDNA3.1-GPR30、sh-NC、sh-GPR30质粒转染BV-2小胶质细胞，OGD 4 h后再灌注12 h。qRT-PCR检测GPR30 mRNA的表达水平，验证其转染效率，Western blot检测细胞中GPR30、硫氧还蛋白相互作用蛋白（thioredoxin-interactingprotein，TXNIP）、NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）、半胱氨酸天冬氨酸蛋白水解酶剪切体-1（cleaved cysteinyl aspartate specific proteinase-1，cleaved Caspase-1）的蛋白表达水平，ELISA检测细胞中活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、白细胞介素-1β（interleukin-1β，IL-1β）、白细胞介素-18（interleukin-18，IL-18）水平。结果 GPR30在OGD/R后显著上升，在6 h时达到峰值，而在12 h时与对照组没有显著差异。随后确定OGD处理4 h，复氧12 h的时间点进行后续实验。qRT-PCR验证慢病毒转染成功，与对照组相比，OGD/R组细胞中ROS、MDA、TNF-α、IL-6、IL-1β、IL-18水平及TXNIP、NLRP3、cleaved-Caspase-1蛋白表达水平显著上升，SOD活性显著下降（P<0.05）；过表达GPR30抑制了ROS、MDA、TNF-α、IL-6、IL-1β、IL-18水平及TXNIP、NLRP3、cleaved-Caspase-1蛋白表达水平，促进了SOD活性；而干扰GPR30表达发挥了相反的作用。结论 GPR30能抑制 ODG/R处理后BV-2细胞内TXNIP/NLRP3信号通路分子的表达，抑制ODG/R诱导的 BV-2细胞氧化应激损伤和炎症反应，这可能是其保护ODG/R细胞的分子机制之一。

Abstract:

Objective To investigate the regulatory effects of estrogen receptor G protein-coupled receptor 30（GPR30） on oxidative stress injury and inflammatory response in oxygen-glucose deprivation and reperfusion（OGD/R） BV-2 cells，and to investigate its protective effect on OGD/R BV-2 cells and its related mechanism.Methods BV-2 cells were treated with ODG for 4 h and then reperfused for 2 h，4 h，6 h or 12 h. The protein expression level of GPR30 was detected by Western blot. BV-2 cells were transfected with packaged pcDNA3.1，PCDNA3.1-GPR30，sh-NC，sh-GPR30 lentiviruses，and then reperfused for 12 h after treated with ODG for 4 h. qRT-PCR was used to detect the expression level of GPR30 mRNA to verify its transfection efficiency. Western blot was used to detect the protein expression levels of GPR30，thioredoxin-interactingprotein（TXNIP），NOD-like receptor thermal protein domain associated protein 3（NLRP3），cleaved cysteinyl aspartate specific proteinase-1（cleaved Caspase-1） in cells. The levels of reactive oxygen species（ROS），malondialdehyde（MDA），superoxide dismutase（SOD），tumor necrosis factor-α（TNF-α），interleukin-6（IL-6），interleukin-1β（IL-1β）and interleukin-18（IL-18） in cells were detected by ELISA.Results The expression level of GPR30 increased significantly after OGD/R，and reached the peak at 6 h. There was no significant difference compared with Control group at 12 h. And then the time points of OGD treatment for 4 h and reoxygenation for 12 h were determined for subsequent experiments. The lentivirus transfection was verified by qRT-PCR. Compared with the control group，the levels of ROS，MDA，TNF-α，IL-6，IL-1β，IL-18 and the protein expression levels of TXNIP，NLRP3，cleaved Caspase-1 in OGD/R group were significantly increased. And SOD activity was significantly decreased（P<0.05）. Overexpression of GPR30 inhibited the levels of ROS，MDA，TNF-α，IL-6，IL-1β，IL-18 and the protein expression levels of TXNIP，NLRP3，cleaved Caspase-1，and it enhanced SOD activity； while interference with GPR30 expression had the opposite effect.Conclusion GPR30 can inhibit the expression of protein of TXNIP/NLRP3 signaling pathway in BV-2 cells，and inhibit the oxidative stress injury and inflammation induced by ODG/R in BV-2 cells，which may be one of its molecular mechanisms for protecting ODG/R cells.