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

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(oxygenglucose 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、cleavedCaspase-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。

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.