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

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.