GPR30对大鼠椎间盘退行性病变的作用及其机制研究

Role of GPR30 in rat intervertebral disc degeneration and its mechanism

目的探讨G蛋白偶联受体30(GPR30)通过Nrf2/ARE信号通路抑制白细胞介素1β(IL-1β)诱导的大鼠椎间盘髓核细胞的凋亡、炎症反应和氧化应激。方法使用IL-1β处理大鼠椎间盘髓核细胞复制体外椎间盘退变模型,将椎间盘髓核细胞分为空白对照(Control)组、IL-1β组、IL-1β+过表达GPR30阴性对照质粒(oe-NC)组、IL-1β+过表达GPR30质粒(oe-GPR30)组、IL-1β+oe-GPR30+干扰Nrf2表达阴性对照质粒(si-NC)组、IL-1β+oe-GPR30+干扰Nrf2表达质粒(si-Nrf2)组,IL-1β的处理浓度为10 ng/mL。实时荧光定量聚合酶链反应检测GPR30 mRNA表达;Western boltting检测GPR30、抗重组与合成蛋白(Nrf2)和抗醌NADH脱氢酶(NQO1)和抗血红素加氧酶1(HO-1)蛋白表达;流式细胞术检测细胞凋亡情况;酶联免疫吸附试验检测肿瘤坏死因子-α(TNF-α)和IL-6水平;使用ELISA法检测活性氧(ROS)、分光光度法检测超氧化物歧化酶(SOD)水平,比色法检测丙二醛(MDA)水平。结果IL-1β组髓核细胞GRP30 mRNA、蛋白相对表达量较Control组降低(P<0.05),IL-1β+oe-GPR30组较IL-1β+oe-NC组升高(P<0.05)。IL-1β组髓核细胞核中Nrf2蛋白相对表达量较Control组升高(P<0.05),IL-1β+oe-GPR30组较IL-1β+oe-NC组升高(P<0.05)。IL-1β组Nrf2、HO-1及NQO1蛋白相对表达量较Control组降低(P<0.05),IL-1β+oe-GPR30组较IL-1β+oe-NC组升高(P<0.05)。IL-1β组髓核细胞Nrf2蛋白相对表达量较Control组降低(P<0.05),IL-1β+oe-GPR30组较IL-1β+oe-NC组升高(P<0.05),IL-1β+oe-GPR30+si-Nrf2组较IL-1β+oe-GPR30+si-NC组降低(P<0.05)。IL-1β组髓核细胞凋亡率较Control组升高(P<0.05),IL-1β+oe-GPR30组较IL-1β+oe-NC组降低(P<0.05),IL-1β+oeGPR30+si-Nrf2组较IL-1β+oe-GPR30+si-NC组升高(P<0.05)。IL-1β组TNF-α、IL-6水平较Control组升高(P<0.05),IL-1β+oe-GPR30组较IL-1β+oe-NC组降低(P<0.05),IL-1β+oe-GPR30+si-Nrf2组较IL-1β+oe-GPR30+si-NC组升高(P<0.05)。IL-1β组ROS、MDA水平较Control组升高(P<0.05),SOD水平较Control组降低(P<0.05),IL-1β+oe-GPR30组ROS、MDA水平较IL-1β+oe-NC组降低,SOD水平较IL-1β+oe-NC组升高(P<0.05),IL-1β+oe-GPR30+si-Nrf2组ROS、MDA水平较IL-1β+oe-GPR30+si-NC组升高,SOD水平较IL-1β+oe-GPR30+si-NC组降低(P<0.05)。结论上调GPR30表达激活Nrf2/ARE信号通路,能抑制IL-1β诱导的髓核细胞凋亡、炎症和氧化应激反应。

Objective To investigate the potential roles of G protein-coupled receptor 30(GPR30)in inhibiting apoptosis,oxidative stress response and inflammation in rat nucleus pulposus cells induced by interleukin-1β(IL-1β)via the nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway.Methods Rat nucleus pulposus cells were treated with IL-1βto establish an in vitro model of intervertebral disc degeneration.The nucleus pulposus cells were divided into blank control(control)group,IL-1βgroup,IL-1β+GPR30 overexpression negative control plasmid(oe-NC)group,IL-1β+GPR30 overexpression plasmid(oe-GPR30)group,IL-1β+oe-GPR30+Nrf2 interference negative control plasmid(si-NC)group and IL-1β+oe-GPR30+Nrf2 interference plasmid(si-Nrf2)group,and the treatment concentration of IL-1βwas 10 ng/ml.Quantitative real-time polymerase chain reaction was used to detect the mRNA expression of GPR30,and Western blotting was used to detect the protein expressions of GPR30,Nrf2,NAD(P)H dehydrogenase quinone 1(NQO1)and heme oxygenase 1(HO-1).The flow cytometry was performed to detect cell apoptosis.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured via enzyme-linked immunosorbent assay,while levels of reactive oxygen species(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA)were measured via corresponding commercial kits.Results Compared with the control group,the mRNA and protein expressions of GRP30 in nucleus pulposus cells were lower in the IL-1βgroup(P<0.05).The mRNA and protein expressions of GRP30 in nucleus pulposus cells in the IL-1β+oe-GPR30 group were higher than those in the IL-1β+oe-NC group(P<0.05).The relative protein expression of Nrf2 in the nucleus of nucleus pulposus cells in the IL-1βgroup was higher than that in the control group(P<0.05),while that in the IL-1β+oe-GPR30 group was higher than that in the IL-1β+oe-NC group(P<0.05).The relative protein expressions of Nrf2,HO-1 and NQO1 in nucleus pulposus cells in the IL-1βgroup were lower than those in the control group(P<0.05),and those in the IL-1β+oe-GPR30 group were higher than those in the IL-1β+oe-NC group(P<0.05).The relative protein expression of Nrf2 in nucleus pulposus cells in the IL-1βgroup was lower(P<0.05),that in the IL-1β+oe-GPR30 group was higher than that in the IL-1β+oe-NC group(P<0.05),and that in the IL-1β+oe-GPR30+si-Nrf2 group was lower than that in the IL-1β+oe-GPR30+si-NC group(P<0.05).The apoptosis rate of nucleus pulposus cells in the IL-1βgroup was higher than that in the control group(P<0.05),that in the IL-1β+oe-GPR30 group was lower than that in the IL-1β+oe-NC group(P<0.05),and that in the IL-1β+oe-GPR30+si-Nrf2 group was higher than that in the IL-1β+oe-GPR30+si-NC group(P<0.05).The levels of TNF-αand IL-6 in the IL-1βgroup were higher than those in the control group(P<0.05),those in the IL-1β+oe-GPR30 group were lower than those in the IL-1β+oe-NC group(P<0.05),and those in the IL-1β+oe-GPR30+si-Nrf2 group were higher than those in the IL-1β+oe-GPR30+si-NC group(P<0.05).Compared with the control group,the levels of ROS and MDA were higher and the level of SOD was lower in the IL-1βgroup(P<0.05).Compared with the IL-1β+oe-NC group,the levels of ROS and MDA were lower and the level of SOD was higher in the IL-1β+oe-GPR30 group(P<0.05).Compared with the IL-1β+oe-GPR30+si-NC group,the levels of ROS and MDA were higher and the level of SOD was lower in the IL-1β+oe-GPR30+si-Nrf2 group(P<0.05).Conclusions Up-regulation of the expression of GPR30 activates the Nrf2/ARE signaling pathway and inhibits IL-1β-induced apoptosis,inflammation and oxidative stress in nucleus pulposus cells.