大黄素上调微小RNA-206减轻缺氧诱导的大鼠肾上腺嗜铬细胞瘤细胞损伤

Emodin up-regulates miR-206 to alleviate hypoxia-induced damage to rat adrenal pheochromocytoma PC12 cells

目的探讨大黄素对缺氧诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞氧化应激、炎症和凋亡的作用机制。方法 2018年6月至2019年6月,用神经生长因子(NGF)处理构建缺氧PC12细胞;大黄素Emodin(2、4、6μg/mL)处理缺氧PC12细胞,筛选最适浓度4μg/mL;用脂质体法将微小RNA阴性对照(miR-con)、微小RNA-206(miR-206)、4μg/mL Emodin+抗-miR-con(anti-miRcon)、4μg/mL Emodin+抗-miR-206(anti-miR-206)转染至缺氧PC12细胞;流式细胞术、蛋白免疫印迹(Western blotting)、酶联免疫吸附试验(ELISA)、实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞的凋亡、裂解的半胱氨酸天冬氨酸蛋白酶3(Cleavedcaspase-3)蛋白表达、丙二醛(MDA)、超氧化物歧化酶(SOD)、活性氧(ROS)、肿瘤坏死因子α(TNF-α)、人白细胞介素-1β(IL-1β)、miR-206的含量。结果与缺氧PC12细胞(37.25±3.75)%相比,大黄素呈浓度依赖性抑制缺氧诱导的PC12细胞凋亡[(30.13±3.15)%、(13.43±1.35)%、(10.36±1.03)%];与缺氧PC12细胞相比,大黄素(4μg/mL)治疗后,细胞氧化应激因子MDA[(1.01±0.10)nmol/mg比(2.56±0.26)nmol/mg]、ROS[(186.28±18.75)比(400.25±40.07)]的表达均明显降低,SOD[(11.75±1.18)U/mg比(2.43±0.24)U/mg]的表达明显升高,炎性因子TNF-α[(4.89±0.50)ng/g比(28.76±2.88)ng/g]、IL-1β[(8.22±0.82)ng/g比(15.72±1.58)ng/g]的表达明显降低,miR-206的表达也出现明显升高(P<0.05)。与hypoxia+miR-con组相比,过表达miR-206后,缺氧诱导PC12细胞中MDA[(2.58±0.27)nmol/mg比(1.01±0.10)nmol/mg]、ROS[(196.32±19.65)比(405.76±40.53)]、TNF-α[(4.56±0.50)ng/g比(28.01±2.75)ng/g]、IL-1β[(8.76±0.88)ng/g比(15.50±1.58)ng/g]的表达均显著降低,SOD[(12.04±0.12)U/mg比(2.55±2.56)U/mg]的表达显著升高,细胞凋亡率[(8.96±0.90)%比(36.25±3.63)%]显著降低,而抑制miR-206能够减弱大黄素对缺氧PC12细胞的保护作用。结论大黄素可抑制缺氧诱导的PC12细胞凋亡,减轻细胞的氧化应激和炎性反应,其机制可能与上调miR-206相关,将可为大黄素的临床应用提供理论支持。

Objective To explore the mechanism of emodin on hypoxia-induced oxidative stress,inflammation and apoptosis of rat adrenal pheochromocytoma PC12 cells.Methods This study was carried out from June 2018 to June 2019.Nerve growth factor(NGF)was used to treat and construct hypoxic PC12 cells.Emodin(2,4,6μg/mL)was used to treat hypoxic PC12 cells,and the optimal concentration was 4μg/mL.The microRNA negative control(miR-con),microRNA-206(miR-206),4μg/mL Emodin+anti-miR-con(antimiR-con),4μg/mL Emodin+anti-miR-206(anti-miR-206)were transfected into hypoxic PC12 cells by liposome method.Flow cytometry,Western blotting,enzyme-linked immunosorbent assay(ELISA)and real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)were used to detect apoptosis,Cleaved-caspase-3 protein expression,and the contents of malondialdehyde(MDA),superoxide dismutase(SOD),reactive oxygen species(ROS),tumor necrosis factorα(TNF-α),human interleukin 1β(IL^-1β),and miR-206.Results Compared with hypoxic PC12 cells(37.25±3.75)%,Emodin inhibited hypoxia-induced PC12 cell apoptosis in a concentration-dependent manner[(30.13±3.15)%,(13.43±1.35)%,(10.36±1.03)%].Compared with hypoxic PC12 cells,after Emodin(4μg/mL)treatment,the expressions of cellular oxidative stress factor MDA[(1.01±0.10)nmol/mg vs(2.56±0.26)nmol/mg]and ROS[(186.28±18.75)vs.(400.25±40.07)]were significantly decreased,the expression of SOD[(11.75±1.18)U/mg vs(2.43±0.24)U/mg]was significantly increased,the expressions of inflammatory factor TNF-α[(4.89±0.50)ng/g vs.(28.76±2.88)ng/g]and IL^-1β[(8.22±0.82)ng/g vs(15.72±1.58)ng/g]were significantly reduced,and the expression of miR-206 increased significantly(P<0.05).Compared with hypoxia+miR-con group,after the overexpression of miR-206,the expressions of hypoxia-induced MDA[(2.58±0.27)nmol/mg vs(1.01±0.10)nmol/mg],ROS[(196.32±19.65)vs.(405.76±40.53)],TNF-α[(4.56±0.50)ng/g vs.(28.01±2.75)ng/g],IL^-1β[(8.76±0.88)ng/g vs.(15.50±1.58)ng/g]in PC12 cells were significantly reduced,the expression of SOD[(12.04±0.12)U/mg vs.(2.55±2.56)U/mg]was significantly increased,and the apoptosis rate[(8.96±0.90)%vs.(36.25)±3.63)%]was significantly reduced.However,inhibition of miR-206 can weaken the protective effect of emodin on hypoxic PC12 cells.Conclusion Emodin can inhibit hypoxia-induced apoptosis of PC12 cells,reduce oxidative stress and inflammatory response of cells,and its mechanism may be related to the up-regulation of miR-206,which will provide theoretical support for the clinical application of emodin.