| 吴坤,张建刚,孙科,等.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[J].安徽医药,2021,25(4):654-658. |
| 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 |
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| DOI：10.3969/j.issn.1009-6469.2021.04.005 |
| 中文关键词: 大黄素 细胞低氧 miR-206 氧化应激 炎症 凋亡 |
| 英文关键词: Emodin Cell hypoxia miR-206 Oxidative stress Inflammation Apoptosis |
| 基金项目: |
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| 摘要点击次数: 1397 |
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| 中文摘要: |
| 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、6 μg/mL)处理缺氧 PC12细胞，筛选最适浓度 4 μg/mL；用脂质体法将微小 RNA阴性对照(miR-con)、微小 RNA-206(miR-206)、 4 μg/mL Emodin+抗-miR-con(anti-miRcon)、4 μg/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(4 μg/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。 |
| 英文摘要: |
| 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, 6 μg/mL) was used to treat hypoxic PC12 cells, and the optimal concentration was 4 μg/mL. The microRNA negative control (miR-con), microRNA-206 (miR-206), 4 μg/mL Emodin+anti-miR-con (anti-miR-con), 4 μg/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 factor α (TNF-α), human interleukin 1β (IL-1β), and miR206.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 (4 μg/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 sig nificantly increased, and the apoptosis rate [(8.96±0.90)% vs. (36.25) ±3.63)%] was significantly reduced. However, inhibition of miR206 can weaken the protective effect of emodin on hypoxic PC12 cells.Conclusion Emodin can inhibit hypoxia-induced apoptosis ofPC12 cells, reduce oxidative stress and inflammatory response of cells, and its mechanism may be related to the up-regulation of miR206, which will provide theoretical support for the clinical application of emodin. |
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