氧化苦参碱改善缺氧缺血引起的HaCaT细胞氧化应激损伤

doi:10.6040/j.issn.1671-7554.0.2020.1557

摘要/Abstract

摘要： 目的探讨氧化苦参碱(OMT)对缺氧缺血环境下角质形成细胞损伤的保护作用。方法选择体外培养的人角质形成细胞系HaCaT细胞,分为正常对照组(NC组)、模型组(HI组)、OMT低剂量组(0.05 g/L OMT组)、OMT高剂量组(0.1 g/L OMT组)4组。采用活细胞数量检测试剂盒CCK8检测HaCaT细胞增殖抑制率和活力,磷脂结合蛋白V(Annexin V)染色检测凋亡率,线粒体膜电位探针JC-1检测HaCaT细胞线粒体膜电位变化,2',7'-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针法检测HaCaT细胞活性氧(ROS)水平,比色法检测HaCaT细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和总抗氧化能力(T-AOC)活力, Western blotting检测凋亡基因天冬氨酸蛋白水解酶3(Caspase 3)、Cleaved-Caspase 3、B淋巴细胞瘤2(Bcl-2)及TGF-β1/SMAD3通路蛋白的表达量。结果与NC组相比,HI组HaCaT细胞Ki67阳性率［(13.52±2.89)%,P<0.001)［和线粒体膜电位降低(0.54±0.03,P<0.001),凋亡细胞数量(13.83±0.81,P<0.001)、ROS水平(164.31±16.93,P<0.001)升高,细胞中GSH-Px(0.96±0.05,P<0.001)、 SOD(0.67±0.06,P<0.001)和T-AOC(1.90±0.02,P<0.001)水平下降。 0.05 g/L OMT干预后,HaCaT细胞Ki67阳性率［(57.98±9.81)%,P<0.001)］和线粒体膜电位升高(0.81±0.04,P<0.001),凋亡细胞数量(8.10±0.53,P<0.001)、ROS水平(175.94±15.75,P<0.001)降低,细胞中GSH-Px(1.04±0.05,P<0.001)、SOD(0.86±0.04,P<0.001)和T-AOC(2.08±0.03,P<0.001)水平升高;Western blotting结果显示:HI处理使HaCaT细胞中TGF-β1信号通路蛋白TGF-β1(1.15±0.14,P=0.010)、 p-SMAD3(0.13±0.03,P=0.112)表达上调,凋亡信号蛋白Caspase-3(0.37±0.045,P=0.001)、Cleaved-Caspase 3(0.54±0.03,P=0.108)蛋白相对表达量较正常培养组均上调;添加0.05 g/L OMT处理后,与HI模型组相比,HaCaT细胞中TGF-β1(0.69±0.13,P=0.005)、 p-SMAD3(0.07±0.01,P<0.001)、 Caspase-3(0.21±0.041,P=0.006)、Cleaved-Caspase 3(0.29±0.054,P=0.016)蛋白相对表达量均下降,Bcl-2蛋白相对表达量升高(0.35±0.013, P=0.015); 0.1 g/L OMT组与0.05 g/L OMT组相比,除Ki67阳性率(P<0.001)、SOD活力(P<0.001)和Bcl-2表达(P=0.045)差异有统计学意义外,其他指标差异均无统计学意义(P>0.05)。结论 0.05~0.1 g/L的OMT能够通过抑制TGFβ1/SMAD3通路来减轻缺氧缺血诱导的细胞线粒体功能障碍、氧化损伤和凋亡,从而保护角质形成细胞的存活。

关键词: 氧化苦参碱, 角质形成细胞, 凋亡, TGFβ1/SMAD3, 氧化应激

Abstract: Objective To explore the protective effect of oxymatrine(OMT)on oxidative damage of keratinocytes in hypoxic-ischemic environment. Methods HaCaT cells cultured in vitro were divided into normal control(NC)group, hypoxia-ischemia(HI)group, low-dose OMT group(0.05 g/L)and high-dose OMT group(0.1 g/L). The proliferation and vitality of keratinocytes were detected with CCK-8 assay; the apoptosis was detected with Annexin V; the mitochondrial membrane potential was detected with mitochondrial membrane potential probe; the reactive oxygen species(ROS)level was detected with DCFH-DA fluorescent probe; the activities of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px)and total antioxidant capacity(T-AOC)in the supernatant of HaCAT cells were determined with colorimetry; the expression levels of Caspase 3, Cleaved-Caspase 3, B lymphoma 2(Bcl-2)and TGF-β1/Smad3 signaling pathway were detected with Western blotting. Results Compared with the NC group, the HI group had reduced Ki67 positive rate ［(13.52±2.89)%, P<0.001］ and lower mitochondrial membrane potential(0.54±0.03, P<0.001), increased apoptotic cells(13.83±0.81, P<0.001)and ROS level(164.31±16.93, P<0.001), and reduced expression levels of GSH-Px(0.96±0.05, P<0.001), SOD(0.67±0.06, P<0.001)and T-AOC(1.90±0.02, P<0.001). Low-dose OMT treatment resulted in increased Ki67 positive rate ［(57.98±9.81)%, P<0.001］, higher mitochondrial membrane potential(0.81±0.04, P<0.001), reduced apoptotic cells(8.10±0.53, P<0.001), reduced ROS level(175.94±15.75, P<0.001), but enhanced expression levels of GSH-Px(1.04±0.05, P<0.001), SOD(0.86±0.04, P<0.001)and T-AOC(2.08±0.03, P<0.001). Western blotting showed that HI treatment increased the expression levels of TGF-β1(1.15±0.14, P=0.010)and p-SMAD3(0.13±0.03, P=0.112), and increased the relative expression levels of Caspase-3(0.37±0.045, P=0.001)and Cleaved-Caspase 3(0.54±0.03, P=0.108). Compared with HI group, low-dose OMT group had reduced relative expressions of TGF-β1(0.69±0.13, P=0.005), p-SMAD3(0.07±0.01, P<0.001), Caspase-3(0.21±0.041, P=0.006)and Cleaved-Caspase 3(0.29±0.054, P=0.016), but increased relative expression of Bcl-2(0.35±0.013, P=0.015). Between the low-dose and high-dose OMT groups, there were only significant differences in Ki67 positive rate(P<0.001), SOD activity(P<0.001)and Bcl-2 expression(P=0.045), and no differences in the other parameters(P>0.05). Conclusion Treatment with 0.05-0.1 g/L can reduce mitochondrial dysfunction, oxidative damage and apoptosis induced by hypoxia-ischemia by inhibiting TGFβ1/ Smad3 pathway, and thus protect the survival of keratinocytes.

Key words: Oxymatrine, Keratinocytes, Apoptosis, TGFβ1/SMAD3, Oxidative stress

中图分类号:

R-932

刘淑丹,张飞燕,郭松林,梁雪云,陈冬梅. 氧化苦参碱改善缺氧缺血引起的HaCaT细胞氧化应激损伤[J]. 山东大学学报 (医学版), 2021, 59(3): 26-34.

LIU Shudan, ZHANG Feiyan, GUO Songlin, LIANG Xueyun, CHEN Dongmei. Oxymatrine ameliorates oxidative stress injury of HaCaT cells induced by hypoxia ischemia[J]. Journal of Shandong University (Health Sciences), 2021, 59(3): 26-34.

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