氧化苦参碱改善缺氧缺血引起的HaCaT细胞氧化应激损伤  

Oxymatrine ameliorates oxidative stress injury of HaCaT cells induced by hypoxia ischemia

作　　者：刘淑丹[1] 张飞燕 郭松林 梁雪云[1] 陈冬梅[1] LIU Shudan;ZHANG Feiyan;GUO Songlin;LIANG Xueyun;CHEN Dongmei(Human Stem Cell Institute,General Hospital of Ningxia Medical University,Ningxia Key Laboratory of Stem Cell and Regenerative Medicine,Yinchuan 750004,Ningxia,China)

机构地区：[1]宁夏医科大学总医院干细胞研究所、宁夏回族自治区干细胞与再生医学重点实验室,宁夏银川750004

出　　处：《山东大学学报（医学版）》2021年第3期26-34,共9页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81960355);宁夏自然科学基金(2018AAC03147)。

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=0.0Objective 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

关 键 词：氧化苦参碱 角质形成细胞 凋亡 TGFβ1/SMAD3 氧化应激 

分 类 号：R-932[医药卫生]