摘要
目的评估迷迭香酸对高糖培养下心肌细胞线粒体自噬水平以及细胞肥大的影响。方法体外培养H9c2大鼠心肌母细胞,分为对照组(葡萄糖5.5 mmol/L)、高糖组(葡萄糖25 mmol/L)、高糖+迷迭香酸组(葡萄糖25 mmol/L+迷迭香酸50μmol/L)、高糖+迷迭香酸+Parkin-siRNA组(葡萄糖25 mmol/L+迷迭香酸50μmol/L+Parkin-siRNA转染);Western blot法测定PINK1、Parkin、LC3II/LC3I蛋白表达水平,透射电子显微镜下观察线粒体自噬体形成情况,流式细胞术检测活性氧物质(ROS)与凋亡水平,分光光度法检测细胞线粒体呼吸链复合酶活性,荧光酶标法检测线粒体膜电位水平,3H-亮氨酸标记法检测细胞蛋白质合成速率,光学显微镜下检测心肌细胞表面积。结果迷迭香酸可提高高糖培养下心肌细胞内PINK1、Parkin、LC3-II/LC3-I蛋白表达水平(P<0.05),增多线粒体自噬体数量,并抑制高糖诱导的ROS生成、恢复线粒体呼吸链复合酶活性与线粒体膜电位水平(P<0.05),抑制高糖诱导的细胞凋亡(P<0.05),并降低心肌细胞表面积与蛋白质合成速率(P<0.05)。利用Parkin-siRNA抑制心肌细胞线粒体自噬,则阻断了迷迭香酸对高糖培养下心肌细胞氧化应激与心肌细胞肥大的保护作用(P<0.05)。结论迷迭香酸可通过激活Parkin介导的线粒体自噬,对高糖诱导心肌细胞的氧化应激损伤有保护作用,并改善心肌细胞肥大。
Objective To evaluate the effect of rosmarinic acid(RA)on mitophagy and hypertrophy of cardiomyocytes exposed to high glucose(HG).Methods Rat cardiomyocytes(H9c2)exposed to HG(25 mmol/L)were treated with 50μmol/L RA or with both RA treatment and Parkin siRNA transfection,with the cells cultured in normal glucose(5.5 mmol/L)and HG as the controls.The expressions of PINK1,Parkin and LC3II/LC3I in the cells were detected by Western blotting.The formation of mitochondrial autophagosomes was observed by transmission electron microscope.Flow cytometry was employed to detect the level of reactive oxygen species(ROS)and apoptotic rate of the cells.The activities of respiratory chain complex enzymes were measured by spectrophotometry.Fluorescence enzyme labeling and 3H-leucine labeling were used for determining the level of membrane potential and protein synthesis rate,respectively.The cell surface area was observed by light microscopy.Results RA treatment significantly increased the expression levels of PINK1,Parkin and LC3-II/I(P<0.05),promoted the formation of mitochondrail autophagosome,inhibited the production of reactive oxygen species(P<0.05),restored the activities of mitochondrial respiratory chain complex enzymes and mitochondrial membrane potential(P<0.05),inhibited apoptosis(P<0.05),and reduced the cell surface area and protein synthesis rate of H9c2 cells induced by HG exposure(P<0.05).The protective effects of RA against HG-induced oxidative stress and cardiomyocyte hypertrophy was obviously blocked by inhibition of mitophagy mediated by transfection with Parkin siRNA(P<0.05).Conclusion RA can protect rat cardiomyocytes against oxidative stress injury and cardiomyocyte hypertrophy induced by HG by activating Parkin-mediated mitophagy.
作者
刁佳宇
赵宏谋
宁玉洁
韩稳琦
王毅
程功
寿锡凌
尤红俊
DIAO Jiayu;ZHAO Hongmou;NING Yujie;HAN Wenqi;WANG Yi;CHENG Gong;SHOU Xiling;YOU Hongjun(Department of Cardiovascular Medicine,Shaanxi Provincial People's Hospital,Xi'an 710068,China,China;Department of Foot and Ankle Surgery,Xi'an Honghui Hospital,Xi'an 710016,China;School of Public Health,Xi'an Jiaotong University Health Science Center,Xi'an 710061,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2020年第11期1628-1633,共6页
Journal of Southern Medical University
基金
陕西省自然科学基金(2020JQ-940)。