基于MAMs探究RVD1对衰老大鼠心肌细胞自噬与凋亡的影响  

To investigate the effects of RVD1 on autophagy and apoptosis of aging rat cardiomyocytes based on MAMs

作　　者：郑扬 黄玉冰[1] 黄文霞[1] 李海涛[1] Zheng Yang;Huang Yubing;Huang Wenxia;Li Haitao(Dept of Cardiovascular Medicine,Hainan Provincial People′s Hospital,Haikou 570311)

机构地区：[1]海南省人民医院心血管内科,海口570311

出　　处：《安徽医科大学学报》2023年第11期1890-1897,共8页Acta Universitatis Medicinalis Anhui

基　　金：海南省卫生健康行业科研项目(编号:22A200204)。

摘　　要：目的探究消退素D1(RVD1)对衰老大鼠心肌细胞自噬与凋亡的调控作用以及对心肌组织线粒体-内质网偶联(MAMs)的影响。方法将30只18月龄SD大鼠按数字随机表法分为模型组、低剂量RVD1组、高剂量RVD1组,每组10只,另取10只6月龄SD大鼠作为对照组,低剂量RVD1组和高剂量RVD1组分别尾静脉注射50、100 ng/ml RVD1,对照组和模型组同时注射等量磷酸盐缓冲液(PBS),持续干预21 d;对-硝基苯酚法测定大鼠心肌组织β-半乳糖苷酶活性,苏木精-伊红(HE)染色检测大鼠心肌组织病理形态学变化,Masson染色观察大鼠心肌组织内胶原沉积情况,末端脱氧核苷酸转移酶介导的dUTP原位末端标记(TUNEL)染色检测大鼠心肌细胞凋亡情况,免疫组化S-P法检测大鼠心肌组织自噬标志物微管相关蛋白3(LC3)表达,蛋白质免疫印迹(Western blot)测定大鼠心肌组织中LC3-Ⅱ/LC3-Ⅰ比值、自噬效应蛋白(Beclin-1、p62)表达水平,透射电镜观察大鼠心肌组织内MAMs结构变化,Western blot测定大鼠心肌组织中葡萄糖调节蛋白75(GRP75)、电压依赖性阴离子通道1(VDAC1)及线粒体融合蛋白2(Mfn2)表达水平。结果与模型组比较,低剂量RVD1组与高剂量RVD1组的大鼠心肌组织β-半乳糖苷酶活性降低(P<0.05),心肌纤维断裂与心肌细胞损伤明显减轻,胶原纤维沉积明显减少,心肌组织内TUNEL阳性细胞比例减少(P<0.05),LC3蛋白阳性率增加(P<0.05),LC3-Ⅱ/LC3-Ⅰ比值升高、Beclin-1蛋白相对表达量上调而p62蛋白相对表达量下调(P<0.05),MAMs结构较为紧密,占线粒体周长百分比也增加(P<0.05),GRP75、VDAC1及Mfn2蛋白相对表达量均上调(P<0.05);与低剂量RVD1组比较,高剂量RVD1组大鼠心肌组织β-半乳糖苷酶活性进一步降低(P<0.05),心肌组织未见明显损伤,胶原纤维沉积进一步减少,心肌组织内TUNEL阳性细胞比例减少(P<0.05),LC3蛋白阳性率增加(P<0.05),LC3-Ⅱ/LC3-Ⅰ比值升高且Beclin-1蛋白相对表达量Objective To explore the regulatory effects of ResolvinD1(RVD1)on autophagy and apoptosis of cardiomyocytes in aging rats,and the effects of RVD1 on mitochondria-associated endoplasmic reticulum membranes(MAMs)of myocardial tissue.Methods Thirty 18-month-old SD rats were randomly divided into model group,low-dose RVD1 group and high-dose RVD1 group,with 10 rats in each group,and another 106-month-old SD rats were selected as control group.The low-dose RVD1 group and high-dose RVD1 group were injected with 50 ng/ml and 100 ng/ml RVD1 through the tail vein,respectively,control group and model group were injected with the same amount of phosphate buffered saline(PBS)for 21 days.Theβ-galactosidase activity of rat myocardial tissue was determined by p-nitrophenol method,the histopathological changes of rat myocardial tissue were detected by hematoxylin-eosin(HE)staining,and the collagen deposition in rat myocardial tissue was observed by Masson staining,apoptosis of rat myocardial cells was detected by dUTP in situ end labeling mediated by terminal deoxynucleotide transferase(TUNEL),the expression of autophagy marker microtubule-associated protein 3(LC3)was detected by immunohistochemical S-P method,the ratio of LC3-Ⅱ/LC3-Ⅰand the expression of Beclin-1,p62 in rat myocardial tissue were determined by Western blot,the structure of MAMs in rat myocardial tissue was observed by transmission electron microscopy,the expression levels of glucose-regulatory protein 75(GRP75),volt-dependent anion channel 1(VDAC1)and mitochondrial fusion protein 2(Mfn2)in rat myocardial tissue were determined by Western blot.Results Compared with model group,β-galactosidase activity of myocardial tissue decreased in low-dose RVD1 group and high-dose RVD1 group(P<0.05),myocardial fiber breakage and myocardial cell damage were significantly alleviated,collagen fiber deposition was significantly reduced,and the proportion of TUNEL positive cells in myocardial tissue decreased(P<0.05),the positive rate of LC3 protein increased(P<0.05),the ra

关 键 词：衰老 ResolvinD1 线粒体-内质网偶联 心肌细胞 自噬 凋亡 

分 类 号：R542.2[医药卫生—心血管疾病]