摘要
目的探索RhoA激酶信号通路介导的自噬障碍在2型糖尿病大鼠(T2DM)心肌纤维化中的作用。方法将60只SD大鼠随机分为对照组、模型组、法舒地尔组(10 mg/kg/d)、3-甲基腺嘌呤(3-MA)组(15 mg/kg/d)和法舒地尔+3-MA组各12只。除对照组的所有大鼠均采用高脂饮食法联合小剂量链脲佐菌素诱导T2DM大鼠模型,均给予相应药物干预4周。RT-PCR检测心肌组织ROCK1、ROCK2、PCⅠ、PCⅢmRNA的表达;Western Blot检测心肌组织肌球蛋白磷酸酶靶向蛋白1(MYPT1)、p-MYPT1、LC3-I、LC3-Ⅱ、Beclin 1、p62、UNC-51样激酶1(ULK1)、p-ULK1的表达;MTT法检测心肌成纤维细胞的增殖;ELISA和RT-PCR检测成纤维细胞培养液和细胞内PCⅠ和PCⅢ水平。结果与模型组相比,法舒地尔组大鼠成纤维细胞的增殖活性明显降低(P<0.05),ROCK1、ROCK2、PCⅠ和PCⅢmRNA的表达均明显上调(P<0.05),p-MYPT1/MYPT1和p62的表达明显下调(P<0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin 1和p-ULK1/ULK1的表达明显上调(P<0.05),成纤维细胞PCⅠ和PCⅢ的水平均明显下降(P<0.05);与法舒地尔组相比,法舒地尔+3-MA组大鼠成纤维细胞的增殖活性明显增加(P<0.05),ROCK1、ROCK2、PCⅠ和PCⅢmRNA的表达均明显下调(P<0.05),p-MYPT1/MYPT1和p62的表达均明显上调(P<0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin 1和p-ULK1/ULK1的表达均明显下调(P<0.05),成纤维细胞PCⅠ和PCⅢ的水平均明显升高(P<0.05)。结论RhoA激酶信号通路介导的自噬障碍可以诱导T2DM大鼠的心肌组织纤维化。
Objective To explore roles of RhoA kinase signaling pathway-mediated autophagy disorder in myocardial fibrosis of type 2 diabetes mellitus(T2DM)rats.Methods 60 SD rats were randomly divided into control group,model group,fasudil group(10 mg/kg/d),3-methyladenine(3-MA)group(15 mg/kg/d)and fasudil+3-MA group,12 cases in each group.Except control group,the other groups were given high-fat diet combined with lowdose streptozotocin to induce T2DM rat models.Rats in each group were given corresponding drugs for 4 weeks.RTPCR was performed to detect expression of ROCK1,ROCK2,PCⅠ and PCⅢ mRNA in myocardial tissue.Western Blot was performed to detect expression of myosin phosphatase targeting protein 1(MYPT1),p-MYPT1,LC3-Ⅰ and LC3-Ⅱ,Beclin 1,p62,Unc-51-like kinase 1(ULK1)and p-ULK1 in myocardial tissue.MTT assay was performed to detect proliferation of myocardial fibroblasts.ELISA and RT-PCR were performed to detect levels of PCⅠ and PCⅢ in fibroblast culture medium and cells.Results Compared with model group,activity of fibroblast proliferation were significantly decreased in fasudil group(P<0.05),expressions of ROCK1,ROCK2,PCⅠ and PCⅢ mRNA was significantly up-regulated(P<0.05),while expression of p-MYPT1/MYPT1 and p62 was significantly down-regulated(P<0.05),expression of LC3-Ⅱ/LC3-Ⅰ,Beclin 1 and p-ULK1/ULK1 was significantly up-regulated(P<0.05),levels of fibroblast PCⅠ and PCⅢ were significantly decreased(P<0.05).Compared with fasudil group,activity of fibroblast proliferation were significantly increased in fasudil+3-MA group(P<0.05),expressions of ROCK1,ROCK2,PCⅠ and PCⅢ mRNA was significantly down-regulated(P<0.05),while expression of p-MYPT1/MYPT1 and p62 was significantly up-regulated(P<0.05),expression of LC3-Ⅱ/LC3-Ⅰ,Beclin 1 and p-ULK1/ULK1 was significantly down-regulated(P<0.05),levels of fibroblast PCⅠ and PCⅢ were significantly increased(P<0.05).Conclusion RhoA kinase signaling pathway-mediated autophagy disorder can induce myocardial fibrosis in T2DM rats.
作者
黄柳
贾妍
郭炳彦
刘素云
李拥军
Huang Liu;Jia yan;Guo Bingyan;Liu Suyun;Li Yongjun(Department of Cardiology,Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)
出处
《中国循证心血管医学杂志》
2019年第12期1446-1450,共5页
Chinese Journal of Evidence-Based Cardiovascular Medicine
基金
国家自然科学基金青年科学基金项目(81400217)
国家自然科学基金面上项目(81570345)
河北省医学科学研究重点课题计划(20180329)
关键词
RhoA激酶
2型糖尿病
心肌纤维化
自噬
RhoA kinase
Type 2 diabetes mellitus
Myocardial fibrosis
Autophagy
