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RhoA激酶信号通路介导的自噬障碍在2型糖尿病大鼠心肌纤维化中的作用

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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、PCⅢ mRNA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PCⅢ mRNA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PCⅢ mRNA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。

Abstract:

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 PC I 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, PC I 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 PC I 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, PC I
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 PC I and PCIII were significantly increased (P<0.05). Conclusion RhoA
kinase signaling pathway-mediated autophagy disorder can induce myocardial fibrosis in T2DM rats.

基金项目:

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)

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