Gao Huikuan, Hou Fei, Dong Ruiqing, Wang Zefeng, Zhao Can, Tang Wurina, Wu Yongquan
Department of Cardiology, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
Department of Infection, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
Cardiovasc Ther. 2016 Oct;34(5):352-9. doi: 10.1111/1755-5922.12206.
Cardiac cell apoptosis plays a crucial role in the progression of diabetic cardiomyopathy. Recent studies have shown that fasudil, a Rho-kinase (ROCK) inhibitor, inhibits cardiac cell apoptosis; however, the underlying mechanism remains unclear.
This study aimed to investigate whether fasudil protects H9c2 cells from high glucose-induced apoptosis via activation of autophagy.
Rat cardiomyocyte H9c2 cells were treated with high glucose and used as a diabetic cardiomyopathy model. Cell survival rate, apoptosis, and subcellular morphology were examined using the MTT assay, flow cytometry, and electron microscopy, respectively. ROCK1 and ROCK2 mRNA levels were determined using quantitative real-time PCR. Bcl-2 and Bax, myosin phosphatase target subunit-1 (MYPT-1), phosphorylated (p)-MYPT1, LC3-II/LC3-I, Beclin-1, soluble and insoluble P62 protein levels were determined by Western blot analysis.
Fasudil reversed the high glucose-induced inhibition of cell proliferation and suppressed high glucose-induced early apoptosis. Fasudil also reversed the high glucose-suppressed Bcl-2 levels and decreased the high glucose-induced Bax levels. Further, Fasudil suppressed ROCK levels, expression, promoted autophagy via increasing the LC3-II/LC3-I ratio, Beclin-1 expression, and the number of autophagosomes in H9c2 cells treated with high glucose. These effects of fasudil were abrogated by 3-methyladenine (3-MA), an autophagy inhibitor.
Fasudil inhibited high glucose-induced apoptosis in rat H9c2 cells through activating autophagy.
心肌细胞凋亡在糖尿病性心肌病的进展中起关键作用。最近的研究表明，Rho激酶（ROCK）抑制剂法舒地尔可抑制心肌细胞凋亡；然而，其潜在机制仍不清楚。
本研究旨在探讨法舒地尔是否通过激活自噬来保护H9c2细胞免受高糖诱导的凋亡。
用高糖处理大鼠心肌细胞H9c2细胞，将其作为糖尿病性心肌病模型。分别使用MTT法、流式细胞术和电子显微镜检测细胞存活率、凋亡情况和亚细胞形态。使用定量实时PCR测定ROCK1和ROCK2 mRNA水平。通过蛋白质免疫印迹分析测定Bcl-2、Bax、肌球蛋白磷酸酶靶向亚基-1（MYPT-1）、磷酸化（p）-MYPT1、LC3-II/LC3-I、Beclin-1、可溶性和不可溶性P62蛋白水平。
法舒地尔逆转了高糖诱导的细胞增殖抑制，并抑制了高糖诱导的早期凋亡。法舒地尔还逆转了高糖抑制的Bcl-2水平，并降低了高糖诱导的Bax水平。此外，法舒地尔抑制ROCK水平和表达，通过增加LC3-II/LC3-I比值、Beclin-1表达以及高糖处理的H9c2细胞中的自噬体数量来促进自噬。法舒地尔的这些作用被自噬抑制剂3-甲基腺嘌呤（3-MA）所消除。
法舒地尔通过激活自噬抑制大鼠H9c2细胞中高糖诱导的凋亡。
