Rho相关激酶在高糖诱导小鼠心脏内皮细胞衰老中的作用研究

doi:10.3969/j.issn.1006-6233.2018.02.001

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摘要 目的: 本研究旨在探讨Rho相关激酶在高糖诱导小鼠心脏内皮细胞衰老中的作用。方法: 采用22mm葡萄糖培养内皮细胞72h构建高糖诱导小鼠心脏内皮细胞衰老模型。空白对照组使用正常葡萄糖浓度培养基,高糖组采用22mm葡萄糖培养内皮细胞,实验组在高糖组基础上添加Rho相关激酶抑制剂Y27632,甘露醇组添加甘露醇作为渗透压对照。采用衰老相关β半乳糖苷酶染色鉴定细胞衰老,流式细胞术分析细胞周期,免疫印迹法分析相关蛋白表达,采用试剂盒检测内皮细胞端粒酶活性和细胞培养基上清液一氧化氮水平。结果: 与空白对照组相比,22mM葡萄糖培养小鼠心脏内皮细胞72h能显著提高衰老相关β半乳糖苷酶染色阳性细胞百分比(44.00%±3.63% vs. 4.17%±0.70%,P<0.01),同时显著增强Rho相关激酶活性,提高G0/G1期细胞比例及衰老相关蛋白p21表达水平,抑制内皮细胞端粒酶活性,并降低细胞培养基中一氧化氮水平(P<0.05)。10μM Y27632能显著抑制Rho相关激酶活性,同时有效抑制高糖诱导内皮细胞衰老(29.17%±3.53% vs. 44.00%±3.63%,P<0.05),降低高糖环境下G0/G1期细胞比例及p21表达水平,同时逆转高糖环境下内皮细胞端粒酶活性和细胞培养基中一氧化氮含量下降(P<0.05)。结论: Rho相关激酶激活参与高糖诱导内皮细胞衰老,抑制Rho相关激酶可降低高糖诱导内皮细胞衰老,为防治糖尿病血管病变提供新的方向。

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	徐昌武
	邱立强
	江洪

关键词 ： Rho相关激酶, 细胞衰老, 高糖, 内皮细胞

Abstract：Objective: To investigate the role of Rho-associated kinase in the high glucose-induced murine heart endothelial cells (MHECs) senescence. Methods: MHECs senescence was induced by culturing with 22 mM glucose for 72 h. MHECs in control group were treated with normal culture media and the MHECs in High glucose (HG) group were treated with 22 mM glucose. MHECs in Y27632 group were pretreated with Rho-associated kinase inhibitor Y27632 before cultured in high glucose. In addition, mannitol was added to exclude the effect of osmotic pressure. The ratio of senescent cells was identified by senescence-associated β -galactosidase (SA-βG) staining and flow cytometry was used to analyze the cell cycle. The expression of senescence associated protein p21, p-MBS(Thr853) and MBS was explored by Western blot. The telomerase activity in endothelial cells and the level of nitric oxide (NO) in culture medium assay kit were detected with respective kits. Results: Compared to the control group, treatment of high glucose could significantly increase the ratio of SA-βG positive cells (44.00%±3.63% vs. 4.17%±0.70%,P＜0.01), enhance ROCK activity, increase the ratio of cells in G0/G1 stage and the expression of p21, inhibit telomerase activity of the cells and NO level in culture medium (all P<0.05). However, treatment of ROCK inhibitor Y27632 could greatly inhibit ROCK activity and effectively decrease the ratio of senescent cells (both P<0.05). Also, treatment of Y27632 could decrease the ratio of cells in G0/G1 stage and the expression of p21 and reverse the decrease of telomerase activity and NO level in cells cultured in high glucose medium (all P<0.05). Conclusion: Rho related kinase activation is involved in high glucose induced endothelial cell senescence, and inhibition of Rho related kinase can reduce the senescence of endothelial cells induced by high glucose, and provide a new direction for the prevention and treatment of diabetic angiopathy.

Key words： Rho-associated kinase Cellular senescence High glucose Endothelial cell

基金资助:国家自然科学基金,(编号:81500228)

通讯作者: 江洪

引用本文:

徐昌武, 邱立强, 江洪. Rho相关激酶在高糖诱导小鼠心脏内皮细胞衰老中的作用研究[J]. 河北医学, 2018, 24(2): 177-181.
XU Changwu, QIU Liqiang, JIANG Hong. Effects of Rho-Associated Kinase on High Glucose-Induced Murine Heart Endothelial Cell Senescence. HeBei Med, 2018, 24(2): 177-181.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2018.02.001 或 http://www.hbyxzzs.cn/CN/Y2018/V24/I2/177

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