Trx通过Sirt3-P53通路促进自噬并改善高糖诱发的CMECs损伤

Thioredoxin Increase the Autophagy to Alleviate Cardiac Microvascular Endothelial Cells Injury Induced by High Glucose via Sirt3-P53 Pathway

目的:明确硫氧还蛋白(Thioredoxin,Trx)通过自噬调节对大鼠心脏微血管内皮细胞损伤的保护作用及相关机制。方法:分离成年大鼠心脏微血管内皮细胞并分为:(1)正常对照组;(2)高糖组;(3)高糖+Trx组;(4)高糖+Trx+Ad-sh Sirt3组;(5)高糖+Trx+Ad-sh P53组;(6)高糖+DMSO空载组。通过In Vitro Vascular Permeability Assay Kit检测单层心脏微血管内皮细胞通透性,TUNEL染色检测细胞凋亡,Western blot法检测Sirt3、P53、Atg5、LC3BI/II等相关自噬相关信号通路关键蛋白的表达水平。结果:与正常对照组相比,高糖引起单层心脏微血管内皮细胞通透功能损伤,增加细胞凋亡,抑制自噬,且Sirt3、Atg5、LC3BI/II表达下降而P53表达上升;给予Trx可以上调Sirt3、Atg5、LC3BI/II蛋白表达水平,抑制P53表达,并显著减轻上述高糖引起的细胞损伤;但是,分别干扰Sirt3和P53表达后,Trx的作用明显减弱。结论:Trx通过Sirt3-P53信号通路促进心脏微血管内皮细胞自噬,降低细胞凋亡,改善高糖诱发的大鼠心脏微血管内皮细胞损伤。

Objective： To investigate the protective role and Sirt3-P53 related mechanism of Thioredoxin in rat cardiac microvascular endothelial cells injury induced by high glucose. Methods： Adult rat cardiac microvascular endothelial cells （CMECs） were cultured and divided to six groups including： ① Control Group; ② High-Glucose Group; ③ HG＋Trx Group; ④ HG＋Trx＋ Ad-shSirt3 Group; ⑤HG＋Trx＋ Ad-sb_P53 Group; ⑥ HG＋Vehicle Group. Permeability ofmonolayer CMECs was tested by In Vitro Vascular Permeability Assay Kit; Cellular apoptosis was detected by TUNEL staining; And Western Blot was used to analyze the protein expression including Sift3, P53, Atg5 and LC3BI/II. Results： Compared with Control Group, the Permeability of monolayer CMECs and cellular apoptosis were increased by high glucose, which also reduced autophagy. The expression of Sift3, Atg5 and LC3BⅠI/Ⅱ were on the decline and the P53 on rise. Trx could alleviate CMECs permeability injury as well as apoptosis and promoted cellular autophagy. At meaning while, Sift3, Atg5 and LC3BⅠ/Ⅱ expression were increased and P53 expression were decreased by Trx. In addition, while Sirt3 or P53 were knockdown by siRNA, the protective role of Trx was interfered obviously. Conclusions： Trx can enhance CMECs autophagy and attenuate apoptosis through Sirt3-P53 pathway, which is helpful to alleviate injury induced by high glucose.