Sulodexide alleviates high glucose-induced cardiac microvascular endothelial cell injury in rat by activating the AMPK/SIRT1 pathway
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摘要 目的：探讨舒洛地特（SDX）减轻高糖（HG）诱导的大鼠心肌微血管内皮细胞（CMEC）氧化应激和凋亡的作用及其机制。方法：分离培养大鼠CMEC，随机分为对照组、HG组、HG+SDX组。CCK-8法检测细胞存活率。利用DCFH-DA作为荧光探针，测定各组DCF荧光强度以判定细胞内活性氧（ROS）水平。比较各组细胞上清液中超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量。采用流式细胞术和TUNEL法检测细胞凋亡率，western blotting法检测AMPK、磷酸化（p-）AMPK、SIRT1蛋白表达。结果：与对照组相比，HG组CMEC细胞存活率降低，细胞上清液中MDA含量增高，SOD活性降低，ROS水平和细胞凋亡率升高，p-AMPK、SIRT1 蛋白表达水平下降（均 P＜0.05）。与 HG 组比较，HG+SDX 组 CMEC 的细胞存活率显著提升，MDA、ROS生成减少，SOD活性升高，CMEC凋亡率下降，p-AMPK、SIRT1蛋白表达水平升高（均P＜0.05）。结论：SDX可能通过激活AMPK/SIRT1信号通路，减轻HG诱导的CMEC的氧化应激及细胞凋亡，从而发挥心血管保护作用。Abstract Objective: To study the effect and mechanism of sulodexide (SDX) on the oxidative stress and apoptosis of cardiac microvascular endothelial cells (CMEC) in rats under high glucose (HG). Methods: CMEC of rats were isolated and cultured, and randomly divided into control group, HG group and HG+SDX group. Cell survival rates were measured by cell counting kit-8 (CCK-8) assay. DCFH-DA was used as a fluorescence probe to determine the fluorescence intensity of DCF in each group to determine the level of intracellular reactive oxygen species (ROS). The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the supernatant were compared. The apoptosis rate was detected by flow cytometry and TUNEL assay. The expression of AMPK, phosphorylated (p-) AMPK and SIRT1 proteins was detected by western blotting. Results: Compared with the control group, the HG group showed decreased cell survival rate, increased cell supernatant level of MDA, reduced SOD activity, elevated ROS level, increased apoptosis rate, and decreased expression of pAMPK、SIRT1 proteins (all P＜0.05). Compared with the HG group, the survival rate of CMEC cells in the HG+ SDX was significantly increased, the production of MDA and ROS was decreased, the activity of SOD was increased, the apoptosis rate of CMEC was decreased, and the expression of p-AMPK and SIRT1 proteins was significantly increased (all P＜0.05). Conclusion: SDX may alleviate HG-induced oxidative stress and apoptosis of CMEC by activating AMPK/SIRT1 signaling pathway, thus playing a cardiovascular protective role.
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