Liu Jiaqi, Wu Jichao, Sun Aili, Sun Yu, Yu Xiangjing, Liu Ning, Dong Shiyun, Yang Fan, Zhang Linxue, Zhong Xin, Xu Changqing, Lu Fanghao, Zhang Weihua
Department of Pathophysiology, Harbin Medical University, Harbin, 150086 China.
Cell Biosci. 2016 May 23;6:33. doi: 10.1186/s13578-016-0099-1. eCollection 2016.
Excessive autophagy induced by extravagant oxidative stress is the main reason for diabetes-induced vascular endothelial cells dysfunction. Hydrogen sulfide (H2S) has anti-oxidative effects but its regulation on excessive autophagy of vascular endothelial cells is unclear.
In this study, aorta of db/db mice (28 weeks old) and rat aortic endothelial cells (RAECs) treated with 40 mM glucose and 500 μM palmitate acted as type II diabetic animal and cellular models, respectively, and 100 μMNaHS was used as an exogenous H2S donor. The apoptosis level was measured by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) staining and Hoechst 33342/PI staining. The activities of SOD, CAT and respiratory complexes were also measured. The mRNA levels of SOD and CAT were detected by real-time PCR. AMPK-siRNA was used to detect the effect of AMPK on autophagy. Western blotting was used to detected the protein level.
H2S production was decreased (p < 0.05, p < 0.01) both in vitro and in vivo; NaHS treatment rescued this impairment (p < 0.05, p < 0.01). The expression of adhesive proteins was increased (p < 0.05, p < 0.01) both in vitro and in vivo; NaHS attenuated (p < 0.05, p < 0.01) these alterations. NaHS could protect endothelial cells against apoptosis induced by type II diabetes (p < 0.05, p < 0.01). Furthermore, the expressions and activities of SOD and CAT were impaired (p < 0.05, p < 0.01) in endothelial cells of diabetes II; NaHS treatment attenuated (p < 0.05) this impairment. NaHS also increased ATP production (p < 0.05) and activities of respiratory complexes (p < 0.05), and the ratio of p-AMPK to AMPK was also decreased by NaHS (p < 0.01). The level of autophagy in endothelial cells was also decreased (p < 0.05, p < 0.01) by NaHS treatment and AMPK-siRNA treatment. The expression of Nrf2 in the nuclei was increased (p < 0.05) by NaHS treatment.
Exogenous H2S might protect arterial endothelial cells by suppressing excessive autophagy induced by oxidative stress through the Nrf2-ROS-AMPK signaling pathway.
过度氧化应激诱导的自噬过度是糖尿病诱导血管内皮细胞功能障碍的主要原因。硫化氢（H2S）具有抗氧化作用，但其对血管内皮细胞自噬过度的调节作用尚不清楚。
在本研究中，分别以28周龄db/db小鼠的主动脉以及用40 mM葡萄糖和500 μM棕榈酸处理的大鼠主动脉内皮细胞（RAECs）作为2型糖尿病动物模型和细胞模型，100 μM硫氢化钠（NaHS）用作外源性H2S供体。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色和Hoechst 33342/碘化丙啶（PI）染色测定细胞凋亡水平。还测定了超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性以及呼吸复合体的活性。通过实时聚合酶链反应（PCR）检测SOD和CAT的信使核糖核酸（mRNA）水平。使用AMPK小干扰RNA（siRNA）检测AMPK对自噬的影响。采用蛋白质免疫印迹法检测蛋白质水平。
体内外H2S生成均减少（p < 0.05，p < 0.01）；NaHS处理可挽救这种损伤（p < 0.05，p < 0.01）。体内外黏附蛋白的表达均增加（p < 0.05，p < 0.01）；NaHS可减轻（p < 0.05，p < 0.01）这些改变。NaHS可保护内皮细胞免受2型糖尿病诱导的细胞凋亡（p < 0.05，p < 0.01）。此外，2型糖尿病内皮细胞中SOD和CAT的表达及活性受损（p < 0.05，p < 0.01）；NaHS处理可减轻（p < 0.05）这种损伤。NaHS还增加了三磷酸腺苷（ATP）生成（p < 0.05）以及呼吸复合体的活性（p < 0.05），并且NaHS还降低了磷酸化AMPK与AMPK的比值（p < 0.01）。NaHS处理和AMPK-siRNA处理也降低了内皮细胞中的自噬水平（p < 0.05，p < 0.01）。NaHS处理使细胞核中核因子E2相关因子2（Nrf2）的表达增加（p < 0.05）。
外源性H2S可能通过Nrf2-活性氧（ROS）-AMPK信号通路抑制氧化应激诱导的自噬过度，从而保护动脉内皮细胞。
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