外源性硫化氢通过调控瘦素/瘦素受体通路抑制高糖诱导的人脐静脉内皮细胞损伤

摘要

Objective To investigate whether exogenous hydrogen sulfide (H2S) inhibits the high-glucose (HG)-induced injury by modulating leptin/leptin receptor (LEPR) signal pathway in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were treated with 40 mmol/L glucose for 3-24 h, and the cell viability was examined by CCK-8 assay. The changes of cell morphology and the number of apoptotic cells were assessed by Hoechst 33258 nuclear staining followed by photofluorography. The intracellular levels of reactive oxygen species (ROS) was detected by DCFH-DA staining followed by photofluorography. Mitochondrial membrane potential (MMP) was determined by Rhodamine 123 (Rh123) staining and photofluorography. The expression levels of leptin and LEPR protein were measured by Western blotting. Results The expression of leptin and LERP in HUVECs began to significantly increase at 3 h after HG exposure and reached the peak levels at 9 h (P<0.01). Pretreatment of HUVECs with 400μmol/L sodium hydrosulfide (H2S donor) for 30 min inhibited HG-induced increase in leptin and leptin receptor expressions in HUVECs (P<0.01). Pretreatment of HUVECs with 400μmol/L NaHS for 30 min or 50 ng/mL leptin antagonists (LA) for 1 h obviously alleviated HG-induced injury by increasing cell viability, decreasing cell apoptosis and lowering accumulation of intracellular ROS and MMP loss (P<0.01). Conclusion Exogenous H2S protects against HG-induced injury by inhibiting leptin/LEPR pathway in HUVECs.%目的：探讨外源性硫化氢（H2S）能否通过调控瘦素/瘦素受体（LEPR）通路抑制高糖引起的人脐静脉内皮细胞（HUVECs）损伤。方法 CCK-8法检测细胞存活率；Hoechst 33258核染色荧光显微镜照相检测凋亡细胞的形态学和数量改变；双氯荧光素（DCFH-DA）染色荧光显微镜照相法检测细胞内活性氧（ROS）水平；罗丹明123（Rh123）染色荧光显微镜照相法检测线粒体膜电位（MMP）水平；Western blotting法测定瘦素及瘦素受体蛋白的表达水平。结果应用40 mmol/L葡萄糖处理HUVECs 3~24 h可以明显上调瘦素、瘦素受体的表达水平，在高糖处理9 h时瘦素、瘦素受体的表达水平达到峰值；在高糖处理HUVECs前，400μmol/L硫氢化钠（NaHS，为H2S的供体）预处理30 min能明显抑制高糖对瘦素及瘦素受体表达的上调作用；400μmol/L NaHS预处理HUVECs 30 min、50 ng/mL瘦素拮抗剂（LA）预处理HUVECs 1 h均可明显抑制高糖引起的HUVECs损伤，使细胞存活率升高，细胞凋亡数量减少，胞内活性氧（ROS）堆积及MMP降低（P<0.01）。结论外源性H2S通过抑制瘦素/瘦素受体通路对抗高糖引起的HUVECs损伤。