香叶木素对高糖诱导的血管内皮细胞损伤的保护作用

Protective effect of diosmetin on vascular endothelial cell injury induced by high glucose

目的探索香叶木素对高糖诱导的血管内皮细胞损伤的保护作用及其机制。方法将人脐静脉血管内皮细胞(HUVEC)暴露于30 mmol·L^(-1)高葡萄糖(HG)中48 h,建立高糖诱导的血管内皮细胞损伤模型。将模型细胞分为模型组(30 mmol·L^(-1) HG干预48 h)和低、中、高3个浓度香叶木素处理组(30 mmol·L^(-1) HG干预48 h+5,10,20 mmol·L^(-1)香叶木素处理24 h)。处理结束后噻唑蓝法检测各细胞活力;相关试剂盒检测各组中一氧化氮(NO),乳酸脱氢酶(LDH)和活性氧(ROS)水平;流式细胞仪检测各组细胞凋亡水平;免疫荧光观察自噬标志物微管相关蛋白1A/1B-轻链3(LC3)和细胞色素c氧化酶亚基IV(COXIV)的共定位比例;蛋白质印迹法测量Yes相关蛋白(YAP)、戴帽蛋白肌Z系Β(CAPZB)和B细胞淋巴瘤-2相关X蛋白(Bax)蛋白的表达水平。结果对照组、模型组和低、中、高3个浓度实验组的细胞活性分别为(100.00±2.43)%,(25.12±1.19)%,(28.34±1.32)%,(45.45±2.68)%,(66.93±2.51)%,(89.46±2.89)%;这5组细胞凋亡率分别为(5.23±0.38)%,(19.41±1.17)%,(13.38±0.84)%,(10.12±0.46)%,(6.15±0.25)%;这5组免疫荧光共定位比例分别为(100.45±2.39)%,(25.31±1.22)%,(24.48±1.17)%,(50.56±1.88)%,(76.28±1.91)%;这5组ROS水平的分别为(100.36±2.59)%,(452.62±14.23)%,(386.27±16.31)%,(267.33±11.37)%,(220.42±12.25)%;这5组YAP蛋白表达水平分别为(100.21±2.17)%,(25.45±1.28)%,(43.36±1.44)%,(60.39±1.53)%,(71.33±3.06)%;这5组CAPZB蛋白表达水平分别为(100.48±2.51)%,(24.13±1.07)%,(35.05±1.38)%,(47.36±2.49)%,(71.42±3.75)%;这5组Bax蛋白表达水平分别为(100.16±1.08)%,(368.73±3.69)%,(271.54±2.47)%,(184.58±1.86)%,(122.47±7.82)%。上述指标,模型组与对照组比较,差异均有统计学意义(均P<0.05);不同浓度实验组与模型组比较,差异均有统计学意义(均P<0.05)。结论香叶木素可通过激活YAP信号通路减轻HG诱导的HUVEC损伤。

Objective To explore the protective effect and mechanism of diosmetin on vascular endothelial cell injury induced by high glucose.Methods Human umbilical vein endothelial cells(HUVEC)were exposed to 30 mmol·L^(-1) high glucose(HG)for 48 h to establish the model of endothelial cell injury induced by HG.Then model cells were divided into model group(30 mmol·L^(-1) HG intervention for 48 h).And low,medium and high concentrations of geranyl lignin(30 mmol·l^(-1) HG intervention for 48 h+5,10,20 mmol·L^(-1) diosmetin treatment for 24 h).After treatment,the cell viability was detected by methyl thiazolyl tetrazolium assay.The levels of nitric oxide(NO),lactate dehydrogenase(LDH)and reactive oxygen species(ROS)in each group were measured by the relevant kits.The apoptosis level of each group was detected by flow cytometry.The co-location ratio of autophagy standard microtubule-associated protein 1A/1B-light chain 3(LC3)and cytochrome C oxidase subunit IV(COXIV)was observed by immunofluorescence.Western blotting was used to measure the expression levels of YES-associated protein(YAP),Cap protein Muscle Z system molar(CAPZB)and pro-apoptotic protein B-cell lymphoma-2 associated x(Bax).Results The cell viability of the control group,model group,and low,medium,and high dose experimental groups were(100.00±2.43)%,(25.12±1.19)%,(28.34±1.32)%,(45.45±2.68)%,(66.93±2.51)%,(89.46±2.89)%;apoptosis rate of these 5 groups were(5.23±0.38)%,(19.41±1.17)%,(13.38±0.84)%,(10.12±0.46)%,(6.15±0.25)%,respectively;the immunofluorescence percentages of these 5 groups were(100.45±2.39)%,(25.31±1.22)%,(24.48±1.17)%,(50.56±1.88)%,(76.28±1.91)%;the protein expression level of ROS in these 5 groups were(100.36±2.59)%,(452.62±14.23)%,(386.27±16.31)%,(267.33±11.37)%,(220.42±12.25)%;the protein expression levels of YAP in these 5 groups were(100.21±2.17)%,(25.45±1.28)%,(43.36±1.44)%,(60.39±1.53)%,(71.33±3.06)%;the protein expression levels of CAPZB in these 5 groups were(100.48±2.51)%,(24.13±1.07)%,(35.05±1.38)%,(47.36±2.49)%,(71.42±3.75)%;the protein expression levels of Bax in these 5 groups were(100.16±1.08)%,(368.73±3.69)%,(271.54±2.47)%,(184.58±1.86)%,(122.47±7.82)%.Above indicators,the differences between the model group and the normal group were statistically significant(all P<0.05);the differences between the experimental group and the model group at different doses were statistically significant(all P<0.05).Conclusion Diosmetin alleviates HG-induced HUVEC injury by activating YAP signaling pathway.