摘要
目的探讨四个半LIM结构域蛋白2(FHL2)对高糖引起内皮细胞功能障碍的影响及其作用机制。方法采用MTT检测含不同浓度葡萄糖培养基培养下的人脐静脉内皮细胞(HUVECs)的细胞活力。将HUVECs分为对照组(5 mmol/L葡萄糖)、高糖组(30 mmol/L葡萄糖)、siNC组(30 mmol/L葡萄糖+siNC)和siFHL2组(30 mmol/L葡萄糖+siFHL2)。利用Western blot检测对照组和高糖组的HUVECs中FHL2蛋白表达水平。采用实时定量PCR法检测转染siFHL2后FHL2的表达水平。运用Annexin V-FITC/PI双染检测各组细胞凋亡情况。利用Western blot检测凋亡相关蛋白FHL2、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、cleaved caspase 3表达水平。采用成管实验检测各组HUVECs的成管能力。利用Western blot检测各组SK-1/1-磷酸鞘氨醇(SK-1/S1P)通路中的蛋白表达水平,酶联免疫吸附(ELISA)检测S1P表达水平变化。结果与对照组相比,高糖组HUVECs内FHL2表达上调(t=4.407,P<0.05)。与对照组相比,高糖组HUVECs凋亡水平升高,Bax和cleaved caspase 3的表达也较对照组显著上调,而下调FHL2表达后,细胞凋亡减少,凋亡相关蛋白Bax和cleaved caspase 3的表达下调(P<0.05)。此外,FHL2表达下调改善了HUVECs的成管能力。分子机制分析显示,与对照组比较,高糖组p-SK-1表达显著下调(P<0.05),而下调FHL2后p-SK-1水平显著上调(P<0.05),S1P水平也显著上调(P<0.05)。结论FHL2可改善高糖引起的内皮细胞功能障碍,其作用机制可能与SK-1/S1P信号通路有关。
Objective To explore the effect of the four-and-a-half LIM domain protein 2(FHL2)on endothelial cell dysfunction induced by high glucose and the underlying mechanism.Methods MTT assay was used to detect cell viability of human umbilical vein endothelial cells(HUVECs)cultured in dextrose culture-medium at various concentrations.HUVECs were divided into control group(5 mmol/L glucose),hyperglycemic group(30 mmol/L glucose),siNC group(30 mmol/L glucose+siNC)and siFHL2 group(30 mmol/L glucose+siFHL2).Western blot was applied to measure FHL2 protein expression level in HUVECs of control group and hyperglycemic group.FHL2 expression level after siFHL2 transfection was detected by real-time quantitative PCR.Annexin V-FITC/PI double staining was used to assess cell apoptosis in each group.Western blot was employed to detect the expression of apoptosis-related proteins[FHL2,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cleaved caspase 3].The tube-forming ability of HUVECs in each group was detected by tubeforming experiment.Protein expression level in SK-1/sphingosine-1-phosphate(S1P)pathway was assessed by Western blot,and changes in S1P expression level were measured using enzyme linked immunosorbent assay(ELISA).Results In the hyperglycemic group,FHL2 expression was up-regulated in HUVECs(t=4.407,P<0.05).Compared with the control group,the apoptosis level of HUVECs in the hyperglycemic group increased,and the expression of Bax and cleaved caspase 3 was also significantly upregulated.However,down-regulation of FHL2 expression led to reduced apoptosis,and decreased expression of apoptosis-related proteins Bax and cleaved caspase 3(P<0.05).In addition,down-regulation of FHL2 expression improved the tube-forming ability of HUVECs.Molecular mechanism analysis showed that compared to the control group,p-SK-1 expression was significantly down-regulated in hyperglycemic group(P<0.05),but the level of p-SK-1 was significantly up-regulated after down-regulating FHL2(P<0.05),and S1P level was also significantly up-regulated(P<0.05).Conclusion FHL2 can ameliorate endothelial cell dysfunction caused by high glucose,and its mechanism of action may be related to the SK-1/S1P signaling pathway.
作者
陈卫卫
廖煌
史振鸿
陈跃武
Chen Weiwei;Liao Huang;Shi Zhenhong;Chen Yuewu(Department of Cardiology,the Second Affiliated Hospital of Hainan Medical College,Haikou570216,China)
出处
《心脑血管病防治》
2023年第9期15-19,共5页
CARDIO-CEREBROVASCULAR DISEASE PREVENTION AND TREATMENT
基金
海南省卫生健康行业科研项目(20A200383)。