CAJ | 학술논문

目的探讨二甲双胍对波动性高糖诱导的内皮细胞损伤的保护作用及其可能机制.方法以体外培养的人脐静脉内皮细胞(HUVECs)作为研究对象,分为6组:正常葡萄糖对照组、高渗对照组、持续性高糖组、波动性高糖组、波动性高糖+二甲双胍组以及波动性高糖+二甲双胍+化合物C组,各组均干预72 h.以硝酸还原酶法检测细胞上清液亚硝酸盐浓度代表一氧化氮(NO)产生水平；流式细胞仪分析测定细胞内活性氧簇(ROS)水平；Western blotting检测单磷酸腺苷激活蛋白激酶(AMPK)、磷酸化AMPK(Thr-172,p-AMPK)及三磷酸鸟苷环化水解酶1(GTPCH1)的蛋白表达水平.组间比较采用单因素方差分析和Q检验.结果 (1)与正常葡萄糖对照组(100％)相比,波动性高糖组细胞内ROS水平增加[(222.4 ±62.0)％],NO水平下降[(70.3±7.1)％]；添加二甲双胍后ROS水平降低[(100.2±17.4)％],NO水平升高[(96.3±9.2)％]；添加AMPK抑制剂化合物C后ROS水平增加[(167.2±19.6)％],NO水平降低[(83.3±8.7)％].差异均有统计学意义(均P＜0.05).(2)与正常葡萄糖对照组相比,波动性高糖组p-AMPK[(1.72±0.08)比(2.34±0.09)]和GTPCH1[(4.07 ±0.17)比(7.83±0.56)]表达水平降低；添加二甲双胍后p-AMPK (2.72±0.22)和GTPCH1(10.24±1.05)表达水平增高；添加化合物C后GTPCH1表达水平降低(2.39 ±0.34).差异均有统计学意义(均P＜0.05).结论二甲双胍可通过激活AMPK信号通路,上调GTPCH1表达,从而改善波动性高糖所致的内皮细胞功能障碍.
목적 탐토이갑쌍고대파동성고당유도적내피세포손상적보호작용급기가능궤제.방법 이체외배양적인제정맥내피세포(HUVECs)작위연구대상,분위6조:정상포도당대조조、고삼대조조、지속성고당조、파동성고당조、파동성고당+이갑쌍고조이급파동성고당+이갑쌍고+화합물C조,각조균간예72 h.이초산환원매법검측세포상청액아초산염농도대표일양화담(NO)산생수평；류식세포의분석측정세포내활성양족(ROS)수평；Western blotting검측단린산선감격활단백격매(AMPK)、린산화AMPK(Thr-172,p-AMPK)급삼린산조감배화수해매1(GTPCH1)적단백표체수평.조간비교채용단인소방차분석화Q검험.결과 (1)여정상포도당대조조(100％)상비,파동성고당조세포내ROS수평증가[(222.4 ±62.0)％],NO수평하강[(70.3±7.1)％]；첨가이갑쌍고후ROS수평강저[(100.2±17.4)％],NO수평승고[(96.3±9.2)％]；첨가AMPK억제제화합물C후ROS수평증가[(167.2±19.6)％],NO수평강저[(83.3±8.7)％].차이균유통계학의의(균P＜0.05).(2)여정상포도당대조조상비,파동성고당조p-AMPK[(1.72±0.08)비(2.34±0.09)]화GTPCH1[(4.07 ±0.17)비(7.83±0.56)]표체수평강저；첨가이갑쌍고후p-AMPK (2.72±0.22)화GTPCH1(10.24±1.05)표체수평증고；첨가화합물C후GTPCH1표체수평강저(2.39 ±0.34).차이균유통계학의의(균P＜0.05).결론 이갑쌍고가통과격활AMPK신호통로,상조GTPCH1표체,종이개선파동성고당소치적내피세포공능장애.
Objective To investigate the effect and mechanism of metformin on endothelial cell damages induced by intermittent high glucose.Methods Cultured human umbilical vein endothelial cells (HUVECs) were divided into six groups:normal glucose control,hyperosmotic control,constant high glucose,fluctuating high glucose,fluctuating high glucose + metformin and fluctuating high glucose +metformin + compound C.After incubation for 72 h,the level of nitric oxide (NO) production was shown as cell supernatant nitrite concentration which was measured by nitrate reductase method; the intracellular level of reactive oxygen species (ROS) was detected by flow cytometry; and the expression levels of adenosine monophosphate activated protein kinase (AMPK),phospho-AMPK (Thr-172,p-AMPK) and guanosine 5'-triphosphate cyclohydrolase-1 (GTPCH1) proteins were determined by western blot.One-way analysis of variance and Q test were applied to analyze the differences among the groups.Results (1) Compared with those in the normal glucose control group (100％),the level of intracellular ROS ((222 ± 62) ％) was increased and the level of NO ((70.3 ± 7.1) ％) was reduced in the fluctuating high glucose group.The level of intracellular ROS ((100 ± 17)％) was decreased and the level of NO ((96.3 ± 9.2) ％) was increased in the fluctuating high glucose group by adding mefformin.The level of intracellular ROS (167.2 ± 19.6) ％) was increased and the level of NO ((83.3 ± 8.7)％) was decreased by further adding compound C,an AMPK inhibitor.All the differences were statistically significant (all P ＜ 0.05).(2) Compared with those in the normal glucose control group,the expression levels of p-AMPK ((1.72 ±0.08) vs (2.34 ±0.09)) and GTPCH1 ((4.07 ±0.17) vs (7.83 ±0.56) ) were significantly downregulated in the fluctuating high glucose group.The expression levels of p-AMPK (2.72 ± 0.22) and GTPCH1 (10.24 ± 1.05) were increased in the fluctuating high glucose group by adding metformin.The expression level of GTPCH1 (2.39 ± 0.34) was decreased by further adding compound C.All the differences were statistically significant (all P ＜0.05).Conclusion Metformin may attenuate intermittent high glucose-induced endothelial dysfunction via upregulating GTPCH1 expression mediated by activation of AMPK signaling pathway.