虫草素对高糖诱导脐静脉内皮细胞凋亡的保护作用研究

目的探讨虫草素对高糖环境下人脐静脉内皮细胞(HUVECs)的保护作用及其机制。方法原代培养HUVECs,实验分为空白对照组、模型组、对照组、抑制剂A组、抑制剂B组、干预A组、干预B组和低、中、高浓度虫草素组。空白对照组给予正常糖浓度(5.5 mmol·L^-1);模型组给予40 mmol·L^-1高糖孵育48 h;对照组给予10μmol·L^-1白藜芦醇+高糖;低、中、高浓度虫草素组分别给予1,10,20μmol·L^-1虫草素+高糖;抑制剂A组给予1μmol·L^-1 CLI-095+高糖;抑制剂B组给予100 nmol·L^-1西罗莫司+高糖;干预A组给予1μmol·L^-1 CLI-095+20μmol·L^-1虫草素+高糖;干预B组给予100 nmol·L^-1西罗莫司+20μmol·L^-1虫草素+高糖。用5-溴脱氧尿嘧啶核苷-酶联免疫吸附法检测光密度值观察细胞增殖能力,用Annexin V-异硫氰酸荧光素/碘化丙啶流式细胞术检测细胞凋亡率,用实时荧光定量聚合酶链反应检测沉默信息调节因子2同源蛋白1(SIRT1)mRNA的表达水平,用Western Blot检测SIRT1蛋白的表达水平。结果模型组、对照组和低、中、高浓度虫草素组以及干预A组、干预B组的增殖能力(光密度值)分别为(1.38±0.11),(1.86±0.06),(1.44±0.03),(1.60±0.05),(1.70±0.08),(1.85±0.04)和(1.85±0.06),细胞凋亡率分别为(6.72±1.00)%,(0.56±0.37)%,(3.86±0.16)%,(2.64±0.23)%,(2.16±0.15)%,(1.57±0.28)%和(0.86±0.20)%,SIRT1 mRNA表达相对倍数分别为(0.24±0.05),(2.97±0.41),(0.62±0.07),(1.37±0.18),(2.14±0.35),(3.45±0.76)和(3.02±0.79)。模型组、对照组、高浓度虫草素组、抑制剂B组和干预B组的SIRT1蛋白表达分别为(0.82±0.11),(1.14±0.29),(0.98±0.13),(0.74±0.19)和(1.30±0.20)。对照组和高浓度虫草素组的上述指标与模型组比较,差异均有统计学意义(均P<0.05)。干预A组和干预B组的细胞增殖力及SIRT1 mRNA表达水平与高浓度虫草素组比较差异均有统计学意义(均P<0.05)。干预B组细胞凋亡率与高浓度虫草素组比较,差异有统计学意义(P<0.05)。干预B组SIRT1蛋白表达与抑制剂B组比较,差异有统计学意义(P<0.05)。结论虫草素可能通过促进SIRT1表达,从而起到保护高糖损伤的内皮细胞作用。

Study on the protective effects of cordycepin on apoptosis of umbilical vein endothelial cells induced by high glucose

Objective To investigate the protective effect of cordycepin on high glucose-induced human umbilical vein endothelial cells(HUVECs). Methods HUVECs were divided into blank control group, model group, control group, low, medium and high concentration cordycepin groups, inhibitor A group, inhibitor B group,intervention A group and intervention B group. Blank control group: normal sugar concentration( 5. 5 mmol·L^-1). Model group:40 mmol·L^-1 was incubated with high glucose for 48 h. Control group: 10 μmol·L^-1 resveratrol with high glucose.Low,medium and high concentration cordycepin groups: 1,10 and 20 μmol·L^-1 cordycepin respectively with high glucose. Inhibitor A group: 1 μmol·L^-1 CLI-095 with high glucose. Inhibitor B group: 100 μmol·L^-1 rapamycin with high glucose. Intervention A group: 1 μmol·L^-1 CLI-095 and 20 μmol·L^-1 cordycepin in high glucose. Intervention B group: 100 μmol·L^-1 rapamycin and 20 μmol·L^-1 cordycepin in high glucose. 5-Bromo-2-deoxy Uridine method was used to detect cell proliferation and annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry was used to detect cell apoptosis. Real-time quantitative polymerase chain reaction( PCR)was used to detect messenger ribose nucleic acid( mRNA) expression of silent mating type information regulation 2 homolog 1( SIRT1). Western bolt was used to detect protein expression of SIRT1. Results Proliferation ability( OD values) of model group,control group,low,medium and high concentration cordycepin groups,intervention A group,intervention B group were( 1. 38 ± 0. 11),( 1. 86 ± 0. 06),( 1. 44 ± 0. 03),( 1. 60 ± 0. 05),( 1. 70 ± 0. 08),( 1. 85 ± 0. 04) and( 1. 85 ± 0. 06),apoptosis rates were( 6. 72 ± 1. 00) %,( 0. 56 ± 0. 37) %,( 3. 86 ± 0. 16) %,( 2. 64 ± 0. 23) %,( 2. 16 ± 0. 15) %,( 1. 57 ± 0. 28) % and( 0. 86 ± 0. 20) %,mRNA expression of SIRT1 were( 0. 24 ±0. 05),( 2. 97 ±0. 41),( 0. 62 ±0. 07),( 1. 37 ± 0. 18),( 2. 14 ± 0. 35),( 3. 45 ± 0. 78) and( 3. 02 ± 0. 79).protein expression of SIRT1 in model group,control group,high-concentration cordycepin group,inhibitor B group and intervention B group were( 0. 82 ± 0. 11),( 1. 14 ± 0. 29),( 0. 98 ± 0. 13),( 0. 74 ± 0. 19) and( 1. 30 ± 0. 20).There were statistically significant differences between model group and control group/high-concentration cordycepin group( all P < 0. 05). There were statistically significant differences in cell proliferation and SIRT1 mRNA expression between high-concentration cordycepin group and intervention A group/intervention B group( all P < 0. 05). The apoptosis rates of intervention B group were significantly different from that of high-concentration cordycepin group( all P < 0. 05). There were statistically significant differences in SIRT1 protein expression between intervention B group and inhibitor B group( all P < 0. 05). Conclusion Cordycepin may protect HUVECs damaged by high glucose through promoting SIRT1 expression.