石榴皮总黄酮对高糖环境下人脐静脉内皮细胞损伤的保护作用及机制

Protective effect of total flavonoids of Pomegranate husk on injuries of human umbilical vein endothelial cells under high glucose

目的探讨石榴皮总黄酮对高糖环境下人脐静脉内皮细胞损伤的保护作用及机制。方法以0、5、10、20、40、80、160 mmol/L葡萄糖诱导人脐静脉内皮细胞损伤后,采用MTT法检测细胞的增殖活力。将对数生长期细胞分成空白组、模型组、阳性对照组、高浓度组、中浓度组、低浓度组。除了空白组外,其余均添加一定量葡萄糖使其终浓度为40 mmol/L,随后阳性对照组加入1×10^(-5)mmol/L氨基胍50μL,高、中、低浓度组分别加入1×10^(-4)mmol/L、1×10^(-5)mmol/L、1×10^(-6)mmol/L石榴皮总黄酮,各50μL,培养48 h,观察人脐静脉内皮细胞的形态,MTT法检测细胞增殖活力、碘化丙啶法检测细胞周期,并检测细胞上清液中乳酸脱氢酶(LDH)、细胞间黏附分子1(ICAM-1)、NO、超氧化物歧化酶(SOD)含量。结果与不加葡萄糖比较,含有≥40 mmol/L葡萄糖培养基培养的人脐静脉内皮细胞吸光度值降低(P<0.05或<0.01)。与空白组比较,除高浓度组外,其余组吸光度值均降低(P<0.05或<0.01);与模型组比较,其余组吸光度值均升高(P<0.05或<0.01)。40 mmol/L葡萄糖可使细胞周期阻滞于G_0~G_1期,而随着石榴皮总黄酮浓度的增加,可有效促进细胞由G_0~G_1期向S期转化,但对G2~M期无影响。与空白组比较,除高浓度组外,其余组细胞上清液中LDH、ICAM-1水平升高,SOD、NO水平降低(P<0.05或<0.01);与模型组比较,阳性对照组、高浓度组、中浓度组细胞上清液中LDH、ICAM-1水平降低,SOD、NO水平升高(P<0.05或<0.01),低浓度组LDH水平降低(P<0.01)。结论石榴皮总黄酮可明显减轻高糖环境下人脐静脉内皮细胞损伤程度,可促使细胞由G_0~G_1期进入S期,促进细胞增殖,机制可能与其降低黏附分子表达,提高SOD及NO水平有关。

To investigate the protective effect of total flavonoids of Pomegranate husk on injuries of human umbilical vein endothelial cells under high glucose.Methods The injuries of human umbilical vein endothelial cells （HUVECs） were induced by 0, 5, 10, 20, 40, 80 and 160 mmol/L glucose.MTT was used to detect the cell viability.The cells in the logarithmic phase were divided into the blank group, model group, positive control group, high concentration group, medium concentration group and low concentration group.In addition to the blank group, cells in the rest of the groups were added with a certain amount of glucose to the final concentration of 40 mmol/L, followed by the positive control group with 1×10-5 mmol/L aminoguanidine 50 μL, high, medium and low concentration groups with 1×10-4, 1×10-5 and 1×10-6 mmol/L total flavonoids of Pomegranate husk （50 μL in each group）, and they were all cultured for 48 h.We observed the morphology of human umbilical vein endothelial cells.MTT assay was used to detect cell viability.The cell clyce, the levels of lactate dehydrogenase （LDH）, intercellular adhesion molecule-1 （ICAM-1）, NO and superoxide dismutase （SOD） were measured by the propidium iodide method.Results The optical density （OD） of HUVECs treated with glucose （≥40 mmol/L） was decreased as compared with that of HUVECs without adding glucose （P〈0.05 or P〈0.01）.Compared with the blank group, in addition to high concentration group, the OD in the other groups was decreased （P〈0.05 or P〈0.01）.Compared with the model group, the OD of the other groups was increased （P〈0.05 or P〈0.01）.Forty mmol/L glucose caused cell cycle arrest in G0-G1 phase, and with the increase of flavonoids from Pomegranate husk, it effectively promoted the cell transformation from G0-G1 to S phase, but it had little effect on cells in the G2-M phase.Compared with the blank group, the levels of LDH and ICAM-1 were higher in the supernatant of the other groups except in the high concentration group, and the levels of SOD and NO were decreased.Compared with the model group, the levels of LDH and ICAM-1 in the supernatant of the positive control group, the high concentration group and the medium concentration group were decreased, the levels of SOD and NO were increased （P〈0.05 or P〈0.01）, and the LDH level in the low concentration group was decreased （P〈0.01）.Conclusion Flavonoids from Pomegranate husk can significantly reduce the injuries of HUVECs under high glucose, promote the cell from G0-G1 to S phase, and promote cell proliferation, which may be related with the reduction of expression of adhesion molecules, and the increase of SOD and NO.