葫芦素B对高糖诱导的人脐静脉内皮细胞损伤的作用

Effect of Cucurbitacin B on High Glucose-Induced Injury of Human Umbilical Vein Endothelial Cells

目的探讨葫芦素B(CuB)对高糖诱导的人脐静脉内皮细胞(HUVECs)损伤的作用。方法培养HUVECs并利用随机数字法进行分组,对照组采用同等渗透压的甘露醇,高糖组采用33.3 mmol/L的葡萄糖刺激细胞,实验组经不同浓度的CuB(1、2、4、8、10 nmol/L)处理12 h后应用高糖刺激,采用CCK-8检测细胞增殖;采用实时聚合酶链反应检测炎症细胞因子[肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1、IL-6]mRNA转录水平;采用2′,7′-二氯荧光黄双乙酸盐探针检测细胞活性氧类(ROS)的水平;采用原位末端缺口标记法(Tunel)检测细胞凋亡水平;采用Matrigel基质胶三维培养内皮细胞,评估其小管形成能力。结果各组细胞的增殖能力逐渐下降;与高糖组相比,8 mmol/L和10 mmol/L CuB组细胞增殖能力提高(P<0.01)。与对照组相比,高糖组促炎因子TNF-α、IL-1、IL-6的mRNA转录水平升高(P<0.05);与高糖组相比,8 nmol/L CuB组和10 nmol/L CuB组促炎因子的mRNA转录水平降低,且10 nmol/L CuB组低于8 nmol/L CuB组(1.98±0.13比2.43±0.11,2.87±0.34比3.45±0.23,1.34±0.21比2.09±0.22)(P<0.05)。与对照组相比,高糖组细胞的ROS荧光强度增强(P<0.05);与高糖组相比,8 nmol/L CuB组和10 nmol/L CuB组的细胞ROS荧光强度减弱,且10 nmol/L CuB组弱于8 nmol/L CuB组(2.12±0.54比3.42±0.23)(P<0.05)。与对照组相比,高糖组的凋亡阳性细胞数目明显增加(P<0.05);与高糖组相比,8 nmol/L CuB组和10 nmol/L CuB组的凋亡阳性细胞数目减少,且10 nmol/L CuB组少于8 nmol/L CuB组[(12.4±4.0)%比(17.8±4.0)%](P<0.05)。培养6 h后,高糖组的平均小管长度明显低于对照组(P<0.05);与高糖组相比,8 mmol/L CuB组和10 mmol/L CuB组的平均小管长度均增长[(254±58)μm、(309±54)μm比(143±65)μm](P<0.05)。结论CuB能保护HUVECs免受高糖刺激损害,减少炎症细胞因子表达,抑制氧化应激,降低细胞凋亡水平,提高小管形成能力。

Objective To investigate the effect of cucurbitacin B(CuB)on high glucose-induced injury of human umbilical vein endothelial cells(HUVECs).Methods HUVECs were cultured and divided into different groups according to random number table method.Mannitol with the same osmotic pressure was used in the control group and 33.3 mmol/L glucose was used in the high glucose group to stimulate cells.The experimental groups received different concentrations of CuB(1,2,4,8,10 nmol/L)to treat human umbilical vein endothelial cells for 12 h followed by high glucose stimulation.The cell proliferation was detected using cell counting kit-8(CKK-8).The inflammation-related factors[tumor necrosis factor-α(TNF-α),interleukin(IL)-1,IL-6]mRNA transcriptional levels were detected by real-time polymerase chain reaction.The cellular reactive oxygen species(ROS)levels were detected using the 2′,7′-dichlorofluorescent yellow diacetate probe.The level of apoptosis was detected by Tunel staining.The ability of tubule formation for endothelial cells was detected by Matrigel three-dimensional culture.Results The cell proliferation ability of each group gradually decreased.Compared with the high glucose group,the cell proliferation ability of the 8 mmol/L and 10 mmol/L CuB group was improved(P<0.01).Compared with the control group,the mRNA transcriptional levels of the pro-inflammatory factors TNF-α,IL-1,and IL-6 in the high glucose group were increased(P<0.05).Compared with the high glucose group,the mRNA transcription levels of proinflammatory factors in the 8 nmol/L CuB group and the 10 nmol/L CuB group were reduced,and the 10 nmol/L CuB group was lower than the 8 nmol/L CuB group(1.98±0.13 vs 2.43±0.11,2.87±0.34 vs 3.45±0.23,1.34±0.21 vs 2.09±0.22)(P<0.05).Compared with the control group,the ROS fluorescence intensity of the cells in the high glucose group was enhanced(P<0.05).Compared with the high glucose group,the ROS fluorescence intensity of the cells in the 8 nmol/L CuB group and the 10 nmol/L CuB group was reduced,and the 10 nmol/L CuB group was weaker than the 8 nmol/L CuB group(2.12±0.54 vs 3.42±0.23)(P<0.05).Compared with the control group,the number of apoptotic positive cells in the high glucose group increased significantly(P<0.05).Compared with the high glucose group,the number of apoptotic positive cells in the 8 nmol/L CuB group and the 10 nmol/L CuB group decreased,and the 10 nmol/L CuB group was less than the 8 nmol/L CuB group[(12.4±4.0)%vs(17.8±4.0)%](P<0.05).After 6 h of culture,the average tubule length of the high-glucose group was significantly lower than that of the control group(P<0.05).Compared with the high-glucose group,the average tubular length of the 8 mmol/L CuB group and the 10 mmol/L CuB group both increased[(254±58)μm,(309±54)μm vs(143±65)μm](P<0.05).Conclusion CuB has a protective effect on high glucose-induced injury of HUVECs,which is related to reducing the expression of inflammatory factors,inhibiting oxidative stress,reducing apoptosis and increasing the ability of tubule formation.