丹皮酚改善氧化低密度脂蛋白诱导的人血管内皮细胞损伤的作用及其机制

Study on the mechanism of paeonol in improving low-density lipoprotein-induced human vascular endothelial cell injury

目的:探讨丹皮酚对氧化低密度脂蛋白(ox-LDL)诱导的人血管内皮细胞损伤的影响及分子机制。方法:人脐静脉血管内皮细胞(HUVECs)分为9组,正常对照(NC)组、ox-LDL组(100 ng/L ox-LDL)、丹皮酚低、中、高剂量组(60μmol/L、120μmol/L、240μmol/L的丹皮酚+100 ng/L ox-LDL)、ox-LDL+小分子干扰RNA阴性对照(si-NC)组、ox-LDL+circ_0003204小分子干扰RNA(si-circ_0003204)组、中剂量+ox-LDL+circ_0003204过表达阴性对照(pcDNA-NC)组、中剂量+ox-LDL+circ_0003204过表达(pcDNA-circ_0003204)组,每组3个复孔。MTT法、流式细胞术法、Western blot法分别对细胞增殖、凋亡、蛋白(CDK2、Bcl2、p27、Bax)表达进行检测;丙二醛(MDA)和超氧化物歧化酶(SOD)试剂盒分别检测MDA含量和SOD活性;实时荧光定量PCR(RT-qPCR)法检测circ_0003204表达水平。结果:与NC组比较,ox-LDL组HUVECs增殖活性、蛋白(CDK2、Bcl2)表达、SOD活性明显降低(P<0.05),细胞凋亡率、蛋白(p27、Bax)表达、MDA含量、circ_0003204表达明显升高(P<0.05)。与ox-LDL组比较,低、中、高剂量丹皮酚组HUVECs增殖活性、蛋白(CDK2、Bcl2)表达、SOD活性明显升高(P<0.05),细胞凋亡率、蛋白(p27、Bax)表达、MDA含量、circ_0003204表达明显降低(P<0.05)。与ox-LDL+si-NC组比较,ox-LDL+si-circ_0003204组HUVECs增殖活性、蛋白(CDK2、Bcl2)表达、SOD活性明显升高(P<0.05),细胞凋亡率、蛋白(p27、Bax)表达、MDA含量明显降低(P<0.05)。与中剂量+ox-LDL+pcDNA-NC组比较,中剂量+ox-LDL+pcDNA-circ_0003204组HUVECs增殖活性、蛋白(CDK2、Bcl2)表达、SOD活性明显降低(P<0.05),circ_0003204水平、细胞凋亡率、蛋白(p27、Bax)表达、MDA含量明显升高(P<0.05)。结论:丹皮酚可抑制ox-LDL诱导的人脐静脉血管内皮细胞凋亡和氧化应激反应,改善人脐静脉血管内皮细胞损伤,其作用机制可能与下调circ_0003204表达有关。

Objective:To investigate the effects of paeonol on low-density lipoprotein-induced human vascular endothelial cell injury and its molecular mechanisms.Methods:Human umbilical vein endothelial cells(HUVECs)were divided into 9 groups,normal control(NC)group,ox-LDL group(100 ng/L ox-LDL),low,medium,and high-dose paeonol groups(60μmol/L,120μmol/L,240μmol/L paeonol+100 ng/L ox-LDL),ox-LDL+small interfering RNA negative control(si-NC)group,ox-LDL+circ_0003204 small interfering RNA(si-circ_0003204)group,middle dose group+ox-LDL+circ_0003204 overexpression negative control(pcDNA-NC)group,middle dose group+ox-LDL+circ_0003204 overexpression(pcDNA-circ_0003204)group,three replicate wells in each group.MTT flow cytometry,and Western blot were used to detect cell proliferation,apoptosis and protein(CDK2,Bcl2,p27,Bax)expressions,respectively.Malondialdehyde(MDA)and superoxide dismutase(SOD)kit were used to detect MDA content and SOD activity;real-time quantitative PCR(RT-qPCR)was used to detect the expression of circ_0003204.Results:Compared with the NC group,the proliferation activity,protein expressions of CDK2 and Bcl2,and SOD activity of HUVECs in the ox-LDL group were decreased significantly(P<0.05),and the apoptosis rate,protein expressions of p27 and Bax,MDA content,and circ_0003204 expression were increased significantly(P<0.05).Compared with the ox-LDL group,the proliferation activity,protein(CDK2,Bcl2)expressions and SOD activity of HUVECs in the low,medium and high dose paeonol groups were increased significantly(P<0.05),and the apoptosis rate,protein(p27,Bax)expressions,MDA content And circ_0003204 expression were decreased significantly(P<0.05).Compared with ox-LDL+si-NC group,the proliferation activity,protein(CDK2,Bcl2)expressions,SOD activity of HUVECs in ox-LDL+si-circ_0003204 group were increased significantly(P<0.05),the apoptosis rate,protein(p27,Bax)expressions,and the content of MDA were decreased significantly(P<0.05).Compared with the middle-dose+ox-LDL+pcDNA-NC group,the HUVECs proliferation activity,protein(CDK2,Bcl2)expressions,and SOD activity in the middle-dose+ox-LDL+pcDNA-circ_0003204 group were decreased significantly(P<0.05),and the levels of circ_0003204,apoptosis rate,protein(p27,Bax)expressions and MDA content were increased significantly(P<0.05).Conclusion:Paeonol can inhibit ox-LDL-induced apoptosis and oxidative stress of human umbilical vein endothelial cells,and alleviate human umbilical vein endothelial cell injury.The mechanism of action may be related to the down-regulation of circ_0003204 expression.