连翘苷通过调控CTRP3表达对高糖诱导的人视网膜血管内皮细胞损伤的影响及其机制研究

Effect of phillyrin on high glucose-induced injury of human retinal vascular endothelial cells by regulating complement C1q/tumor necrosis factor-related protein-3 expression and its mechanism

目的比较不同剂量连翘苷(PHN)对高糖(HG)诱导的人视网膜血管内皮细胞损伤的影响,并分析其对补体C1q/肿瘤坏死因子相关蛋白3(CTRP3)表达的调控作用及可能的作用机制。方法采用HG培养人视网膜血管内皮细胞并建立细胞损伤模型(HG组)。HG+PHN-L组、HG+PHN-M组、HG+PHN-H组人视网膜血管内皮细胞分别用1μmol·L^(-1)、10μmol·L^(-1)、100μmol·L^(-1)PHN处理后进行HG诱导。HG+pcDNA组、HG+pcDNA-CTRP3组分别用pcDNA、pcDNA-CTRP3转染至人视网膜血管内皮细胞后进行HG诱导。HG+PHN-H+sh-NC组、HG+PHN-H+sh-CTRP3组分别用sh-NC、sh-CTRP3转染后,用100μmol·L^(-1)PHN处理HG诱导的人视网膜血管内皮细胞。检测细胞丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平。采用流式细胞术检测细胞凋亡率。采用实时荧光定量聚合酶链反应(qRT-PCR)、Western blot分别检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)、CTRP3蛋白表达水平。结果与Con组比较,HG组细胞凋亡率、MDA水平、Bax蛋白水平均升高,SOD、GSH-Px、Bcl-2蛋白、CTRP3 mRNA及蛋白水平均降低(均为P<0.05)。与HG组比较,HG+PHN-L组、HG+PHN-M组、HG+PHN-H组细胞凋亡率、MDA水平、Bax蛋白水平均降低,且HG+PHN-H组<HG+PHN-M组<HG+PHN-L组(均为P<0.05),SOD、GSH-Px、Bcl-2蛋白、CTRP3 mRNA及蛋白水平均升高,且HG+PHN-H组>HG+PHN-M组>HG+PHN-L组(均为P<0.05)。与HG+pcDNA组比较,HG+pcDNA-CTRP3组细胞凋亡率、MDA水平、Bax蛋白水平均降低,SOD、GSH-Px、CTRP3蛋白、Bcl-2蛋白水平均升高(均为P<0.05)。与HG+PHN-H+sh-NC组比较,HG+PHN-H+sh-CTRP3组细胞凋亡率、MDA水平、Bax蛋白水平均升高,SOD水平、GSH-Px水平、CTRP3蛋白、Bcl-2蛋白水平均降低(均为P<0.05)。结论PHN可减轻HG诱导的人视网膜血管内皮细胞损伤,其作用机制可能与上调CTRP3表达有关。

Objective To compare the effects of different doses of phillyrin(PHN)on the injury of human retinal vascular endothelial cells(RVECs)induced by high glucose(HG)and analyze its regulatory effect on the expression of complement C1q/tumor necrosis factor-related protein-3(CTRP3)and its possible mechanism.Methods RVECs were cultured with HG to establish cell injury models(HG group).RVECs in the HG+PHN-L group,HG+PHN-M group,and HG+PHN-H group were treated with 1μmol·L^(-1),10μmol·L^(-1),and 100μmol·L^(-1)PHN,respectively,followed by HG induction.RVECs in the HG+pcDNA group and HG+pcDNA-CTRP3 group were transfected with pcDNA and pcDNA-CTRP3,respectively,followed by HG induction.RVECs in the HG+PHN-H+sh-NC group and HG+PHN-H+sh-CTRP3 group were transfected with sh-NC and sh-CTRP3,respectively,then induced by HG and treated with 100μmol·L^(-1)PHN.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione peroxidase(GSH-Px)were measured.Cell apoptosis was detected by flow cytometry.The expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X(Bax)and CTRP3 protein were determined by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot,respectively.Results Compared with the Con group,the cell apoptosis rate and the levels of MDA and Bax protein in the HG group increased,while the levels of SOD,GSH-Px,Bcl-2 protein,CTRP3 mRNA and protein decreased(all P<0.05).Compared with the HG group,the cell apoptosis rate and the levels of MDA and Bax protein in the HG+PHN-L,HG+PHN-M,and HG+PHN-H groups decreased(HG+PHN-H group<HG+PHN-M group<HG+PHN-L group),while the levels of SOD,GSH-Px,Bcl-2 protein,CTRP3 mRNA and protein increased(HG+PHN-H group>HG+PHN-M group>HG+PHN-L group)(all P<0.05).Compared with the HG+pcDNA group,the cell apoptosis rate and the levels of MDA and Bax protein in the HG+pcDNA-CTRP3 group decreased,while the levels of SOD,GSH-Px,CTRP3 protein,and Bcl-2 protein increased(all P<0.05).Compared with the HG+PHN-H+sh-NC group,the HG+PHN-H+sh-CTRP3 group showed an increase in the cell apoptosis rate and the levels of MDA and Bax protein and a decrease in the levels of SOD,GSH-Px,CTRP3 protein,and Bcl-2 protein(all P<0.05).Conclusion PHN can alleviate HG-induced damage to RVECs,which may be related to the upregulation of the CTRP3 expression.