利拉鲁肽通过circ-sirt1/NF-κB信号通路抑制血管平滑肌细胞炎症的机制研究

Mechanism of lilarutide inhibiting inflammation of vascular smooth muscle cells via circ-sirt1/NF-κB signaling pathway

目的探究利拉鲁肽(liraglutide,LIR)对肿瘤坏死因子α(tumour necrosis factor-α,TNF-α)诱导的血管平滑肌细胞炎症反应的作用及其可能的分子机制。方法实验分为空白对照组(NC组)、TNF-α组(10μg/L)和TNF-α+LIR组(10μg/L TNF-α+100 nmol/L LIR)。酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测细胞培养基中的白细胞介素1β(interleukin-1β,IL-1β)和白细胞介素6(interleukin-6,IL-6)。实时荧光定量核酸扩增检测系统(real-time quantitative polymerase chain reaction detecting system,qPCR)检测circ-sirt1及细胞间黏附分子1(inter-cellular adhesion molecule 1,ICAM-1)、单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)、抑制性卡巴蛋白α(inhibitor kppa B-α,IκB-α)mRNA的表达。Western Blot检测细胞ICAM-1、MCP-1和IκB-α蛋白表达。免疫荧光检测核因子κB(nuclear factor kappa-B,NF-κB)p65蛋白分布。结果与NC组相比,TNF-α组细胞培养基中的IL-1β和IL-6含量显著升高,ICAM-1、MCP-1 mRNA及蛋白表达显著升高,IκB-αmRNA及蛋白表达显著减低,circ-sirt1表达显著降低,NF-κB p65蛋白主要定位在细胞核。与TNF-α组相比,TNF-α+LIR组IL-1β和IL-6含量显著减少,ICAM-1、MCP-1蛋白表达显著降低,IκB-α蛋白表达显著增加,circ-sirt1表达显著升高,NF-κB p65蛋白细胞核定位减少。结论利拉鲁肽能够抑制血管平滑肌细胞的炎性反应,可能是通过抑制炎症介质释放及circ-sirt1/NF-κB信号通路的激活发挥作用。

Objective To investigate the effect of liraglutide(LIR) on tumor necrosis factor(TNF)-α-induced vascular smooth muscle cell inflammation and its possible molecular mechanism. Methods The experiment was divided into three groups: blank control group(NC group), TNF-α group(10 μg/L) and TNF-α +LIR group(10 μg/L TNF-α + 100 nmol/L LIR). IL-1β and IL-6 in cell culture medium were detected by enzyme linked-immunosorbent assay(ELISA). The mRNA expressions of ICAM-1, MCP-1 and IκB-α and circ-sirt1 were detected by real-time quantitative polymerase chain reaction detecting system(qPCR). The protein expressions of ICAM-1, MCP-1 and IκB-α were detected by Western blot. The protein distribution of NF-κB p65 was detected by immunofluorescence. Results Compared with NC group, the levels of IL-1β and IL-6 in TNF-α group were significantly increased, and the mRNA and protein expressions of ICAM-1 and MCP-1 were significantly increased. The mRNA and protein expression of IκB-α was significantly decreased, and the expression of circ-sirt1 was significantly decreased. NF-κB p65 protein was mainly localized in the nucleus. Compared with TNF-α group, the levels of IL-1β and IL-6 and the protein expressions of ICAM-1 and MCP-1 were significantly decreased, while the protein expression of IκB-α was significantly increased in TNF-α+LIR group. Meanwhile, the expression of circ-sirt1 was significantly increased and nuclear localization of NF-κB p65 protein was decreased in TNF-α+LIR group. Conclusion Lilarutide can inhibit the inflammatory response of vascular smooth muscle cells, possibly by inhibiting the release of inflammatory mediators and the activation of circ-sirt1/NF-κB signaling pathway.