NCX-4016通过下调终末化糖基产物受体表达抑制高糖内环境诱导的动脉粥样硬化

NCX-4016 represses the atherosclerosis in diabetic atherosclerotic mouse through inhibition of RAGE

目的探讨一氧化氮(NO)供体药物NCX-4016在2型糖尿病动脉粥样硬化模型小鼠中的保护作用,并研究其与终末化糖基产物受体(RAGE)表达相关的分子机制。方法10只apoE-/-小鼠和db/db杂交后的2型糖尿病动脉粥样硬化模型小鼠给予高脂饮食诱导动脉粥样硬化形成后,均分为实验组和对照组。实验组给予NO供体药物NCX-4016,对照组给予等量生理盐水,处理4周后,油红染色检测主动脉根部动脉粥样硬化斑块面积,Western bolt法检测RAGE及炎症信号通路蛋白高迁移率族蛋白B1(HMGB1)、核转录因子-κB(NF-κB)p65、RAGE和炎症因子表达水平。采用酶联免疫吸附试验(ELISA)检测模型小鼠可溶性晚期糖基化终末产物受体(sRAGE)的血清学水平。人脐静脉内皮细胞(hUVECs)经NCX-4016加或不加eNOS合酶抑制剂、PKG抑制剂诱导后,观察炎症通路信号蛋白和炎症因子的表达水平。结果与对照组比较,实验组小鼠主动脉根部动脉粥样硬化斑块面积明显减小,差异有统计学意义(P<0.05)。实验组与对照组小鼠ELISA检测的血清sRAGE水平比较,差异无统计学意义(P>0.05)。与对照组比较,实验组主动脉根部斑块组织内RAGE和NF-κB p65相对表达水平明显减少,eNOS相对表达水平增加,差异均有统计学意义(P<0.05)。同时,与对照组比较,实验组小鼠主动脉根部斑块组织内TNF-α、IL-6、IL-1β水平明显减少,差异均有统计学意义(P<0.05)。与单独NCX-4016处理比较,eNOS抑制剂+NCX-4016处理hUVECs中IL-6表达水平无明显改变(P>0.05),但NF-κB p65、RAGE、IL-1β、TNF-α表达水平明显升高,差异有统计学意义(P<0.05),PKG抑制剂+NCX-4016处理细胞中NF-κB p65、IL-1β、IL-6和TNF-α表达水平均明显升高,差异有统计学意义(P<0.05)。结论NCX-4016可以明显在体外抑制hUVECs中RAGE的表达,在体外高糖刺激环境下抑制炎性反应,在体内对糖尿病动脉粥样硬化模型小鼠的血管发挥保护作用。

Objective To investigate the protective effect of NCX-4016 as nitric oxide(NO)donor in type 2 diabetic atherosclerosis model mice and explore the underlying molecular mechanism associated with the receptor of advanced glycation end products(RAGE).Methods A total of 10 type 2 diabetic atherosclerosis model mice were obtained by crossing apoE-/-mice and db/db mice.Atherosclerosis induced by high fat diet,and all the mice were divided into experimental group and control group.Experimental group was given NO donor drug NCX-4016,control group was given equivalent normal saline.The area of aortic root atherosclerotic plaques in type 2 diabetic atherosclerosis mice were observed by oil red staining after 4 weeks.Western blot analysis was used to detect the expression levels of HMGB1,NF-κB p65,RAGE and inflammatory factors.Serological levels of sRAGE in model mice were determined by ELISA.Human umbilical vein endothelial cells(hUVECs)were treated by NCX-4016 with or without inhibitor of eNOS,PKG.Then,inflammatory signaling pathway proteins,inflammatory factors were determined.Results Compared with that in control group,the area of atherosclerotic plaque in the aortic root in experimental group reduced significantly(P<0.05).There was no significant difference on serum sRAGE level between the two groups detected by ELISA.Compared with the control group,the expression of RAGE and NF-κB p65 in aortic root plaque tissue in experimental group decreased significantly,and the expression of eNOS increased,with statistical significance(P<0.05).At the same time,the expression of local inflammatory factors TNF-α,IL-6 and IL-1βdecreased significantly(P<0.05).Compared with NCX-4016 alone,expression of NF-κB p65,RAGE,IL-1β,TNF-αincreased significantly(P<0.05),whereas no difference on IL-6 was observed after eNOS inhibitor+NCX-4016 treatment(P>0.05).Expression of NF-κB p65,IL-1β,IL-6 and TNF-αincreased significantly after PKG inhibitor+NCX-4016 treatment(P<0.05).Conclusion NCX-4016 could significantly inhibit the expression of RAGE in hUVECs in vitro,which could repress inflammatory responses under high glucose stimulating conditions,and play a protective effects in diabetic atherosclerotic model mice.