摘要
探讨晚期糖基化终产物受体(RAGE)表达对血管平滑肌细胞线粒体动力学变化的影响机制。细胞水平以小鼠RAGE基因mRNA序列作为干扰靶点,设计3组靶向RAGE的shRNA,转染离体培养的小鼠主动脉血管平滑肌细胞,并通过PCR和Western blot方法验证转染是否成功;采用CML-BSA分别干预正常血管平滑肌细胞(VSMCs)和转染的VSMCs,采用细胞免疫荧光检测血管平滑肌细胞线粒体动力相关蛋白(DRP1)分布和表达情况;Western blot分析VSMCs的双联RNA依赖蛋白激酶(PKR)、线粒体动力相关蛋白(DRP1)、线粒体融合蛋白(Mfn2)以及相关通路JAK2/STAT3磷酸化蛋白表达水平。动物水平上,通过高脂饲喂和腹腔STZ注射建立2型糖尿病模型;后经尾静脉注射RAGE基因敲降(RAGE-/-)质粒,采用Western blot法测定RAGE基因敲除对糖尿病小鼠主动脉PKR、JAK2、STAT3磷酸化水平以及对线粒体动力学相关蛋白表达水平的影响;采用HE和油红O病理学染色观察敲降RAGE基因后AGEs-RAGE轴诱导糖尿病小鼠血管壁的厚度变化。细胞水平上特异性敲降RAGE基因后,小鼠主动脉血管平滑肌细胞RAGE转录水平明显降低,细胞免疫荧光显示VSMCs细胞中线粒体分裂蛋白DRP1表达水平明显下降,Western blot分析DRP1、PKR蛋白水平也呈现下降趋势,Mfn2和JAK2/STAT3磷酸化水平明显升高,同时在动物水平也得到了相同的结果。体外实验表明,和阳性对照组相比,敲降RAGE基因的细胞能减缓AGEs-RAGE轴诱导的线粒体动力学变化。体内实验表明,和糖尿病模型小鼠相比,RAGE基因敲除后的小鼠组织病理学染色后,其动脉内膜厚度(IMT)明显减少。研究结果表明,AGEs-RAGE轴可能通过JAK2/STAT3信号通路来调节血管平滑肌细胞线粒体动力学变化。
The purpose of this paper is to investigate the mechanism of the receptor of advanced glycation endproducts(RAGE)expression on mitochondrial dynamics of vascular smooth muscle cells.The mRNA sequence of mouse RAGE gene as the interference target,three groups of RAGE targeted shRNA were designed and transfected into cultured mouse aortic vascular smooth muscle cells.PCR and Western blot were used to verify it.Normal vascular smooth muscle cells(VSMCs)and transfected VSMCs were interfered with CML-BSA,and the distribution and expression of mitochondrial motion-related protein(DRP1)in vascular smooth muscle cells(VSMCs)were detected by cellular immunofluorescence.The experssions of the phosphorylated proteins JAK2/STAT3,PKR,DRP1 and Mfn2 in VSMCs were analyzed by Western blot.At the individual level,male C57BL/6 mice with wild type and RAGE gene knockout(RAGE-/-)were respectively given high-fat feeding and intra-abdominal STZ injection to establish a type 2 diabetes model.Western blot was used to determine the effect of RAGE gene knockout on the phosphorylation levels of JAK2 and STAT3 in diabetic mice and the expression levels of mitochondrial kinetic-related proteins.HE and oil red O pathological staining were used to observe the thickness change of blood vessel wall induced by AGEs-RAGE axis in diabetic mice after the RAGE gene knockout.In vitro,after gene RAGE was specificly knockout,aortic vascular smooth muscle cells in mice had a significantly lower transcription,and cell immune fluorescent showed VSMCs cell mitochondria mitotic protein expression level decreased obviously.Meanwhile,DRP1,PKR protein levels also showed a trend of decline,Mfn2 and JAK2/STAT3 phosphorylation level increased significantly,while at the individual level also got the same result.In vitro,cells with RAGE gene knockout out slowed AGE-RAGE axis induced mitochondrial dynamics changes compared to controls.In vivo,compared with diabetic model mice,histopathological staining of RAGE gene knockout mice showed a significant decrease in arter
作者
孙晟甲
黄清昱
阿力木江·买买提江
SUN Shengjia;HUANG Qingyu;Alimujiang Maimaitijiang(Department of Cardiology,Huashan Hospital,Fudan University,Shanghai 200040,China)
出处
《甘肃科学学报》
2022年第3期38-44,共7页
Journal of Gansu Sciences
