血管软化丸调控lncRNA-TUG1防治动脉粥样硬化的分子机制

Molecular mechanism of vascular softening pill regulating lncRNA-TUG1 to prevent atherosclerosis

目的观察血管软化丸调控长链非编码RNA牛磺酸上调基因1(lncRNA-TUG1)抑制p38丝裂原活化蛋白激酶(p38 MAPK)信号通路活化及血管炎症反应的分子机制。方法体内实验:采用高脂饮食法建立动脉粥样硬化小鼠模型。按照随机数字表法将75只ApoE-/-小鼠分为模型组、牛磺酸上调基因1(TUG1)抑制剂组(10μL TUG1干扰慢病毒)、阴性对照组(10μL空载慢病毒)、血管软化丸组(43.2 g/kg)、联合组(10μL TUG1干扰慢病毒+43.2 g/kg血管软化丸),每组各15只,连续干预8周。采用免疫组化法检测各组小鼠主动脉TUG1的蛋白表达;酶联免疫吸附测定(ELISA)法检测各组小鼠外周血清中细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)、白细胞介素-8(IL-8)、单核细胞趋化蛋白-1(MCP-1)的含量;实时荧光PCR法检测各组小鼠主动脉TUG1、p38总蛋白(T-p38)的mRNA表达水平。体外实验:建立血管内皮细胞(VEC)功能紊乱模型,采用血管软化丸含药血清进行干预。干预后采用MTT法检测细胞存活率;透射电镜观察细胞超微结构;ELISA法检测细胞液中ICAM-1、VCAM-1、IL-8和MCP-1的含量;实时荧光PCR法检测细胞TUG1、T-p38的mRNA表达水平;蛋白质印迹法检测细胞T-p38、磷酸化p38(p-p38)蛋白表达水平。结果体内实验发现,免疫组化结果显示,TUG1蛋白表达阳性信号为棕黄色或棕褐色,模型组和阴性对照组的动脉粥样硬化斑块形成明显,且TUG1蛋白表达阳性信号强;与模型组相比,血管软化丸组主动脉TUG1平均光密度降低(P<0.05),外周血清中ICAM-1、VCAM-1、IL-8和MCP-1含量降低(P<0.05),主动脉TUG1和T-p38 mRNA表达水平降低(P<0.05)。体外实验发现,与模型组相比,血管软化丸血清组的VEC细胞存活率升高(P<0.05);透射电镜显示,血管软化丸血清组细胞内线粒体间距离较模型组小,细胞内纤维排列较模型组整齐;血管软化丸血清组细胞液中ICAM-1、VCAM-1、IL-8和MCP-1含量降低(P<0.05),细胞TUG1和T-p38 mRNA表达水平降低(P<0.05),细胞T-p38、p-p38蛋白表达水平降低(P<0.05)。结论血管软化丸防治动脉粥样硬化的分子机制可能与通过lncRNA-TUG1调控p38 MAPK信号通路、抑制血管炎症反应、抑制VEC凋亡,保护血管内皮有关。

Objective We aimed to investigate the molecular mechanism by which vascular softening pill regulate the activation of the p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway and the vascular inflammatory response to prevent atherosclerosis by long-stranded non-coding RNA taurine upregulated gene 1(lncRNA-TUG1).Methods In vitro,a high-fat diet was used to establish an atherosclerosis mouse model.Seventy-five ApoE-/-mice were randomly divided into the model group,the TUG1 inhibitor group(10μL TUG1-interfering lentivirus),the negative control group(10μL empty lentivirus),the vascular softening pill group(43.2 g/kg)and the combination group(10μL TUG1-interfering lentivirus+43.2 g/kg vascular softening pill),15 mice in each group.The intervention was continued for 8 weeks.Protein expression of TUG1 were measured by immunohistochemistry;the levels of intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),interleukin-8(IL-8)and monocyte chemotactic protein-1(MCP-1)in peripheral serum were measured by ELISA;the mRNA expression levels of TUG1 and total p38 protein(T-p38)in mouse aorta were detected by real-time PCR.In vivo,a vascular endothelial cell(VEC)dysfunction model was established and the vascular softening pill drug serum was used for intervention.After the intervention,survival rate of cell was measured by MTT assay,the cell ultrastructure was observed by transmission electron microscopy,the levels of ICAM-1,VCAM-1,IL-8 and MCP-1 in the serum were measured by ELISA,the mRNA expression levels of TUG1 and T-p38 were measured by real-time PCR,and the protein levels of T-p38 and phosphorylated p38(p-p38)were measured by Western blotting.Results After the intervention,Immunohistochemical detection showed that the positive signal of TUG1 mean optical density was brown yellow or brown.The most obvious atherosclerotic plaque formation and the most obvious positive signal of TUG1 protein expression were found in the model and negative control groups.Compared with the model group,the aortic TUG1 mean optical density was reduced in the vascular softening pill group(P<0.05),the peripheral serum levels of ICAM-1,VCAM-1,IL-8 and MCP-1 were reduced(P<0.05),and aortic TUG1 and T-p38 mRNA expression levels were reduced(P<0.05).Cell culture experiments revealed that the survival rate of VEC cells was increased in the vascular softening pill serum group compared with the model group(P<0.05).Transmission electron microscopy showed that the distance between mitochondria in the cells in the vascular softening pill serum group was smaller than that in the model group,and the arrangement of intracellular fibers was more orderly than that in the model group.Compared with the model group,the levels of ICAM-1,VCAM-1,IL-8 and MCP-1 in the cell sap of the vascular softening pill serum group were reduced(P<0.05),the cellular TUG1 and T-p38 mRNA expression levels were reduced(P<0.05)and the cellular T-p38 and p-p38 protein expression levels were reduced(P<0.05).Conclusion The molecular mechanism by which vascular softening pills protect against atherosclerosis may be related to the regulation of the p38 MAPK signaling pathway through lncRNA-TUG1,inhibition of the vascular inflammatory response,inhibition of vascular endothelial cells apoptosis and protection of vascular endothelium.