周期性拉伸通过组蛋白乳酸化修饰调控平滑肌细胞增殖功能  

Cyclic stretch regulates smooth muscle cell proliferation through histone lactylation modification

作　　者：陈鑫 吕慧珍 艾玎 CHEN Xin;LYU Hui-zhen;AI Ding(Department of Physiology and Pathophysiology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院生理学与病理生理学系,天津300070

出　　处：《天津医科大学学报》2023年第5期494-499,531,共7页Journal of Tianjin Medical University

基　　金：国家自然科学基金青年科学基金项目(82000423)。

摘　　要：目的:利用细胞单轴周期性拉伸模型,探究静脉移植手术后的周期性拉伸应力对血管平滑肌细胞(VSMC)组蛋白乳酸化修饰的影响。方法:使用细胞单轴周期性拉伸装置,对大鼠原代VSMC给予拉伸幅度15%、频率1 Hz的拉伸刺激来模拟动脉的周期性拉伸,对照组以细胞静止于拉伸小室来模拟静脉的拉伸状态。细胞收样后,使用Western印迹检测VSMC周期性拉伸24 h前后的增殖细胞核抗原(PCNA)和α平滑肌肌动蛋白(α-SMA)的蛋白水平,qPCR检测平滑肌细胞增殖和分化基因的mRNA水平。随后用比色法检测周期性拉伸前后细胞内的乳酸含量,并提取在两种不同力学条件下处理24 h的VSMC的组蛋白来检测泛和位点特异性的乳酸化修饰水平。结果:Western印迹结果说明,和静息状态相比,VSMC周期性拉伸24 h后的PCNA蛋白水平没有明显变化(P=0.777),α-SMA的蛋白水平下降(t=4.715,P<0.01)。qPCR结果显示,和静息状态相比,PCNA的mRNA水平在VSMC周期性拉伸12 h后上升,24 h恢复到初始水平;细胞周期蛋白Ccnd1和Cdk6的mRNA水平呈时间依赖性上升,细胞周期依赖性激酶抑制剂Cdkn1a和分化相关基因Acta2、Tagln的mRNA水平呈时间依赖性下降。比色法结果显示,和静息状态相比,VSMC周期性拉伸24 h后的乳酸积累增加(t=5.554,P<0.01)。Western印迹结果显示,和静息状态相比,VSMC周期性拉伸24 h后的泛乳酸化修饰水平上升(t=3.603,P<0.01),特异性位点乳酸化修饰如H3K9la、H3K14la和H3K18la的水平均增加。与静息状态(0 h)相比,H3K9la、H3K14la和H3K18la的水平显著增加(t=6.001、6.966、12.750,均P<0.0001)。结论:周期性拉伸后VSMC的代谢状态发生改变,糖酵解的最终代谢产物乳酸积累增加,组蛋白乳酸化修饰水平也增加,这可能是引起VSMC增殖的原因。Objective:To investigate the effect of cyclic stretch(CS)stress on histone lactylation modification in vascular smooth muscle cells(VSMC)after vein graft surgery using a cellular uniaxial cyclic stretching model.Methods:Using a uniaxial cyclic stretching device,the primary VSMCs of rats were subjected to stretch stimulus with a stretch amplitude of 15%and a frequency of 1 Hz to simulate the cyclic stretch of arteries,while cells static in the stretch chamber were to simulate the cyclic stretch of veins and served as control.After cell collection,Western blotting was used to detect PCNA andα-SMA protein levels before and after 24 h of cyclic stretching in VSMC.The mRNA levels of VSMC proliferation and differentiation-related genes were detected by qPCR.Subsequently,the intracellular lactate levels before and after cyclic stretch were detected by colorimetric assay.Histones of VSMC treated under two different mechanical conditions for 24 h were extracted for detection of pan-and site-specific lysine lactonization levels.Results:Western blotting results showed that the protein level of PCNA did not change significantly after 24 hours of cyclic stretch of VSMC compared with the static state(P=0.777),butα-SMA decreased(t=4.715,P<0.01).The qPCR results showed that the mRNA levels of PCNA increased after 12 h of VSMC cyclic stretch and returned to basal level at 24 h.The mRNA levels of cell cycle proteins Ccnd1 and Cdk6 increased in a timedependent manner,and the mRNA levels of cell cycle-dependent kinase inhibitor Cdkn1a,differentiation-related genes Acta2 and Tagln decreased in a time-dependent manner.Colorimetric assay showed the accumulation of lactic acid in VSMC after 24 hours of periodic stretching elevated compared to static state(t=5.554,P<0.01).Western blotting results showed an increase in the level of pan-lysine lactation of VSMC after 24 hours of cyclic stretching compared to the static state(t=3.603,P<0.01),including significant elevation of site-specific lactation modifications such as H3K9la,H3K14la an

关 键 词：周期性拉伸 乳酸 组蛋白乳酸化修饰 血管平滑肌细胞 

分 类 号：R363.1[医药卫生—病理学]