摘要
目的探讨冬虫夏草提取物虫草菌液(Cordyceps sinensis,CS)对高磷诱导的血管平滑肌细胞钙化的影响机制。方法用细胞增殖与活性检测试剂盒(cell counting kit-8,CCK-8)检测CS对细胞生存活力的影响;用β-甘油磷酸(β-GP,10mmol/L))建立大鼠血管平滑肌细胞(VSMC)钙化模型,加入CS(10mg/L)、自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA,5mmol/L)及一磷酸腺苷(AMP)活化的蛋白激酶(AMPK)抑制剂化合物C(CC,10μmol/L)进行干预,实验分为5组:对照组(Ctrl)、β-GP组、β-GP+CS组、3-MA+β-GP+CS组、CC+β-GP+CS组。通过茜素红S染色及钙测定试剂盒检测各组细胞钙沉积量;透射电镜观察VSMC内自噬体的形成;免疫荧光检测细胞质内微管相关蛋白1轻链3(LC3)的表达;Western印迹法检测血管平滑肌标志物α-SMA蛋白、成骨指标Runx2、自噬相关蛋白LC3Ⅱ/LC3Ⅰ和Beclin1及AMPK/雷帕霉素靶蛋白(mTOR)信号通路蛋白。结果虫草菌液能使高磷环境中血管平滑肌细胞的自噬体、LC3荧光点状聚集及LC3Ⅱ/LC3Ⅰ、Beclin1、α-SMA、磷酸化(p)-AMPK蛋白表达增多,Runx2、p-mTOR蛋白表达及钙沉积减少(均P<0.01)。用3-MA抑制自噬后,β-GP+CS环境中的血管平滑肌细胞α-SMA蛋白表达减少(P<0.01),Runx2蛋白表达及钙沉积增多(均P<0.01),提示虫草菌液是通过激活自噬减轻了β-GP诱导的血管平滑肌细胞的钙化。用CC抑制AMPK/mTOR信号通路后,β-GP+CS组的血管平滑肌细胞的p-AMPK蛋白表达明显减少(P<0.01),且LC3Ⅱ/LC3Ⅰ、Beclin1、α-SMA蛋白表达量及自噬体、LC3荧光点状聚集也减少,p-mTOR、Runx2蛋白表达量及钙沉积增多(均P<0.01),提示虫草菌液是通过激活依赖AMPK/mTOR信号通路的自噬而减轻了β-GP诱导的血管平滑肌细胞的钙化。结论虫草菌液能有效减轻高磷诱导的血管平滑肌细胞钙化,其机制可能是通过激活AMPK/mTOR信号通路增强自噬。
Objective To investigate the influence mechanism of Cordyceps sinensis (CS) on β-glycerophosphate-induced vascular smooth muscle cell (VSMC) calcification. Methods The effect of CS on VSMC cell viability was detected by CCK-8. The cellular models of rat VSMC calcification were established by treating with β-glycerophosphate (β-GP, 10 mmol/L);then CS (10 mg/L), autophagy inhibitor 3-methyladenine (3-MA, 5 mmol/L), and AMPK inhibitor compound C (CC, 10 μmol/L) were added to the cell cultures. There were a total of 5 experiment groups: VSMC cultured in normal medium (Control), VSMC treated with β-GP, VSMC treated with β-GP and CS, VSMC treated with 3-MA, β-GP and CS, and VSMC treated with CC, β-GP and CS. The calcium nodules and calcium content were examined with alizarin red S staining and the O-cresolphthaleincomplexone method, respectively. The autophagosomes within the VSMC were observed using transmission electron microscope (TEM). Immunofluorescence showed the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta. In addition, levels of osteogenic related proteins, autophagy related proteins, and AMPK/mTOR pathway related proteins were evaluated by Western blotting. Results CS increased the number of autophagosomes and the accumulation of LC3 puncta within VSMC. It also up-regulated the protein levels of LC3Ⅱ/LC3Ⅰ, beclin1, α-SMA, and p-AMPK;whereas, the protein levels of Runx2 and p-mTOR, as well as calcium nodules and calcium content were reduced (all P<0.01). When the cells were pretreated with 3-MA before treating with β-GP and CS, the autophagosomes, accumulation of LC3 puncta, and protein levels of LC3Ⅱ/LC3Ⅰ, beclin1, and α-SMA were decreased (all P<0.01);however, the protein level of Runx2, and the calcium nodules and calcium content were increased (all P<0.01). Nevertheless, when the cells were pretreated with CC before giving β-GP and CS, the autophagosomes, the accumulation of LC3 puncta, and the expression levels of p-AMPK, LC3Ⅱ/LC3Ⅰ, beclin1, and α-SMA
作者
邱小波
盛子桐
韩怡然
徐天华
田滨瑶
赵翡
于艳秋
姚丽
Qiu Xiaobo;Sheng Zitong;Han Yiran;Xu Tianhua;Tian Binyao;Zhao Fei;Yu Yanqiu;Yao Li(Department of Nephrology,the First Hospital of China Medical University,Shenyang 110000,China;Department of Pathophysiology,College of Basic Medical Sciences,China Medical University,Shenyang 110013,China)
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2019年第2期119-126,共8页
Chinese Journal of Nephrology
基金
国家自然科学基金(81770766)
国家重点研发计划(2016YFC0901500)
辽宁省自然科学基金(20170540999).
关键词
冬虫夏草
自噬
肌
平滑
血管
磷
肾功能不全
慢性
钙化
Cordyceps sinensis
Autophagy
Muscle, smooth, vascular
Phosphorus
Renal insufficiency, chronic
Calcification
