Study on effects of phorbol ester on inducing autophagy of C2C12 myotubes and protective effects of corylifol A

Jixia KUANG; Qiang SHEN; Xuan LIU

doi:10.16306/j.1008-861x.2022.02.009

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Study on effects of phorbol ester on inducing autophagy of C2C12 myotubes and protective effects of corylifol A

Experiment Research | 更新时间：2022-08-29

- Study on effects of phorbol ester on inducing autophagy of C2C12 myotubes and protective effects of corylifol A
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 36, Issue 2, Pages: 49-58(2022)
- 作者机构：
  
  1.上海中医药大学交叉科学研究院（上海　201203）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2022.02.009
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邝计霞,沈强,刘璇.佛波酯诱导C2C12肌管自噬及corylifol A的保护作用研究[J].上海中医药大学学报,2022,36(02):49-58.

KUANG Jixia,SHEN Qiang,LIU Xuan.Study on effects of phorbol ester on inducing autophagy of C2C12 myotubes and protective effects of corylifol A[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(02):49-58.
邝计霞,沈强,刘璇.佛波酯诱导C2C12肌管自噬及corylifol A的保护作用研究[J].上海中医药大学学报,2022,36(02):49-58. DOI： 10.16306/j.1008-861x.2022.02.009.

KUANG Jixia,SHEN Qiang,LIU Xuan.Study on effects of phorbol ester on inducing autophagy of C2C12 myotubes and protective effects of corylifol A[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(02):49-58. DOI： 10.16306/j.1008-861x.2022.02.009.

摘要

目的,2,探索佛波酯（PMA）诱导C2C12肌管自噬的作用机制并考察补骨脂成分次苷酸查尔酮（CYA）对肌管的保护作用。,方法,2,构建能够准确分析细胞自噬状态的mRFP-GFP-LC3B双荧光C2C12成肌细胞，将已分化C2C12细胞分为对照组、PMA组（1 μmol/L）、自噬阳性对照组（饥饿组）、CYA组（10 μmol/L）及PMA+CYA组。处理时间为48 h。Cytation 5 测定细胞在不同波长下的荧光强度，Western blot检测自噬相关蛋白Beclin-1及微管相关蛋白1轻链3B（LC3B）的表达水平，qPCR技术检测蛋白激酶C（PKC）-ε、PKC-β，PKC-θ和缺氧诱导因子-2α（HIF-2α）的表达水平。用海马能量代谢分析仪（Seahorse XFe 24分析仪）检测细胞线粒体呼吸能力。,结果,2,①与对照组比较，PMA （1 μmol/L）可使C2C12肌管的黄色荧光斑点显著增加，红色荧光信号强度也显著增强（,P,<,0.01），即自噬及自噬溶酶体均增加。而CYA（10 μmol/L）可以显著降低PMA诱导的C2C12肌管黄色荧光斑点和红色荧光信号强度（,P,<,0.01）。②与对照组比较，PMA可以上调Beclin-1和LC3BⅡ蛋白表达水平（,P,<,0.05，,P,<,0.01）；与PMA组比较，PMA+CYA组可以降低自噬相关蛋白Beclin-1及LC3BⅡ的蛋白表达水平（,P,<,0.05，,P,<,0.01）。③与对照组比较，PMA显著降低C2C12肌管的基础呼吸、最大呼吸和ATP的生成（,P,<,0.01）。④与对照组比较，PMA组可以显著上调P,KC,-,ε,、,PKC,-,β,、,PKC,-,θ,和,HIF,-,2α,基因表达水平（,P,<,0.05，,P,<,0.01）。,结论,2,PMA可能通过激活C2C12肌管的PKC通路来抑制细胞线粒体呼吸，促进HIF-2α的表达，HIF-2α上调自噬相关蛋白Beclin-1和LC3BⅡ的表达，从而诱导C2C12肌管自噬。CYA则可能通过下调Beclin-1和LC3BⅡ的蛋白表达来保护肌管对抗PMA诱导的自噬。

Abstract

Objective： To investigate the mechanisms of phorbol ester （PMA） in inducing autophagy of C2C12 myotubes and the protective effects of psoralen corylifol A （CYA） on myotubes.,Methods,2,The mRFP-GFP-LC3B dual-fluorescence C2C12 myoblast cell strain， whose autophagy status can be accurately analyzed， was constructed. The differentiated C2C12 cells were divided into control group， PMA group （1 μmol/L）， autophagy positive control group （starvation group），CYA group （10 μmol/L），and PMA+ CYA group. The treatment time was 48 h. Cytation 5 was used to detect the fluorescence intensity of cells at different wavelengths. Western blot was implied to detect the protein levels of autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3B （LC3B）. qPCR technology was used to detect the expression levels of protein kinase C（PKC）-ε， PKC-β， PKC-θ and hypoxia-inducible factor-2α （HIF-2α）. Cellular mitochondrial respiration capacity was detected with a hippocampal energy metabolism analyzer （Seahorse XFe24 Analyzer）.,Results,2,①Compared with the control group， the PMA group （1 μmol/L） significantly increased the yellow fluorescent spots and red fluorescence signal intensity of C2C12 myotubes （,P,<,0.01）， which indicated the increase of both autophagy and autolysosomes. CYA （10 μmol/L） significantly reduced the yellow fluorescent spots and red fluorescence signal intensity of C2C12 myotubes induced by PMA （,P,<,0.01）.②Compared with the control group， the PMA group up-regulated the protein expression levels of Beclin-1 and LC3B-Ⅱ （,P,<,0.05，,P,<,0.01）. Compared with the PMA group， the PMA+CYA group significantly down-regulated the protein expression levels of autophagy-related proteins Beclin-1 and LC3BⅡ （,P,<,0.05，,P,<,0.01）.③Compared with the control group， the PMA group significantly reduced the basal respiration， maximum respiration and ATP generation of the C2C12 myotubes （,P,<,0.01）. ④Compared with the control group， the PMA group significantly up-regulated the gene expression levels of PKC-ε， PKC-β， PKC-θ and hypoxia ,HIF,-,2α, （,P,<,0.05，,P,<,0.01）.,Conclusions,2,PMA might inhibit cellular mitochondrial respiration and promote the expression of HIF-2α by activating the PKC pathway of C2C12 myotubes. HIF-2α could up-regulate the expression of autophagy-related proteins Beclin-1 and LC3BⅡ， and thus induce the autophagy of C2C12 myotubes. CYA might protect myotubes against PMA-induced autophagy by down-regulating the protein expressions of Beclin-1 and LC3BⅡ.

关键词

佛波酯次苷酸查尔酮自噬C2C12肌管

Keywords

phorbol estercorylifol AautophagyC2C12 myotubes

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