佛波酯诱导C2C12肌管自噬及corylifol A的保护作用研究

邝计霞; 沈强; 刘璇

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

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佛波酯诱导C2C12肌管自噬及corylifol A的保护作用研究

实验研究 | 更新时间：2022-08-29

- 佛波酯诱导C2C12肌管自噬及corylifol A的保护作用研究
- Study on effects of phorbol ester on inducing autophagy of C2C12 myotubes and protective effects of corylifol A
- 上海中医药大学学报 2022年36卷第2期页码：49-58
- 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：
  
  邝计霞，女，在读硕士生，主要从事中药小分子抗肿瘤恶病质作用研究
  沈强，男，在读博士生，主要从事肿瘤恶病质的病理机制研究（本文贡献与第一作者等同
  刘璇，研究员，博士生导师；E-mail：xuanliu@shutcm.edu.cn
- 基金信息：
  
  国家自然科学基金资助项目(81873056)
- DOI：10.16306/j.1008-861x.2022.02.009
  中图分类号：
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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

references

LEVINE B，KROEMER G. Biological functions of autophagy genes：a disease perspective［J］. Cell， 2019， 176（1-2）： 11-42.

MIZUSHIMA N，KOMATSU M. Autophagy：renovation of cells and tissues［J］. Cell， 2011， 147（4）： 728-741.

KIM K H，LEE M S. Autophagy-a key player in cellular and body metabolism［J］. Nat Rev Endocrinol， 2014， 10（6）： 322-337.

MASIERO E，AGATEA L，MAMMUCARI C，et al. Autophagy is required to maintain muscle mass［J］. Cell Metab， 2009， 10（6）： 507-515.

RABEN N，HILL V，SHEA L，et al. Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease［J］. Hum Mol Genet， 2008， 17（24）： 3897-3908.

DE PALMA C，MORISI F，CHELI S，et al. Autophagy as a new therapeutic target in Duchenne muscular dystrophy［J］. Cell Death Dis， 2012， 3（11）： e418.

GARCÍA-PRAT L， MARTÍNEZ-VICENTE M， PERDIGUERO E，et al. Autophagy maintains stemness by preventing senescence［J］. Nature， 2016， 529（7584）： 37-42.

PENNA F，BALLARÒ R，MARTINEZ-CRISTOBAL P，et al. Autophagy exacerbates muscle wasting in cancer cachexia and impairs mitochondrial function［J］. J Mol Biol，2019，431（15）：2674-2686.

WANG T，LIU C，JIA L L. The roles of PKCs in regulating autophagy［J］.J Cancer Res Clin Oncol， 2018， 144（12）： 2303-2311.

YU J H， LIU C Y，ZHENG G B，et al. Pseudolaric acid B induced cell cycle arrest， autophagy and senescence in murine fibrosarcoma l929 cell［J］. Int J Med Sci， 2013， 10（6）： 707-718.

ROBERT G，BEN SAHRA I，PUISSANT A，et al. Acadesine kills chronic myelogenous leukemia （CML） cells through PKC-dependent induction of autophagic cell death［J］ .PLoS One， 2009， 4（11）： e7889.

CLAVEL S，SIFFROI-FERNANDEZ S，COLDEFY A S，et al. Regulation of the intracellular localization of Foxo3a by stress-activated protein kinase signaling pathways in skeletal muscle cells［J］ .Mol Cell Biol， 2010， 30（2）： 470-480.

白洁，孟军，刘艳辉，等. Ox-LDL对PMA诱导的THP-1巨噬细胞自噬的影响［J］. 中南医学科学杂志，2014， 42（3）： 217-221.

HAN Y，LEE H，LI H，et al. Corylifol A from Psoralea corylifolia L. enhances myogenesis and alleviates muscle atrophy［J］.Int J Mol Sci， 2020， 21（5）： 1571.

李亚静，沈强，邝计霞，等. C26结肠癌细胞与RAW264.7巨噬细胞共培养体系诱导C2C12细胞肌管萎缩及甘草多糖的保护作用［J］. 上海中医药大学学报，2021， 35（4）： 45-53.

WANG Y，HAO C L，ZHANG Z H，et al. Valproic acid increased autophagic flux in human multiple myeloma cells in vitro［J］. Biopha， 2020， 127： 110167.

罗胜男，苏震，黄佩佩，等. 高剂量葡萄糖对小鼠肌管细胞自噬表达的影响［J］. 温州医科大学学报，2019， 49（5）： 313-320.

SANDRI M. Autophagy in skeletal muscle［J］.FEBS Let， 2010， 584（7）： 1411-1416.

ZHAO J，BRAULT J J，SCHILD A，et al. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells［J］. Cell Metab，2007， 6（6）： 472-483.

DOYLE A，ZHANG G，ABDEL FATTAH E A，et al. Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways［J］. FASEB J， 2011， 25（1）： 99-110.

LOKIREDDY S， WIJESOMA I W， BONALA S， et al. Myostatin is a novel tumoral factor that induces cancer cachexia［J］. Biochem J， 2012， 446（1）： 23-36.

COSPER P F，LEINWAND L A. Cancer causes cardiac atrophy and autophagy in a sexually dimorphic manner［J］. Cancer Res， 2011， 71（5）： 1710-1720.

PAUL P K，GUPTA S K，BHATNAGAR S，et al. Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice［J］. Cell Biol， 2010， 191（7）： 1395-1411.

BROWN D I，GRIENDLING K K. Nox proteins in signal transduction［J］. Free Radic Biol Med， 2009， 47（9）： 1239-1253.

JOSEPH L C，BARCA E，SUBRAMANYAM P，et al. Inhibition of NAPDH oxidase 2 （NOX2） prevents oxidative stress and mitochondrial abnormalities caused by saturated fat in cardiomyocytes［J］. PLoS One， 2016， 11（1）： e0145750.

JIN Q，JIANG Y H，FU L Z，et al. Wenxin Granule Ameliorates Hypoxia/Reoxygenation-Induced Oxidative Stress in Mitochondria via the PKC-δ/NOX2/ROS Pathway in H9c2 Cells［J］. Oxid Med Cell Longev， 2020， 2020： 3245483.

WANG Y，BISWAS G，PRABU S K，et al. Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Calpha in C2C12 myocytes［J］. Biochem Pharmacol， 2006， 72（7）： 881-892.

TORMOS K V，CHANDEL N S. Inter-connection between mitochondria and HIFs［J］. J Cell Mol Med，2010，14（4）：795-804.

LI H，SATRIANO J，THOMAS J L，et al. Interactions between HIF-1α and AMPK in the regulation of cellular hypoxia adaptation in chronic kidney disease［J］. Am J Physiol Renal Physiol， 2015， 309（5）： F414-F428.

WALTER K M，SCHÖNENBERGER M J，TRÖTZMÜLLER M，et al. Hif-2α promotes degradation of mammalian peroxisomes by selective autophagy［J］. Cell Metab， 2014， 20（5）： 882-897.

SCHÖNENBERGER M J，KREK W，KOVACS W J. EPAS1/HIF-2α is a driver of mammalian pexophagy［J］. Autophagy， 2015， 11（6）： 967-969.

LEE S J，KIM H P，JIN Y， et al. Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis［J］. Autophagy，2011， 7（8）： 829-839.

KOUL B， TAAK P， KUMAR A， et al. Genus Psoralea： A review of the traditional and modern uses， phytochemistry and pharmacology［J］. J Ethnopharmacol， 2019（232）： 201-226.

PAN X，XU K，QIU X，et al. The Extract of Fructus Psoraleae promotes viability and cartilaginous formation of rat chondrocytes In Vitro［J］. Evid Based Complement Alternat Med， 2016， 2016： 2057631.

周倚墨，张建宁，单中书. 补骨脂提取物干预骨质疏松模型大鼠骨密度及骨生物力学的变化［J］. 中国组织工程研究，2020， 24（2）： 165-170.

TSAI M H， HUANG G S， HUNG Y C，et al. Psoralea corylifolia extract ameliorates experimental osteoporosis in ovariectomized rats［J］. Am J Chin Med， 2007， 35（4）： 669-680.

YIN S，FAN C Q，WANG Y，et al. Antibacterial prenylflavone derivatives from Psoralea corylifolia，and their structure-activity relationship study［J］. Bioorg Med Chem，2004，12（16）： 4387-4392.

LI N，MIAO J，LI J，et al. Enzymatic synthesis of novel corylifol A glucosides via a UDP-glycosyltransferase［J］. Carbohydr Res，2017（446-447）： 61-67.

LEE A，YANG H，KIM T，et al. Identification and pharmacokinetics of bioavailable anti-resorptive phytochemicals after oral administration of Psoralea corylifolia L.［J］. Biomed Pharmacother， 2021（144）： 112300.

MATSUDA H，KIYOHARA S，SUGIMOTO S，et al. Inhibitors from the seeds of psoralea corylifolia on production of nitric oxide in lipopolysaccharide-activated macrophages［J］. Biol Pharm Bull， 2009， 32（1）： 147-149.

LEE S W，YUN B R，KIM M H，et al. Phenolic compounds isolated from Psoralea corylifolia inhibit IL-6-induced STAT3 activation［J］. Planta Med， 2012， 78（9）： 903-906.

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