Chinese Journal of Integrative Medicine

, Volume 25, Issue 7, pp 521–528 | Cite as

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway

  • Sheng-nan Dai
  • Ai-jie Hou
  • Shu-mei Zhao
  • Xiao-ming Chen
  • Hua-ting Huang
  • Bo-han Chen
  • Hong-liang KongEmail author
Original Article



To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway.


Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 μmol/L CoCl2), Gs-Rb1 group (200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group (500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group (500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot.


Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A (P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A (P<0.01) and was not affected by AICAR =0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B II, the LC3B II/I ratio and cathepsin B activity of hypoxia cardiomyocytes (P<0.05), each of these protein levels was significantly enhanced by Ara A (all P<0.01), but was not affected by AICAR (all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871).


Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.


cardiomyocytes ginsenosides-Rb1 hypoxia adenosine 5′-monophosphate-activated protein kinase autophagic flux 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science 2004;306:990–995.CrossRefGoogle Scholar
  2. 2.
    Yan L, Vatner DE, Kim SJ, Ge H, Masurekar M, Massover WH, et al. Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A 2005;12:13807–13812.CrossRefGoogle Scholar
  3. 3.
    Hamacher-Brady A, Brady NR, Gottlieb RA. Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes. J Biol Chem 2006;28:29776–29787.CrossRefGoogle Scholar
  4. 4.
    Dosenko VE, Nagibin VS, Tumanovska LV, Moibenko AA. Protective effect of autophagy in anoxia-reoxygenation of isolated cardiomyocyte. Autophagy 2006;2:305–306.CrossRefGoogle Scholar
  5. 5.
    Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, et al. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res 2007;100:914–922.CrossRefGoogle Scholar
  6. 6.
    Gui L, Liu B, Lu G. Hypoxia induces autophagy in cardiomyocytes via a hypoxia-inducible factor 1-dependent mechanism. Exp Ther Med 2016;11:2233–2239.CrossRefGoogle Scholar
  7. 7.
    Jiang QS, Huang XN, Yang GZ, Jiang XY, Zhou QX. Inhibitory effect of ginsenoside Rb1 on calcineurin signal pathway in cardiomyocyte hypertrophy induced by prostaglandin F2alpha. Acta Pharmacol Sin (Chin) 2007;28:1149–1154.CrossRefGoogle Scholar
  8. 8.
    Kong HL, Wang JP, Li ZQ, Zhao SM, Dong J, Zhang WW. Anti-hypoxic effect of ginsenoside Rb1 on neonatal rat cardiomyocytes is mediated through the specific activation of glucose transporter-4 ex vivo. Acta Pharmacol Sin (Chin) 2009;30:396–403.CrossRefGoogle Scholar
  9. 9.
    Kong HL, Li ZQ, Zhao YJ, Zhao SM, Zhu L, Li T, et al. Ginsenoside Rb1 protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening. Acta Pharmacol Sin (Chin) 2010;31:687–695.CrossRefGoogle Scholar
  10. 10.
    Kong HL, Li ZQ, Zhao SM, Yuan L, Miao ZL, Liu Y, et al. Apelin-APJ effects of ginsenoside-Rb1 depending on hypoxia-induced factor 1α in hypoxia neonatal cardiomyocytes. Chin J Integr Med 2015;21:139–146.CrossRefGoogle Scholar
  11. 11.
    Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005;25:1025–1040.CrossRefGoogle Scholar
  12. 12.
    Kim I, Rodriguez-Enriquez S, Lemasters JJ. Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys 2007;462:245–253.CrossRefGoogle Scholar
  13. 13.
    Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest 2005;115:2679–2688.CrossRefGoogle Scholar
  14. 14.
    Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis 2008;13:1–9.CrossRefGoogle Scholar
  15. 15.
    Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Selfeating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007;8:741–752.CrossRefGoogle Scholar
  16. 16.
    Espert L, Denizot M, Grimaldi M, Robert-Hebmann V, Gay B, Varbanov M, et al. Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4. J Clin Invest 2006;116: 2161–2172.CrossRefGoogle Scholar
  17. 17.
    Moretti L, Cha YI, Niermann KJ, Lu B. Switch between apoptosis and autophagy: radiation-induced endoplasmic reticulum stress? Cell Cycle 2007;6:793–798.CrossRefGoogle Scholar
  18. 18.
    Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ 2006;13:363–373.CrossRefGoogle Scholar
  19. 19.
    Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 2007;3:452–460.CrossRefGoogle Scholar
  20. 20.
    Xie Z, Nair U, Klionsky DJ. Atg8 controls phagophore expansion during autophagosome formation. Mol Biol Cell 2008;19:3290–3298.CrossRefGoogle Scholar
  21. 21.
    Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 2001;152:657–668.CrossRefGoogle Scholar
  22. 22.
    Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T. Beclinphosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep 2001;2:330–335.CrossRefGoogle Scholar
  23. 23.
    Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, et al. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res 2007;100:914–922.CrossRefGoogle Scholar
  24. 24.
    Valentim L, Laurence KM, Townsend PA, Carroll CJ, Soond S, Scarabelli TM, et al. Urocortin inhibits Beclin1- mediated autophagic cell death in cardiac myocytes exposed to ischaemia/reperfusion injury. J Mol Cell Cardiol 2006;40:846–852.CrossRefGoogle Scholar
  25. 25.
    Ma X, Liu H, Foyil SR, Godar RJ, Weinheimer CJ, Hill JA, et al. Impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury. Circulation 2012;125:3170–3181.CrossRefGoogle Scholar
  26. 26.
    Zeng M, Wei X, Wu Z, Li W, Li B, Zhen Y, et al. NF-κBmediated induction of autophagy in cardiac ischemia/ reperfusion injury. Biochem Biophys Res Commun 2013;436:180–185.CrossRefGoogle Scholar
  27. 27.
    Feng Y, Yao Z, Klionsky DJ. How to control self-digestion: transcriptional, post-transcriptional, and post-translational regulation of autophagy. Trends Cell Biol 2015;25:354–363.CrossRefGoogle Scholar
  28. 28.
    Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 2009;10:458–467.CrossRefGoogle Scholar
  29. 29.
    Shintani T, Huang WP, Stromhaug PE, Klionsky DJ. Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway. Dev Cell 2002;3:825–837.CrossRefGoogle Scholar
  30. 30.
    Xie Z, Nair U, Klionsky DJ. Atg8 controls phagophore expansion during autophagosome formation. Mol Biol Cell 2008;19:3290–3298.CrossRefGoogle Scholar
  31. 31.
    Kaufmann A, Beier V, Franquelim HG, Wollert T. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell 2014;156:469–481.CrossRefGoogle Scholar
  32. 32.
    Zhang J, He Z, Xiao W, Na Q, Wu T, Su K, et al. Overexpression of BAG3 attenuates hypoxia-induced cardiomyocyte apoptosis by inducing autophagy. Cell Physiol Biochem 2016;39:491–500.CrossRefGoogle Scholar
  33. 33.
    Gao YH, Qian JY, Chen ZW, Fu MQ, Xu JF, Xia Y, et al. Suppression of Bim by microRNA-19a may protect cardiomyocytes against hypoxia-induced cell death via autophagy activation. Toxicol Lett 2016;257:72–83.CrossRefGoogle Scholar
  34. 34.
    Hsieh DJ, Kuo WW, Lai YP, Shibu MA, Shen CY, Pai P, et al. 17β-Estradiol and/or estrogen receptor β attenuate the autophagic and apoptotic effects induced by prolonged hypoxia through HIF-1α-mediated BNIP3 and IGFBP-3 signaling blockage. Cell Physiol Biochem 2015;36:274–284.CrossRefGoogle Scholar
  35. 35.
    Komatsu M, Kageyama S, Ichimura Y. P62/SQSTM1/A170: physiology and pathology. Pharmacol Res 2012;66:457–462.CrossRefGoogle Scholar
  36. 36.
    Layfield R, Cavey JR, Najat D, Long J, Sheppard PW, Ralston SH, et al. p62 mutations, ubiquitin recognition and Paget’s disease of bone. Bio Soc Trans 2006;34:735–737.CrossRefGoogle Scholar
  37. 37.
    Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, et al. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell 2010;141:1146–1158.CrossRefGoogle Scholar
  38. 38.
    Moscat J, Diaz-Meco MT, Wooten MW. Signal integration and diversification through the p62 scaffold protein. Trends Biol Sci 2007;32:95–100.CrossRefGoogle Scholar
  39. 39.
    Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007;282:24131–24145.CrossRefGoogle Scholar
  40. 40.
    Mizushima N, Hara T. Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration? Autophagy 2006;2:302–304.CrossRefGoogle Scholar
  41. 41.
    Bjørkøy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 2005;171:603–614.CrossRefGoogle Scholar
  42. 42.
    Wang Q, Yang L, Hua Y, Nair S, Xu X, Ren J. AMP-activated protein kinase deficiency rescues paraquat-induced cardiac contractile dysfunction through an autophagy-dependent mechanism. Toxicol Sci 2014;142:6–20.CrossRefGoogle Scholar
  43. 43.
    Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003;115:577–590.CrossRefGoogle Scholar
  44. 44.
    Tripathi DN, Chowdhury R, Trudel LJ, Tee AR, Slack R S, Walker CL, et al. Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1. Proc Natl Acad Sci U S A 2013;110: E2950–E2957.CrossRefGoogle Scholar

Copyright information

© The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Sheng-nan Dai
    • 1
  • Ai-jie Hou
    • 1
  • Shu-mei Zhao
    • 2
  • Xiao-ming Chen
    • 1
  • Hua-ting Huang
    • 1
  • Bo-han Chen
    • 3
  • Hong-liang Kong
    • 1
    Email author
  1. 1.Department of Cardiology, the People’s Hospital of China Medical Universitythe People’s Hospital of Liaoning ProvinceShenyangChina
  2. 2.International Education CollegeShenyang Normal UniversityShenyangChina
  3. 3.Department of Cardiologythe First Affiliated Hospital of Dalian Medical UniversityDalianChina

Personalised recommendations