Cell Stress and Chaperones

, Volume 24, Issue 6, pp 1091–1099 | Cite as

Protodioscin protects PC12 cells against oxygen and glucose deprivation-induced injury through miR-124/AKT/Nrf2 pathway

  • Kun Shu
  • Yuelin ZhangEmail author
Original Paper


The purpose of the current study was to demonstrate the neuroprotective effect of protodioscin (Prot) in an in vitro model of ischemia/reperfusion (I/R) and investigate the underlying molecular mechanism. After PC12 cells were exposed to oxygen and glucose deprivation (OGD) reperfusion, PI staining by flow cytometry was used to quantify the rate of apoptosis. The levels of hypoxia-inducible factor 1-alpha (HIF-1α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using commercially available kits. Intracellular reactive oxygen species (ROS) level was detected using the 20,70-dichlorodihy-drofluorescein diacetate (DCFH-DA) fluorescence assay. The expression levels of heat-shock proteins (HSP), PI3K, AKT, Nrf2, and miR-124 were tested by western blot or quantitative PCR. Prot significantly attenuated oxygen–glucose deprivation/reperfusion (OGD/R)-induced apoptotic death. Prot also reduced the oxidative stress as revealed by increasing the activities of SOD and GSH-Px, decreasing the levels of ROS and MDA. Moreover, mechanism investigations suggested that Prot prevented the decrease of HSP70, HSP32 (hemeoxygenase-1, HO-1), and PI3K protein expression, phosphorylation of AKT, and the accumulation of nuclear Nrf2. The level of miR-124 was decreased in PC12 cells, which was also effectively reversed by Prot treatment. Prot protected PC12 cells against OGD/R-induced injury through inhibiting oxidative stress and apoptosis, which could be associated with increasing HSP proteins expression via activating PI3K/AKT/Nrf2 pathway and miR-124 modulation.


Protodioscin Antioxidant OGD/R Heat-shock protein miR-124 PI3K/AKT Nrf2 





Oxygen–glucose deprivation/reperfusion


Hypoxia-inducible factor 1-alpha


Superoxide dismutase


Glutathione peroxidase




Reactive oxygen species


Heat shock proteins




Nuclear factor (erythroid-derived 2)-like 2


Phosphatidylinositol-3 kinase


Funding information

This work is supported by the Xi’an Jiaotong University, Xi’an, Shanxi, China.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Chan PH (1996) Role of in ischemic brain damage. Stroke 27(6):1124–1129PubMedGoogle Scholar
  2. Chen K, Gunter K, Maines MD (2000) Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death. J Neurochem 75:304–313PubMedGoogle Scholar
  3. Chen SD, Yang DI, Lin TK, Shaw FZ, Liou CW, Chuang YC (2011) Roles of oxidative stress, apoptosis, PGC-1α and mitochondrial biogenesis in cerebral ischemia. Int J Mol Sci 12:7199–7215PubMedPubMedCentralGoogle Scholar
  4. Chen M, Yan R, Zhou K, Li X, Zhang Y, Liu C, Jiang M, Ye H, Meng X, Pang N, Zhao L, AKT-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia. Proc Natl Acad Sci U S A 2018a Nov 06;115(45)Google Scholar
  5. Chen G, Chen X, Niu C, Huang X, An N, Sun J, Huang S, Ye W, Li S, Shen Y, Liang J (2018b) Baicalin alleviates hyperglycemia-induced endothelial impairment 1 via Nrf2. J Endocrinol 01Google Scholar
  6. Cheng LC, Pastrana E, Tavazoie M, Doetsch F (2009) miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci 12:399–408PubMedPubMedCentralGoogle Scholar
  7. Chu S-f, Zhang Z, Zhou X, He W-b, Chen C, Luo P, Liu D-d, Ai Q-d, Gong H-f, Wang Z-z, Sun H-s, Feng Z-p, Chen N-h (2019) Ginsenoside Rg1 protects against ischemic/reperfusion-induced neuronal injury through miR-144/Nrf2/ARE pathway. Acta Pharmacol Sin 40:13–25PubMedGoogle Scholar
  8. Cui HY, Zhang XJ, Yang Y, Zhang C, Zhu CH, Miao JY, Chen R (2018) Rosmarinic acid elicits neuroprotection in ischemic stroke Nrf2 and heme oxygenase 1 signaling. Neural Regen Res 13(12)Google Scholar
  9. Diener HC, Katsarava Z, Weimar C (2008) Headache associated with ischemic cerebrovascular disease. Rev Neurol (Paris) 164(10):819–824Google Scholar
  10. Elnaz MA, Mahdavi M, Fard FJ, Chamani S, Farajdokht F, Karimi P (2018) Metformin protects PC12 cells against oxygenglucose deprivation/reperfusion injury. Toxicol Mech Methods:28(8)Google Scholar
  11. Fei L, Huang X, Luo Z, He J, Haider F, Song C, Peng L, Chen T, Buling W (2019) Hypoxia-activated PI3K/Akt inhibits oxidative stress via the regulation of reactive oxygen species in human dental pulp cells. Oxidative Med Cell Longev 2019:6595189Google Scholar
  12. Foresti R, Sarathchandra P, Clark JE, Green CJ, Motterlini R (1999) Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis. Biochem J 339:729–736PubMedPubMedCentralGoogle Scholar
  13. Gao FB (2008) Posttranscriptional control of neuronal development by microRNA networks. Trends Neurosci 31:20–26PubMedGoogle Scholar
  14. Grell AS, Thigarajah R, Edvinsson L, Samraj AK (2014) Regulatory mechanism of endothelin receptor B in the cerebral arteries after focal cerebral ischemia. PLoS One 9:e113624PubMedPubMedCentralGoogle Scholar
  15. Haiying L, Gao A, Feng D, Yang W, Zhang L, Cui Y, Li B, Wang Z, Chen G (2014) Evaluation of the protective potential of brain microvascular endothelial cell autophagy on blood–brain barrier integrity during experimental cerebral ischemia–reperfusion injury. Transl Stroke Res 5:618–626Google Scholar
  16. Haleh M, Najafzadeh N, Vardin MM (2018) miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9. J Cell Physiol:1–10Google Scholar
  17. Hamzei Taj S, Kho W, Riou A, Wiedermann D, Hoehn M (2016) MiRNA-124 induces neuroprotection and functional improvement after focal cerebral ischemia. Biomaterials 91Google Scholar
  18. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB (2005) Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4(12):988–1004PubMedGoogle Scholar
  19. Hu S, Wu Y, Zhao B, Hu H, Zhu B, Sun Z, Li P, Du S (2018a) Saponins protect cerebral microvascular endothelial cells against oxygen-glucose deprivation/reperfusion-induced barrier dysfunction via activation of PI3K/AKT/Nrf2 antioxidant signaling pathway. Molecules 26, 23(11)PubMedCentralGoogle Scholar
  20. Hu X, Liu J, Zhao G, Zheng J, Qin X (2018b) Long non-coding RNA GAS5 aggravates hypoxia injury in PC-12 cells via down-regulating miR-124. J Cell Biochem 119(8)PubMedGoogle Scholar
  21. Jiang W, Meng L, Xu G, Lv C, Wang H, Tian H, Chen R, Jiao B, Wang B, Huang C (2018) Wentilactone A induces cell apoptosis by targeting AKR1C1 gene via the IGF-1R/IRS1/PI3K/AKT/Nrf2/FLIP/Caspase-3 signaling pathway in small cell lung cancer. Oncol Lett 16(5)Google Scholar
  22. Jin H, Li Q, Cao F, Wang SN, Wang RT, Wang Y, Tan QY, Li CR, Zou H, Wang D (2017) Xu CX, miR-124 inhibits lung tumorigenesis induced by K-ras mutation and NNK. Mol Ther Nucleic Acids 15:9Google Scholar
  23. Kilic U, Caglayan AB, Beker MC, Gunal MY, Caglayan B, Yalcin E, Kelestemur T, Gundogdu RZ, Yulug B, Yilmaz B, Kerman BE, Kilic E (2017) Particular phosphorylation of PI3K/Akt on Thr308 via PDK-1 and PTEN mediates melatonin’s neuroprotective activity after focal cerebral ischemia in mice. Redox Biol 12:657–665PubMedPubMedCentralGoogle Scholar
  24. Lee HA, Kim JE, Sung JE, Yun WB, Kim DS, Lee HS, Hong JT, Hwang DY (2018) Asparagus cochinchinensis stimulates release of nerve growth factor and abrogates oxidative stress in the Tg2576 model for Alzheimer’s disease. BMC Complement Altern Med 06:18(1)Google Scholar
  25. Leker RR, Gai N, Mechoulam R, Ovadia H (2003) Drug-induced hypothermia reduces ischemic damage: effects of the cannabinoid HU-210. Stroke. 34(8):2000–2065PubMedGoogle Scholar
  26. Liu HJ, Yang JP, Wang CH, Liu RC, Li Y, Li CY (2009) Endoplasmic reticulum in the penumbra following middle cerebral artery occlusion in the rabbit. Neurol Sci 30(3):227–232PubMedGoogle Scholar
  27. Liu X, Li F, Zhao S, Luo Y, Kang J, Zhao H, Yan F, Li S, Ji X, MicroRNA-124-mediated regulation of inhibitory member of apoptosis-stimulating protein of p53 family in experimental stroke. Stroke 2013;44(7)PubMedGoogle Scholar
  28. Liu X, Zhao J, Liu Q, Xiong X, Zhang Z, Jiao Y, Li X, Liu B, Li Y, Lu Y (2016) MicroRNA-124 promotes hepatic triglyceride accumulation through targeting tribbles homolog 3. Sci Rep 11(15):6Google Scholar
  29. Liu JY, Hou YL, Cao R, Qiu HX, Cheng GH, Tu R, Wang L, Zhang JL, Liu D (2017) Protodioscin ameliorates oxidative stress, inflammation and histology outcome in Complete Freund’s adjuvant induced arthritis rats. Apoptosis 22(11)PubMedGoogle Scholar
  30. Martin E, Tosi R, Bocanegra V, Manucha W, Lorenzo AG, Patricia G (2011) VallésThe Nrf2–Keap1 cellular defense pathway and heat shock protein 70 (Hsp70) response. Role in protection against oxidative stress in early neonatal unilateral ureteral obstruction (UUO). Cell Stress Chaperones 16:57–68Google Scholar
  31. Nanda YP, Chatterjee A, Purohit AK, Dia llo A, Innui K, Sharma RN, Lib eau G, Theva saga yam JA, Bruning A, Kitching RP, Anderson J, Barrett T, Taylor WP (1996) The isolation of peste des petits ruminants virus from northern India. Vet Microbiol 51:207–216PubMedGoogle Scholar
  32. Panahian N, Yoshiura M, Maines MD (1999) Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice. J Neurochem 72:1187–1203PubMedGoogle Scholar
  33. Qiu LL, Niu H, Huang W (2011) Ultrasonic and fermented pretreatment technology for diosgenin production from Dioscorea zingiberensis C.H. Wright. Chem Eng Res Des 89:239–247Google Scholar
  34. Qiu L, Jin Z, Xu Z, Yang H, Liang L, Li G, Li F, Shaoli G, Zong S, Zhou J, Liang C, Wang Z, Xiao W (2019) Antioxidant effects of ginkgolides and bilobalide against cerebral ischemia injury by activating the Akt/Nrf2 pathway in vitro and in vivo. Cell Stress Chaperones 24:441–452Google Scholar
  35. Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR, Sharp FR (2000) Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Ann Neurol 47:782–791PubMedGoogle Scholar
  36. Santana PM, Miranda M, Gutiérrez Y, García G, Orellana T, Orellana A (2009) Antinflammatory effect and chemical composition of bursera graveolens Triana & Planch.branch oil (palo santo) from Ecuador. Rev Cuba Plantas Med 14:45–53Google Scholar
  37. Santos TMM, Sinzato YK, Gallego FQ, Iessi IL, Volpato T, Dallaqua B, Damasceno DC (2015) Extracellular HSP70 levels in diabetic environment in rats. Cell Stress Chaperones 20:595–603PubMedPubMedCentralGoogle Scholar
  38. Shaonan H, Wu Y, Zhao B, Hu H, Zhu B, Sun Z, Pengyue L, Du S (2018) Saponins protect cerebral microvascular endothelial cells against oxygen-glucose deprivation/reperfusion-induced barrier dysfunction via activation of PI3K/AKT/Nrf2 antioxidant signaling pathway. Molecules 23:2781PubMedCentralGoogle Scholar
  39. Sharp FR, Massa SM, Swanson RA (1999) Heat-shock protein protection. Trends Neurosci 22:97–99PubMedGoogle Scholar
  40. Song S, Fajol A, Chen Y, Ren B, Shi S (2018) Anticonvulsive effects of protodioscin against pilocarpine-induced epilepsy. Eur J Pharmacol 15:833Google Scholar
  41. Sun Y, Gui H, Li Q, Luo ZM, Zheng MJ, Duan JL, Liu X (2013) MicroRNA-124 protects neurons against apoptosis in cerebral ischemic stroke. CNS Neurosci Ther 19(10)Google Scholar
  42. Sun M, Hou X, Guang R, Zhang Y, Cheng H (2018a) Dynamic changes in miR-124 levels in patients with acute cerebral infarction. Int J NeurosciGoogle Scholar
  43. Sun P, Nie X, Chen X, Yin L, Luo J, Sun L, Wan C, Jiang S (2018b) Nrf2 signaling elicits a neuroprotective role against PFOS-mediated oxidative damage and apoptosis. Neurochem Res 43(12)PubMedGoogle Scholar
  44. Szliszka E, Czuba ZP, Bronikowska J, Mertas A, Paradysz A, Krol W (2011) Ethanolic extract of propolis augments TRAIL-induced apoptotic death in prostate cancer cells. Evid Based Complement Alternat Med eCAM 2011:535172PubMedGoogle Scholar
  45. Tang W, Dong W, Xie P, Cheng P, Bai S, Ren Y, Wang G, Chen X, Cui C, Zhuang Y, Huang W (2015) The effect of pre-condition cerebella fastigial nucleus electrical stimulation within and beyond the time window of thrombolytic on ischemic stroke in the rats. PLoS One 10:e0128447PubMedPubMedCentralGoogle Scholar
  46. Tulis DA, Durante W, Peyton KJ, Evans AJ, Schafer AI (2001) Heme oxygenase-1 attenuates vascular remodeling following balloon injury in rat carotid arteries. Atherosclerosis 155:113–122PubMedGoogle Scholar
  47. Wang TJ, Liu ZB, Li J, Zhong M, Li JP et al (2007) Determination of protodioscin in rat plasma by liquid chromatography–tandem mass spectrometry. J Chromatogr B 848:363–368Google Scholar
  48. Wang C, Zhijie W, Jiang G, Liu H (2017) Neuroprotective mechanisms of miR-124 activating PI3K/AKT signaling pathway in ischemic stroke. Exp Ther Med 13(6)PubMedPubMedCentralGoogle Scholar
  49. Wu DP, Zhang JL, Wang JY, Cui MX, Jia JL, Liu XH, Liang QD (2017) MiR-1246 promotes LPS-induced inflammatory injury in chondrogenic cells ATDC5 by targeting HNF4γ. Cell Physiol Biochem 43(5)PubMedGoogle Scholar
  50. Xanthoudakis S, Nicholson DW (2000) Heat-shock proteins as death determinants. Nat Cell Biol 2:E163–E165PubMedGoogle Scholar
  51. Xi JS, Wang YF, Long XX, Ma Y (2018) Mangiferin potentiates neuroprotection by isoflurane in neonatal hypoxic brain injury by reducing oxidative stress and activation of Phosphatidylinositol-3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling. Med Sci Monit 19:24Google Scholar
  52. Xiao J, Chen B, Wang Q, Yang L, Guo H (2018) Paeonin extracted from potatoes protects gastric epithelial cells from HO-induced oxidative damage in vitro by PI3K/AKT-mediated Nrf2 signaling pathway. Sci Rep 18:8(1)Google Scholar
  53. Xu X, Kim JJ, Li Y, Xie J, Shao C, Wei JJ (2018) Oxidative stress-induced miRNAs modulate AKT signaling and promote cellular senescence in uterine leiomyoma. J Mol Med 96(10)PubMedGoogle Scholar
  54. Xue Q, Yu C, Wang Y, Liu L, Zhang K, Fang C, Liu F, Bian G, Song B, Yang A, Ju G, Wang J (2016) miR-9 and miR-124 synergistically affect regulation of dendritic branching via the AKT/GSK3β pathway by targeting Rap2a. Sci Rep 05(25):6Google Scholar
  55. Yao L, Ye Y, Mao H, Lu F, He X, Lu G, Zhang S (2018) MicroRNA-124 regulates the expression of MEKK3 in the inflammatory pathogenesis of Parkinson’s disease. J Neuroinflammation 12:15(1)Google Scholar
  56. Yenari MA, Giffard RG, Sapolsky RM, Steinberg GK (1999) The neuroprotective potential of heat shock protein 70 (HSP70). Mol Med Today 5:525–531PubMedGoogle Scholar
  57. Zhang X, Xue X, Xian L, Guo Z, Ito Y, Sun W (2016) Potential neuroprotection of protodioscin against cerebral ischemia-reperfusion injury in rats through intervening inflammation and apoptosis. Steroids 09:113Google Scholar
  58. Zhang R, Xu M, Wang Y, Xie F, Zhang G, Qin X (2017) Nrf2-a promising therapeutic target for defensing against oxidative stress in stroke. Mol Neurobiol 54:6006–6017PubMedGoogle Scholar
  59. Zhang W, Song JK, Yan R, Li L, Xiao ZY, Zhou WX, Wang ZZ, Xiao W, Du GH (2018) Diterpene ginkgolides protect against cerebral ischemia/reperfusion damage in rats by activating Nrf2 and CREB through PI3K/AKT signaling. Acta Pharmacol Sin 39(8)PubMedPubMedCentralGoogle Scholar
  60. Zhong Y, Zhu Y, He T, Li W, Li Q, Miao Y (2019) Brain-derived neurotrophic factor inhibits hyperglycemia-induced apoptosis and downregulation of synaptic plasticity-related proteins in hippocampal neurons via the PI3K/AKT pathway. Int J Mol Med 43(1)Google Scholar

Copyright information

© Cell Stress Society International 2019

Authors and Affiliations

  1. 1.Department of MedicineXi’an Jiaotong UniversityXi’anChina
  2. 2.Department of NeurosurgeryThe Third Affiliated Hospital of Xi’an Jiaotong UniversityXi’an CityChina

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