Naringin Attenuates Cerebral Ischemia-Reperfusion Injury Through Inhibiting Peroxynitrite-Mediated Mitophagy Activation

  • Jinghan Feng
  • Xingmiao Chen
  • Shengwen Lu
  • Wenting Li
  • Dan Yang
  • Weiwei Su
  • Xijun Wang
  • Jiangang Shen


Excessive autophagy/mitophagy plays important roles during cerebral ischemia-reperfusion (I/R) injury. Peroxynitrite (ONOO), a representative reactive nitrogen species, mediates excessive mitophagy activation and exacerbates cerebral I/R injury. In the present study, we tested the hypothesis that naringin, a natural antioxidant, could inhibit ONOO-mediated mitophagy activation and attenuate cerebral I/R injury. Firstly, we demonstrated that naringin possessed strong ONOO scavenging capability and also inhibited the production of superoxide and nitric oxide in SH-SY5Y cells exposed to 10 h oxygen-glucose-deprivation plus 14 h of reoxygenation or ONOO donor 3-morpholinosydnonimine conditions. Naringin also inhibited the expression of NADPH oxidase subunits and iNOS in rat brains subjected to 2 h ischemia plus 22 h reperfusion. Next, we found that naringin was able to cross the blood-brain barrier, and naringin decreased neurological deficit score, reduced infarct size, and attenuated apoptotic cell death in the ischemia-reperfused rat brains. Furthermore, naringin reduced 3-nitrotyrosine formation, decreased the ratio of LC3-II to LC3-I in mitochondrial fraction, and inhibited the translocation of Parkin to the mitochondria. Taken together, naringin could be a potential therapeutic agent to prevent the brain from I/R injury via attenuating ONOO-mediated excessive mitophagy.


Cerebral ischemia-reperfusion injury Mitophagy Naringin Nitrative stress Peroxynitrite 



This work was supported by the National Natural Science Foundation of China (No. 31570855), the Research Grants Council, Hong Kong SAR (No. 17118717, No. 17102915), and Areas of Excellence Scheme 2016/17 AoE/P-705/16.

Author Contributions

J.-H.F. designed and performed experiments, analyzed the data, and wrote the manuscript. J.-G.S. received funding, conceived and supervised the research designs and experiments, and co-wrote the manuscript. X.-M.C. detected ONOO with probes and analyzed data. S.-W.L. conducted pharmacokinetics study of naringin with UPLC-MS/MS. W.-T.L. detected ONOO scavenging capability of naringin with UPLC. D.Y. contributed to HKYellow-AM probe design and development. W.-W.S. supervised naringin compound extraction and designed animal experiments about naringin. X.-J.W. received funding, supervised and designed pharmacokinetics study, and designed the experiments.

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing interests.

Supplementary material

12035_2018_1027_MOESM1_ESM.docx (302 kb)
ESM 1 (DOCX 301 kb)


  1. 1.
    Mukherjee D, Patil CG (2011) Epidemiology and the global burden of stroke. World Neurosurg 76(6):S85–S90CrossRefPubMedGoogle Scholar
  2. 2.
    Tsivgoulis G, Katsanos AH, Alexandrov AV (2014) Reperfusion therapies of acute ischemic stroke: potentials and failures. Front Neurol 5:215. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Winquist RJ, Kerr S (1997) Cerebral ischemia-reperfusion injury and adhesion. Neurology 49(5 Suppl 4):S23–S26CrossRefPubMedGoogle Scholar
  4. 4.
    Gomis M, Dávalos A (2014) Recanalization and reperfusion therapies of acute ischemic stroke: what have we learned, what are the major research questions, and where are we headed? Front Neurol 5:226. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ohsumi Y (2014) Historical landmarks of autophagy research. Cell Res 24(1):9–23. CrossRefPubMedGoogle Scholar
  6. 6.
    Boya P, Reggiori F, Codogno P (2013) Emerging regulation and functions of autophagy. Nat Cell Biol 15(7):713–720. CrossRefPubMedGoogle Scholar
  7. 7.
    Zhu C, Wang X, Xu F, Bahr BA, Shibata M, Uchiyama Y, Hagberg H, Blomgren K (2005) The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia. Cell Death Differ 12(2):162–176. CrossRefPubMedGoogle Scholar
  8. 8.
    Liu C, Gao Y, Barrett J, Hu B (2010) Autophagy and protein aggregation after brain ischemia. J Neurochem 115(1):68–78. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tian F, Deguchi K, Yamashita T, Ohta Y, Morimoto N, Shang J, Zhang X, Liu N et al (2010) In vivo imaging of autophagy in a mouse stroke model. Autophagy 6(8):1107–1114CrossRefPubMedGoogle Scholar
  10. 10.
    Li H, Qiu S, Li X, Li M, Peng Y (2015) Autophagy biomarkers in CSF correlates with infarct size, clinical severity and neurological outcome in AIS patients. J Transl Med 13:359. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Dolman NJ, Chambers KM, Mandavilli B, Batchelor RH, Janes MS (2013) Tools and techniques to measure mitophagy using fluorescence microscopy. Autophagy 9(11):1653–1662. CrossRefPubMedGoogle Scholar
  12. 12.
    Chen W, Sun Y, Liu K, Sun X (2014) Autophagy: a double-edged sword for neuronal survival after cerebral ischemia. Neural Regen Res 9(12):1210–1216. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wei K, Wang P, Miao CY (2012) A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury. CNS Neurosci Ther 18(11):879–886. CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang XM, Zhang L, Wang G, Niu W, He Z, Ding L, Jia J (2015) Suppression of mitochondrial fission in experimental cerebral ischemia: The potential neuroprotective target of p38 MAPK inhibition. Neurochem Int 90:1–8. CrossRefPubMedGoogle Scholar
  15. 15.
    Baek SH, Noh AR, Kim KA, Akram M, Shin YJ, Kim ES, Yu SW, Majid A et al (2014) Modulation of mitochondrial function and autophagy mediates carnosine neuroprotection against ischemic brain damage. Stroke 45(8):2438–2443. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Feng J, Chen X, Shen J (2017) Reactive nitrogen species as therapeutic targets for autophagy: implication for ischemic stroke. Expert Opin Ther Targets 21(3):305–317. CrossRefPubMedGoogle Scholar
  17. 17.
    Feng J, Chen X, Guan B, Li C, Qiu J, Shen J (2018) Inhibition of Peroxynitrite-induced mitophagy activation attenuates cerebral ischemia-reperfusion injury. Mol Neurobiol.
  18. 18.
    Ferrer-Sueta G, Radi R (2009) Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. ACS Chem Biol 4(3):161–177. CrossRefPubMedGoogle Scholar
  19. 19.
    Chen XM, Chen HS, Xu MJ, Shen JG (2013) Targeting reactive nitrogen species: a promising therapeutic strategy for cerebral ischemia-reperfusion injury. Acta Pharmacol Sin 34(1):67–77. CrossRefPubMedGoogle Scholar
  20. 20.
    Ding R, Chen Y, Yang S, Deng X, Fu Z, Feng L, Cai Y, Du M et al (2014) Blood-brain barrier disruption induced by hemoglobin in vivo: involvement of up-regulation of nitric oxide synthase and peroxynitrite formation. Brain Res 1571:25–38. CrossRefPubMedGoogle Scholar
  21. 21.
    Liu B, Tewari AK, Zhang L, Green-Church KB, Zweier JL, Chen YR, He G (2009) Proteomic analysis of protein tyrosine nitration after ischemia reperfusion injury: mitochondria as the major target. Biochim Biophys Acta 1794(3):476–485. CrossRefPubMedGoogle Scholar
  22. 22.
    Vattemi G, Mechref Y, Marini M, Tonin P, Minuz P, Grigoli L, Guglielmi V, Klouckova I et al (2011) Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement. Mol Cell Proteomics 10(4):M110 002964. CrossRefPubMedGoogle Scholar
  23. 23.
    Liu K, Sun Y, Gu Z, Shi N, Zhang T, Sun X (2013) Mitophagy in ischaemia/reperfusion induced cerebral injury. Neurochem Res 38(7):1295–1300. CrossRefPubMedGoogle Scholar
  24. 24.
    Chen Y, Nie YC, Luo YL, Lin F, Zheng YF, Cheng GH, Wu H, Zhang KJ et al (2013) Protective effects of naringin against paraquat-induced acute lung injury and pulmonary fibrosis in mice. Food Chem Toxicol 58:133–140. CrossRefPubMedGoogle Scholar
  25. 25.
    Li P, Wang S, Guan X, Liu B, Wang Y, Xu K, Peng W, Su W et al (2013) Acute and 13 weeks subchronic toxicological evaluation of naringin in Sprague-Dawley rats. Food Chem Toxicol 60:1–9. CrossRefPubMedGoogle Scholar
  26. 26.
    Li P, Wang S, Guan X, Cen X, Hu C, Peng W, Wang Y, Su W (2014) Six months chronic toxicological evaluation of naringin in Sprague-Dawley rats. Food Chem Toxicol 66:65–75. CrossRefPubMedGoogle Scholar
  27. 27.
    Sharma M, Akhtar N, Sambhav K, Shete G, Bansal AK, Sharma SS (2015) Emerging potential of citrus flavanones as an antioxidant in diabetes and its complications. Curr Top Med Chem 15(2):187–195CrossRefPubMedGoogle Scholar
  28. 28.
    Rajadurai M, Stanely Mainzen Prince P (2006) Preventive effect of naringin on lipid peroxides and antioxidants in isoproterenol-induced cardiotoxicity in Wistar rats: biochemical and histopathological evidences. Toxicology 228(2–3):259–268. CrossRefPubMedGoogle Scholar
  29. 29.
    Nie YC, Wu H, Li PB, Luo YL, Long K, Xie LM, Shen JG, Su WW (2012) Anti-inflammatory effects of naringin in chronic pulmonary neutrophilic inflammation in cigarette smoke-exposed rats. J Med Food 15(10):894–900. CrossRefPubMedGoogle Scholar
  30. 30.
    Luo YL, Zhang CC, Li PB, Nie YC, Wu H, Shen JG, Su WW (2012) Naringin attenuates enhanced cough, airway hyperresponsiveness and airway inflammation in a guinea pig model of chronic bronchitis induced by cigarette smoke. Int Immunopharmacol 13(3):301–307. CrossRefPubMedGoogle Scholar
  31. 31.
    Gil M, Kim YK, Hong SB, Lee KJ (2016) Naringin decreases TNF-alpha and HMGB1 release from LPS-stimulated macrophages and improves survival in a CLP-induced Sepsis mice. PLoS One 11(10):e0164186. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Rajadurai M, Prince M, Stanely P (2006) Preventive effect of naringin on lipids, lipoproteins and lipid metabolic enzymes in isoproterenol-induced myocardial infarction in Wistar rats. J Biochem Mol Toxicol 20(4):191–197CrossRefPubMedGoogle Scholar
  33. 33.
    Han Y, Su J, Liu X, Zhao Y, Wang C, Li X (2016) Naringin alleviates early brain injury after experimental subarachnoid hemorrhage by reducing oxidative stress and inhibiting apoptosis. Brain Res Bull 133:42–50. CrossRefPubMedGoogle Scholar
  34. 34.
    Cui QJ, Wang LY, Wei ZX, Qu WS (2014) Continual naringin treatment benefits the recovery of traumatic brain injury in rats through reducing oxidative and inflammatory alterations. Neurochem Res 39(7):1254–1262. CrossRefPubMedGoogle Scholar
  35. 35.
    Gaur V, Aggarwal A, Kumar A (2009) Protective effect of naringin against ischemic reperfusion cerebral injury: possible neurobehavioral, biochemical and cellular alterations in rat brain. Eur J Pharmacol 616(1–3):147–154. CrossRefPubMedGoogle Scholar
  36. 36.
    Qin X, Sun ZQ, Zhang XW, Dai XJ, Mao SS, Zhang YM (2013) TLR4 signaling is involved in the protective effect of propofol in BV2 microglia against OGD/reoxygenation. J Physiol Biochem 69(4):707–718. CrossRefPubMedGoogle Scholar
  37. 37.
    Croslan DR, Schoell MC, Ford GD, Pulliam JV, Gates A, Clement CM, Harris AE, Ford BD (2008) Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia. Brain Res 1210:39–47. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, Kalyanaraman B (2003) Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 34(11):1359–1368CrossRefPubMedGoogle Scholar
  39. 39.
    Kalyanaraman B, Hardy M, Podsiadly R, Cheng G, Zielonka J (2017) Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling. Arch Biochem Biophys 617:38–47. CrossRefPubMedGoogle Scholar
  40. 40.
    Zhou X, He P (2011) Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate. Am J Physiol Heart Circ Physiol 301(1):H108–H114. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Lepiller S, Laurens V, Bouchot A, Herbomel P, Solary E, Chluba J (2007) Imaging of nitric oxide in a living vertebrate using a diamino-fluorescein probe. Free Radic Biol Med 43(4):619–627. CrossRefPubMedGoogle Scholar
  42. 42.
    Peng T, Chen XM, Gao L, Zhang T, Wang W, Shen JG, Yang D (2016) A rationally designed rhodamine-based fluorescent probe for molecular imaging of peroxynitrite in live cells and tissues. Chem Sci 7(8):5407–5413. CrossRefGoogle Scholar
  43. 43.
    Zhou L, Li F, Xu HB, Luo CX, Wu HY, Zhu MM, Lu W, Ji X et al (2010) Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat Med 16(12):1439–1443. CrossRefPubMedGoogle Scholar
  44. 44.
    Cipolla MJ, Chan SL, Sweet J, Tavares MJ, Gokina N, Brayden JE (2014) Postischemic reperfusion causes smooth muscle calcium sensitization and vasoconstriction of parenchymal arterioles. Stroke 45(8):2425–2430. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Mao L, Jia J, Zhou X, Xiao Y, Wang Y, Mao X, Zhen X, Guan Y et al (2013) Delayed administration of a PTEN inhibitor BPV improves functional recovery after experimental stroke. Neuroscience 231:272–281. CrossRefPubMedGoogle Scholar
  46. 46.
    Tsikas D (2007) Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: ppraisal of the Griess reaction in the L-arginine/nitric oxide area of research. J Chromatogr B Analyt Technol Biomed Life Sci 851(1–2):51–70. CrossRefPubMedGoogle Scholar
  47. 47.
    Chen HS, Chen XM, Feng JH, Liu KJ, Qi SH, Shen JG (2015) Peroxynitrite decomposition catalyst reduces delayed thrombolysis-induced hemorrhagic transformation in ischemia-reperfused rat brains. CNS Neurosci Ther 21(7):585–590. CrossRefPubMedGoogle Scholar
  48. 48.
    Kuhn DM, Sakowski SA, Sadidi M, Geddes TJ (2004) Nitrotyrosine as a marker for peroxynitrite-induced neurotoxicity: the beginning or the end of the end of dopamine neurons? J Neurochem 89(3):529–536. CrossRefPubMedGoogle Scholar
  49. 49.
    Li Q, Atochin D, Kashiwagi S, Earle J, Wang A, Mandeville E, Hayakawa K, d'Uscio LV et al (2013) Deficient eNOS phosphorylation is a mechanism for diabetic vascular dysfunction contributing to increased stroke size. Stroke 44(11):3183–3188. CrossRefPubMedGoogle Scholar
  50. 50.
    Chen J, Cui X, Zacharek A, Roberts C, Chopp M (2009) eNOS mediates TO90317 treatment-induced angiogenesis and functional outcome after stroke in mice. Stroke 40(7):2532–2538. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Grivennikova VG, Kareyeva AV, Vinogradov AD (2010) What are the sources of hydrogen peroxide production by heart mitochondria? Biochim Biophys Acta 1797(6–7):939–944. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Kang J, Pervaiz S (2012) Mitochondria: redox metabolism and dysfunction. Biochem Res Int 2012:896751. CrossRefPubMedPubMedCentralGoogle Scholar

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Authors and Affiliations

  1. 1.School of Chinese Medicine, LKS Faculty of MedicineThe University of Hong KongHong KongChina
  2. 2.National TCM Key Laboratory of Serum Pharmacochemistry, Key Lab of Chinmedomics, Department of Pharmaceutical AnalysisHeilongjiang University of Chinese MedicineHarbinChina
  3. 3.Morningside Laboratory for Chemical Biology and Department of ChemistryThe University of Hong KongHong KongChina
  4. 4.Guangzhou Quality R&D Center of Traditional Chinese Medicine, Guangdong Key Laboratory of Plant Resources, School of Life SciencesSun Yat-sen UniversityGuangzhouChina

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