Journal of Physiology and Biochemistry

, Volume 74, Issue 2, pp 335–343 | Cite as

Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury

  • Jing Cai
  • Yu Jiang
  • Meng Zhang
  • Hongting Zhao
  • Huihui Li
  • Kuanyu Li
  • Xin ZhangEmail author
  • Tong QiaoEmail author
Original Article


Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically.


SS-31 Hind limb ischemia-reperfusion injury Reactive oxygen species Mitochondria Inflammation 


Funding information

This work was supported by the National Natural Science Foundation of China (grant number: 81370387).

Compliance with ethical standards

Experiments were approved by the Animal Investigation Ethic Committee of Nanjing University and were carried out in accordance with the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13105_2018_617_MOESM1_ESM.pdf (401 kb)
ESM 1 (PDF 400 kb)


  1. 1.
    Atahan E, Ergun Y, Kurutas EB, Alici T (2010) Protective effect of zinc aspartate on long-term ischemia-reperfusion injury in rat skeletal muscle. Biol Trace Elem Res 137:206–215CrossRefPubMedGoogle Scholar
  2. 2.
    Becker LB (2004) New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res 61:461–470CrossRefPubMedGoogle Scholar
  3. 3.
    Beiras-Fernandez A, Thein E, Chappel D, Gallego R, Fernandez-Roel D, Kemming G, Hammer C (2004) Polyclonal anti-thymocyte globulins influence apoptosis in reperfused tissues after ischaemia in a non-human primate model. Transpl Int : Off J Eur Soc Organ Transplant 17:453–457CrossRefGoogle Scholar
  4. 4.
    Birk AV, Liu S, Soong Y, Mills W, Singh P, Warren JD, Seshan SV, Pardee JD, Szeto HH (2013) The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin. J Am Soc Nephrol : JASN 24:1250–1261CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chen Q, Moghaddas S, Hoppel CL, Lesnefsky EJ (2006) Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion. J Pharmacol Exp Ther 319:1405–1412CrossRefPubMedGoogle Scholar
  6. 6.
    Cho J, Won K, Wu D, Soong Y, Liu S, Szeto HH, Hong MK (2007) Potent mitochondria-targeted peptides reduce myocardial infarction in rats. Coron Artery Dis 18:215–220CrossRefPubMedGoogle Scholar
  7. 7.
    Chovatiya R, Medzhitov R (2014) Stress, inflammation, and defense of homeostasis. Mol Cell 54:281–288CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Comu FM, Kilic Y, Ozer A, Kirisci M, Dursun AD, Tatar T, Zor MH, Kartal H, Kucuk A, Boyunaga H et al (2016) Effect of picroside II on erythrocyte deformability and lipid peroxidation in rats subjected to hind limb ischemia reperfusion injury. Drug Des Dev Ther 10:927–931Google Scholar
  9. 9.
    Dai DF, Chen T, Szeto H, Nieves-Cintron M, Kutyavin V, Santana LF, Rabinovitch PS (2011) Mitochondrial targeted antioxidant peptide ameliorates hypertensive cardiomyopathy. J Am Coll Cardiol 58:73–82CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Del Rio D, Stewart AJ, Pellegrini N (2005) A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovas 15:316–328CrossRefGoogle Scholar
  11. 11.
    Dolegowska B, Pikula E, Safranow K, Olszewska M, Jakubowska K, Chlubek D, Gutowski P (2006) Metabolism of eicosanoids and their action on renal function during ischaemia and reperfusion: the effect of alprostadil. Prostaglandins Leukot Essent Fat Acids 75:403–411CrossRefGoogle Scholar
  12. 12.
    Du LL, Chai DM, Zhao LN, Li XH, Zhang FC, Zhang HB, Liu LB, Wu K, Liu R, Wang JZ et al (2015) AMPK activation ameliorates Alzheimer’s disease-like pathology and spatial memory impairment in a streptozotocin-induced Alzheimer’s disease model in rats. J Alzheimers Dis 43:775–784PubMedCrossRefGoogle Scholar
  13. 13.
    Duehrkop C, Denoyelle J, Shaw S, Rieben R (2014) Use of dextran sulfate in tourniquet-induced skeletal muscle reperfusion injury. J Surg Res 187:150–161CrossRefPubMedGoogle Scholar
  14. 14.
    Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15:1583–1606CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Gokce EC, Kahveci R, Gokce A, Sargon MF, Kisa U, Aksoy N, Cemil B, Erdogan B (2016) Curcumin attenuates inflammation, oxidative stress, and ultrastructural damage induced by spinal cord ischemia-reperfusion injury in rats. J Stroke Cerebrovasc Dis : Off J Natl Stroke Assoc 25:1196–1207CrossRefGoogle Scholar
  16. 16.
    Hao S, Ji J, Zhao H, Shang L, Wu J, Li H, Qiao T, Li K (2015) Mitochondrion-targeted peptide SS-31 inhibited oxidized low-density lipoproteins-induced foam cell formation through both ROS scavenging and inhibition of cholesterol influx in RAW264.7 cells. Molecules 20:21287–21297CrossRefPubMedGoogle Scholar
  17. 17.
    Kang WL, Xu GS (2016) Atrasentan increased the expression of klotho by mediating miR-199b-5p and prevented renal tubular injury in diabetic nephropathy. Sci Rep 6:19979CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lejay A, Meyer A, Schlagowski AI, Charles AL, Singh F, Bouitbir J, Pottecher J, Chakfe N, Zoll J, Geny B (2014) Mitochondria: mitochondrial participation in ischemia-reperfusion injury in skeletal muscle. Int J Biochem Cell Biol 50:101–105CrossRefPubMedGoogle Scholar
  19. 19.
    Li J, Li RJ, Lv GY, Liu HQ (2015) The mechanisms and strategies to protect from hepatic ischemia-reperfusion injury. Eur Rev Med Pharmacol Sci 19:2036–2047PubMedGoogle Scholar
  20. 20.
    Manczak M, Mao PZ, Calkins MJ, Cornea A, Reddy AP, Murphy MP, Szeto HH, Park B, Reddy PH (2010) Mitochondria-targeted antioxidants protect against amyloid-beta toxicity in Alzheimer’s disease neurons. J Alzheimers Dis 20:S609–S631CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Medzhitov R (2008) Origin and physiological roles of inflammation. Nature 454:428–435CrossRefPubMedGoogle Scholar
  22. 22.
    Pang YWC, Yu L (2015) Mitochondria-targeted antioxidant SS-31 is a potential novel ophthalmic medication for neuroprotection in glaucoma. Med Hypothesis Discov Innov Ophthalmol 4:120–126PubMedPubMedCentralGoogle Scholar
  23. 23.
    Paradis S, Charles AL, Meyer A, Lejay A, Scholey JW, Chakfe N, Zoll J, Geny B (2016) Chronology of mitochondrial and cellular events during skeletal muscle ischemia-reperfusion. Am J Physiol Cell Physiol 310:C968–C982CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Peralta C, Jimenez-Castro MB, Gracia-Sancho J (2013) Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu. J Hepatol 59:1094–1106CrossRefPubMedGoogle Scholar
  25. 25.
    Petri S, Kiaei M, Damiano M, Hiller A, Wille E, Manfredi G, Calingasan NY, Szeto HH, Beal MF (2006) Cell-permeable peptide antioxidants as a novel therapeutic approach in a mouse model of amyotrophic lateral sclerosis. J Neurochem 98:1141–1148CrossRefPubMedGoogle Scholar
  26. 26.
    Rodriguez-Lara SQ, Cardona-Munoz EG, Ramirez-Lizardo EJ, Totsuka-Sutto SE, Castillo-Romero A, Garcia-Cobian TA, Garcia-Benavides L (2016) Alternative interventions to prevent oxidative damage following ischemia/reperfusion. Oxidative Med Cell Longev 2016:7190943CrossRefGoogle Scholar
  27. 27.
    Shen J, Xu S, Zhou H, Liu H, Jiang W, Hao J, Hu Z (2017) IL-1beta induces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells. Sci Rep 7:41067CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Siegel MP, Kruse SE, Percival JM, Goh J, White CC, Hopkins HC, Kavanagh TJ, Szeto HH, Rabinovitch PS, Marcinek DJ (2013) Mitochondrial-targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice. Aging Cell 12:763–771CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Skulachev VP (1996) Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants. Q Rev Biophys 29:169–202CrossRefPubMedGoogle Scholar
  30. 30.
    Szeto HH (2008) Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury. Antioxid Redox Signal 10:601–619CrossRefPubMedGoogle Scholar
  31. 31.
    Szeto HH (2014) First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics. Brit J Pharmacol 171:2029–2050CrossRefGoogle Scholar
  32. 32.
    Szeto HH, Liu S, Soong Y, Wu D, Darrah SF, Cheng FY, Zhao Z, Ganger M, Tow CY, Seshan SV (2011) Mitochondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury. J Am Soc Nephrol : JASN 22:1041–1052CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Szeto HH, Schiller PW (2011) Novel therapies targeting inner mitochondrial membrane-from discovery to clinical development. Pharm Res-Dordr 28:2669–2679CrossRefGoogle Scholar
  34. 34.
    Takhtfooladi HA, Hesaraki S, Razmara F, Takhtfooladi MA, Hajizadeh H (2016) Effects of N-acetylcysteine and pentoxifylline on remote lung injury in a rat model of hind-limb ischemia/reperfusion injury. Jornal brasileiro de pneumologia : publicacao oficial da Sociedade Brasileira de Pneumologia e Tisilogia 42:9–14CrossRefGoogle Scholar
  35. 35.
    Toledo-Pereyra LH, Lopez-Neblina F, Toledo AH (2004) Reactive oxygen species and molecular biology of ischemia/reperfusion. Ann Transplant 9:81–83PubMedGoogle Scholar
  36. 36.
    Tran TP, Tu H, Pipinos II, Muelleman RL, Albadawi H, Li YL (2011) Tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles: involvement of superoxide. Eur J Pharmacol 650:328–334CrossRefPubMedGoogle Scholar
  37. 37.
    Tsubota H, Marui A, Esaki J, Bir SC, Ikeda T, Sakata R (2010) Remote postconditioning may attenuate ischaemia-reperfusion injury in the murine hindlimb through adenosine receptor activation. Eur J Vasc Endovasc Surg : Off J Eur Soc Vasc Surg 40:804–809CrossRefGoogle Scholar
  38. 38.
    Wang WZ, Fang XH, Stephenson LL, Zhang X, Khiabani KT, Zamboni WA (2011) Melatonin attenuates I/R-induced mitochondrial dysfunction in skeletal muscle. J Surg Res 171:108–113CrossRefPubMedGoogle Scholar
  39. 39.
    Wang Y, Wei Y, Zhang H, Shi Y, Li Y, Li R (2012) Arsenic trioxide induces apoptosis of p53 null osteosarcoma MG63 cells through the inhibition of catalase. Med Oncol 29:1328–1334CrossRefPubMedGoogle Scholar
  40. 40.
    Wu J, Zhang M, Li H, Sun X, Hao S, Ji M, Yang J, Li K (2016) BDNF pathway is involved in the protective effects of SS-31 on isoflurane-induced cognitive deficits in aging mice. Behav Brain Res 305:115–121CrossRefPubMedGoogle Scholar
  41. 41.
    Yassin MM, Harkin DW, Barros D'Sa AA, Halliday MI, Rowlands BJ (2002) Lower limb ischemia-reperfusion injury triggers a systemic inflammatory response and multiple organ dysfunction. World J Surg 26:115–121CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang M, Zhao H, Cai J, Li H, Wu Q, Qiao T, Li K (2017) Chronic administration of mitochondrion-targeted peptide SS-31 prevents atherosclerotic development in ApoE knockout mice fed Western diet. PLoS One 12:e0185688CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Zhao H, Li H, Hao S, Chen J, Wu J, Song C, Zhang M, Qiao T, Li K (2017) Peptide SS-31 upregulates frataxin expression and improves the quality of mitochondria: implications in the treatment of Friedreich ataxia. Sci Rep 7:9840CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 279:34682–34690CrossRefPubMedGoogle Scholar
  45. 45.
    Zhao WY, Han S, Zhang L, Zhu YH, Wang LM, Zeng L (2013) Mitochondria-targeted antioxidant peptide SS31 prevents hypoxia/reoxygenation-induced apoptosis by down-regulating p66Shc in renal tubular epithelial cells. Cell Physiol Biochem : Int J Exp Cell Physiol Biochem Pharmacol 32:591–600CrossRefGoogle Scholar
  46. 46.
    Zhu C, Hu W, Wu H, Hu X (2014) No evident dose-response relationship between cellular ROS level and its cytotoxicity—a paradoxical issue in ROS-based cancer therapy. Sci Rep 4:5029CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© University of Navarra 2018

Authors and Affiliations

  1. 1.Department of Vascular SurgeryThe Affiliated Drum Tower Hospital of Nanjing University Medical School, Medical School of Nanjing UniversityNanjingChina
  2. 2.Department of Vascular SurgeryDrum Tower Hospital, Medical School of Southeast UniversityNanjingChina
  3. 3.Department of Vascular SurgeryDrum Tower Clinical Medical College of Nanjing Medical UniversityNanjingChina
  4. 4.Jiangsu Key Laboratory for Molecular MedicineMedical School of Nanjing UniversityNanjingChina
  5. 5.Department of Neurosurgery, Jinling Hospital, School of MedicineNanjing UniversityNanjingChina

Personalised recommendations