Cell Stress and Chaperones

, Volume 14, Issue 2, pp 207–217 | Cite as

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

  • Han-Yao Chiu
  • Lon-Yen Tsao
  • Rei-Cheng Yang
Original Paper


The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H2O2-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H2O2. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H2O2-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H2O2 treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H2O2-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H2O2-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H2O2-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.


Heat-shock response Rat Superoxide Oxidative stress Mitochondrion 







heat-shock protein


mitochondrial membrane potential


peripheral blood mononuclear cell


Reactive oxygen species


propidium iodium


TdT-mediated dUTP nick end-labeling assay


terminal deoxynucleotidyl transferase


5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide


  1. Andreyev AY, Kushnareva YE, Starkov AA (2005) Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70:200–214 doi: 10.1007/s10541-005-0102-7 CrossRefGoogle Scholar
  2. Arya R, Mallik M, Lakhotia SC (2007) Heat shock genes—integrating cell survival and death. J Biosci 32:595–610 doi: 10.1007/s12038-007-0059-3 PubMedCrossRefGoogle Scholar
  3. Aslan M, Horoz M, Kocyigit A, Ozgonul S, Celik H, Celik M, Erel O (2006) Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia. Mutat Res 601:144–149 doi: 10.1016/j.mrfmmm.2006.06.013 PubMedGoogle Scholar
  4. Beere HM (2005) Death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways. J Clin Invest 115:2633–2639 doi: 10.1172/JCI26471 PubMedCrossRefGoogle Scholar
  5. Cases N, Sureda A, Maestre I, Tauler P, Aguilo A, Cordova A, Roche E, Tur JA, Pons A (2006) Response of antioxidant defences to oxidative stress induced by prolonged exercise: antioxidant enzyme gene expression in lymphocytes. Eur J Appl Physiol 98:263–269 doi: 10.1007/s00421-006-0273-y PubMedCrossRefGoogle Scholar
  6. Chen HW, Hsu C, Lu TS, Wang SJ, Yang RC (2003) Heat shock pretreatment prevents cardiac mitochondrial dysfunction during sepsis. Shock 20:274–279 doi: 10.1097/00024382-200309000-00013 PubMedCrossRefGoogle Scholar
  7. Chen HW, Hsu C, Lue SI, Yang RC (2000) Attenuation of sepsis-induced apoptosis by heat shock pretreatment in rats. Cell Stress Chaperones 5:188–195 doi: 10.1379/1466-1268(2000)005<0188:AOSIAB>2.0.CO;2 PubMedCrossRefGoogle Scholar
  8. Chen HW, Kuo HT, Lu TS, Wang SJ, Yang RC (2004) Cytochrome c oxidase as the target of the heat shock protective effect in septic liver. Int J Exp Pathol 85:249–256 doi: 10.1111/j.0959-9673.2004.00393.x PubMedCrossRefGoogle Scholar
  9. Chen HW, Kuo HT, Wang SJ, Lu TS, Yang RC (2005) In vivo heat shock protein assembles with septic liver NF-kappaB/I-kappaB complex regulating NF-kappaB activity. Shock 24:232–238 doi: 10.1097/01.shk.0000174020.87439.f2 PubMedCrossRefGoogle Scholar
  10. Choi S, Park KA, Lee HJ, Park MS, Lee JH, Park KC, Kim M, Lee SH, Seo JS, Yoon BW (2005) Expression of Cu/Zn SOD protein is suppressed in hsp 70.1 knockout mice. J Biochem Mol Biol 38:111–114PubMedGoogle Scholar
  11. Clarkson PM, Thompson HS (2000) Antioxidants: what role do they play in physical activity and health? Am J Clin Nutr 72:637S–646SPubMedGoogle Scholar
  12. Csiszar A, Toth J, Peti-Peterdi J, Ungvari Z (2007) The aging kidney: role of endothelial oxidative stress and inflammation. Acta Physiol Hung 94:107–115 doi: 10.1556/APhysiol.94.2007.1-2.10 PubMedCrossRefGoogle Scholar
  13. Fatokun AA, Stone TW, Smith RA (2006) Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: the effects of glutamate and protection by purines. Bone 39:542–551 doi: 10.1016/j.bone.2006.02.062 PubMedCrossRefGoogle Scholar
  14. Fedeli D, Falcioni G, Olek RA, Massi M, Cifani C, Polidori C, Gabbianelli R (2007) Protective effect of ethyl pyruvate on msP rat leukocytes damaged by alcohol intake. J Appl Toxicol. 27(6):561–570 doi: 10.1002/jat.1236 PubMedCrossRefGoogle Scholar
  15. Gogvadze V, Zhivotovsky B (2007) Alteration of mitochondrial function and cell sensitization to death. J Bioenerg Biomembr 39:23–30 doi: 10.1007/s10863-006-9054-x PubMedCrossRefGoogle Scholar
  16. Guo S, Wharton W, Moseley P, Shi H (2007) Heat shock protein 70 regulates cellular redox status by modulating glutathione-related enzyme activities. Cell Stress Chaperones 12:245–254 doi: 10.1379/CSC-265.1 PubMedCrossRefGoogle Scholar
  17. Hansen JM, Go YM, Jones DP (2006) Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling. Annu Rev Pharmacol Toxicol 46:215–234 doi: 10.1146/annurev.pharmtox.46.120604.141122 PubMedCrossRefGoogle Scholar
  18. Ji LL (1999) Antioxidants and oxidative stress in exercise. Proc Soc Exp Biol Med 222:283–292 doi: 10.1046/j.1525-1373.1999.d01-145.x PubMedCrossRefGoogle Scholar
  19. Jiang B, Xiao W, Shi Y, Liu M, Xiao X (2005) Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells. Cell Stress Chaperones 10:252–262 doi: 10.1379/CSC-124R.1 PubMedCrossRefGoogle Scholar
  20. Kakkar P, Singh BK (2007) Mitochondria: a hub of redox activities and cellular distress control. Mol Cell Biochem 305:235–253 doi: 10.1007/s11010-007-9520-8 PubMedCrossRefGoogle Scholar
  21. Kwon JH, Kim JB, Lee KH, Kang SM, Chung N, Jang Y, Chung JH (2007) Protective effect of heat shock protein 27 using protein transduction domain-mediated delivery on ischemia/reperfusion heart injury. Biochem Biophys Res Commun 363:399–404 doi: 10.1016/j.bbrc.2007.09.001 PubMedCrossRefGoogle Scholar
  22. Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157 doi: 10.1016/S0092-8674(00)80085-9 PubMedCrossRefGoogle Scholar
  23. Narula J, Haider N, Arbustini E, Chandrashekhar Y (2006) Mechanisms of disease: apoptosis in heart failure—seeing hope in death. Nat Clin Pract Cardiovasc Med 3:681–688 doi: 10.1038/ncpcardio0710 PubMedCrossRefGoogle Scholar
  24. Nicolls MR, Haskins K, Flores SC (2007) Oxidant stress, immune dysregulation, and vascular function in type I diabetes. Antioxid Redox Signal 9:879–889 doi: 10.1089/ars.2007.1631 PubMedCrossRefGoogle Scholar
  25. Ohtsuka K, Kawashima D, Gu Y, Saito K (2005) Inducers and co-inducers of molecular chaperones. Int J Hyperthermia 21:703–711 doi: 10.1080/02656730500384248 PubMedCrossRefGoogle Scholar
  26. Onyango IG, Khan SM (2006) Oxidative stress, mitochondrial dysfunction, and stress signaling in Alzheimer’s disease. Curr Alzheimer Res 3:339–349 doi: 10.2174/156720506778249489 PubMedCrossRefGoogle Scholar
  27. Pandey P, Farber R, Nakazawa A, Kumar S, Bharti A, Nalin C, Weichselbaum R, Kufe D, Kharbanda S (2000) Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3. Oncogene 19:1975–1981 doi: 10.1038/sj.onc.1203531 PubMedCrossRefGoogle Scholar
  28. Sakaguchi S, Furusawa S (2006) Oxidative stress and septic shock: metabolic aspects of oxygen-derived free radicals generated in the liver during endotoxemia. FEMS Immunol Med Microbiol 47:167–177 doi: 10.1111/j.1574-695X.2006.00072.x PubMedCrossRefGoogle Scholar
  29. Saleh A, Srinivasula SM, Balkir L, Robbins PD, Alnemri ES (2000) Negative regulation of the Apaf-1 apoptosome by Hsp70. Nat Cell Biol 2:476–483 doi: 10.1038/35019510 PubMedCrossRefGoogle Scholar
  30. Song HJ, Lee TS, Jeong JH, Min YS, Shin CY, Sohn UD (2005) Hydrogen peroxide-induced extracellular signal-regulated kinase activation in cultured feline ileal smooth muscle cellsJ Pharmacol Exp Ther 312:391–398 doi: 10.1124/jpet.104.074401 PubMedCrossRefGoogle Scholar
  31. Soti C, Nagy E, Giricz Z, Vigh L, Csermely P, Ferdinandy P (2005) Heat shock proteins as emerging therapeutic targets. Br J Pharmacol 146:769–780 doi: 10.1038/sj.bjp.0706396 PubMedCrossRefGoogle Scholar
  32. Sureda A, Tauler P, Aguilo A, Cases N, Fuentespina E, Cordova A, Tur JA, Pons A (2005) Relation between oxidative stress markers and antioxidant endogenous defences during exhaustive exercise. Free Radic Res 39:1317–1324 doi: 10.1080/10715760500177500 PubMedCrossRefGoogle Scholar
  33. Wardle EN (2005) Cellular oxidative processes in relation to renal disease. Am J Nephrol 25:13–22 doi: 10.1159/000083477 PubMedCrossRefGoogle Scholar
  34. Zalata A, Yahia S, El-Bakary A, Elsheikha HM (2007) Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress. Mutat Res 629(2):140–147PubMedGoogle Scholar
  35. Zheng KC, Ariizumi M (2007) Modulations of immune functions and oxidative status induced by noise stress. J Occup Health 49:32–38 doi: 10.1539/joh.49.32 PubMedCrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2008

Authors and Affiliations

  1. 1.Department of Respiratory CareChang Jung Christian UniversityTainanTaiwan
  2. 2.Department of PediatricsChanghua Christian HospitalChanghuaTaiwan
  3. 3.Department of Pediatrics, College of MedicineKaohsiung Medical UniversityKaohsiung CityTaiwan

Personalised recommendations