Hemin attenuated oxidative stress and inflammation to improve wound healing in diabetic rats

  • Dhirendra KumarEmail author
  • Geeta Rani Jena
  • Mahendra Ram
  • Madhu Cholenahalli Lingaraju
  • Vishakha Singh
  • Raju Prasad
  • Sanjay Kumawat
  • Vinay Kant
  • Priyanka Gupta
  • Surendra Kumar Tandan
  • Dinesh Kumar
Original Article


Oxidative stress and persistent inflammation play crucial role in the progression of diabetic wound complications. Hemeoxgenase-1 (HO-1) by degrading hemin has been shown to display anti-oxidant and anti-inflammatory effects. Further, hemin is a potent HO-1 inducer. Thus, the current study was aimed to evaluate the effect of topical application of hemin on diabetic wound in rats. Four hundred square millimeter open excision wound were created 2 weeks after induction of diabetes with single intraperitoneal injection of streptozotocin (60 mg/kg), and the diabetic rats were divided into three groups namely diabetic control, hemin, and tin protoporphyrin (SnPPIX). Ointment base, hemin (0.5% in ointment base), and SnPPIX (0.5% in ointment base) were applied topically to wounded area in diabetic control, hemin, and SnPPIX group rats, respectively, twice daily for 19 days. Hemin significantly increased the wound contraction in comparison to control and SnPPIX-treated rats. Time-dependent analysis revealed significant increase in anti-oxidants with concomitant decrease in oxidants in hemin-treated rats as compared to diabetic control rats. Further, mRNA expression decreased for inflammatory cytokine and increased for anti-inflammatory cytokine in hemin group as compared to diabetic control rats. Expression of HO-1 also increased in hemin group as compared to diabetic control rats. However, SnPPIX group results were in disagreement with results of hemin which is clearly reflected in histopathology. Results indicate the ability of hemin to accelerate wound healing in diabetic rats by combating inflammation and oxidative stress probably via HO-1.


Diabetes Wound Hemin Tin protoporphyrin Oxidative stress Inflammation 


Authors’ contribution

DK, MR, VK, and DK conceived and designed the research program. DK, MCL, VS, RP, and SK conducted the experiments. RP, VK, and PG conducted the mRNA studies. DK, GRJ, and SKT analyzed the data. DK and SKT interpreted the results. DK, GRJ, and DK wrote the manuscript. All authors read and approved the manuscript.

Funding information

The Director the Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India, provided the necessary funds and facilities for conducting the present study to the first author.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All the experimental procedures conducted on animals (Wistar rats) were in compliance with at least the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals. The experimental procedures and protocols were duly approved by the Institutional Animal Ethics Committee of Indian Veterinary Research Institute, Izatnagar, India.

This article does not contain any studies with human participants performed by any of the authors.


  1. Abraham NG, Drummond GS, Lutton JD, Kappas A (1996) The biological significance and physiological role of heme oxygenase. Cell Physiol Bioche 6:129–168. CrossRefGoogle Scholar
  2. Abraham NG, Kappas A (2008) Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev 60:79–127. CrossRefGoogle Scholar
  3. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126. CrossRefGoogle Scholar
  4. Ahanger AA, Prawez S, Leo MD, Kathirvel K, Kumar D, Tandan SK, Malik JK (2010) Pro-healing potential of hemin: an inducer of heme oxygenase-1. Eur J Pharmacol 645:165–170. CrossRefGoogle Scholar
  5. Alcaraz MJ, Fernández P, Guillén MI (2003) Anti-inflammatory actions of the heme oxygenase-1 pathway. Curr Pharm Des 9:2541–2551. CrossRefGoogle Scholar
  6. An L, Liu CT, Qin XB, Liu QH, Liu Y, Yu SY (2011) Protective effects of hemin in an experimental model of ventilator-induced lung injury. Eur J Pharmacol 661:102–108. CrossRefGoogle Scholar
  7. Berlanga-Acosta J, Valdéz-Pérez C, Savigne-Gutiérrez W, Mendoza Mari Y, Franco Pérez N, Vargas Machiran E, Poll Marrón N, Alvarez Duarte H, Echeverría Requeijo H, Pérez Aguilar RM (2010) Cellular and molecular insights into the wound healing mechanism in diabetes. Biotecnología Aplicada 27:255–261Google Scholar
  8. Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820. CrossRefGoogle Scholar
  9. Cano SM, Lancel S, Boulanger E, Neviere R (2018) Targeting oxidative stress and mitochondrial dysfunction in the treatment of impaired wound healing: a systematic review. Antioxidants (Basel) 7:98. CrossRefGoogle Scholar
  10. Chan JC, Malik V, Jia W, Kadowaki T, Yajnik CS, Yoon KH, Hu FB (2009) Diabetes in Asia: epidemiology, risk factors, and pathophysiology. JAMA 301:2129–2140. CrossRefGoogle Scholar
  11. Chen QY, Wang GG, Li W, Jiang YX, Lu XH, Zhou PP (2016) Heme oxygenase-1 promotes delayed wound healing in diabetic rats. J Diabetes Res 2016:9726503. Google Scholar
  12. Choi AM, Alam J (1996) Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15:9–19. CrossRefGoogle Scholar
  13. Collino M, Pini A, Mugelli N, Mastroianni R, Bani D, Fantozzi R, Papucci L, Fazi M, Masini E (2013) Beneficial effect of prolonged heme oxygenase 1 activation in a rat model of chronic heart failure. Dis Model Mech 6:1012–1020. CrossRefGoogle Scholar
  14. Fadini GP, Albiero M, Menegazzo L, Boscaro E, Pagnin E, Iori E, Cosma C, Lapolla A, Pengo V, Stendardo M, Agostini C, Pelicci PG, Giorgio M, Avogaro A (2010) The redox enzyme p66Shc contributes to diabetes and ischemia-induced delay in cutaneous wound healing. Diabetes 59:2306–2314. CrossRefGoogle Scholar
  15. Giacco F, Brownlee M (2010) Oxidative stress and diabetic complications. Circ Res 107:1058–1070. CrossRefGoogle Scholar
  16. Greenhalgh DG, Sprugel KH, Murray MJ, Ross R (1990) PDGF and FGF stimulate healing in the genetically diabetic mouse. Am J Pathol 136:1235–1246Google Scholar
  17. Grochot-Przeczek A, Lach R, Mis J, Skrzypek K, Gozdecka M, Sroczynska P, Dubiel M, Rutkowski A, Kozakowska M, Zagorska A, Walczynski J, Was H, Kotlinowski J, Drukala J, Kurowski K, Kieda C, Herault Y, Dulak J, Jozkowicz A (2009) Heme oxygenase-1 accelerates cutaneous wound healing in mice. PLoS One 4:e5803. CrossRefGoogle Scholar
  18. Hassan N, El-Bassossy HM, Zakaria MN (2013) Heme oxygenase-1 induction protects against hypertension associated with diabetes: effect on exaggerated vascular contractility. Naunyn Schmiedeberg's Arch Pharmacol 386:217–226. CrossRefGoogle Scholar
  19. Hyvelin JM, Maurel B, Uzbekov R, Motterlini R, Lermusiaux P (2010) Hemin prevents in-stent stenosis in rat and rabbit models by inducing heme-oxygenase-1. J Vasc Surg 51:417–428.
  20. Juckett MB, Balla J, Balla G, Jessurun J, Jacob HS, Vercellotti GM (1995) Ferritin protects endothelial cells from oxidized low density lipoprotein in vitro. Am J Pathol 147:782–789Google Scholar
  21. Kakkar R, Mantha SV, Radhi J, Prasad K, Kalra J (1998) Increased oxidative stress in rat liver and pancreas during progression of streptozotocin-induced diabetes. Clin Sci 94:623–632CrossRefGoogle Scholar
  22. Khanna S, Biswas S, Shang Y, Collard E, Azad A, Kauh C, Bhasker V, Gordillo GM, Sen CK, Roy S (2010) Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One 5:e9539.
  23. Kowluru RA, Chan PS (2007) Oxidative stress and diabetic retinopathy. Exp Diabetes Res 2007:43603. Google Scholar
  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408. CrossRefGoogle Scholar
  25. Madesh M, Balasubramanian KA (1998) Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide. Indian J Biochem Biophys 35(3):184–188Google Scholar
  26. Maines MD, Kappas A (1977) Enzymatic oxidation of cobalt protoporphyrin IX: observations on the mechanism of heme oxygenase action. Biochemistry 16:419–423. CrossRefGoogle Scholar
  27. Mishima Y, Kuyama A, Tada A, Takahashi K, Ishioka T, Kibata M (2001) Relationship between serum tumor necrosis factor alpha and insulin resistance in obese men with type 2 diabetes mellitus. Diabetes Res Clin Pract 52:119–123. CrossRefGoogle Scholar
  28. Morse D, Choi AM (2005) Heme oxygenase-1: from bench to bedside. Am J Respir Crit Care Med 172:660–670.
  29. Naguib G, Al Mashat H, Desta T, Graves DT (2004) Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation. J Invest Dermatol 123:87–92. CrossRefGoogle Scholar
  30. Ndisang JF, Lane N, Jadhav A (2009) The heme oxygenase system abates hyperglycemia in Zucker diabetic fatty rats by potentiating insulin-sensitizing pathways. Endocrinology 150:2098–2108.
  31. Nwomeh BC, Yager DR, Cohen IK (1998) Physiology of the chronic wound. Clin Plast Surg 25:341–356. Google Scholar
  32. Pham HT, Rich J, Veves A (2002) Using living skin equivalents for diabetic foot ulceration. Int J Low Extrem Wounds 1:27–32. CrossRefGoogle Scholar
  33. Ram M, Singh V, Kumar D, Kumawat S, Gopalakrishnan A, Lingaraju MC, Gupta P, Tandan SK, Kumar D (2014) Antioxidant potential of bilirubin-accelerated wound healing in streptozotocin-induced diabetic rats. Naunyn Schmiedeberg's Arch Pharmacol 387:955–961. CrossRefGoogle Scholar
  34. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590. CrossRefGoogle Scholar
  35. Sato Y, Ohshima T, Kondo T (1999) Regulatory role of endogenous interleukin-10 in cutaneous inflammatory response of murine wound healing. Biochem Biophys Res Commun 265(1):194–199. CrossRefGoogle Scholar
  36. Schäfer M, Werner S (2008) Oxidative stress in normal and impaired wound repair. Pharmacol Res 58(2):165–171. CrossRefGoogle Scholar
  37. Schonfelder U, Abel M, Wiegand C, Klemm D, Elsner P, Hipler UC (2005) Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro. Biomaterials 26:6664–6673. CrossRefGoogle Scholar
  38. Sedlak J, Lindsay RHC (1968) Estimation of total, protein bound and nonprotein sulfhydryl groups in tissue with Ellmann’s reagent. Anal Biochem 25:192–205. CrossRefGoogle Scholar
  39. Sedlak TW, Saleh M, Higginson DS, Paul BD, Juluri KR, Snyder SH (2009) Bilirubin and glutathione have complementary antioxidant and cytoprotective roles. Proc Natl Acad Sci U S A 106(13):5171–5176. CrossRefGoogle Scholar
  40. Shafiq-Ur-Rehman (1984) Lead-induced regional lipid peroxidation in brain. Toxicol Lett 21:333–337. CrossRefGoogle Scholar
  41. Shukla A, Rasik AM, Patnaik GK (1997) Depletion of reduced glutathione, ascorbic acid, vitamin E and antioxidant defence enzymes in a healing cutaneous wound. Free Radic Res 26:93–101Google Scholar
  42. Taye A, Ibrahim BM (2013) Activation of renal haeme oxygenase-1 alleviates gentamicin-induced acute nephrotoxicity in rats. J Pharm Pharmacol 65:995–1004.
  43. Wang HD, Pagano PJ, Du Y, Cayatte AJ, Quinn MT, Brecher P, Cohen RA (1998) Superoxide anion from the adventitia of the rat thoracic aorta inactivates nitric oxide. Circ Res 82:810–818. CrossRefGoogle Scholar
  44. Wetzler C, Kampfer H, Stallmeyer B, Pfeilschifter J, Frank S (2000) Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol 115:245–253. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Dhirendra Kumar
    • 1
    Email author
  • Geeta Rani Jena
    • 2
  • Mahendra Ram
    • 1
  • Madhu Cholenahalli Lingaraju
    • 1
  • Vishakha Singh
    • 1
  • Raju Prasad
    • 1
  • Sanjay Kumawat
    • 1
  • Vinay Kant
    • 1
  • Priyanka Gupta
    • 1
  • Surendra Kumar Tandan
    • 1
  • Dinesh Kumar
    • 1
  1. 1.Division of Pharmacology and ToxicologyIndian Veterinary Research InstituteIzatnagarIndia
  2. 2.Department of Clinical Medicine, College of Veterinary Science and Animal HusbandryOUATBhubaneswarIndia

Personalised recommendations