Adrenomedullin protects rat dorsal root ganglion neurons against doxorubicin-induced toxicity by ameliorating oxidative stress

Document Type : Original Article


1 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

3 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran


Objective(s): Despite effective anticancer effects, the use of doxorubicin (DOX) is hindered due to its cardio and neurotoxicity. The neuroprotective effect of adrenomedullin (AM) was shown in several studies. The present study aimed to evaluate the possible protective effects of AM against DOX-induced toxicity in dorsal root ganglia (DRGs) neurons.
Materials and Methods: Rat embryonic DRG neurons were isolated and cultured. The effect of various concentrations of DOX (0.0 to 100 µM) in the absence or presence of AM (3.125 -100 nM) on cell death, apoptosis, oxidative stress, expression of tumor necrosis-α (TNF-α), interleukin1- β (IL-1β), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP) 3 and 13, and SRY-related protein 9 (SOX9) were examined.
Results: Based on MTT assay data, DOX decreased the viability of DRG neurons in a dose and time-dependent manner (IC50=6.88 µm) while dose-dependently, AM protected DRG neurons against DOX-induced cell death. Furthermore, results of annexin V apoptosis assay revealed the protective effects of AM (25 nm) against DOX (6.88 µM)-induced apoptosis and necrosis of DRG neurons. Also, AM significantly ameliorated DOX-induced oxidative stress in DRG neurons. Real-time PCR results showed a significant increase in the expression of TNF-α, IL-1β, iNOS, MMP 3, and MMP 13, and a decrease in the expression of SOX9 following treatment with DOX. Treatment with AM (25 nM) significantly reversed the effects of DOX on the above-mentioned genes expression.
Conclusion: Our findings suggest that AM can be considered a novel ameliorating drug against DOX-induced neurotoxicity.


1. McGowan JV, Chung R, Maulik A, Piotrowska I, Walker JM, Yellon DM. Anthracycline chemotherapy and cardiotoxicity. Cardiovasc Drugs Ther 2017; 31:63-75.
2. Zhao L, Zhang B. Doxorubicin induces cardiotoxicity through upregulation of death receptors mediated apoptosis in cardiomyocytes. Sci Rep 2017; 7:44735-44745.
3. Thomas TC, Beitchman JA, Pomerleau F, Noel T, Jungsuwadee P, Butterfield DA, et al. Acute treatment with doxorubicin affects glutamate neurotransmission in the mouse frontal cortex and hippocampus. Brain Res 2017; 1672:10-17.
4. Yarmohmmadi F, Rahimi N, Faghir-Ghanesefat H, Javadian N, Abdollahi A, Pasalar P, et al. Protective effects of agmatine on doxorubicin-induced chronic cardiotoxicity in rat. Eur J Pharmacol 2017; 796:39-44.
5. Wu S-N, So EC, Liao Y-K, Huang Y-M. Reversal by ranolazine of doxorubicin-induced prolongation in the inactivation of late sodium current in rat dorsal root ganglion neurons. Pain Med 2015; 16:1032-1034.
6. Saito T, Okamura A, Inoue J, Makiura D, Yakushijin K, Matsuoka H, et al. Anemia is a novel predictive factor for the onset of severe chemotherapy-induced peripheral neuropathy in lymphoma patients receiving rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone therapy. Oncol Res 2019; 27:469-474.
7. Cappetta D, De Angelis A, Sapio L, Prezioso L, Illiano M, Quaini F, et al. Oxidative stress and cellular response to doxorubicin: a common factor in the complex milieu of anthracycline cardiotoxicity. Oxid Med Cell Longev 2017; 2017:1521020-1521020.
8. Farias JG, Molina VM, Carrasco RA, Zepeda AB, Figueroa E, Letelier P, et al. Antioxidant therapeutic strategies for cardiovascular conditions associated with oxidative stress. Nutrients 2017; 1: 9-16.
9. Pintér E, Pozsgai G, Hajna Z, Helyes Z, Szolcsányi J. Neuropeptide receptors as potential drug targets in the treatment of inflammatory conditions. Br J Clin Pharmacol. 2014; 77:5-20.
10. Mukhopadhyay P, Rajesh M, Batkai S, Patel V, Kashiwaya Y, Liaudet L, et al. CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes. Oxid Med Cell  Longev 2009; 85:773-784
11. Heeba GH, Mahmoud ME. Dual effects of quercetin in doxorubicin‐induced nephrotoxicity in rats and its modulation of the cytotoxic activity of doxorubicin on human carcinoma cells. Environ Toxicol 2016; 31:624-636.
12. Mohammed WI, Radwan RA, Elsayed HM. Prophylactic and ameliorative effect of N-acetylcysteine on doxorubicin-induced neurotoxicity in Wister rats. Egypt J Basic Clin Pharmacol 2019; 9: 1-13.
13. Chan B, Roczkowsky A, Poirier M, Moser N, Ilarraza R, Granzier H, et al. Matrix metalloproteinase inhibitors attenuate doxorubicin-induced heart failure by preventing cardiac titin proteolysis. FASEB J 2018; 32:864.810-864.810.
14. Chan B, Hughes B, Schulz R. Doxorubicin-induced activation of intracellular matrix metalloproteinase-2 by oxidative stress. FASEB J 2015; 29:955.955.
16. Hong Y, Liu Y, Chabot JG, Fournier A, Quirion R. Upregulation of adrenomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats. Pain 2009; 146:105-113.
17. Takhshid MA, Owji AA, Vasei M, Panjehshahin MR, Tabei SMB, Tabatabaee HR, et al. Expression of spinal cord Fos protein in response to intrathecal adrenomedullin and CGRP in conscious rats. Brain Res 2004; 1020:30-36.
18. Ma W, Chabot J-G, Quirion R. A role for adrenomedullin as a pain-related peptide in the rat. Proc Natl Acad Sci USA 103:16027-16032.
19.Watkins HA, Chakravarthy M, Abhayawardana RS, Gingell JJ, Garelja M, Pardamwar M, et al. Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties. J Biol Chem 2016; 291:11657-11675.
20. Nishikimi T, Kuwahara K, Nakagawa Y, Kangawa K, Nakao K. Chapter 2 - Adrenomedullin. In: Schisler JC, Lang CH, Willis MS. Endocrinology of the Heart in Health and Disease. Academic Press; 2017. p. 41-58.21.
21. Lee EG, Lee SI, Chae HJ, Park SJ, Lee YC, Yoo WH. Adrenomedullin inhibits IL-1beta-induced rheumatoid synovial fibroblast proliferation and MMPs, COX-2 and PGE2 production. Inflammation 2011; 34:335-343.
22. Nagata S, Yamasaki M, Kitamura K. Polyethylene glycol-conjugated human adrenomedullin as a possible treatment for vascular dementia. Peptides 2019:170133.
23. Li MY, Zhu XL, Zhao BX, Shi L, Wang W, Hu W, et al. Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis. Cell Death Dis 2019; 10:489.
24. Voors AA, Kremer D, Geven C, Ter Maaten JM, Struck J, Bergmann A, et al. Adrenomedullin in heart failure: pathophysiology and therapeutic application. Eur J Heart Fail 2019; 21:163-171.
25. Holmes D, Campbell M, Harbinson M, Bell D. Protective effects of intermedin on cardiovascular, pulmonary and renal diseases: comparison with adrenomedullin and CGRP. Curr Protein Pept Sci 2013; 14:294-329.
26. Geven C, Kox M, Pickkers P. Adrenomedullin and adrenomedullin-targeted therapy as treatment strategies relevant for sepsis. Front Immunol 2018; 9:292-305.
27. Martinez-Herrero S, Larrayoz IM, Narro-Iniguez J, Rubio-Mediavilla S, Martinez A. Lack of adrenomedullin aggravates acute TNBS-induced colitis symptoms in mice, especially in females. Front Physiol 2017; 8:1058-1074.
28. Dogru MI, Dogru AK, Gul M, Esrefoglu M, Yurekli M, Erdogan S, et al. The effect of adrenomedullin on rats exposed to lead. J Appl Toxicol 2008; 28:140-146.
29. Onur OE, Guneysel O, Akoglu H, Denizbasi A, Onur E. Adrenomedullin reduces the severity of cerulein-induced acute pancreatitis. Peptides 2007; 28:2179-2183.
30. Yoshizawa T, Takizawa S, Shimada S, Tokudome T, Shindo T, Matsumoto K. Effects of adrenomedullin on doxorubicin-induced cardiac damage in mice. Biol Pharm Bull 2016; 39:737-746.
31. Burkey TH, Hingtgen CM, Vasko MR. Isolation and culture of sensory neurons from the dorsal-root ganglia of embryonic or adult rats.  Pain Research: Springer; 2004. p. 189-202.
32. Takhshid MA, Poyner DR, Chabot JG, Fournier A, Ma W, Zheng WH, et al. Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture. Br J Pharmacol 2006; 148:459-468.
33. Chen X, Zhong Z, Xu Z, Chen L, Wang Y. 2’,7’-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radic Res 2010; 44:587-604.
34. Hare JM, Beigi F, Tziomalos K. Chapter Twenty-One - Nitric Oxide and Cardiobiology; Methods for Intact Hearts and Isolated Myocytes. In: Cadenas E, Packer L, editors. Methods in Enzymology. 441: Academic Press; 2008. p. 369-392.
35. Tsikas D. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges. Anal Biochem 2017; 524:13-30.
36. Baracca A, Sgarbi G, Solaini G, Lenaz G. Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis. Biochim Biophys Acta 2003; 1606:137-146.
37. Schwarz N, Renshaw D, Kapas S, Hinson JP. Adrenomedullin increases the expression of calcitonin-like receptor and receptor activity modifying protein 2 mRNA in human microvascular endothelial cells. J Endocrinol 2006; 190:505-514.
38. Manchon JFM, Dabaghian Y, Uzor N-E, Kesler SR, Wefel JS, Tsvetkov AS. Levetiracetam mitigates doxorubicin-induced DNA and synaptic damage in neurons. Sci Rep 2016; 6:25705-25705.
39. Deák É, Rosta J, Boros K, Kis G, Sántha P, Messlinger K, et al. Chronic adriamycin treatment impairs CGRP-mediated functions of meningeal sensory nerves. Neuropeptides 2018; 69:46-52.
40. Salas-Ramirez KY, Bagnall C, Frias L, Abdali SA, Ahles TA, Hubbard K. Doxorubicin and cyclophosphamide induce cognitive dysfunction and activate the ERK and AKT signaling pathways. Behav Brain Res 2015; 292:133-141.
41. Bi GR, Zhang HM, Bai LJ, Zhou HJ, Hai H, Zhang H, et al. Effect of adrenomedullin on neuron apoptosis, infarction volume and expression of Egr-1 mRNA after focal ischemia-reperfusion in rats. Neurosci Bull 2006; 22:323-330.
42. Liao D, Xiang D, Dang R, Xu P, Wang J, Han W, et al. Neuroprotective effects of dl-3-n-butylphthalide against doxorubicin-induced neuroinflammation, oxidative stress, endoplasmic reticulum stress, and behavioral changes. Oxid Med Cell Longev 2018; 2018.
43.Yoshimoto T, Gochou N, Fukai N, Sugiyama T, Shichiri M, Hirata Y. Adrenomedullin inhibits angiotensin II-induced oxidative stress and gene expression in rat endothelial cells. Hypertension  2005; 28:165-172.
44. Hu W, Zhou Ph, Rao T, Zhang Xb, Wang W, Zhang Lj. Adrenomedullin attenuates interleukin-1β-induced inflammation and apoptosis in rat Leydig cells via inhibition of NF-κB signaling pathway. Exp Cell Res 2015; 339:220-230.
45. Kim SM, Kim JY, Lee S, Park JH. Adrenomedullin protects against hypoxia/reoxygenation-induced cell death by suppression of reactive oxygen species via thiol redox systems. FEBS Lett 2010; 584:213-218.
46. Pecoraro M, Del Pizzo M, Marzocco S, Sorrentino R, Ciccarelli M, Iaccarino G, et al. Inflammatory mediators in a short-time mouse model of doxorubicin-induced cardiotoxicity. Toxicol Appl Pharmacol 2016; 293:44-52.
47. Scott CE, Wynn SL, Sesay A, Cruz C, Cheung M, Gomez Gaviro MV, et al. SOX9 induces and maintains neural stem cells. Nat Neurosci 2010; 13:1181-1189.
48. Pevny L, Placzek M. SOX genes and neural progenitor identity. Curr Opin Neurobiol 2005; 15:7-13.
49. Roche KC, Gracz AD, Liu XF, Newton V, Akiyama H, Magness ST. SOX9 maintains reserve stem cells and preserves radioresistance in mouse small intestine. Gastroenterology 2015; 149:1553-1563.e1510.
50. Piera-Velazquez S, Hawkins DF, Whitecavage MK, Colter DC, Stokes DG, Jimenez SA. Regulation of the human SOX9 promoter by Sp1 and CREB. Exp Cell Res 2007; 313:1069-1079.
51. Murakami S, Lefebvre V, de Crombrugghe B. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem 2000; 275:3687-3692.
52. Pan Q, Yu Y, Chen Q, Li C, Wu H, Wan Y, et al. Sox9, a key transcription factor of bone morphogenetic protein-2-induced chondrogenesis, is activated through BMP pathway and a CCAAT box in the proximal promoter. J Cell Physiol 2008; 217:228-241.
53. Al-Dasooqi N, Wardill HR, Gibson RJ. Gastrointestinal mucositis: the role of MMP-tight junction interactions in tissue injury. Pathol Oncol Res 2014; 20:485-491.
54. Sakamaki Y, Sasamura H, Hayashi K, Ishiguro K, Takaishi H, Okada Y, et al. Absence of gelatinase (MMP-9) or collagenase (MMP-13) attenuates adriamycin-induced albuminuria and glomerulosclerosis. Nephron Exp Nephrol 2010; 115:e22-32.
55. Nishida K, Kuchiiwa S, Oiso S, Futagawa T, Masuda S, Takeda Y, et al. Up-regulation of matrix metalloproteinase-3 in the dorsal root ganglion of rats with paclitaxel-induced neuropathy. Cancer Sci 2008; 99:1618-1625.
56. Teja KV, Ramesh S, Priya V. Regulation of matrix metalloproteinase-3 gene expression in inflammation: A molecular study. J Conserv Dent 2018; 21:592-596.
57. Lawenda BD, Kelly KM, Ladas EJ, Sagar SM, Vickers A, Blumberg JB. Should ssupplemental antioxidant administration be avoided during chemotherapy and radiation therapy? J Natl Cancer Inst 2008 4;100:773-783.