The Cannabinoid WIN 55,212-2 Reduces Delayed Neurologic Sequelae After Carbon Monoxide Poisoning by Promoting Microglial M2 Polarization Through ST2 Signaling

  • Jing-Jing Du
  • Zhi-Qin Liu
  • Yue Yan
  • Jing Xiong
  • Xiao-Tao Jia
  • Zheng-Li Di
  • Jing-Jing RenEmail author


Delayed neurologic sequelae (DNS) are among the most serious complications of carbon monoxide (CO) poisoning caused partly by elevated neuroinflammation. WIN 55,212-2, a non-selective agonist of cannabinoid receptors, has been demonstrated to have anti-inflammatory properties in various brain disorders. The anti-inflammatory action of WIN 55,212-2 is potentially associated with driving microglial M2 polarization. ST2 signaling is important in regulating inflammatory responses and microglial polarization. Therefore, we aimed to investigate the neuroprotective effect of WIN 55,212-2 on DNS after CO poisoning and elucidate its relationship with ST2-mediated microglial M2 polarization. The behavioral tests showed that treatment with WIN 55,212-2 significantly ameliorates the cognitive impairment induced by CO poisoning. This behavioral improvement was accompanied by reduced neuron loss, decreased production of pro-inflammatory cytokines, and a limited number of microglia in the hippocampus. Moreover, WIN 55,212-2 elevated the protein expression of IL-33 (the ligand of ST2) and ST2, increased the ratio of CD206-positive (M2 phenotype) and ST2-positive microglia, and augmented production of M2 microglia-associated cytokines in the hippocampus of CO-exposed rats. Furthermore, we observed that the WIN 55,212-2-mediated increases in ST2 protein expression, CD206-positive and ST2-positive microglia, and microglia-associated cytokines were blocked by the cannabinoid receptor 2 (CB2R) antagonist AM630 but not by the cannabinoid receptor 1 (CB1R) antagonist AM251. In contrast, the WIN 55,212-2-induced upregulation of the IL-33 protein expression was inhibited by AM251 but not by AM630. Altogether, these findings reveal cannabinoid receptors as promising therapeutic agents for CO poisoning and identify ST2 signaling-related microglial M2 polarization as a new mechanism of cannabinoid-induced neuroprotection.


Carbon monoxide poisoning WIN 55,212-2 Microglia ST2 Neuroinflammation 







Cannabinoid type 1 receptor


Cannabinoid type 2 receptor


Carbon monoxide


Delayed neurologic sequelae


Enhanced chemiluminescence












Morris water maze


Nitric oxide


Reactive oxygen species


Standard deviation


Tumor necrosis factor-α


Terminal deoxytransferase-mediated dUTP nick end labeling


WIN 55,212-2


Funding Information

This work was supported by a grant from the National Natural Science Foundation of China (grant number 81501139.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Aguirre-Rueda D et al (2015) WIN 55,212-2, agonist of cannabinoid receptors, prevents amyloid beta1-42 effects on astrocytes in primary culture. PLoS One 10:e0122843. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Akyol S, Yuksel S, Pehlivan S, Erdemli HK, Gulec MA, Adam B, Akyol O (2016) Possible role of antioxidants and nitric oxide inhibitors against carbon monoxide poisoning: having a clear conscience because of their potential benefits. Med Hypotheses 92:3–6. CrossRefPubMedGoogle Scholar
  3. Alonso-Alconada D, Alvarez FJ, Alvarez A, Mielgo VE, Goñi-de-Cerio F, Rey-Santano MC, Caballero A, Martinez-Orgado J, Hilario E (2010) The cannabinoid receptor agonist WIN 55,212-2 reduces the initial cerebral damage after hypoxic–ischemic injury in fetal lambs. Brain Res 1362:150–159. CrossRefPubMedGoogle Scholar
  4. Aso E, Ferrer I (2014) Cannabinoids for treatment of Alzheimer’s disease: moving toward the clinic. Front Pharmacol 5:37. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Betterman K, Patel S (2014) Neurologic complications of carbon monoxide intoxication. Handb Clin Neurol 120:971–979. CrossRefPubMedGoogle Scholar
  6. Bitterman H (2009) Bench-to-bedside review: oxygen as a drug. Crit Care (London, England) 13:205. CrossRefGoogle Scholar
  7. Bosier B, Bellocchio L, Metna-Laurent M, Soria-Gomez E, Matias I, Hebert-Chatelain E, Cannich A, Maitre M, Leste-Lasserre T, Cardinal P, Mendizabal-Zubiaga J, Canduela MJ, Reguero L, Hermans E, Grandes P, Cota D, Marsicano G (2013) Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes. Mol Metab 2:393–404. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Buckley NA, Juurlink DN, Isbister G, Bennett MH, Lavonas EJ (2011) Hyperbaric oxygen for carbon monoxide poisoning. Cochrane Database Syst Rev:Cd002041.
  9. Cherry JD, Olschowka JA, O'Banion MK (2014) Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation 11:98. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chiurchiu V, Leuti A, Maccarrone M (2015) Cannabinoid signaling and neuroinflammatory diseases: a melting pot for the regulation of brain immune responses. J NeuroImmune Pharmacol 10:268–280. CrossRefPubMedGoogle Scholar
  11. Choi IY, Ju C, Anthony Jalin AM, Lee DI, Prather PL, Kim WK (2013) Activation of cannabinoid CB2 receptor-mediated AMPK/CREB pathway reduces cerebral ischemic injury. Am J Pathol 182:928–939. CrossRefPubMedGoogle Scholar
  12. Dong G, Ren M, Wang X, Jiang H, Yin X, Wang S, Wang X, Feng H (2015) Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats. Neurotoxicology 48:171–179. CrossRefPubMedGoogle Scholar
  13. Facchinetti F, Del Giudice E, Furegato S, Passarotto M, Leon A (2003) Cannabinoids ablate release of TNFalpha in rat microglial cells stimulated with lypopolysaccharide. Glia 41:161–168. CrossRefPubMedGoogle Scholar
  14. Fernandez-Lopez D, Faustino J, Derugin N, Wendland M, Lizasoain I, Moro MA, Vexler ZS (2012) Reduced infarct size and accumulation of microglia in rats treated with WIN 55,212-2 after neonatal stroke. Neuroscience 207:307–315. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gadani SP, Walsh JT, Smirnov I, Zheng J, Kipnis J (2015) The glia-derived alarmin IL-33 orchestrates the immune response and promotes recovery following CNS injury. Neuron 85:703–709. CrossRefPubMedGoogle Scholar
  16. Garg J, Krishnamoorthy P, Palaniswamy C, Khera S, Ahmad H, Jain D, Aronow WS, Frishman WH (2018) Cardiovascular abnormalities in carbon monoxide poisoning. Am J Ther 25:e339–e348. CrossRefPubMedGoogle Scholar
  17. Gonzalez C et al (2011) Cannabinoid/agonist WIN 55,212-2 reduces cardiac ischaemia-reperfusion injury in Zucker diabetic fatty rats: role of CB2 receptors and iNOS/eNOS. Diabetes Metab Res Rev 27:331–340. CrossRefPubMedGoogle Scholar
  18. Hillard CJ, Manna S, Greenberg MJ, DiCamelli R, Ross RA, Stevenson LA, Murphy V, Pertwee RG, Campbell WB (1999) Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid receptor (CB1). J Pharmacol Exp Ther 289:1427–1433PubMedGoogle Scholar
  19. Hu X, Li P, Guo Y, Wang H, Leak RK, Chen S, Gao Y, Chen J (2012) Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia. Stroke 43:3063–3070. CrossRefPubMedGoogle Scholar
  20. Huo J et al (2019) Inhibiting a spinal cord signaling pathway protects against ischemia injury in rats. J Thorac Cardiovasc Surg 157:494–503.e491. CrossRefPubMedGoogle Scholar
  21. Icme F, Kozaci N, Ay MO, Avci A, Gumusay U, Yilmaz M, Satar S (2014) The relationship between blood lactate, carboxy-hemoglobin and clinical status in CO poisoning. Eur Rev Med Pharmacol Sci 18:393–397. CrossRefPubMedGoogle Scholar
  22. Jiang M, Liu X, Zhang D, Wang Y, Hu X, Xu F, Jin M, Cao F, Xu L (2018) Celastrol treatment protects against acute ischemic stroke-induced brain injury by promoting an IL-33/ST2 axis-mediated microglia/macrophage M2 polarization. J Neuroinflammation 15:78. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Koch M, Kreutz S, Bottger C, Grabiec U, Ghadban C, Korf HW, Dehghani F (2011) The cannabinoid WIN 55,212-2-mediated protection of dentate gyrus granule cells is driven by CB1 receptors and modulated by TRPA1 and Cav 2.2 channels. Hippocampus 21:554–564. CrossRefPubMedGoogle Scholar
  24. Kossatz E, Maldonado R, Robledo P (2016) CB2 cannabinoid receptors modulate HIF-1alpha and TIM-3 expression in a hypoxia-ischemia mouse model. Eur Neuropsychopharmacol 26:1972–1988. CrossRefPubMedGoogle Scholar
  25. Lara-Celador I, Castro-Ortega L, Alvarez A, Goni-de-Cerio F, Lacalle J, Hilario E (2012) Endocannabinoids reduce cerebral damage after hypoxic-ischemic injury in perinatal rats. Brain Res 1474:91–99. CrossRefPubMedGoogle Scholar
  26. Li D et al (2014) IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice. J Allergy Clin Immunol 134:1422–1432.e1411. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lin L, Yihao T, Zhou F, Yin N, Qiang T, Haowen Z, Qianwei C, Jun T, Yuan Z, Gang Z, Hua F, Yunfeng Y, Zhi C (2017) Inflammatory regulation by driving microglial M2 polarization: neuroprotective effects of cannabinoid receptor-2 activation in intracerebral hemorrhage. Front Immunol 8:112. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lin MS, Lin CC, Yang CC, Weng SC, Wang SM, Chen CY, Huang N, Chou YH (2018) Myocardial injury was associated with neurological sequelae of acute carbon monoxide poisoning in Taiwan. J Chin Med Assoc 81:682–690. CrossRefPubMedGoogle Scholar
  29. Marchalant Y, Rosi S, Wenk GL (2007) Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation. Neuroscience 144:1516–1522. CrossRefPubMedGoogle Scholar
  30. Marchalant Y, Brothers HM, Wenk GL (2009) Cannabinoid agonist WIN-55,212-2 partially restores neurogenesis in the aged rat brain. Mol Psychiatry 14:1068–1069. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mecha M, Feliu A, Carrillo-Salinas FJ, Rueda-Zubiaurre A, Ortega-Gutierrez S, de Sola RG, Guaza C (2015) Endocannabinoids drive the acquisition of an alternative phenotype in microglia. Brain Behav Immun 49:233–245. CrossRefPubMedGoogle Scholar
  32. Pang L, Zhang N, Dong N, Wang DW, Xu DH, Zhang P, Meng XW (2016) Erythropoietin protects rat brain injury from carbon monoxide poisoning by inhibiting Toll-like receptor 4/NF-kappa B-dependent inflammatory responses. Inflammation 39:561–568. CrossRefPubMedGoogle Scholar
  33. Ransohoff RM (2016) A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19:987–991. CrossRefPubMedGoogle Scholar
  34. Rose JJ, Wang L, Xu Q, McTiernan CF, Shiva S, Tejero J, Gladwin MT (2017) Carbon monoxide poisoning: pathogenesis, management, and future directions of therapy. Am J Respir Crit Care Med 195:596–606. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sanchez AJ, Garcia-Merino A (2012) Neuroprotective agents: cannabinoids. Clin Immunol (Orlando, Fla) 142:57–67. CrossRefGoogle Scholar
  36. Shen MH, Cai JM, Sun Q, Zhang DW, Huo ZL, He J, Sun XJ (2013) Neuroprotective effect of hydrogen-rich saline in acute carbon monoxide poisoning. CNS Neurosci Ther 19:361–363. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Shen M et al (2016) Neuroprotective effects of methane-rich saline on experimental acute carbon monoxide toxicity. J Neurol Sci 369:361–367. CrossRefPubMedGoogle Scholar
  38. Shin JA, Lim SM, Jeong SI, Kang JL, Park EM (2014) Noggin improves ischemic brain tissue repair and promotes alternative activation of microglia in mice. Brain Behav Immun 40:143–154. CrossRefPubMedGoogle Scholar
  39. Slavic S et al (2013) Cannabinoid receptor 1 inhibition improves cardiac function and remodelling after myocardial infarction and in experimental metabolic syndrome. J Mol Med (Berlin, Germany) 91:811–823. CrossRefGoogle Scholar
  40. Stella N (2010) Cannabinoid and cannabinoid-like receptors in microglia, astrocytes, and astrocytomas. Glia 58:1017–1030. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Su BX, Chen X, Huo J, Guo SY, Ma R, Liu YW (2017) The synthetic cannabinoid WIN55212-2 ameliorates traumatic spinal cord injury via inhibition of GAPDH/Siah1 in a CB2-receptor dependent manner. Brain Res 1671:85–92. CrossRefPubMedGoogle Scholar
  42. Tang Y, Le W (2016) Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol 53:1181–1194. CrossRefPubMedGoogle Scholar
  43. Tao Y et al (2016) Cannabinoid receptor-2 stimulation suppresses neuroinflammation by regulating microglial M1/M2 polarization through the cAMP/PKA pathway in an experimental GMH rat model. Brain Behav Immun 58:118–129. CrossRefPubMedGoogle Scholar
  44. Thom SR, Bhopale VM, Fisher D, Zhang J, Gimotty P (2004) Delayed neuropathology after carbon monoxide poisoning is immune-mediated. Proc Natl Acad Sci U S A 101:13660–13665. CrossRefPubMedPubMedCentralGoogle Scholar
  45. van Praag H, Shubert T, Zhao C, Gage FH (2005) Exercise enhances learning and hippocampal neurogenesis in aged mice. J Neurosci 25:8680–8685. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Wang W, Tian L, Li Y, Wang X, Xia F, Li L, Li J, Zhang Z (2013) Effects of hydrogen-rich saline on rats with acute carbon monoxide poisoning. J Emerg Med 44:107–115. CrossRefPubMedGoogle Scholar
  47. Wang Y, Ma S, Wang Q, Hu W, Wang D, Li X, Su T, Qin X, Zhang X, Ma K, Chen J, Xiong L, Cao F (2014) Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart. Sci China Life Sci 57:201–208. CrossRefPubMedGoogle Scholar
  48. Wilson JL, Bouillaud F, Almeida AS, Vieira HL, Ouidja MO, Dubois-Randé JL, Foresti R, Motterlini R (2017) Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus. Free Radic Biol Med 104:311–323. CrossRefPubMedGoogle Scholar
  49. Yang Y, Liu H, Zhang H, Ye Q, Wang J, Yang B, Mao L, Zhu W, Leak RK, Xiao B, Lu B, Chen J, Hu X (2017) ST2/IL-33-dependent microglial response limits acute ischemic brain injury. J Neurosci 37:4692–4704. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Yasuoka S, Kawanokuchi J, Parajuli B, Jin S, Doi Y, Noda M, Sonobe Y, Takeuchi H, Mizuno T, Suzumura A (2011) Production and functions of IL-33 in the central nervous system. Brain Res 1385:8–17. CrossRefPubMedGoogle Scholar
  51. Zarruk JG et al (2012) Cannabinoid type 2 receptor activation downregulates stroke-induced classic and alternative brain macrophage/microglial activation concomitant to neuroprotection. Stroke 43:211–219. CrossRefPubMedGoogle Scholar
  52. Zhang M, Martin BR, Adler MW, Razdan RK, Jallo JI, Tuma RF (2007) Cannabinoid CB2 receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model. J Cereb Blood Flow Metab 27:1387–1396. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zhao N, Liang P, Zhuo X, Su C, Zong X, Guo B, Han D, Yan X, Hu S, Zhang Q, Tie X (2018) After treatment with methylene blue is effective against delayed encephalopathy after acute carbon monoxide poisoning. Basic Clin Pharmacol Toxicol 122:470–480. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Neurology, Xi’an Central HospitalXi’an Jiaotong UniversityXi’anChina
  2. 2.Department of Hematology, Xi’an Central HospitalXi’an Jiaotong UniversityXi’anChina

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