Skip to main content
SpringerLink
Log in

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways

  • Research Article
  • Published:
European Cytokine Network Aims and scope

Abstract

Background and Objective

Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro.

Methods

Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000 ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10−5 M) alone, treated with 100 ng/mL LPS alone, or treated with different concentrations of ACh (10−9/10−8/10−7/10−6/10−5 M) before LPS stimulation. HUVECs were also pre-treated with 10−6 M ACh with or without mecamylamine (an nAChR blocker) (10 µM) and methyllycaconitine (a specific α7 nAChR blocker) (10 µM) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways.

Results

LPS (at 10 ng/mL, 100 ng/mL and 1,000 ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100 ng/mL and 1,000 ng/mL). ACh (10−9 M–10−5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10−5 and 10−6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10−6 M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10−6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38 MAPK in HUVECs, which was blocked by mecamylamine.

Conclusions

ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

References

  1. Castro-Ferreira R, Cardoso R, Leite-Moreira A, Mansilha A. The role of endothelial dysfunction and inflammation in chronic venous disease. Ann Vasc Surg 2018; 46: 380–93. doi: https://doi.org/10.1016/j.avsg.2017.06.131.

    Article  PubMed  Google Scholar 

  2. Chen PY, Schwartz MA, Simons M. Endothelial-to-mesenchymal transition, vascular inflammation, and atherosclerosis. Front Cardiovasc Med 2020; 7: 53. doi: https://doi.org/10.3389/fcvm.2020.00053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bui TM, Wiesolek HL, Sumagin R. ICAM-1: a master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis. J Leukoc Biol 2020; 108: 787–99. doi: https://doi.org/10.1002/JLB.2MR0220-549R

    Article  CAS  PubMed  Google Scholar 

  4. Cerda A, Pavez M, Manriquez V, et al.. Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: role of nitric oxide mediating pleiotropic effects. Cardiovasc Ther 2017; 35. doi: https://doi.org/10.1111/1755-5922.12261.

  5. Chang KC. Cilostazol inhibits HMGB1 release in LPS-activated RAW 264.7 cells and increases the survival of septic mice. Thromb Res 2015; 136: 456–64. doi: https://doi.org/10.1016/j.thromres.2015.06.017.

    Article  CAS  PubMed  Google Scholar 

  6. Lv Y, Kim K, Sheng Y, et al. YAP controls endothelial activation and vascular inflammation through TRAF6. Circ Res 2018; 123: 43–56. doi: https://doi.org/10.1161/CIRCRESAHA.118.313143.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tracey KJ. The inflammatory reflex. Nature 2002; 420: 853–9. doi: https://doi.org/10.1038/nature01321.

    Article  CAS  PubMed  Google Scholar 

  8. Sun P, Zhou K, Wang S, et al.. Involvement of MAPK/NF-κB signaling in the activation of the cholinergic anti-inflammatory pathway in experimental colitis by chronic vagus nerve stimulation. PLoS One 2013; 8: e69424. doi: https://doi.org/10.1371/journal.pone.0069424.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Borovikova LV, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 2000; 405: 458–62. doi: https://doi.org/10.1038/35013070.

    Article  CAS  PubMed  Google Scholar 

  10. Wang H, Yu M, Ochani M, et al.. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003; 421: 384–8. doi: https://doi.org/10.1038/nature01339.

    Article  CAS  PubMed  Google Scholar 

  11. Chen J, Qiu M, Huang Z, et al.. Nicotine suppresses the invasiveness of human trophoblasts by downregulation of CXCL12 expression through the alpha-7 subunit of the nicotinic acetylcholine receptor. Reprod Sci 2020; 27: 916–24. doi: https://doi.org/10.1007/s43032-019-00095-4.

    Article  CAS  PubMed  Google Scholar 

  12. Wedn AM, El-Gowilly SM, El-Mas MM. Nicotine reverses the enhanced renal vasodilator capacity in endotoxic rats: role of α7/αB2;2 nAChRs and HSP70. Pharmacol Rep 2019; 71: 782–93. doi: https://doi.org/10.1016/j.pharep.2019.04.013.

    Article  CAS  PubMed  Google Scholar 

  13. Middlebrook AJ, Martina C, Chang Y, Lukas RJ, DeLuca D. Effects of nicotine exposure on T cell development in fetal thymus organ culture: arrest of T cell maturation. J Immunol 2002; 169: 2915–24. doi: https://doi.org/10.4049/jimmunol.169.6.2915.

    Article  CAS  PubMed  Google Scholar 

  14. Han T, Wang Q, Lai R, Zhang D, Diao Y, Yin Y. Nicotine induced neurocognitive protection and anti-inflammation effect by activating a4β2 nicotinic acetylcholine receptors in ischemic rats. Nicotine Tob Res 2020; 22: 919–24. doi: https://doi.org/10.1093/ntr/ntz126.

    Article  CAS  PubMed  Google Scholar 

  15. Wang H, Liao H, Ochani M, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004; 10: 1216–21. doi: https://doi.org/10.1038/nm1124.

    Article  CAS  PubMed  Google Scholar 

  16. Crotty Alexander LE, Drummond CA, Hepokoski M, et al. Chronic inhalation of e-cigarette vapor containing nicotine disrupts airway barrier function and induces systemic inflammation and multiorgan fibrosis in mice. Am J Physiol Regul Integr Comp Physiol 2018; 314: R834–47. doi: https://doi.org/10.1152/ajpregu.00270.2017.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cortés MP, Alvarez R, Sepúlveda E, et al. A new isoxazolic compound acts as alpha7 nicotinic receptor agonist in human umbilical vein endothelial cells. Z Naturforsch C J Biosci 2014; 69: 291–9. doi: https://doi.org/10.5560/znc.2012-0176.

    Article  PubMed  Google Scholar 

  18. Fu X, Zong T, Yang P, et al. Nicotine: regulatory roles and mechanisms in atherosclerosis progression. Food Chem Toxicol 2021; 151: 112154. doi: https://doi.org/10.1016/j.fct.2021.112154.

    Article  CAS  PubMed  Google Scholar 

  19. Macklin KD, Maus AD, Pereira EF, Albuquerque EX, Conti-Fine BM. Human vascular endothelial cells express functional nicotinic acetylcholine receptors. J Pharmacol Exp Ther 1998; 287: 435–9.

    CAS  PubMed  Google Scholar 

  20. Whitehead AK, Erwin AP, Yue X. Nicotine and vascular dysfunction. Acta Physiol (Oxf) 2021; 231: e13631. doi: https://doi.org/10.1111/apha.13631.

    Article  CAS  PubMed  Google Scholar 

  21. Zoli M, Pucci S, Vilella A, Gotti C. Neuronal and extraneuronal nicotinic acetylcholine receptors. Curr Neuropharmacol 2018; 16: 338–49. doi: https://doi.org/10.2174/1570159X15666170912110450.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Saeed RW, Varma S, Peng-Nemeroff T, et al. Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation. J Exp Med 2005; 201: 1113–23. doi: https://doi.org/10.1084/jem.20040463.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhou K, Sun P, Zhang Y, You X, Li P, Wang T. Estrogen stimulated migration and invasion of estrogen receptor-negative breast cancer cells involves an ezrin-dependent crosstalk between G protein-coupled receptor 30 and estrogen receptor beta signaling. Steroids 2016; 111: 113–20. doi: https://doi.org/10.1016/j.steroids.2016.01.021

    Article  CAS  PubMed  Google Scholar 

  24. Li P, Liu H, Sun P, et al. Chronic vagus nerve stimulation attenuates vascular endothelial impairments and reduces the inflammatory profile via inhibition of the NF-κB signaling pathway in ovariectomized rats. Exp Gerontol 2016; 74: 43–55. doi: https://doi.org/10.1016/j.exger.2015.12.005

    Article  CAS  PubMed  Google Scholar 

  25. Moreau KL, Hildreth KL, Meditz AL, Deane KD, Kohrt WM. Endothelial function is impaired across the stages of the menopause transition in healthy women. J Clin Endocrinol Metab 2012; 97: 4692–700. doi: https://doi.org/10.1210/jc.2012-2244.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Oliveira PW, de Sousa GJ, Caliman IF, et al. Metformin ameliorates ovariectomy-induced vascular dysfunction in non-diabetic Wistar rats. Clin Sci (Lond) 2014; 127: 265–75. doi: https://doi.org/10.1042/CS20130553.

    Article  CAS  PubMed  Google Scholar 

  27. Chen Z, Chen Y, Pan L, et al. Dachengqi decoction attenuates inflammatory response via inhibiting HMGB1 mediated NF-κB and P38 MAPK signaling pathways in severe acute pancreatitis. Cell Physiol Biochem 2015; 37: 1379–89. doi: https://doi.org/10.1159/000430403.

    Article  CAS  PubMed  Google Scholar 

  28. Kim YM, Kim HJ, Chang KC. Glycyrrhizin reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and endotoxemic mice by p38/Nrf2-dependent induction of HO-1. Int Immunopharmacol 2015; 26: 112–8. doi: https://doi.org/10.1016/j.intimp.2015.03.014.

    Article  CAS  PubMed  Google Scholar 

  29. Mai J, Nanayakkara G, Lopez-Pastrana J, et al. Interleukin-17A promotes aortic endothelial cell activation via transcriptionally and post-translationally activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway. J Biol Chem 2016; 291: 4939–54. doi: https://doi.org/10.1074/jbc.M115.690081.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li X, Liu Y, Wang L, Li Z, Ma X. Unfractionated heparin attenuates LPS-induced IL-8 secretion via PI3K/Akt/NF-KB signaling pathway in human endothelial cells. Immunobiology 2015; 220: 399–405. doi: https://doi.org/10.1016/j.imbio.2014.10.008.

    Article  CAS  PubMed  Google Scholar 

  31. Kanarek N, Ben-Neriah Y. Regulation of NF-κB by ubiquitination and degradation of the IκBs. Immunol Rev 2012; 246: 77–94. doi: https://doi.org/10.1111/j.1600-065X.2012.01098.x.

    Article  PubMed  Google Scholar 

  32. Majdalawieh A, Ro HS. Regulation of IkappaBalpha function and NF-kappaB signaling: AEBP1 is a novel proinflammatory mediator in macrophages. Mediators Inflamm 2010; 2010: 823821. doi: https://doi.org/10.1155/2010/823821.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Gaestel M. MAPK-activated protein kinases (MKs): novel insights and challenges. Front Cell Dev Biol 2016; 3: 88. doi: https://doi.org/10.3389/fcell.2015.00088

    Article  PubMed  PubMed Central  Google Scholar 

  34. Maik-Rachline G, Hacohen-Lev-Ran A, Seger R. Nuclear ERK: mechanism of translocation, substrates, and role in cancer. Int J Mol Sci 2019; 20: 1194. doi: https://doi.org/10.3390/ijms20051194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Oh HJ, Magar TBT, Pun NT, Lee Y, Kim EH, Lee ES, Park PH. YJI-7 suppresses ROS production and expression of inflammatory mediators via modulation of p38MAPK and JNK signaling in RAW 264.7 macrophages. Biomol Ther (Seoul) 2018; 26: 191–200. doi: https://doi.org/10.4062/biomolther.2016.276.

    Article  CAS  PubMed  Google Scholar 

  36. Zhou H, Sun Y, Zhang L, Kang W, Li N, Li Y. The RhoA/ROCK pathway mediates high glucose-induced cardiomyocyte apoptosis via oxidative stress, JNK, and p38MAPK pathways. Diabetes Metab Res Rev 2018; 34: e3022. doi: https://doi.org/10.1002/dmrr.3022. Epub 2018 Jun 4.

    Article  PubMed  Google Scholar 

  37. O’Neil JD, Ammit AJ, Clark AR. MAPK p38 regulates inflammatory gene expression via tristetraprolin: doing good by stealth. Int J Biochem Cell Biol 2018; 94: 6–9. doi: https://doi.org/10.1016/j.biocel.2017.11.003.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Corre I, Paris F, Huot J. The p38 pathway, a major pleiotropic cascade that transduces stress and metastatic signals in endothelial cells. Oncotarget. 2017; 8: 55684–714. doi: https://doi.org/10.18632/oncotarget.18264.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Joko T, Shiraishi A, Akune Y, et al. Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β(2). Exp Eye Res 2013; 108: 23–32. doi: https://doi.org/10.1016/j.exer.2012.11.018.

    Article  CAS  PubMed  Google Scholar 

  40. Li L, Hu J, He T, et al. P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury. Sci Rep 2015; 5: 8895. doi: https://doi.org/10.1038/srep08895.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Cicenas J, Zalyte E, Rimkus A, Dapkus D, Noreika R, Urbonavicius S. JNK, p38, ERK, and SGK1 inhibitors in cancer. Cancers (Basel) 2017; 10: 1. doi: https://doi.org/10.3390/cancers10010001.

    Article  PubMed  Google Scholar 

  42. Mei F, Zuo T, Zhao L, et al.. Differential JNK, p38 and ERK response to renal injury in a rat model of acute pancreatitis in pregnancy. Arch Gynecol Obstet 2018; 297: 933–942. doi: https://doi.org/10.1007/s00404-018-4668-x.

    Article  CAS  PubMed  Google Scholar 

  43. Kim EK, Choi EJ. Compromised MAPK signaling in human diseases: an update. Arch Toxicol 2015; 89: 867–82. doi: https://doi.org/10.1007/s00204-015-1472-2.

    Article  CAS  PubMed  Google Scholar 

  44. Wilund KR, Rosenblat M, Chung HR, et al. Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: a possible link between the nervous system and atherosclerosis development. Biochem Biophys Res Commun 2009; 390: 148–54. doi: https://doi.org/10.1016/j.bbrc.2009.09.088.

    Article  CAS  PubMed  Google Scholar 

  45. Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: the nonneuronal cholinergic system in humans. Br J Pharmacol 2008; 154: 1558–71. doi: https://doi.org/10.1038/bjp.2008.185.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. These authors contributed equally

Authors and Affiliations

  1. Department of Pathology, Peking University Shenzhen Hospital, 1120 Lianhua Road, Shenzhen, Guangdong Province, 518036, People’s Republic of China

    Ping Li

  2. Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road 2, Guangzhou, Guangdong Province, 510080, People’s Republic of China

    Ping Li, Kewen Zhou, Jiehao Li, Xiaodan Xu, Ling Wang & Tinghuai Wang

Authors
  1. Ping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kewen Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiehao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaodan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ling Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tinghuai Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tinghuai Wang.

Ethics declarations

Disclosure. Financial support: this work was supported by the National Natural Science Foundation of China (no. 81572585 and no. 81800848), Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515110011) and the Research Foundation of Peking University Shenzhen Hospital (No. JCYJ2020006).

Conflict of interest: none.

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, P., Zhou, K., Li, J. et al. Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways. Eur Cytokine Netw 33, 79–89 (2022). https://doi.org/10.1684/ecn.2023.0481

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1684/ecn.2023.0481

Key words

Search

Navigation