title: Carbachol alleviates rat cytokine release and organ dysfunction induced by lipopolysaccharide.
abstract: BACKGROUND
  To observe the influence of carbachol on inflammatory cytokine release and its protective role on organ function in rat endotoxemia model, and, furthermore, to investigate its receptor mechanism in rat peritoneal macrophages in vitro.
  
  
  METHODS
  In the animal experiments, Wistar rats were subjected to lipopolysaccharide (LPS) injection (5 mg/kg body weight) to establish an endotoxemia animal model, and carbachol/nicotine was given 15 minutes after LPS injection. Serum contents of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined with enzyme-linked immunosorbent assay 4 hours after LPS injection. Plasma alanine aminotransferase, creatine kinase-MB, and diamine oxidase contents were detected 24 hours after LPS injection. In cell experiments, rat peritoneal macrophages were collected and initially pretreated with atropine (muscarinic cholinergic receptor antagonist) or α-Bungarotoxin (an antagonist that specifically binds α7 subunit of nicotinic cholinergic receptor), then with carbachol or nicotine, and finally stimulated with LPS. Contents of TNF-α, IL-6, and IL-10 in supernatant were assayed by enzyme-linked immunosorbent assay. Furthermore, macrophages were exposed to nicotine and carbachol of high concentration and then stained with fluorescein isothiocyanate-labeled α-bungarotoxin and observed with fluorescent confocal microscopy.
  
  
  RESULTS
  Carbachol inhibited expression of TNF-α and IL-6 after LPS injection and had no significant effect on IL-10 in rat endotoxemia model. It also inhibited the increase of plasma alanine aminotransferase and creatine kinase-MB contents whereas restored the inhibited plasma diamine oxidase activity. Cell experiments also showed that increases of TNF-α and IL-6 after LPS stimulation could be significantly inhibited by carbachol or nicotine, whereas IL-10 was not apparently altered. Atropine did not downregulate the inhibitive effects of both carbachol and nicotine, whereas α-bungarotoxin significantly downregulated these effects. Fluorescent confocal microscopy showed that nicotine and carbachol pretreatment markedly reduced the intensity of binding between fluorescein isothiocyanate-labeled α-bungarotoxin and macrophages.
  
  
  CONCLUSIONS
  The results suggested that both carbachol and nicotine play a role in the anti-inflammatory process and organ function protection through the α7 subunit of nicotinic cholinergic receptor.
authors.affiliation: Laboratory of Shock and Organ Dysfunction, the First Hospital Affiliated to the People's Liberation Army General Hospital, Beijing, China.
authors.forename: Guoyong
authors.initials: G
authors.lastname: Zhou
authors.forename: Sen
authors.initials: S
authors.lastname: Hu
authors.forename: Yi
authors.initials: Y
authors.lastname: Lv
authors.forename: Qi
authors.initials: Q
authors.lastname: Song
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Zou
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Sheng
mesh_terms: D000818:Animals; D002217:Carbachol / Q000008:administration & dosage / Q000627:therapeutic use*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000008:administration & dosage / Q000627:therapeutic use; D016207:Cytokines / Q000187:drug effects / Q000378:metabolism*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D008070:Lipopolysaccharides / Q000633:toxicity; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D018613:Microscopy, Confocal; D009102:Multiple Organ Failure / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015996:Survival Rate; D018746:Systemic Inflammatory Response Syndrome / Q000139:chemically induced / Q000188:drug therapy* / Q000378:metabolism; D016896:Treatment Outcome
chemical_list: D018679:Cholinergic Agonists; D016207:Cytokines; D008070:Lipopolysaccharides; D009538:Nicotine; D002217:Carbachol