title: C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38-MAPK activation in acute liver failure.
abstract: OBJECTIVE
  To investigate the role of the complement 5a (C5a)/C5a receptor (C5aR) pathway in the pathogenesis of acute liver failure (ALF) in a mouse model.
  
  
  METHODS
  BALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide (LPS)/D-galactosamine (D-GalN) (600 mg/kg and 10 μg/kg) were used to induce ALF. The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5 (C5), C5a, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, high-mobility group protein B1 (HMGB1) and sphingosine-1-phosphate levels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5aR, sphingosine kinase 1 (SphK1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells (PBMCs) and peritoneal exudative macrophages (PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5aR mRNA levels were detected by quantitative real-time PCR.
  
  
  RESULTS
  Activation of C5 and up-regulation of C5aR were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5aR with a C5aR antagonist (C5aRa C5aRa) significantly reduced the levels of serum ALT, inflammatory cytokines (TNF-α, IL-1β and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates (P< 0.01 for all). Blockade of C5aR decreased SphK1 expression in both liver tissue and PBMCs significantly at 0.5 h after ALF induction. C5aRa pretreatment significantly down-regulated the phosphorylation of p38-MAPK in liver tissues of ALF mice and C5a stimulated PEMs or RAW 264.7 cells. Moreover, inhibition of p38-MAPK activity with SB203580 reduced SphK1 protein production significantly in PEMs after C5a stimulation.
  
  
  CONCLUSIONS
  The C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Yan-Chang
authors.initials: YC
authors.lastname: Lei
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Chun-Lei
authors.initials: CL
authors.lastname: Lu
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Ge
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: Yang
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Li
authors.affiliation: Yan-Chang Lei, Ling Chen, Ke Ge, Department of Infectious Diseases, Zhejiang Hospital, Hangzhou 310013, Zhejiang Province, China.
authors.forename: Yuan-Hua
authors.initials: YH
authors.lastname: Wu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015936:Complement C5a / Q000378:metabolism*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005688:Galactosamine / Q000633:toxicity; D024243:HMGB1 Protein / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D053208:Kaplan-Meier Estimate; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D017114:Liver Failure, Acute / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D008246:Lysophospholipids / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011897:Random Allocation; D060888:Real-Time Polymerase Chain Reaction; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000099157:Sphingosine Kinase
chemical_list: D015936:Complement C5a; D005688:Galactosamine; D024243:HMGB1 Protein; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D012333:RNA, Messenger; D044087:Receptor, Anaphylatoxin C5a; D013110:Sphingosine; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D000099157:Sphingosine Kinase; C582808:HMGB1 protein, mouse; C587672:IL1B protein, mouse; C577686:interleukin-6, mouse; C060506:sphingosine 1-phosphate
keywords: Acute liver failure; C5a/C5aR; Sphingosine kinase 1; p38-MAPK