title: Antisense inhibition of secretory and cytosolic phospholipase A2 reduces the mortality in rats with sepsis*.
abstract: OBJECTIVE
  Phospholipase A(2) has been implicated to play a pivotal role in the pathogenesis of sepsis syndrome. The two major forms of phospholipase A(2) isoenzymes, secretory phospholipase A(2) and cytosolic phospholipase A(2), are overexpressed during sepsis. The objective of this study was to test the hypothesis that inhibition of the overexpressed secretory phospholipase A(2) and cytosolic phospholipase A(2) during sepsis benefits the disease's eventual outcome.
  
  
  METHODS
  Short-chain antisense oligonucleotide molecules were designed with the aid of computer software programs, and their in vitro efficacies were assessed in cell culture systems based on inhibition of target protein expression. The in vivo efficacies were determined in intact sepsis rats using 35-day survival rate as a primary efficacy end point.
  
  
  METHODS
  Animal research laboratory at a university.
  
  
  METHODS
  Male Sprague-Dawley rats (180-200 g).
  
  
  METHODS
  Sepsis was induced by cecal ligation and puncture. Antibiotics were administered subcutaneously once daily at 12 mg/kg, for 20 days. Oligonucleotides (antisense or mismatch) were administered intravenously once daily at 2 mg/kg to 0.8 mg/kg in a decreasing order, for 20 days.
  
  
  RESULTS
  In cell culture systems, 21 of the 105 antisense constructs were found to be efficacious in inhibiting secretory phospholipase A(2) IIa and cytosolic phospholipase A(2) IVa protein expression. In sepsis rats, antisense oligonucleotides were capable of reducing their target protein expression by 18%-61% in major organs such as liver, heart, and kidney. In animal experiments, sepsis without any treatment (Group 1) had a median survival time of 2 days and a zero (0) percent survival rate at day 14. Sepsis with antibiotic treatment (Group 2) had a median survival time of 6 days and a 35-day survival rate of 28%. Sepsis with cotreatment of antibiotics and antisense oligonucleotides (one against secretory phospholipase A2 IIa and the other against cytosolic phospholipase A(2) IVa) (Group 4) increased the median survival time from 6 to 35 days and the 35-day survival rate from 28% to 58.8% as compared with antibiotics alone (Group 4 vs. Group 2; p <.05). Sepsis with cotreatment of antibiotics and mismatch oligonucleotides (Group 3) did not affect the median survival time and the 35-day survival rate as compared to antibiotics alone (Group 3 vs. Group 2; p >.05).
  
  
  CONCLUSIONS
  The results demonstrate that antisense strategy against secretory phospholipase A(2) IIa and cytosolic phospholipase A(2) IVa can inhibit their target protein expression in major organs and greatly improve the clinical outcome, i.e., an absolute reduction in 35-day mortality of 30.8%, in rats with sepsis. Our studies, thus, provide an improved method for the treatment of sepsis by targeting multiple forms of phospholipase A(2) isoenzymes with DNA antisense oligomers.
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St Louis, MO, USA. lium@slu.edu
authors.forename: Maw-Shung
authors.initials: MS
authors.lastname: Liu
authors.forename: Chia-Hsiung
authors.initials: CH
authors.lastname: Liu
authors.forename: Guang
authors.initials: G
authors.lastname: Wu
authors.forename: Yuefang
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D004195:Disease Models, Animal; D015536:Down-Regulation; D054513:Group IV Phospholipases A2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006321:Heart / Q000187:drug effects; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D054497:Phospholipases A2, Secretory / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018805:Sepsis / Q000188:drug therapy* / Q000401:mortality*
chemical_list: D000900:Anti-Bacterial Agents; D016376:Oligonucleotides, Antisense; D054513:Group IV Phospholipases A2; D054497:Phospholipases A2, Secretory