title: In vitro and in vivo therapeutics of β-thujaplicin on LPS-induced inflammation in macrophages and septic shock in mice.
abstract: β-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of β-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-a gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of β-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of β thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-α were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-κB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, β-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, β-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-α production as well as iNOS, COX2, and NF- κB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, β-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-α production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of β-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-α and iNOS production. β-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications. β-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of β-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-alpha gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of β-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of β-thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-alpha were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-kB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, β-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, β-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-alpha production as well as iNOS, COX2, and NF-kB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, β-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-alpha production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of β-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-alpha and iNOS production. β-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications.
authors.affiliation: Department of Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan.
authors.forename: M-F
authors.initials: MF
authors.lastname: Shih
authors.forename: L-Y
authors.initials: LY
authors.lastname: Chen
authors.forename: P-J
authors.initials: PJ
authors.lastname: Tsai
authors.forename: J-Y
authors.initials: JY
authors.lastname: Cherng
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000627:therapeutic use*; D002478:Cells, Cultured; D015232:Dinoprostone / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D039821:Monoterpenes / Q000494:pharmacology / Q000627:therapeutic use*; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009569:Nitric Oxide / Q000096:biosynthesis; D012772:Shock, Septic / Q000188:drug therapy*; D014334:Tropolone / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D039821:Monoterpenes; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D014334:Tropolone; D015232:Dinoprostone; C009479:beta-thujaplicin