(SASA-Health®) inhibited NO and prostaglandin E2 (PGE2) production by activated macrophages via inhibition of inducible NO synthase and cyclooxygenase-2 expression at both protein and mRNA

Background: We have previously reported that azulene-related compounds, and alkaline extract of Sasa senanensis Rehder potently inhibited nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophages. We investigated here whether they can inhibit pro-inflammatory cytokine production, by activated human gingival fibroblast (HGF). Materials and Methods: HGF was established from the periodontal tissues of extracted tooth. Viable cell number was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Production of Prostaglandin E2 (PGE2) and cytokines was determined by enzyme immunoassay, and enzyme-linked immunosorbent assay, respectively. Results: Interleukin (IL)1β did not inhibit, but rather slightly stimulated the growth of HGF cells. IL-1β stimulated the production of PGE2, IL-6, IL8 and monocyte chemotactic protein-1 very potently, but not that of nitric oxide and tumor necrosis factor-α. Native LPS and synthetic lipid A from E. coli and P. gingivalis was much less stimulatory. Dexamethasone, not indomethacin, was an efficient inhibitor of IL-8 production. Among five azulenerelated compounds, benzo[b]cyclohepta[e][1,4]thiazine most potently inhibited the IL-8 production by HGF cells, as well as NO production by activated RAW264.7 cells. The alkaline extract of Sasa senanensis Rehder significantly inhibited IL-8 production, without affecting the cell viability. Conclusion: The present system may be applicable for use in the search for anti-gingivitis substances. Chemotherapy and radiotherapy, while highly effective in the treatment of neoplast, adversely affect the epithelia of oral mucosa, causing severe inflammation, lesioning, ulceration and bleeding in the mouth (1). The cause of stomatitis is unknown and thought to be multifactorial with many triggers or precipitating factors. The risk factors include the poor oral hygiene, secondary infection of both Gram-positive and Gram-negative bacteria, low rates of salivary production, poor production of mucins, antimicrobial-, anti-fungal and anti-viral factors, impaired local or systemic immunity, and age/gender (2). Since some risk factors for stomatitis and those for oral cancer are common between the two diseases overlapped with each other (3), there is a possibility that stomatitis may trigger the carcinogenesis of oral cancer. It is therefore very important to establish an in vitro assay system for the measurement of anti-stomatitis activity. For this purpose, as the first part of the present study, we investigated here several stimulators for their ability to induce the production of pro-inflammatory substances in human gingival fibroblast (HGF) cells. We have previously reported that benzo[b]cyclohepta[e][1,4]thiazine [1], 6,8-dibromobenzo[b]cyclohepta [e][1,4]thiazine [2] (4), benzo[b]cyclohept[e][1,4]oxazin6(11H)-one [5] (5), 3-methyl-1-trichloroacetylazulene [2b] and 3-ethyl-1-trichloroacetylazulene [3b] (6) (Figure 1) inhibited nitric oxide (NO) production by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells (Table I). Similarly, alkaline extract of the leaves of Sasa senanensis Rehder or Sasa albo-marginata Makino et Shibata (SE) (SASA-Health®) inhibited NO and prostaglandin E2 (PGE2) production by activated macrophages via inhibition of inducible NO synthase and cyclooxygenase-2 expression at both protein and mRNA levels (7) (Table I). As the second part of our study, we investigated here whether these compounds and SE can effectively inhibit the production of pro-inflammatory cytokines by HGF. 763 Correspondence to: Professor Hidetsugu Wakabayashi, Faculty of Science, Josai University, Sakado, Saitama 350-0295, Japan. Tel: +81 0492717959, Fax: +81 0492717985, e-mail: [email protected]/ [email protected]/[email protected]