Induction of Apoptosis in Activated RAW 264.7 Cells and Inhibition of Pro-Inflammatory Mediators in Rat Air Pouch by Ethylacetate Fraction of Ocimum Gratissimum Leaves

Ocimum gratissimum L. has attracted substantial consideration from researchers because of its anti-inflammatory uses in ethnomedicine in Sub-Saharan Africa and Asia. This study investigated the effect of flavonoid-rich ethylacetate fraction of O. gratissimum (EAFOg) in apoptosis induction of activated macrophages and inflammatory response in LPS-induced air pouch in rats. Apoptotic effect of EAFOg in LPS-stimulated RAW 264.7 cells was evaluated using flow cytometry after staining with annexin-V and 7-aminoactinomycin D. Its effects on inflammatory cells and mediators were investigated utilizing 6 day old subcutaneous air pouch-rats. Sterile saline (0.9%) or LPS (100 ng/mL) was injected into the air pouch on 6th day after EAFOg (25, 50 and 100 mg/kg) pretreatment. Rectal body temperature was recorded hourly for 5 h after LPS injection. Thereafter, the neutrophil count, nitrite, TNF-α, PGE2, nitrite, malondialdehyde and reduced glutathione (GSH) levels were determined in the pouch lavage. The activities of myeloperoxidase and superoxide dismutase (SOD) as well as immunohistochemical staining for cyclooxygenase-2 were also performed. EAFOg (10, 30 and 100 µg/mL) induced apoptosis in LPS-stimulated RAW 264.7 macrophages. The EAFOg reduced hyperthermia and decreased neutrophil counts, TNF-α, PGE2, nitrite, myeloperoxidase as well as cyclooxygenase-2 expression evoked by LPS in rats. It also reduced malondialdehyde, and increased GSH and SOD levels in LPS-induced air pouch in vivo. The results of this study suggest that the EAFOg induces apoptosis in activated macrophages and reduces inflammatory responses provoked by LPS via down regulation of cyclooxygenase-2 expression in rats.