Elucidation of anti-inflammatory effect and osteogenesis of Inchinkoto in human osteosarcoma cell line Saos-2

Abstract

Inchinkoto, a medical kampo formulation, is widely used in Japan for the treatment of chronic liver disease and stomatitis. In this study, we investigated the effects of Inchinkoto on the inflammatory cytokines interleukin (IL)-6 and IL-8, and the secretion of type I collagen and alkaline phosphatase (ALP), markers of bone formation, using the human osteosarcoma cell line (Saos-2). The proliferative ability of Saos-2 cells was analyzed using MTT assay and BrdU Cell Proliferation Assay Kit, while collagen production was analyzed using ELISA. ALP production was assessed using the LabAssay ALP kit. We used a system in which Saos-2 produces IL-6 and IL-8 in response to lipopolysaccharide (LPS) stimulation from the periodontal pathogen Actinobacillus actinomycetemcomitans. IL-6, IL-8, type I collagen production, and ALP secretion levels were evaluated using ELISA and ALP assay. Saos-2 cells were cultured for 24 hours in the presence or absence of Inchinkoto at concentrations of 0.1, 1, 10, 100, and 1000 μg/mL. Inchinkoto significantly induced the proliferation of Saos-2 cells in a concentration-dependent manner. In this study, Inchinkoto at 1000 μg/mL suppressed IL-6 and IL-8, promoting type I collagen production and ALP secretion. These results suggest that Inchinkoto has potential anti-inflammatory and bone-forming effects.