Effects of Tokishakuyakusan on Regeneration of Murine Olfactory Neurons In Vivo and In Vitro

Post-upper respiratory tract infection related olfactory dysfunction typically occurs due to neural damage after an upper respiratory tract infection associated with a common cold or influenza. At present, Tokishakuyakusan, a Japanese traditional Kampo medicine, has been found to be effective for post-viral olfactory dysfunction. However, the pharmacodynamics of Tokishakuyakusan in the treatment of post-viral olfactory dysfunction remains unresolved. We investigated the effects of Tokishakuyakusan on the regeneration of olfactory neurons and expression of nerve growth factor (NGF) in neural systems, using in vivo murine studies and in vitro cell culture studies. Eight-week-old BALB/C female mice were fed a pellet diet with or without Tokishakuyakusan. Degeneration of cells in olfactory epithelium was induced by intraperitoneal methimazole injection. Regeneration of olfactory neurons was observed by histological and immunohistochemical procedures. NGF expression in the olfactory bulb was measured by enzyme-linked immunosorbent assay. NGF gene and protein expression were measured using rat primary cultured astrocytes by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We found that olfactory marker protein, Ki-67, and NGF were more highly expressed in the olfactory epithelium during the regeneration period in mice receiving Tokishakuyakusan. In cultured astrocytes, Tokishakuyakusan as well as its individual components, Atractylodes lancea rhizome and Japanese angelica root, increased NGF expression. Screening assays revealed that NGF production was increased by atractylodin and levistolide A, which are ingredients in Atractylodes lancea rhizome and Japanese angelica root, respectively. These results suggest that Tokishakuyakusan promotes regeneration of olfactory neurons by increasing NGF expression in the olfactory bulb.