Immunomodulatory effects & mode of action of ginsenoside Rb-1

Abstract

Immune system protects the body against pathogens while spleen is crucial to the development of both innate and adaptive immune system. Upon activation, immune cells (e.g. B cells and T cells) communicate with each other through secretion of cytokines. Examples are interleukin-2, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 alpha, interleukin-6 and interleukin-10. On another front, ginseng (Panax quinquefolium) is one of the most commonly used medicinal herbs. It is believed to have diverse pharmacological effects including immunomodulation. However, its mode of actions is unclear. In this study, we aimed to investigate the immunomodulatory effects of ginsenoside Rb-1 and its mode of actions. With MTT assay, Rb-1 would neither induce growth nor inhibit proliferation of LPS-stimulated splenocytes. On the other hand, by ELISA assay, it was found that (a) Rb-1 alone did not induce IFN-y and TNF-a production in splenocytes cultures; (b) various pre-incubation periods of Rb-1 prior to LPS stimulation did not alter the secretion of IFN-y and TNF-a; (c) cytokines secretions were inhibited at highest dosage of Rb-1 such as 100 ug/ml significantly. Beside cytokines, LPS-induced nitrite production was also lowed after addition of Rb-1. It should be stressed that both cytokines and iNOS expression share a universal transcription factor - nuclear factor kb (NF-kb). Hence, with a liquid-protein suspension array system, we found that: (a) there was no difference in terms of total protein expression of I-kb in either Rb-1 treated or Rb-1 untreated splenocytes; (b) however, phosphorylation of I-kb was decreased in splenocytes culture treated with Rb-1; (c) ratio of (phosphorylated I-kb : total I-kb) in Rb-1-supplemented splenocytes was lowered than those without Rb-1. Furthermore, Western blotting experiment showed that translocation of NF-kb into nucleus was decreased in Rb-1-treated splenocytes culture. These results explained why there was suppression of cytokines secretion and lower level of nitrite produced in Rb-1 treated splenocytes culture. Therefore, Rb-1 was concluded to have immunosuppressive effects on immune system. Lastly, differential proteome expression of Rb-1-added splenocytes was investigated in order to exploit other possible action pathways involved by Rb-1. According to 2D-PAGE analysis, 32 protein spots were found to be differentially expressed in response to LPS and Rb-1. Of those 32 spots, 12 proteins could be identified by MALDI-TOF MS. Further comparison between LPS-stimulated splenocytes with or without Rb-1 incubation, 2 proteins (gelsolin and myovirus resistance 1 protein) (out of 12 spots) were found to be differentially expressed upon the addition of Rb-1. However, their exact involvements in the actions of Rb-1 on splenocytes have to be further studied.