Effects Of HB-13 And HP-13 On NF-κB And P38MAPK Signaling Pathways

Inflammatory skin diseases is a common kind of dermatoses represented by psoriasis, atopic eczema and etc.Nowadays this kind of disease is considered to be immunological disordered dermatoses mediated by T lymphocytes.It is also tightly related with Th1-type cytokines which are produced by T lymphocytes(represented by TNF-α,IFN-γ).Cytokines released by T lymphocytes and keratinocytes form a complicated network in inflammatory skin diseases,resulting in their specific immunopathologic injury.Recently it has been found that the abnormal nuclear factor-kappaB(NF-κB) and p38 mitogen-activated protein kinase(p38MAPK) signaling pathways are closely involved in inflammatory response.Thereby these two pathways have become two of the hot topics in the pathogensis of inflammatory skin diseases and targets for drug treatment. While TNF-αand IFN-γ,originated from T lymphocytes,are crucial pathogenic factors in skin inflammatory response,which can lead to the cascade reactions of intracellular kinases,activate the above mentioned pathways and participate in regulating the pathogensis and development of inflammatory response.13-hexyl-berberine hydrochloride(HB-13) and 13-hexyl-palmatine hydrochloride (HP-13) were two active compounds respectively from berberine and palmatine derivatives and our previous studies showed that both of them had obvious effcets in anti-bacterium,anti-herpes simplex virus(HSV) and anti-inflammation.The aim of this study was to investigate the effects of HB-13 and HP-13 on the activation of NF-κB and the phosphorylation of p38MAPK in a spontaneous transformed human epidermal cell line HaCaT and the effects of them on the activation of mouse T lymphocytes and the effects of them on the expression of IL-8 in HaCaT cells and the effects of them on the expression of IL-1βin mouse macrophages.PartⅠThe effects of HB-13 and HP-13 on the activation of T lymphocytes and on the expression of IL-8 in human keratinocytes and the effects of them on the expression of IL-1βin mouse macrophagesObjective To investigate the effects of HB-13 and HP-13 on the activation of mouse T lymphocytes and the effects of them on the expression of IL-8 in HaCaT cells and the effects of them on the expression of IL-1βin mouse macrophages.Methods The activation of T lymphocytes was induced by ConA in mouse and was detected by MTT; The expression of IL-8 was induced by recombinant human TNF-α(rhTNF-α) in HaCaT cells and was determined by ELISA;The expression of IL-1βwas induced by LPS in mouse macrophages and was determined by ELISA.Results HB-13 and HP-13 could inhibit the activation of mouse T lymphocytes during the experimental concentration 0.04875μg/ml～0.39μg/ml and 0.78μg/ml～3.12μg/ml,respectively.Their rate of inhibition for the activation of T lymphocytes were respectively 12.7%-78.2%and 14.3%-92.5%.Both HB-13 and HP-13 could significantly inhibit the expression of IL-8 in HaCaT cells stimulated by rhTNF-αduring the experimental concentration 0.39μg/ml～3.12μg/ml.Their rate of inhibition for IL-8 were respectively 85.7%-99.5% and 68.4%-95.1%.HB-13 could inhibit the expression of IL-1βin mouse macrophages during the experimental concentration 0.01μg/ml～1μg/ml.1μg/ml HP-13 could inhibit the expression of IL-1βin mouse macrophages with the inhibition rate over 90%. Conclusion HB-13 and HP-13 can inhibit the activation of T lymphocytes and the expression of their cytokines.PartⅡThe effects of HB-13 and HP-13 on signal transduction pathway of NF-κB in human keratinocytesObjective To investigate the effects of HB-13 and HP-13 on the activation of NF-κB in HaCaT keratinocytes stimulated by tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) in vitro.Methods HaCaT cells were cultured and were used as target cells.After treatment by either HB-13 or HP-13 for two hours,cells were induced by recombinant human TNF-α(rhTNF-α) or by recombinant human IFN-γ(rhIFN-γ) for two hours.The expression of phosphorylated IκBα(p-IκB-α) was detected by the Western blot technique.Results Both HB-13 and HP-13 could significantly inhibit the expression of p-IκB-αin HaCaT cells stimulated by rhTNF-αduring the experimental concentration 0.39μg/ml～1.56μg/ml.Their rate of inhibition for p-IκB-αwere respectively 47.4%-67.5%and 32%-67.2%.HB-13 and HP-13 could also inhibit the expression of p-IκB-αin HaCaT cells stimulated by rhIFN-γ.Their rate of inhibition for p-IκB-αwere respectively 37.8%-57.4%and 37.5%-43.6%.Conclusion HB-13 and HP-13 can inhibit the activation of NF-κB in HaCaT keratinocytes stimulated by TNF-αand IFN-γ.PartⅢThe effects of HB-13 and HP-13 on signal transduction pathway of p38MAPK in human keratinocytesObjective To investigate the effects of HB-13 and HP-13 on the phosphorylation of p38MAPK in HaCaT keratinocytes stimulated by TNF-αin vitro.Methods HaCaT cells were cultured and were used as target cells.After treatment by either HB-13 or HP-13 for two hours,cells were induced by rhTNF-αfor fifteen minutes.The expression of phosphorylated p38MAPK(p-p38) was detected by the Western blot technique. Results Neither HB-13 nor HP-13 during the experimental concentration had significant effect on the expression of p-p38 in HaCaT cells stimulated by rhTNF-α. Conclusion None of HB-13 and HP-13 can inhibit the phosphorylation of p38MAPK in HaCaT keratinocytes stimulated by TNF-α.