Therefore the objective of the present study was to further

Therefore, the objective of the present study was to further evaluate the effect of the 70% hydromethanolic extract of D. alata underground tuber for modulating TH1 (IL-2 and IFN-γ) and TH2 (IL-4 and IL-10) cytokine expression and its effect on the proliferation pattern of murine splenic T-lymphocytes. In addition, the correlation patterns of the cytokine expressions and the cell proliferation were also studied.
Materials and methods
Results
Discussion The complex interactions between the lymphoid, inflammatory and hematopoietic cells are chiefly governed by the cytokines which are generally divided into TH1 and TH2 sub-sets. TH1 cells are the primary source of IL-2, IFN-γ, lymphotoxins and other factors which characterize the cell-mediated immune responses [22]. IL-4, IL-5, and IL-10 are produced by the TH2 cells which not only mediate the humoral immune responses, but also promote allergic reactions by inducing the function of IgE formation [7]. In the present study, hydro-methanolic extract of D. alata underground tuber was found to stimulate the splenic T-lymphocyte proliferation and modulate cytokine expressions in vitro. Spleen functions primarily against the order moexipril in the blood stream. The outer red pulp of spleen is abundant in lymphocytes. Con A, which is a typical T-lymphocyte mitogen, responsible for blastoid differentiation of the T-lymphocytes [23], must have stimulated T-lymphocyte population out of the whole splenocyte cells to secret the cytokines in our experiments as well. This is evidenced from the increase in the number of cells. Previously, Liu et al. [24] have also demonstrated the mitogenic effect of dioscorin, the storage protein of D. alata on the phytohemagglutinin induced cell proliferation. Besides 70% methanolic extract of D. alata formerly showed the capacity to increase the splenic cellularity in murine model in vivo[19]. Here, we have shown that the degree of stimulation of splenocytes by D. alata extract at 80μg/mL concentration along with con A was 178.46±6.08%, much higher than that of the stimulation of con A alone in group I (0μg/mL). It may be hypothesized that D. alata hydro-methanolic extract acted synergistically with the external mitogenic stimulators to trigger mitosis of the splenic cells. Therefore, D. alata possess potent immunogenicity towards the activation and proliferation of the splenic cells. Profound increases in the TH1 cytokine (IL-2, IFN-γ) levels and decrease in TH2 cytokine (IL-4 and IL-10) levels were evident by the stimulation of D. alata. Similar findings [25,26] also suggest the potentiality of various other plant materials to up-regulate the TH1 response. In the present study, dose-dependent increase in the splenic T-lymphocyte was evident and the radius of proliferation was much higher in case of con A along with the plant extract than con A alone induced stimulation. This could possibly be due to the fact that the D. alata induced increased expression of IL-2, which is a natural T-lymphocyte stimulating factor or the plant extract itself may posses mitogenic effect, perhaps, which have mimicked the function of IL-2 in the stimulation of splenic T-lymphocytes. IL-4 and IL-10 are known to suppress the expansion of TH1 subsets. Thus in the present study (Fig. 3), high negative correlation between IFN-γ and IL-4 (R2=0.9053) and IL-10 (R2=0.8073) were evident and positive correlation between IL-2 and IFN-γ (R2=0.9496) were found, both of which are TH1 cytokines. Considering the dose-dependent expression patterns of the four cytokines, the correlation matrix (Table 1) also displayed very high positive correlation (0.974) between IL-2 and IFN-γ and very high negative correlation of IL-4 with IFN-γ (−0.951) and IL-2 (−0.955). Principal component analysis is a multivariate data reduction technique which statistically allows visualizing the pattern of inter-relations among the variables of the experiments [27]. Groups of data with correlated immunological parameters can be visualized in a simplified manner using this data reduction technique [28]. Under univariate correlation analysis, bias may emerge due to antagonist or stimulatory relations of different cytokines. This bias is eliminated and the overall inter-correlation patterns are visualized using this technique. Previously, PCA analysis have been extensively used to analyse the cytokine correlation patterns in different immunological studies [29,30].