Inflammatory cytokines stimulate multiple signaling cascades leading to ß cell apoptosis  in T1D . By suppressing inflammatory cytokines, WP increases the capacity of diabetic [15, 17] animals to heal wounds. Potential effects of WP on immune processes, including the regulation of cytokines  and enhancement of the proliferation capability of PBMCs  have been observed. WP, therefore, can not only suppresses the inflammatory cytokines from autoreactive T cells but also restore normal ß-cell mass and function during diabetes.
In this study diabetes was found significantly to impair the proliferative response of splenic lymphocytes in vivo and antigen-stimulated lymphocytes in vitro. Indeed, we did not observe any significant proliferation activities in the PALS zone, which are provoked by CD4+ T cells. Similarly, Aarnisalo et al.  found a marked reduction in the proliferative response of CD4+ T cells among patients with T1D.
It is evident from our results that, in WP-treated rats, lymphocytes in splenic follicles showed highly significant proliferating activity, and this was confirmed in vitro. Although WP activates cell proliferation, therefore, staining with anti-PCNA antibodies showed that this proliferation was located in the B cell zone but not in the T cell zone of the splenic tissue. The proliferation activity stimulated by WP, therefore, was directed toward B cells rather than T cells. This finding was confirmed by staining with anti-CD20+ and anti-CD3+ antibodies which demonstrated that the number of B cells was greatly increased but that the number of T cells was reduced in the tissues of the WP-treated diabetic rats compared with untreated diabetic rats. This provides further evidence that WP is associated with B cell stimulation and T cell suppression during diabetes.
Our results suggest that WP may suppress the Th1 type of T cell that has a critical role in diabetic complications. This suggestion was practically confirmed by the up-regulation of Cdc42 expression, which maintains the homeostasis of naïve T cells by promoting cell survival and suppressing T cell activation . Cdc42-deficient naïve T cells display impaired actin polymerization and show an enhanced differentiation to Th1 and CD8+ effector and memory cells . Restoring Cdc42 by WP may, therefore, maintain the balance between the Th1 and Th2 subsets of T cell by specifically suppressing T cell activation and differentiation to Th1 and CD8+ effector and memory cells. Ultimately, this T cell homeostasis results in an improvement in the condition of the pancreatic ß cells.
Akt is critical for cell survival and is triggered by different stimuli . WP supplementation was found to restore mRNA expression of Akt1 to the normal level. It was also shown in this work that up-regulation of Akt1 was accompanied by a significant increase of lymphocyte proliferation in the splenic tissues. The mRNA expression of the activated lymphocytes showed that diabetes had decreased the Akt1 signaling. Inhibition of cell migration has also been shown to be effected by inhibition of phosphorated PI3K/Akt, resulting in rapid Cdc42 proteolysis . These studies confirm that Akt depletion impairs the mRNA expression of Cdc42 by activated lymphocytes during diabetes. Taken together, the results of our study confirm that WP linked up-regulation of Akt1 significantly elevates Cdc42 and this enhances T cell homeostasis but not ß cells attacking Th1 cells.
Cell death stimuli signals are either an intrinsic, mitochondrial pathway of apoptosis or can kill the cell through one of the six death receptors such as Fas . Fas was significantly up-regulated in diabetic rats, as shown by the high number of dead lymphocytes in the diabetic group, suggesting that diabetic complications and oxidative stress induce cell apoptosis via Fas up-regulation [34, 35]. The increased expression of Fas in the diabetic lymphocytes was, however, significantly inhibited by WP treatment. This indicates that WP restored T cell homeostasis by suppressing T cell proliferating activity rather than through the Fas-mediated apoptotic pathway.
Turning now to CD28, we found a lower level of CD28 expression on antigen-stimulated lymphocytes in the diabetic group while this level was restored in diabetic rats treated with WP. Recent studies have demonstrated that a significantly lower level of CD28 surface expression on T cells was detected in diabetic rats, children with T1D, cell leukaemia, chronic lymphocytic leukaemia and colorectal cancer [36–38]. CD28 may contribute to T cell viability by increasing glucose metabolism in activated T cells . CD28 signals are also required to protect T cells from Fas-mediated apoptosis by activating the PI3K/Akt pathway . Thus, a lower level of CD28 surface expression on T cells from diabetic rats could explain the observed two-fold higher level of dead cells in the diabetic group. In the absence of co-stimulation, cytokine secretion and T cell expansion, proliferation, survival and memory development are affected [41, 42]. The diabetic rats in our study also exhibited decreased IL-2 or IFN-γ expression, which is a major T cell function. On the other hand, it is likely that the higher level of CD28 surface expression in the WP-treated animals causes the higher viability and proliferating capacity, and the restoration of IL-2 and IFN-γ levels.
WP suppresses TNF-α production, which is the key factor in ß cell-destruction in T1D since TNF-α controls the expression of the inflammatory gene network and contributes to the pathological complications observed in many inflammatory diseases such as schizophrenia and diabetes [43, 44]. Therefore, while the higher expression of TNF-α caused pancreatic damage and dysfunction in the diabetic group in this study, WP was found to regulate expression of TNF-α and its apoptosis receptor, Fas to normal levels. By increasing glutathione, WP also induces oxidative stability leading to suppression of the inflammatory cascade . Since blocking the production of TNF-α improves blood glucose concentrations, targeting TNF-α could effectively reduce expressions of the primary factors behind the complications associated with diabetes . Thus, normal morphological features of the pancreatic ß-cells and glucose clearance were restored in the diabetic rats treated with WP due to the suppression of TNF-α. The hypoglycaemic effect of WP in individuals with T1D has recently been confirmed [46, 47] and WP has also been shown to restore the capacity of the pancreatic islet to secrete insulin . Here, we add to this picture of the beneficial effects of WP in the context of diabetes by showing that WP supplementation for five weeks in normalized glucose clearance in diabetic rats.
In conclusion, we have shown here that there is a strong, positive correlation between WP and immune function during diabetes in an animal model. WP restored the normal immune response as follows: 1) by activating the Akt1 pathway, it activated the CD28 signals required to protect T cells from Fas-mediated apoptosis; 2) by stimulating Cdc42, which maintains naïve T cell homeostasis by promoting cell survival and suppressing T cell activation. This leads to the restoration of the polyfunctional T cells (IFN-γ and IL-2 production), increased cell viability, and may restore the T cell subset balance; 3) by suppressing TNF-α and its receptor, Fas. The positive outcome of this range of effects at the cellular level are confirmed by our evidence on improvements of the pancreatic ß cell mass and function. There is a need, however, to investigate more intensively the potential role of WP in the treatment of diabetic immune impairment.