Research On Immunosuppressive Activity And Anti-Autoimmune Disease Of Withanolides

Withanolides are structurally diverse steroids with an ergostane skeleton in which C22 and C26 are oxidized to form aδ-lactone. Withanolides represent not only aδ-lactone with C22 and C26 oxidized, but also all biosynthectic pathways related compounds. These compounds show anti-inflammatory, antibacterial, antiviral, anti-microbial, anti-oxidation, anti-tumor, anti-convulsions, as well as inhibition activity of acetylcholinesterase. In this study, the immunosuppressive fraction of withanolides from Solanaceae plant were screened, the result demonstrated that this fraction showed significant immunosuppression activity. In order to clarify its effective chemicals relation to immunosuppression, the thirty compounds were isolated out from the effective fractions by our cooperative Lab. We found that Withangulatin A (WA), Physagulin L, Physalin H, and et al, exhibited significant immunosuppression activity. According to the results, the structure-immunosuppression activity relationship was summarized. In order to further explore its immunosuppressive mechanisms, WA was selected as typical compounds for its significance activity.In this study, we evaluated the cytotoxicity and immunosuppressive activity of WA in normal lymphocyte, ab-normal proliferation and mixed lymphocyte. The results illustrated that WA showed more effective and lower cytotoxicty than positive drug of cyclosporine A (CsA). At the same time, we measured immunosuppressive activity of WA in Xylene-induced ear tumefaction mice, the result also showed WA was more effective than CsA in inhibiting xylene-induced ear tumefaction while the weight of mice was not significantly different. These results provide an important scientific evidence of WA for further research.In this study, we investigated the effect on mice T lymphocytes function of WA. FCM data documented that WA obstructed DNA replication in G1-S1 stages to regulate cell cycle and decreased CD4+/CD8+ and CD25+ to inhibit the overproduced lymphocyte. In addition, the results of ELISA showed that WA regulated Thl/Th2 balance in vitro and in vivo to show immunosuppressive activity.In this study, further immunosuppression mechanism of WA was studied through a molecular target of protective gene Heme oxygenase-1(HO-1), which plays immune regulation and protects cell from injury. So induction of HO-1 expression would become a new type of treatment in the immune regulation. The results of Western blot and RT-PCR displayed that WA induced the expression of HO-1 in the LPS-induced mice T lymphocytes through ERK/MAPK pathway in a dose- and time-dependent manner. Furthermore, WA inhibited LPS-induced nuclear factorκB (NF-κB p65) translocation directly or indirectly mediated by HO-1, which probably involved in increasing PPAR-y while decreasing COX-2 expression.In this study, we established a model for screening COX-2 specific inhibitor on account of dual-luciferase report gene system. Dual-luciferase report gene is an effective method in promoter activity assay, and this method is rapid, sensitive, economic and without pollution of isotope. We constructed recombination luciferase report gene vector dependent COX-1 or COX-2 gene promoter, which the recombination vector and control vector were co-transfected into cells and analyzed activity luciferase. The activity luciferase was index of activity of COX-1 and COX-2 gene promoter. In addition, we used COX-2 specific inhibitor Celecoxib to confirm this model for its regulation in COX-2 gene expression, and the results showed that this model was precise and sensitive. Therefore, this model could be used to screen COX-2 specific inhibitor, and also used to probe mechanism of targeted inhibiting COX-2 expression.Based on dual-luciferase reporter gene analysis, our studies further explore the molecular mechanism of targeted inhibiting COX-2. The results of Western blot and RT-PCR displayed that WA significantly inhibited COX-2 expression and PGE2 production through ERK/MAPK, p38/MAPK pathways and NF-κB nuclear translocation, while the expression level of COX-1 was not influenced markedly. The results showed that MAPK participated in the translocation of NF-κB, which possiblely regulated some other transcription factors.In order to further verify the immunosuppressive activity of WA, the present studies preliminary investigate the therapeutic effect of WA on the disease systemic lupus erythematosus with the MRL/lpr lupus mice model. The results showed that WA effectively improved immune status and reduced the splenomegaly and lymphadenopathy possibly through BAFF/BAFF-R pathway. Moreover, WA significantly improved kidneys and lung function of MRL/lpr mice, reduced proteinuria symptoms and anti-dsDNA level of serum, as well as to improve their lives function, which might provide new clues for the immunosuppressive activity of WA in vivo.In this paper, we investigated the immunosuppressive activity of WA based on a series of cellular, molecular level and mice experiments, The results showed that WA suppressed the over-proliferation of mice T lymphocytes through blocking cell mitosis and suppressing CD4+/CD8+ and CD25+ cell subsets, as well as regulated Th1/Th2 balance. WA might play immune suppression activity by increasing protective genes HO-1 expression and targeted inhibiting the expression of COX-2. At the same time, the results of inflammation mice and MRL/lpr lupus mice model showed a very good therapeutic effect of WA. The above results in vitro and in vivo further provide scientific evidences for the potential of new immunosuppressive activity of WA and Withanolide compound.