Study Of Astragalus Polysaccharides On Maturationof Dendritic Cell And Promotion Of Specific Anti-tumor Immunity

Backgroud: According to the traditional view, we treat tumors depend on three methods: surgery, chemotherapy and radiotherapy. However, these methods have its own limitations in application. Traditional Chinese Medicine(TCM) is one of the oldest healing systems. Astragalus mongholicus(AMs) that is a common herbal medicine, animportant part of TCM, have been proved to be effective in treating cancers from lots of clinical cases. However, we have not fully understood the anti-tumor mechanism of APS, and this has lead to some doubt from some Western-Medicine scholars, which restricts its widely use. Dendritie cells(DCs),the most effective antigen presenting cell in vivo that we have ever known,can ingest and process antigens then initiate specific-immune response in vivo.With DCs biological functions in the tumor immunity,transplantation immunity,anti-infection immunity are realized gradually by people these years, clinical doctors increasingly pay attention to DCs.The main objective of this research is to discuss the effect and mechanism of APS to human stomach cancer. Objecctive: To observe the effect of APS on tumor growth and its mechanism.Materials and Methods: we did the research from three major aspects: the influence of APS on DCs, the inhibition of tumor growth in vitro as well as the animal studies in vivo after treatment. First, we cultured the mouse dendritic cells(DCs) from bone marrow of mouse hind legs according to the method using Interleukin-4(IL-4) and Granulocyte-macrophage colony stimulating factor(GM-CSF), which referred to the way established by Inaba(Inaba K, 1992). And then, we investigated the growth-rate of the DCs co-cultured with APS injection. We analyzed the expression of the Toll-like-receptor 4(TLR4) with SYBR-Green I Real-time PCR and the I-kappa-B-alpha(IκB-α) with Western-Blot, the main regulatory protein to control nuclear factor NFκB-p65 nuclear translocation. Second, we choosed the human gastric cancer cell lines MKN 45 as the target cell, which was co-cultured with DCs,T cells from spleen of mouse and APS injection, and used MTT assay to judge the amount of cell lines and immnunoflurescene to analyze the expression of anti-active caspase 3 p Ab anti-PARP P85 fragment p Ab, the mark of apoptosis of cells.Finally,we had conducted the animal studies experiment in vitro.The nude mouse developed stomach cancer, was established by axillary injection of human stomachcancer MKN45 cells, and then were randomly divided into three groups:control group, cytokine group, low concentration of APS group, highconcentration of APS group, with 8 mice in each group. APS in low and high concentration group were intravenously injected at 10 mg/kg and 30 mg/kg, respectively.The control group was injected with the same volume ofsaline for continuous 15 days. In the experiment, tumor volume was measured every 2 days; at the end of the experiment, mice were killed, and the tumor was removed, then tumor volume and weight were measured.The expression of Ki67 protein was detected by IHC, and tumor cell apoptosis was detected by TUNEL method to studythe inhibitory effect of APS on tumor. Results: All results showed that APS were effective in treating human stomach cancer and the mechanism might be regulated by TLR4 mediated signal transduction of DCs. The results were briefly introduced as follows: First, we succeeded in culturing the DCs induced by IL-4 and GM-CSF and found the positive rate of CD11 c expression, the mark of DCs, was beyond 90%. We detected APS could precipitate DCs maturation via up regulating TLR4 by SYBR-Green I Real-time PCR and suppressing IκB-α by Western-Blot. Second, after the MKN45 co-cultured with DCs, T cells and APS injection, the result showed that APS could reduce the amount of cell lines by MTT assay and lead to nduce apoptosis with immunofluorescence. Finally, we had conducted animal studies in vitro, and the results in vivo showed that APS caused significant decease in the weight and diameter of the tumor, anti-tumor rate, apoptosis rate were significantlyincreased, compared to controls. Besides, the expression of Ki67 proteinwas dose-dependentlyreduced. Conclusion: As a good chinese herbal medicine, APS could effectively treat human stomach cancers by precipitating DCs maturation and inhibiting the tumor growth, which is probably due to its effects on regulating the TLR4 mediated signal transduction.