Astragalus mongholicus regulate the Toll-like-receptor 4 meditated signal transduction of dendritic cells to restrain stomach cancer cells.

BACKGROUND According to the traditional view, we depend on three methods to treat tumors; 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 the common herbal medicine, the biggest 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 AMs, and this has lead to some doubt for some Western-Medicine scholars and restricts its wide use. The main objective of this research is to discuss the effect and mechanism of AMs to human stomach cancer. MATERIALS AND METHODS To observe the effect and mechanism of tumor treatment by AMs, we have done the research from three major aspects, the influence of DCs, the inhibition of tumor in vitro as well as the animal studies in vivo after treatment. First, we culture 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 refer to the way established by Inaba (Inaba K, 1992). And then we investigate the growth-rate of the DCs co-cultured with AMs injection. We analyze 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 choose 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 AMs injection, and use MTT assay to judge the amount of cell lines and Immnunoflurescene to analyze the expression of anti-active caspase 3 pAb anti-PARP P85 fragment pAb, the mark of apoptosis of cells. Third, we have conducted the animal studies beside the basic experiment in vitro. The nude mouse developed stomach cancer, due to intra-preritoneal injection with MKN45 have been divided into two groups: the treatment group challenged with AMs injection and the control group with saline injection. We took the average of the diameter of each group as the y axis and the days after administered with AMs as x axis. After 40 days, all animals were killed by detruncation, and the tumor were removed and measured. We compare the diameter (<40 days) and weight (>40 days) of the tumor as well as the survival days between different groups to investigate the effect of inhibition of cancer. RESULTS All results show that AMs is effective in treating human stomach cancer and the mechanism might be regulated by TLR4 mediated signal transduction of DCs. The results are briefly introduced as follows: First, we succeed in culturing the DCs induced by IL-4 and GM-CSF and find the positive rate of CD11c expression, the mark of DCs, is beyond 90% (Fig-1). We detect AMs can precipitate DCs maturation by upregulating TLR4 in SYBR-Green I Real-time PCR (Fig-2) and suppressing I.B-aby Western-Blot (Fig-3). Second, after the MKN45 co-cultured with DCs, T cells and AMs injection, the result show that AMs can great reduce the amount of cell lines by MTT assay (Fig-4) and induce apoptosis with Immunofluorescence (Fig-5). Finally, we have conducted animal studies beside the experiment in vitro, and the result in vivo show that AMs can delay tumor development from the diameter and weight of the tumor (Fig-6, Fig-7), prolong life-span and improve life-quality. Figure 1the morphology and phenotypic identification of DCs.The form of DCs observed by microscope with field 20*.The isotype antibody control using FCM.The positive rate of CD11c expression.Figure 2the melting curve and the chart of TLR4 expressiona) the melting curve of beta-actin; b)the melting curve of TLR4;c)the TLR4 expression of DCs stimulated with AM at different dose. There is significant statistic difference between the 60ng/mL and 80ng/mL group and other group (P<0.05 by rank test)Figure 3the IκB-α expression of DCs with different dose of AMsL0: 0ng/mL; L1:20ng/mL; L2:40ng/mL; L3:60ng/mL; L4:80ng/mLFigure 4MTT assay to analyse the viability and proliferation of the two cell lines (P<0.05 between the group with the dose of 60ng/mL and 80ng/mL and other group). the horizontal axis is the group treated with AM and saline at different dose, the vertical axis is the cell number.Figure 5the anti-active caspase-3 pAb (a) and anti-PARP P85 fragment pAb (b) actived by immunofluorescence.The cell mix were treated with 100uL anti-active caspase-3 pAb at a 1:250 dilution and anti-PARP P85 fragment pAb at a 1:100 dilution, and the secondary Ab was donkey anti-rabbit Cy®3 conjugate diluted 1:500 in PBS (Jackson Cat#711-165-152). From the photo, we find that anti-active caspase-3 pAb and anti-PARP P86 fragment pAb can express which is very important to indicate cell-apoptosis.Figure 6The difference of tumor between treatment group and control group. CONCLUSION Ams Can play a great role in treating human stomach cancers as a good Chinese herbal medicine by precipitating DCs maturation, which is probably due to its effects by regulating the TLR4 mediated signal transduction.