| Chinese medicine has become a research hotspot in cancer treatment, its therapeutic mechanism may be considered as enhancing the immune system. This study aimed to analyze Yigong capsules to reveal its anti-tumor mechanism so as to provide an objective and scientific evidence for its clinical studies.This paper first analyzed the pharmacokinetic process of YiGong capsules. A solid-phase extraction method by using high performance liquid chromatography (SPE-HPLC) was developed for simultaneous determination of four coumarins contained by YiGong capsules in rat plasma. Then, this paper analyzed the anti-tumor effect in vivo, immunoloregulation effect and possible mechanism of YiGong capsules. Yigong capsules were orally administered to mouse S180 solid tumor models, ascites tumor models and immune suppressed models by cyclophosphamide to observe its drugs anti-tumor activity, life extension rate and impact on thymocytes in immune suppressed mice, weight of thymus and level of serum IgG. Based on the isolation of mouse peritoneal macrophages, spleen lymphocytes and natural killer cells, dinitrochlorobenzene induced delayed-type cutaneous reaction model was established. Then the carbon particle clearance method was used to study the impact of YiGong capluses on the immune system of mice. Application of luciferase reporter system, MTT method, CCK-8 method, Brdu method, RT-PCR, Western-Bloting and flow cytometry was taken to explore the molecular mechanisms of immunoenhancement of YiGong capsules. HMEC-1 cells were observed to evaluate the impact of YiGong capsules on angiogenesis of cells and migration of cells.The results showed that peak times of four courmarins in rat plasma were respectively 0.75±0.21 h,1.00±0.32 h,2.00±0.27 h and 1.00±0.36 h. The anti-tumor inhibition rate of single use of YiGong capsules (1.6,3.0,5.0 g-kg-1) on S180 was 20.99%,26.54% and 35.98%, while the survival time rate on S180 ascites tumor of mice was 5.15%,11.85% and 35.15% respectively. The anti-tumor inhibition rate of YiGong capsules combined with cyclophosphamide was 29.44%, 38.79% and 44.39%, while the the survival time rate of YiGong capsules combined with fluorouracil was 14.12%,22.70% and 41.42%. YiGong capsules could significantly improve the immune suppressed thymus in mice, spleen weight index and serum IgG levels. The kill tumor rates of YiGong capsules (1.6,3.0,5.0 g·kg-1) on peritoneal macrophages, lymphocytes and natural killer cells in normal mice and transplanted mice were separately 20.99%,29.04%,36.08%; 26.69%,30.94%, 37.97%; 19.09%,27.15%,35.04%; 28.53%,31.87%,40.10%; 24.26%,26.12%, 28.80%; 24.54%,29.04%,42.00%. YiGong capsules had no effect on the phagocytic activity of peritoneal macrophages. The phagocytic index K and the corrected phagocytic index in mice were increased. YiGong capsules (0.1,1,10μg/ml) could activate the transcriptional activity of NF-κB. YiGong capsules could increase the S180 cells in mice, peritoneal macrophage in mice, lymphocyte expression of p65 mRNA in mice and protein content, while it could not activate the S180 cells in mice, peritoneal macrophages in mice, NF-κB in spleen lymphocytes, MAPKs and PI3K/Akt signaling pathway. YiGong capsules could delay tumor cells in mice S180, it had no effect on the migration of HMEC-1 cell and angiogenic process.This study established an appropriate detection method of four courmarin in rat plasma after it was orally administered with YiGong capsules. The result showed that YiGong capsules could significantly inhibit tumor growth in mice, enhance the anti-tumor effect of cyclophosphamide and fluorouracil and may alleviate the cyclophosphamide induced immune suppression. YiGong capsules could increase the kill tumor activity of peritoneal macrophages and lymphocytes in mice and enhance the killing ability of natural killer cells, but it could not enhance the phagocytosis ability of macrophage phagocytosis. YiGong capsules could enhance delayed-type hypersensitivity in mice induced by DNCB and promote the phagocytosis ability of the reticuloendothelial system. It could activate the transcriptional activity of NF-κB, increase level of p65 expression in cells and it has no effect on NF-κB, MAPKs and PI3K/Akt signaling pathway. It could delay tumor cells in mice S180, while it could not promote the migration of endothelial cells HMEC-1 in vitro and the angiogenic process. This study revealed the anti-tumor mechanism of Yigong capsules, which lay a great foundaction for its clinical studies in the future. |
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