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Effect of BieJiaJianWan(BJJW) in H22Tumor-bearing Mice and in Bel-7402Human Hepatoma Cells and the Research of Its Mechanism

Author: ChangXinFeng
Tutor: HanLiMin
School: Hunan University of Traditional Chinese Medicine
Course: Traditional Chinese Medicine
Keywords: BieJiaJianWan(BJJW) primary liver cancer H22 cells Bel-7402cells Cell apoptosis Angiogenesis
CLC: R735.7
Type: PhD thesis
Year: 2014
Downloads: 1
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Background:Primary liver cancer is a common malignancy and harm human health seriously. At present, to explore effective treatment methods to improve the quality of life and survival of patients is becoming a serious problem, because that the overall efficacy is not optimistic. Bie Jia Jian Wan(BJJW) is created by Zhang Zhongjing, with the effect of activating blood, nourishing, and relieving congestion. Studies have shown that BJJW has better inhibition of liver cancer growth and prevents metastasis of liver cancer, but its exact mechanisms of anti-cancer remain unclear. This project is to study the effect of BJJW and its anti-cancer mechanism by using in vivo animal models and in vitro cell models. It can provide a theoretical basis for using TCM in treatment of liver cancer.Objective:To observe the effect of BJJW in H22tumor-bearing mice and in Bel-7402human hepatoma cells through in vivo and in vitro experiments, and explore the possible anti-cancer mechanisms of BJJW by promoting apoptosis of tumor cells and inhibition of tumor angiogenesis so as to lay the foundation for further clinical application of the drug in treatment of liver cancer.Methods:Systematically study the ancient and modern document, to set forth the understanding of traditional Chinese medicine for primary liver cancer and clinical application and advances in experimental study from the aspects of name of the disease, etiology, pathogenesis, syndromes and treatment. Through different research methods like serum pharmacology, morphology, flow cytometry, MTT method, Western blot and so on, to study the inhibitory effect of BJJW on the growth of liver cancer and investigate its mechanism in vitro and in vivo. Results:1. Literature study:Liver cancer whose main cause is "deficiency of Qi"," blood stasis" and "heat" is the result of interaction of internal and external factors of the human body. Liver cancer basses on’’deficiency of Qi" and results from "Qi stagnation and blood stasis","phlegm-dampness cross knot" and "accumulation of wet and hot". BJJW has the effect of activating blood, nourishing, and relieving congestion. It is clinically used to treat liver cancer and has achieved good results.2. In vivo experiments:Effect of BJJW in H22tumor-bearing mice.(1) The weight of the tumor in BJJWL, BJJWM and BJJWH was significantly reduced, which prompts BJJW has a significant inhibitory effect.(2) Compared with the Model, the spleen index of BJJWM and BJJWH were significantly increased while the thymus index of BJJWL, BJJWM and BJJWH were not significantly different. It shows that BJJW could improve immune function in tumor-bearing mice and inhibit the growth of tumor.(3) Flow cytometry showed compared with Model the content of CD4+and the ratio of CD4+/CD8+in BJJWL, BJJWM and BJJWH were significantly reduced while the content of CD8+was increased. It intimates that BJJW could enhance immunity to inhibit tumor.(4) Western bolt showed BJJWL, BJJWM, and BJJWH reduced the expression of Bcl-2, increased the expression of Bax and decreased the ratio of Bcl-2/Bax. Besides, BJJW could inhibit the synthesis of p-ERK and promote the synthesis of p-JNK. This prompts that BJJW could block phosphorylation of ERK1protein and promote phosphorylation of JNK protein, so as to reduce the expression of anti-apoptosis protein Bcl-2, enhance the expression of apoptosis protein Bax and adjust the ratio of Bcl-2/Bax to achieve the aim of promoting apoptosis of tumor, and it has a certain dose-effect relationship. (5) Western bolt showed BJJWL, BJJWM, and BJJWH reduced the expression of VEGF, Delta-4and NICD. It implies that BJJW could inhibit tumor growth by reducing the expression of VEGF, Delta-4and NICD and inhibiting the formation of tumor angiogenesis.3. In vitro experiments:Effect of serum containing BJJW in Bel-7402human hepatoma cells.(1) Under an inverted microscope, cell morphology changed, some cells became smaller and round, the amount of cytoplasm reduced, pulp was visible within a large number of transparent particles, growth of cells was into a single state, adherent ability decreased, shedding increased and some cells were grown in suspension.(2) MTT showed different doses of serum containing BJJW had a certain inhibition on the growth of Bel-7402cells and as the dosage increased, inhibition increased. It implies serum containing BJJW has a certain inhibition on the growth of Bel-7402cells.(4) Annexin V/PI double staining flow cytometry results showed with the dose of BJJW increased, the proportion of apoptosis increased. It hints serum containing BJJW could promote the apoptosis of Bel-7402cells.(5) Western blot showed BJJW in different concentration group could inhibit the generation of p-ERK, promote phosphorylation of JNK, raise Bax and reduce Bcl-2. It indicates that BJJW may inhibit tumor growth by blocking ERK1/2signal pathway, activating JNK signal pathway.(6) Western blot showed compared with Control the expression of VEGF and Delta4in BJJWL, BJJWM, and BJJWH were significantly reduced, and the expression of NICD in BJJWM and BJJWH were significantly reduced. It hints BJJW may inhibit tumor by blocking the VEGF-D114-Notch signal transduction pathway.Conclusions:Combination of in vivo and in vitro experimental results, we came to the conclusion:BJJW can effectively inhibit the growth of liver cancer, enhance immune function and improve quality of life. The inhibition mechanism may be related to signal transduction as follows:(1) Blocking ERK1/2signal transduction, inhibiting the activation of ERK1, thereby promoting apoptosis of tumor cells.(2) Activating JNK signal transduction, promoting JNK phosphorylation, indirectly inhibiting the activity of Bcl-2, and promoting tumor cell apoptosis.(3) Blocking VEGF-D114-Notch signal transduction, inhibiting tumor angiogenesis and reducing tumor blood supply, ultimately to control tumor growth.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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