| objectivesPrimary hepatocellular carcinoma (PHC) is one of the most common malignant diseases in digestive system, which has strong invasive and metastatic features, and high fatality rate, thus making it the second most common malignancy in China. Due to its occult onset, most people suffering from PHC are already in the late stage and cannot be fully cured by operation when they go to see doctors’. Meanwhile, imbalances of helper T-cells differentiation, decrease in number or function defect of dendritic cells, leading to poor curative effect of chemotherapy. Previous studies suggested that body’s immunity plays an important role in regulating the occurrence and development of malignant tumors, and immunotherapy has become a top priority in cancer treatment. With the deepening research of immunotherapy, how to boost the immunity of a bearing body has become one of the core issues of immunotherapy. Polysaccharide of Chinese medicine have advantages of preventing occurrence of liver cancer and reducing recurrence, antagonism the side effects of radiotherapy and chemotherapy, enhancing immunity to extend the survival period, making it widely used in clinic.Lycium barbarum polysaccharides (LBP) is the key constituent in Barbary Wolfberry Fruit Fructus Lycii, which has been shown to have significant immunomodulatory effects, though regulating of immunologic effector cells (T cells, cytotoxic T cells and macrophages) and inducing anticancer cytokines. However, its molecular and immunological mechanisms of antitumor effects still remain unclear. In addition, studies indicated that Chemokines 10 (CXCL10) has the capacity to affect T cell, mononuclear cells, natural killer cells and other inflammatory cells to the tumor location to kill tumor cells. And it has been proven that expression of CXCL10 is closely related to liver diseases such as hepatitis, liver cirrhosis and liver cancer. The combination of polysaccharides (LBP) and Chemokines 10 will be a new attempt to treat liver cancer. On the basis of our previous research studies, we try to investigate the effects of LBP on HepG2 cell lines, and then, explore the antitumor effects of polysaccharides alone or combined with CXCL10 in experimental mice hepatoma.Methods:1. In vivo studies:the killing effect of LBP on hepatocellular carcinoma cell line HepG2 and the enhancing sensitization effect of LBP with fluorouracil:1.1 Inhibition effect of LBP on HepG2 cell line:to seed HepG2 cell suspension on 96-well plates in a density of 3500/well. Groups were divided into control group, and LBP intervention groups (200 mg/L,400 mg/L,800 mg/L and 1000 mg/L). The relative inhibition rates of drugs were detected by MTT assay.1.2 Inhibition effect of LBP combined with fluorouracil (5 FU):Two doses of LBP (200 mg/L,400 mg/L) were chosen to combine with fluorouracil (1umol/L). Groups were divided into control group, LBP groups, and LBP combined with fluorouracil. The relative inhibition rates of drugs were detected by MTT assay and Jin’s Q formula was used to evaluate the interaction of mixtures.2.In vivo experiments:A mouse H22 subcutaneous tumor model was established for our study. The mice were randomly divided into model group, the LBP (50 mg/kg) lavage group, LBP+CXCL10 (50 mg/kg LBP lavage+right right flank subcutaneous injection 15 ug/kg) group, the CXCLIO (right flank subcutaneous injection 15 ug/kg), cyclophosphamide (20 mg/kg) and fluorouracil (12 mg/kg) intraperitoneal injection group, with 12 mice in each group. Serum was drawn from eye ball after two weeks of successive administration. Two weeks later, tumors, spleen, thymus were dissected and calculated anti-tumor rate, thymus index and spleen index.Thl/TH2 differentiation in peripheral blood were detected by flow cytometry, and expression of IFN-yand IL-4 were detected by ELISA. The IL12 in peripheral blood and TNF-a mRNA expression were detected by fluorescence quantitative PCR, S-100 protein in tumor tissues were detected by immunohistochemical method. The density of tumor cells and lymphocyte infiltration were examined by HE staining. Expression of S-100 in tumor interstitial infiltrates DC were detected by immunohistochemical method.Results:1. LBP has the killing effects on HepG2 cell line, and the effects can be enhanced when combined with fluorouracil (5 FU). Compared with control group, the suppression rate in LBP groups (200 mg/L,400 mg/L,800 mg/L and 1000 mg/L) are 9.23%,22.80%, 36.84% and 76.25% respectively. Q value of groups of LBP (200 mg/L,400 mg/L) combined with fluorouracil (5 FU) are 1.12 and 1.62.2. Tumor suppression effects of LBP alone or combined with CXCL10, and their effects on immune organs. Compared with model group, quality of tumor body in intervention groups decrease significantly, especially in groups of LBP, LBP+CXCL10 and fluorouracil, reaching the suppression rate of 55.90%,52.82% and 65.58%, with statistical difference.3. LBP alone or joint CXCL10 with the result of mice with H22 liver cancer peripheral blood T helper cells differentiation. LBP alone and combined CXCLIO group on H22 mice made a tumor-burdened obvious trend increasing peripheral blood Th1 cells (model group were raised from 10.61±4.72 to 28.38±21.98,24.26±13.86) and reducingthe Th2 cells (model group were reduced respectively from 21.48±23.23 to 7.71 ±5.24,5.46±2.90). Thl/Th2 ratio had also improved, the model group were raised by0.87±0.42 to 6.05±7.83,6.123.89.4. LBP alone or combined CXCL10 on H22 liver cancer in mice peripheral blood INF-γ, the influence of IL4 protein expression. Compared with model group.LBP alone or combined CXCL10 can make the mice with H22 liver cancer peripheral blood INF-y respectively up to 134.26±19.62,19.62±24.47 ng/mL, the peripheral blood IL4 were reduced to 62.77±8.79,8.79±8.54 ng/mL, ratio of INF-y/IL4 were raised to 2.14 ±0.10,2.22±0.13, and P< 0.05.5. LBP alone or combined CXCL10 on H22 liver cancer peripheral blood IL12, TNF-α mRNA expression in mice. Compared with model group, LBP alone or combined CXCL10 made a tumor-burdened mice peripheral blood IL12 mRNA expression raised 2.94 and 3.39 times respectively (P< 0.05), while the TNF-α raised 1.55 and 4.74 times respectively (P< 0.05).6 LBP alone or combined CXCL10 with H22 liver cancer infiltration of DC in mice S-100 protein expression. Detected the S-100 protein expression in tumor infiltrating DC by immunohistochemical method,found that, compared with model group, LBP alone or combined CXCL10 had more S-100+DC,more performance for highly invasive, statistical difference(P< 0.05).ConclusionIn vitro experiments, Chinese wolfberry polysaccharides could inhibit growth of human liver cell line (HepG2) inhibition, and enhance sensitization effect joint positive antitumor medicine 5fu by MTT method. Based on the clear LBP has anti-tumor effect in vitro, vivo experiments was designed,through H22 liver ascites tumor mice transplanted tumor growth curve, tumor inhibition rate in quality and pathological morphology observation, flow cytometry and ELISA, fluorescence quantitative PCR and immunohistochemical techniques, to discuss the influence on tumor growth in mice of LBP alone and joint CXCL10, and preliminary reveals the molecular and immunological mechanism of its action, we draw the following conclusion: LBP can resist Liver ascites carcinoma, its effect is related to the following immunological mechanisms:1. LBP and LBP+CXCL10 can promote a tumor-burdened immune organs such as spleen and thymus index in mice, improve the body’s immune function as a whole.2. LBP and LBP+CXCL10 type can promote Thl cytokine secretion and restore the Thl/Th2 balance, improve DC cell number and function, intervention tumor-burdened the immunosuppression state and improve the body anti tumor immune function. |
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