The Study Of Lycium Barbarum Polysaccharide(LBP) On Anti-tumor Effects Through Quantitive Pathological Analysis And Immune Mechanism In H22 Bearing Mice.

Hepatocellular carcinoma is one of the most familiar malignant tumors that hazard human's health severely. Because of the special anatomic and histological character of liver it's easy to invade and metastasize so that most patients have been in advanced stage when the disease is diagnosed, its prognosis is very poor. Although the diagnosis and treatment methods develop fast, the mortality of hepatocellular carcinoma is still high. Because its surgical resection rate is only 10% and tumor relapses easily, non-surgical therapy is still the main method for hepatocellular carcinoma until now. However, chemical and irradiational therapy can cause severe immune system and bone marrow suppression. The emphasis of modern therapy for hepatocellular carcinoma is to combine western medicine, Chinese traditional medicine and other useful methods. Chinese traditional medicine has particular advantages at preventing neoplasia, decreasing relapse, reducing side effects of chemical and irradiational therapy and extending survival time and so on. At present its curative effects keep on improving constantly and its action mechanisms are being widely researched.It is reported that immune deficiency and several mechanisms of rumor immune escape play a great role in neoplasia and tumor development. Recent research of tumor therapy is focus on how to improve deficient immune function and interfere with mechanisms of tumor immune escape in tumor-bearing hosts.Researches have revealed that there are many characters of cell-mediated immunity deficiency in tumor-bearing hosts such as decrease of T lymphocyte counts, abnormal T lymphocyte subsets, activation suppression, decrease of dendritic cell(DC) counts and deficiency of its function, depression of NK cell and so on. It has been proved that counts of CD3+,CD4+cells in tumor-bearing hosts decrease, counts of CD8+cells increase and CD4+/CD8+ ratio is inverted. Dendritic cell (DC) is the most powerful antigen presenting cell (APC) that has been discovered until now and activated DC plays a key role in, anti-tumor immune response. But in tumor-bearing hosts low expression of major histocompatible complex (MHC) class II molecules and costimulatory molecules on surface of DC usually result in its function deficiency so that DC can't present tumor antigen effectively, moreover can't induce sufficient production of cytotoxic T lymphocytes (CTL)to kill tumor cells and thoroughly inhibit immune response of anti-tumor. Some researchers think low expression of costimulatory molecules B7 is possibly the main reason that makes DC lose its ability to activate CTL response.Tumor cells can escape from immune surveillance in tumor-bearing hosts by several mechanisms. They include interaction of Fas and FasL;immune suppression factors produced and secreted by tumor cells;missing or transformation of MHC class I antigen on surface of tumor cells and so on. The interaction of Fas and FasL is one of important pathways in cell apoptosis process. When tumor cells expressing FasL touch activated Fas(+) lymphocytes, they can induce apoptosis of active immunocytes by transmitting death signal, then keep themselves living. Experiments in vitro prove that Fas(+) hepatocellular carcinoma cells can induce apoptosis of T lymphocytes through Fas-FasL system, so it is possibly one of the reasons that result in decrease of TIL counts and immune tolerance within tumor tissue. Tumor cells can produce directly or induce body to secret many immune suppression factors such as transforming growth factor-6(TGF-6), interleukinlO(IL-lO), vessel endothelium growth factor(VEGF) and so on that can suppress proliferation and activation of immunocytes, cause missing or transformation of MHC-I class antigen on surface of tumor cells so that they can't be recognized by T lymphocytes. By these mechanisms, tumor cells can escape from immune surveillance and keep on growth, then invade and metastasize.Clinical and experimental researches have demonstrated that many Chinese medicine can positively enhance and regulate immune function. It is reported that Lycium barbarum polysaccharide(LBP) can improve immune function of body and kill tumor cells directly. It can increase counts of nucleated cell and CFU-GM in bone marrow, induce several kinds of cytokines that can improve proliferation, differentiation and mature of active immunocytes. In this research, we observed the effects of LBP on T lymphocyte subsets and dendritic cells in H22-bearing mice and initially investigated its molecular mechanism of anti-tumor effect from the view of interfering with tumor immune escape.Objective: The study is aimed to observe the effects of LBP on tumor growth suppression and influence on immune function of H22-bearing mice in vivo, moreover investigate the molecular immune mechanism of its anti-tumor effect.Method: We used H22-bearing mice model. The mice were divided into two groups: high and low LBP dose group, respectively, which had been successively treated with LBP for two weeks. The diameter and weight of tumor were assayed every day to establish tumor growth curve. Two weeks later the mice were killed, their thymus and tumor were weighed and pathological examination was done to calculate thymus index and tumor inhibiting rate. The counts and subsets of T lymphocyte in peripheral blood and infiltrating within tumor, counts of tumor-infiltrating dendritic cell (TIDC) and expression of B7-1 in spleen and tumor tissue were detected by flow cytometry analysis. Moreover the levels of serum VEGF, TGF- 3 1 were measured by ELIS A and expressions of FasL,VEGF in tumor tissue were detected by immunohistochemistry, respectively. The normal group with LBPwas established in order to compare with the influence on immune function in normal mice. Results:1. Results of the experiment in vivo and quantitive pathological analysisLBP could suppress tumor growth evidently after being used in different dose. The volume and average weight of tumor decreased, the inhibiting rate of tumor growth was 41.12% in high LBP dose group, significantly higher than model control group (P<0.05). LBP could slightly affect tumor cell density and necrosis, but pathological nuclear cleavage decerased significantly. Moreover, LBP could improve fibro-connective tissue proliferation and infiltrating lymphocytes within tumor, there was significant difference between high LBP dose group and model group (P<0.05).2. The study of immune mechanism of anti-tumor effects in H22-bearing mice after treatment with LBP2.1 Influence of LBP on thymus in normal mice and H22-bearing miceLBP could improve thymus index and thymus quality of normal mice in some degree. In H22 -bearing mice their thymus index and thymus quality decreased obviously, they could be recovered by LBP in some degree, so did conventional pathological examination prove, there was significant difference between high dose group and model control group(P<0.05).2.2 Influence of LBP on counts of CD4+, CD8+cells and CD4+/CD8+ ratio in mice2.2.1 Influence of LBP on counts of CD4+,CD8+cells and CD4+/CD8+ ratio in peripheral blood of normal mice and H22 -bearing miceLBP could increase the counts of CD4+cells and CD4VCD8+ ratio in peripheral blood of normal mice. In H22-bearing mice, the counts of CD4+,CD8+cells and CD4+/CD8+ ratio were all lower than normal mice, the decrease of CD4+cell counts was significant (P<0.05). After treatment with LBP in different dose for two weeks, the counts of CD4+cells were significantly higher than model control group (P<0.05), so were the CD4+/CD8+ ratios, but there was no significant increase in the counts of CD8+cells.2.2.2 Influence of LBP on counts of CD4+,CD8+cells and CD4+/CD8+ ratio infiltrating within tumor.The counts of CD4+, CD8+cells infiltrating within tumor decreased apparently. After treatment with LBP for two weeks, the counts of CD4+,CD8+cells infiltrating within tumor increased, especially in high LBP dose group. The counts of CD4+,CD8+cells in high LBP dose group were (23.39±2.43)%,(14.36±1.16)%, respectively, significantly higher than model control group ((12.89±1.25%), (7.72±0.48%), respectively, P<0.05)).2.3 Influence of LBP on the counts and function of DC.2.3.1 Influence of LBP on the counts and function of DC in spleen.In model control group, the percentages of CDllc1", CD80+and CDllc+CD80+cells ki spleen were higher than normal mice, especially the percentage of CDllc+CD80+jcells (P<0.05). LBP could increase the percentages of CDllc+,CD80+and CDllc+CD80+sells in spleen of normal and H22-bearing mice, especially counts of CDllc+CD80+cells jricreasedsignificantly in high LBP dose group(P<0.05).2.3.2 Influence of LBP on the counts and function of DCs infiltrating within tumorThe percentages of CDllc+,CD80+ and CDllc+CD80+cells infiltrating within tumor were (23.07±2.36)%,(15.15±1.47)% and (11.65+1.73)%, respectively, in model control group. They all increased slightly in different LBP dose groups, parallel with increase of counts of CD4+,CD8+cells infiltrating within tumor in some degree, but there was no significant difference among model control group and different LBP dose groups. 2.4 Interfering with the mechanism of tumor immune escape in H22 -bearing mice after treatment with LBP2.4.1 Influence of LBP on the expression of FasL in tumor cellsThere was general expression of FasL in tumor tissue of model control group, the percentage of FasL(+) cells was (76.63±3.93)%. LBP could downregulate the expression of FasL in different degree, the percentages of FasL(+) cells were (51.83±3.17)%, (45.10±3.40)%, respectively, in high and low LBP dose group, significantly lower than model control group(P<0.05).Comparing the percentage of FasL(+) cells with counts of lymphocyte infiltrating within tumor, we found that there was negative relationship between them. After treatment with LBP the expression of FasL in tumor tissue was downregulated, the amount of lymphocytes infiltrating within tumor contrarily increased.2.4.2 Influence of LBP on the secretion of immune suppression cytokines from tumor cells in H22-bearing miceIn model control group, the densities of VEGF, TGF-61 in serum were (49.44±4.03)ng/L, (39.79±1.54)ug/L, respectively, significantly higher than in normal group(P<0.01). After treatment with LBP in different dose for two weeks, the serum densities of VEGF, TGF-61 decreased evidently, especially in high LBP dose group the density of VEGF was significantly lower than model control group and so were the densities of TGF-81 in different LBP dose groups(P<0.05). Moreover we inspected the expression of VEGF in tumor tissue by immunohistochemistry and found the percentages of cells expressing VEGF in model control group were higher than different LBP dose groups, especially compared with high dose group(P<0.05).Conclusion: We observed the effects of LBP on tumor growth suppression in H22 -bearing mice, investigated its mechanism and target initially by conventional pathological examination, immunohistochemistry, flow cytometry analysis and ELISA serological inspection, respectively. It is concluded that:LBP can suppress tumor growth in H22-bearing mice and have powerful anti-tumor effects. The possible mechanism is:1. LBP can promote anti-tumor activity by improving immune function of tumor-bearing hosts. It mainly shows:1.1 LBP can improve thymus index and thymus quality in normal and H22 -bearing mice, then it can provide certain protection for thymus atrophy due to immune suppressionin tumor-bearing hosts.1.2 LBP can increase the counts of CD4+cells and CD4+/CD8+ ratio in peripheral blood of normal and H22-bearing mice. Moreover it can increase the counts of CD4+, CD8+cells infiltrating within tumor, resume CD4+/CD8+ratio in some degree, recover immune function of tumor region and whole body.1.3 LBP can increase the counts of DC and expression of B7-1 within spleen and tumor tissue in some degree, but it's not sure whether LBP can improve the function of DC.2. LBP can improve anti-tumor activity of H22-bearing mice by interfering with ways that tumor cells escape from immune surveillance.2.1 LBP can downregulate the expression of FasL on tumor cell surface, weaken the attack to FasL(+) lymphocytes by tumor cells and suppress apoptosis of immune active lymphocytes. Because there is negative relationship between percentage of FasL(+) tumor cells and counts of lymphocyte infiltrating within tumor, the increase of lymphocytes within tumor by LBP is perhaps related to interfering with signal transmission through the Fas-FasL pathway then LBP can relieve immune suppression in the microenviroment of tumor in some degree.2.2 LBP can decrease the secretion of VEGF and TGF-B1, intervene their suppression fuction on proliferation and activation in immune cells, then recover the quantity and activity of immune active cells.