The Study Of Artesunate On Anti-tumor Effects In H₂₂ Bearing Mice And Influence On Expression Of VEGF And FasL In Tumor

Primary hepatic carcinoma(PHC)is one of the cancers with severe malignance and very poor prognosis,90%of which is mainly referring to hepatocellular carcinoma(HCC),and it is the third letalis cause in the global population of cancer deaths.Most patients have been in advanced stage when the disease is diagonosed and the median survival time is less than 12 months commonly.There are about 110,000 people died of PHC every year in China and mortality of PHC is in third place in malignant tumors.There are almost 260,000 New cases worldwide every year and more than half in China,and further more, the morbility is of a rising trend.Despite the diagnosis and treatment of hepatocellular carcinoma has been improving,the morbility and mortality remains high,the prevention and treatment study of PHC is a very important task.Currently,The hotspots of anti-hepatic carcinoma mainly focus on these aspects such as inducing cells apoptosis,resisting tumor immune escape, depressing the growth of tumor angiogenesis and prolonging patients life time research,on the basis of which many anticancer drugs are investigated and studied.The active participation of Chinese Medicine(CM)in PHC is a feature that promote PHC prevention and treatment study and improve clinical efficacy in China.It develops widely in experiments and clinical research including determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs,compatibility in complex prescription, abstracting effctive component from Chinese medicinal materials et al.CM has shown some advantages that it can reduce recrudescence and metastasis,relieve side effects of radiotherapy or chemotherapy,enhance body immune,as well as improve quality of life and prolong survival time of patients.CM is of a very important option in anti-hepatic carcinoma.It become hot topics that in searching of effective components and clarifying its material basis and mechanism with high-tech to anti-tumors in CM.Although research in domestic and overseas shows that Artesunate has good effect against liver cancer by inducing apoptosis of hepatoma cells,more systematic studies are needed to open out its role and mechanism.As artesunate has multiple targets and more pharmacological action,it is possible that artesunate has multiple mechanisms against liver cancer,To explore its possible mechanisms will be conducive to developing its new use.ObjectiveTo observe the effects of different doses of Artesunate on tumor inhibition in H₂₂bearing hepatic carcinoma mice in vivo,and to reveal its potential mechanisms against liver cancer by investigating contribution to apoptosis in tumor cells,expression of VEGF and FasL in tumor tissue and impact to body immune.MethodsModel was H₂₂bearing mice by subcutaneous xenografts.NIH mice under SPF was selected and randomly divided into six groups.They were cyclophosphamide group(CTX),different doses of ATS groups(ATS 15mg/kg,ATS 30mg/kg,ATS 45mg/kg and ATS 60mg/kg)and model control group(Model).The CTX group was given cyclophosphamide 20mg/kg,qod,and normal saline instead on next day, the different dose of ATS groups were given Artesunate 15mg/kg,30mg/kg, 45mg/kg and 60mg/kg respectively,qd,and the Control group was given equivalent normal saline,qd.Normal mice were used to be Normal group in these experiments.Each group was given drugs by intraperitoneal injection for 15 days.The general situation was observed and survival time in mice was compared. The diameter was measured to calculate tumor volume.The tumors were weighted after their complete dissection to calculate tumor inhibitory rate.The tumor cells apoptosis was detected by TUNEL,expression of VEGF and FasL in tumor tissue were detected byimmunohistochemistry,respectively.The T lymphocyte subpopulations and NK cells percentage in peripheral blood of the mice were detected by Flow Cytometry.Results1.1 The general situation and survival time of miceThe Model was successful with rate of 100%.Mass could be touched with slightly protuberant from skin around the site of subcutaneous injection from D3 in Control group and D5 in other groups.The masses growed quickly with spherical or hemispherical appearance,and they could be moved under skin and became hard gradually.The masses were not in the same size and part of them ulcerated in the surface for their oversize in the late stage.Withthe growth of the masses,the mice were in poor state with less eated and drinked and reduced activities and fur lost luster gradually.The mice lost weight in the advanced stage.From D16,the tumor beared mice were dead gradually and the Model group died quickly.No obviously toxic and adverse reaction appeared in the different doses of ATS groups.The medium survival time of Control group was 20.30±3.95 days,while 27.90±7.96d of ATS 30mg/kg group,28.60±6.36d of ATS 45mg/kg group and 26.30±5.1d of CTX group,they were longer than of Model group(P＜0.05,P＜0.01).The survival time of ATS 30mg/kg group and ATS 45mg/kg group were longer than of ATS 15mg/kg group also(P=0.04 and P=0.02), while no significant difference between ATS 60mg/kg group and ATS 15mg/kg group (P＞0.05).Campared with life prolonging rate,the rate was 40.89%in ATS 45mg/kg group and 37.44%in ATS 30mg/kg group,longer than it of 29.56%in CTX group.1.2 Tumor volume,tumor quality and tumor inhibitory rateTumor volume in All treatment groups was less than in Model group on D16 (P＜0.05,P＜0.01).Compared among the treatment groups,the tumor volume in ATS 45mg/kg was significantly less than in ATS 15mg/kg(P=0.003).while no difference between in ATS 45mg/kg,ATS 30mg/kg and ATS 60mg/kg(P＞0.05).No statistic difference between in ATS 45mg/kg group and in CTX group(P=0.29).Tumor quality in Model group was 2.35±0.16g,and it is larger than in every treatment group(P＜0.01).Compared among the treatment groups,tumor quality in ATS 45mg/kg group was evidently less than in ATS 15mg/kg group and ATS 60mg/kg group(P=0.000 and P=0.037 respectively).Tumor quality in ATS 45mg/kg group was equal to in ATS 30mg/kg group(P=0.269).It was no difference between in CTX group with in ATS 45mg/kg group(P=0.428)and between in CTX group with in ATS 30mg/kg group(P=0.06).Tumor inhibitory rate in CTX group, in ATS 45mg/kg group,and in ATS 30mg/kg group was not significant(P＞0.05), while it was better than in ATS 15mg/kg group and in ATS 60mg/kg group.1.3 tumor cells apoptosis index(AI)Campared with in Model group,apoptosis index(AI)in the other treatment groups increased in varying degrees.The AI increased when the H₂₂bearing mice were treated by ATS different doses or CTX(P＜0.05,P＜0.01).There was not significantly different between in ATS 30mg/kg group with in ATS 45mg/kg group(P＞0.05),the AI in ATS 30mg/kg group with in ATS 45mg/kg group were higher than in ATS 60mg/kg group and in ATS 15mg/kg group(P＜0.05).and what's more,there were no difference among in ATS 30mg/kg group,in ATS 45mg/kg group and in CTX troup(P＞0.05).1.4 The expression of VEGF in tumor tissueIt showed that all mice in experiment were positive expression of VEGF in tumor tissues.And the expression was strong positive in Model group.The strong positive expression decreased in ATS different doses group and in CTX group,and the weak positive expression increased.It showed the expression was statistically different in the six groups after calculated by Kruskal-Wallis Test(P=0.04).The suppression of VEGF expression in ATS 45mg/kg group was obvious,which was significant different compared with in Model group(P＜0.05).In addition,the suppression was shown in CTX group too(P＜0.05),there was not different compared with in ATS 45mg/kg group(P＞0.05).1.5 The expression of FasL in tumor tissueExpression of FasL in tumor tissues was detected positive in All mice in the experiment.It showed that expression in Model group was in higher degree of positive classification,while some depression was shown in ATS different doses groups.Compared between the 6 groups,the expression of FasL in ATS 45mg/kg was obviously lower than in Model group and in CTX group(P＜0.05).1.6 T Lymphocytes subpopulation in peripheral blood of the miceIt showed that CD₃⁺ T Lymphocytes and CD+4⁺T T Lymphocytes percentage in Model group was obviously lower than in Normal group(P＜0.01),while there was no statistical difference in CD₈⁺T T Lymphocytes between the two groups,which resulted in the ratio of CD₄⁺/CD₈⁺ in Model group lower than in Normal group (P＜0.05).The percentages of CD₃⁺ T Lymphocytes and CD₄⁺ T Lymphocytes in ATS 30mg/kg group,ATS 45mg/kg group were increased(P＜0.05,P＜0.01),while the percentage of CD₈⁺ T Lymphocytes was not statistically different compared with Model group,and the ratio of CD₄⁺/ CD₈⁺ increased(P＜0.01).There was no difference in ATS 15mg/kg group and ATS 60mg/kg compared with in Model group(P＞0.05).The results of CD₃⁺,CD₄⁺,CD₈⁺ in ATS 30mg/kg group and ATS 45mg/kg group were not statistically different compared with CTX group(P＞0.05),even compared with Normal group(P＞0.05).1.7 percentage of NK ceils in peripheral blood of the miceThe result showed that the percentage of NK cells in peripheral blood in the Model group was obviously lower than in Normal group(P＜0.05).The percentage of NK cells in CTX group was not different compared with that in Control group(P＞0.05).Percentage of NK cells in peripheral blood could increase after the mice treated by ATS different doses,and the percentage was obviously higher in ATS 60mg/kg group,ATS 45mg/kg group,and ATS 30mg/kg group than in Model group and in CTX group,which was statistically significant (P＜0.05).What's more,the percentages in ATS 60mg/kg group,ATS 45mg/kg group, and ATS 30mg/kg group were near with Normal group(P＞0.05).Conclution1.1 Artesunate prolonging the mean survival time of H₂₂bearing miceArtesunate 30mg/kg and 45mg/kg doses can prolong the survival time of H₂₂ bearing mice,with no obviously side effect to the quality of life in the mice.1.2 Artesunate inhibiting the growth of tumor in H₂₂bearing mice and inducing apoptosis in tumor cells.Different doses of Artesunate can inhibit the growth of tumor in H₂₂bearing mice and induce the apoptosis of the tumor cells to a certain extent,what's more,ATS 45mg/kg and ATS 30mg/kg are the appropriate doses to inhibit the growth of tumor,and the tumor inhibitory rate is 65.26%,57.39%respectively.1.3 Artegunate depressing the expressionof VEGF and FasL in tumor tissue of H₂₂bearing miceATS 45mg/kg dose can depress the expression of VEGF in tumor tissue of H₂₂bearing mice and growth of tumor angiogenesis,which results in reduction of invasion,metastasis and recurrence of hepatic carcinoma.ATS 45mg/kg dose can reduce the expression of FasL in tumor tissue and resist tumor immune escape conduced by the expression of FasL in tumor tissue.1.4 Artesunate enhancing immune in H₂₂bearing miceATS 45mg/kg and 30mg/kg doses can increase the percentage of CD₃⁺ and CD₄⁺T T Lymphocytes in peripheral blood of the mice and enhance cell immune.ATS 60mg/kg,45mg/kg and 30mg/kg doses can increase the percentage of NK cells and enhance the activity of NK cells,which indicates that Artesunate can reactivate the anti-tumor effect of body immune,and recover its effect against tumor.1.5 Artesunate 30mg/kg and 45mg/kg doses are appropriate doses against hepatic carcinoma in H₂₂bearing mice by subsutaneous xenografts.The dose 45mg/kg was a prefer dose in suppressing expression of VEGF and FasL in tumor.In a word,the research showed the inhibition effects of different doses of Artesunate against tumor growth in vivo,and investigated its mechanism from different study hotspots.