The Effect And Mechanism Of Hydroxysafflor Yellow A On The Tumor Immune Microenvironment Of Balb/c Liver Cancer Mice

Objective:To explore the impact of HSYA on anticancer and immune organs in Balb/c mice,to investigate the effect of HSYA on regulatory T cells and impacts of HSYA on related factors of HCC tumor immune microenvironment inBalb/c mice.Methods:Experiment 1:Firstly,we establishedan orthotopic liver cancermodelin Balb/c mouse,then we observed the effect of different doses of HSYA on cancergeneral growth conditions and change of body weight in Balb/c mouse,thymus and spleen index were used to evaluate the effects of HSYA on immune organs,the anti-tumor effect of HYSA in liver cancer mice was observed by HE stain method.Experiment 2:Immunofluorescencewas used to detect the proportion of CD4+CD25+Foxp3+ T cells in spleen of liver cancer mice,studying the effect of HSYA on regulatory T cells in the mice overall immune system;Real-time PCR and immunohistochemical was used to test the mRNA and protein of FOXP3 in tumor tissue,researchingthe effect of HSYA on regulatory T cells in localtumor tissuesof liver cancer mice.Experiment 3:Thl and Th2 cytokines inserum weredetected by CBA assay,and the mRNA expression of TLR4,TGF-?1,RORytin tumor tissue were tested by real-time PCR method,exploringthe effect of HSYA on the related cytokinesin tumor immune microenvironment.Results:Experiment 1:There was an upward trend in the weight of HCC mice who were in the Control group,Model group and HSYA groups,while theweight of mice in Cisplatin group and Combination groupwas decreased,which was significantlydecreased from the 7th day of intervention(P<0.05);Compared with Model group,there was no significant diffeerence among different HSYA groups;Compared with Cisplatin group,the weight of micein Combination group was increased,and there was statistically significant onthe 4th,5th and 6th day ofintervention(P<0.05).Compared with Control group,there was an increasing trend in thymus and spleen indexof HCC mice inModel group,buthad no significant difference(P>0.05);the index of spleen and thymus were significantly decreased in Cisplatin group(P<0.05);Compared with Model group,the different-dose HSYA significantlyincreased the thymus index of HCC mice(P<0.05),while had no statistically significant in the index of spleen,both the thymus and spleen indexwere significantly decreased in Cisplatin group and Combination group(P<0.05).Compared with Cisplatin group,there was an increasing trendin the thymus and spleen indexinCombination group,buthad no significant difference(P>0.05).Pathologic results showed that the lobularstructure was clear,liver cells arranged in neat rows showing cords in Control group of HCC mice.In Model group,there were cancer tissues in solid sheets and the cancer cells were in different sizes and irregular arrangement.In the middle-dose HSYAgroup,low-dose HSYA group and Cisplatin group,there were aggregated scattered and sheet cancer cells,whose chip area and number decreased significantly compared with the Model group,showing precancerous pathological changes.Compared with Cisplatin group,in the Combination group there was almost no solid sheet carcinoma,just some scattered hepatoma cells.Experiment 2:Compared with Control group,the proportion of CD4+CD25+Foxp3+T cells in spleen of HCC mice,the expression level of Foxp3 mRNA and protein in liver tissue were significantly increasedin Model group(P<0.05);Compared with Model group,middle-dose HSYA significantly decreased the proportion of CD4+CD25+Foxp3+T cells in spleen of HCC mice,and also significantly reduce the expression level of Foxp3 mRNA and protein in liver tissue(P<0.05);Compared with Cisplatin group,there was no effect on the proportion of CD4+CD25+Foxp3+T cells and the expression level of Foxp3 mRNAinCombination group,but there wassignificantly difference inthe expression level of Foxp3 protein(P<0.05).Experiment 3:Compared with Control group,there was no significant difference in the content of TH1-type(IL-2,IFN-y,TNF)and TH2-type(IL-4,IL-5)in serum of HCC mice(P>0.05);Compared with Model group,each dose of HSYA had no significant effect onThland Th2 cytokinein mouse serum(P>0.05).Compared with Control group,the mRNA expression of TLR4 and RORytin liver tissue was significantly increased in Model group(P<0.01),but there was no statistical difference in the mRNA expression TGF-?1(P>0.05);Compared with Model group,middle-dose HSYA could reduce the mRNA expression of TLR4and RORyt,and the differences were statistically significant(P<0.05).Conclusion:Firstly,a certain concentrationof HS YA had an anticancer effect on orthotopic HCC in Balb/c mice,and could enhance the body's immune system.In addition,HSYA with cisplatin hadmore effective effect in anticancer,and particularly improve the situation of cisplatin-induced immune dysfunction.Secondly,HSYA could improve the tumor immune microenvironment by reducing the proportion of CD4+CD25+Foxp3+T cells in spleen and decreasing the expressionof Foxp3 mRNA and protein in tumor tissue,and thensuppresstumor growth.Thirdly,a certain concentration of HSYA was able to reduce the mRNA expressionof TLR4 and RORyt in tumor tissue,however,it had little effect on Thl/Th2 cytokinesand the mRNA expressionof TGF-?1.