Basic Study On The Application Of QHF Compound Against Immune Escape Of Liver Cancer By Chemotactic Tregs Cells

Background:Hepatocellular carcinoma(HCC)is the fifth most common malignancy in the world,and its morbidity and mortality rank the sixth and third among malignancies,respectively.The morbidity and mortality of HCC in developing countries are much higher than those in developed countries.Objective To investigate the inhibitory effect of TCM QHF compound on immune escape of liver cancer through chemotactic Tregs,and further clarify that this compound inhibits the chemotactic effect of mouse liver cancer cells Hepa1-6 on Tregs by reducing the expression of chemotactic factor CCL20 throμgh the CCL20/CCR6 signal axis,and reduces the accumulation of Tregs around the tumor.Methods:1.In vitro experiments:Methods:(1)The splenic lymphocytes of C57BL/6mice and hepatocellular carcinoma cells of HEPA1-6 were obtained by grinding and sorting,and the two kinds of cells were co-cultured;(2)ELISA kit was used to detect the expression of IL-10 in the system of co-culture of two kinds of cells,and the best co-culture time and the proportion of two kinds of cells were determined by the speed and time of IL-10 decline;(3)CCK8 method and cell counting method were used to detect the promoting effect of QHF compound on lymphocyte proliferation and the inhibitory effect of QHF compound on hepatoma cell proliferation;(4)Fluorescence quantitative PCR was used to detect the expression level of Foxp3~+gene in lymphocytes in the co-culture system.(5)PCR was used to detect the gene expression levels of CCL20 and CCR6 in the process of co-culture of two kinds of cells.2.In vivo experiments:(1)Ready to need to use Hepa1-6 liver cancer cells in mice,by in situ injection of liver cancer cell C57BL/6 mice liver in situ tumor cell model is established,the experimental level of 50 SPF C57BL/6 mice were grouped:divided into DDP,QHF group,CCL20 transfection group,group,and Hepa1 idle-6,and then choose 10 SPF level of experimental C57BL/6 as were injected with normal saline control group;(2)The body weight,hair,food intake and mental state of mice were measured every two days during15 days of intragastric administration;(3)Through the mice in the liver tumor growth,compared with cisplatin group to determine drug QHF compound tumor inhibitory effect on the in situ tumor mice,by measuring the quality of thymus and spleen calculate thymus and spleen index to judge whether drugs can improve the immune function in mice;(4)HE staining to observe the change of tumor tissue and tumor cells,compared with not observed group,determine how QHF compound effect;(5)Immunohistochemical double staining(CCl20+Foxp3~+)was performed to observe the localization of CCL20 and Foxp3~+in tumor tissues and to clarify the relationship between them;(6)ELISA kit was used to detect the contents of IL-2,IFN-γ,TGF-βand IL-10 in the serum of mice,and to judge the effect of QHF compound on the immune system;(7)PCR was used to detect the gene expression of CCL20 and CCR6 in the peripheral blood of mice to determine whether the drug could regulate CCL20/CCR6;(8)Western Blot was used to measure the contents of CCL20 and CCR6 in tumor tissues.Results 1.(1)The co-culture model of mouse spleen lymphocytes and hepatoma cells Hepa1-6 was successfully established;(2)According to the ELISA results,the optimal co-culture time of QHF compound to inhibit the release of IL-10 from lymphocytes was 48h,and the optimal concentration ratio of spleen lymphocytes and hepatoma cells was10:1.Moreover,different concentrations of QHF could inhibit the content of IL-10 in the supernatant of the co-culture model(p<0.05);(3)According to the results of CCK8,different concentrations of QHF could significantly promote the proliferation of lymphocytes and lymphocytes after co-culture,and there were statistically significant differences between the two at 24h(p<0.05),different concentrations of QHF had significant inhibitory effects on HCC cell lines Hepa1-6 and HCC cell lines Hepa1-6 after co-culture compared with model group(p<0.05);(4)The expression level of Foxp3~+gene in lymphocytes in the co-culture system(Hepa1-6+LY)was detected by fluorescence quantitative PCR,and it was found that QHF could inhibit the expression of Foxp3~+gene in the co-culture system(Hepa1-6+LY).When the concentration of QHF was 100μg/m L,there was a statistical difference compared with the model group(p<0.05);(5)QHF(50μg/m L、100μg/m L)could inhibit the expression of CCR6 gene in lymphocytes in co-culture(Hepa1-6+LY),and the difference was statistically significant compared with model group(p<0.05);QHF(50μg/m L、100μg/m L)inhibits co-culture CCL20 gene expression in Hepa1-6 liver cancer cells cultured(Hepa1-6+LY)was significantly different from that in model group(p<0.05).2.The C57BL/6 mouse model of axillary tumor transplantation was successfully established,and the experiment was divided into 5 groups:DDP group,QHF group,CCL20transfection group,idling group,and Hepa1-6 group.The results were as follows:(1)The body weight and food intake of mice in QHF group were significantly higher than those in DDP group,with statistical significance(p<0.05、0.01);The spleen index and thymus index in QHF group were increased,but the spleen index in DDP group was significantly decreased,with statistical significance(p<0.05、0.01);(2)The tumor mass of mice in the QHF and DDP groups was significantly lower than that in the Hepa1-6-CCL20 group,with statistical significance(p<0.05、0.01).The tumor inhibition rates of QHF group and DDP group were56.98%、66.28%,respectively;(3)The results of HE staining showed that there were a lot of vacuolar degeneration,cell pyroconstriction and obvious signs of necrosis in the QHF and DDP groups;(4)Immunofluorescence double staining showed that the QHF group and DDP group could inhibit the expression of Tregs specific marker Foxp3~+and cancer cell CCL20 at the same time,and CCL20 and Foxp3~+co-expressed.The inhibition effect of QHF was more obvious than DDP;(5)ELISA assay showed that QHF group could promote the secretion of IL-2 and INF-γ,the difference was statistically significant compared with Hepa1-6-CCL20group(p<0.05、0.01);QHF group could inhibit the secretion of IL-10 and TGF-βand the difference was statistically significant compared with Hepa1-6-CCL20 group(p<0.05、0.01),while DDP did not have this function,indicating that QHF compound could enhance the immune function of tumor-bearing mice;(6)PCR results showed that QHF group could down-regulate the gene expression levels of CCL20 m RNA and CCR6 m RNA;(7)Western Blot results showed that the expression of CCL20 and CCR6 could be decreased in the QHF group,while the DDP group could only reduce the expression of CCL20,but the effect on CCR6 was not obvious.Conclusion This study illustrates the molecular mechanism of QHF compound inhibiting the migration of hepatocellular carcinoma cells to Tregs throμgh the CCL20/CCR6signal axis by inhibiting the secretion of CCL20.This study provides a theoretical basis for the application of QHF compound in the screening of immune escape from liver cancer and the search for new therapeutic targets.