| Background Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death.Occult onset,rapid progression and high postoperative recurrence rate are the reasons why hepatocellular carcinoma is difficult to be eliminated.With the increasing number of HCC patients in China,liver cancer has become an important public health problem.Objective The co-culture model of macrophages and hepatocellular carcinoma cells in vitro was established to simulate the tumor microenvironment.Tumor associated macrophages(TAMs)were induced to be M2-type macrophages by IL-4 cytokines,and the transplanted tumor model of liver cancer in mice was established.The mechanism of traditional Chinese medicine QHF formula regulating the phenotype transformation of macrophages from M2-type to M1-type through IL-4R/STATA6 pathway was discussed through in vitro and in vivo experiments,so as to regulate the tumor microenvironment and inhibit the tumor.Methods 1.In vitro experiment:(1)IL-4 cytokines were used to induce mouse peritoneal macrophage cell line RAW264.7 to be M2-TAM in vitro,and MTT was used to detect the drug toxicity of different concentrations of QHF formula to RAW264.7.At the same time,the effects of different concentrations of QHF formula and M2-TAM conditioned medium containing different concentrations of QHF formula on the proliferation ability of mouse hepatoma cells Hepa1-6 were detected;(2)Establish a non-contact Co-culture system of RAW264.7 and Hepa1-6,which is divided into Control group(Control),Co-culture group(Co-culture),IL-4 group(IL-4),QHF formula group(QHF)and QHF+IL-4 group(QHF+IL-4);(3)Using immunofluorescence double staining technique,the M2 macrophage marker CD206 and M1 macrophage marker CD80 were labeled respectively to observe the effect of QHF formula on the polarization phenotype of TAM;(4)Real-time PCR was used to detect the effect of QHF formula on the m RNA expression of IL-4R,STAT6,SOCS1,M1(i NOS)and M2(Arg-1)macrophage markers in TAM;(5)Western Blot was used to detect the effect of QHF formula on the expression of IL-4R,STAT6,p-STAT6,SOCS1 protein and the ratio of STAT6/p-STAT6 in TAM;(6)ELISA experiment was used to detect the effect of QHF formula on the secretion of M2(IL-10)and M1(TNF-α)cytokines.2.In vivo experiment:(1)hepatocellular carcinoma cell line Hepa1-6 was selected to establish the axillary transplanted tumor model of C57BL/6 mice.mice were randomly divided into Model group(M),QHF formula low dose group(QHF-L),QHF formula high dose group(QHF-H),Oxaliplatin group(OXA)and STAT6 inhibitor group(AS1517499);(2)during the period of drug treatment,closely observe the survival of mice in each group,regularly measure and record the weight and tumor volume of mice,calculate the tumor inhibition rate of drugs in each group,and evaluate the tumor inhibition effect of drugs;(3)H&E staining was used to observe the morphological changes of cells in tumor tissues;(4)Immunofluorescence double staining(Label with F4/80+CD206 and F4/80+CD80)was used to observe the polarization of TAM in tumor tissues of mice;(5)the expression of IL-4R,STAT6,SOCS1 and the m RNA expression and M2(Arg-1,CD206,IL-10)and M1(i NOS,CD86,TNF-α)macrophage markers in tumor tissue were detected by Real-time PCR;(6)Western Blot detection of IL-4R,STAT6,p-STAT6,STAT6/p-STAT6,SOCS1 and the expression of tumor invasion and migration(Snail,Twist-1),angiogenesis(VEGF)and immune escape(PD-L1)proteins;(7)ELISA was used to detect the secretion of M2(IL-10)and M1(TNF-α)cytokines in the peripheral blood of tumor mice.Results 1.In vitro experiment:(1)when the concentration of QHF formula is less than or equal to 200 μg/ml,it has no drug toxicity to RAW264.7 cells,but it has a proliferation inhibitory effect on Hepa1-6 cells.Further intervention with M2-TAM conditioned medium containing QHF formula has a more obvious proliferation inhibitory effect,and QHF formula can affect TAM to enhance the inhibitory effect on hepatoma cells;(2)in the co-culture group and IL-4 group,the average fluorescence intensity of CD206 and CD80 of TAM was higher than that of the control group.After the intervention of 100 μg/m L QHF formula,the average fluorescence intensity of CD206 decreased,the average fluorescence intensity of CD80 further increased,and the polarization of TAM decreased to M2 type and increased to M1type;(3)in the co-culture group and IL-4 group,the expression of M2 and M1 related genes Arg-1 and i NOS increased compared with the control group.After the intervention of 100μg/m L QHF formula,the expression of Arg-1 decreased,the expression of i NOS further increased,and the tumor-promoting function of TAM was inhibited and turned into tumor inhibition;(4)the expressions of IL-4R/STAT6 pathway-related genes IL-4R,STAT6,SOCS1 and proteins IL-4R,p-STAT6,SOCS1 in IL-4 group were higher than those in control group.After the intervention of 100 μg/m L QHF formula,the expression of pathway-related genes and proteins decreased,but the expression of STAT6 protein had no effect,and the IL-4R/STAT6 pathway in TAM was inhibited;(5)compared with the control group,the secretion of IL-10 increased and TNF-α decreased in the supernatant of cell culture medium of co-culture group and IL-4 group.After the intervention of 100 μg/m L QHF formula,the secretion of IL-10 decreased,the secretion of TNF-α increased,and the positive regulatory factors increased and the negative regulatory factors decreased in the immune microenvironment.2.In vivo experiment:(1)the axillary transplanted tumor model of C57BL/6 mice with liver cancer was established.After the treatment,the tumors of each mouse were peeled off,and the tumor weight was weighed to calculate the tumor inhibition rate,which was respectively: QHF-L group: 36.775%,QHF-H group: 62.264%,OXA group: 49.109%,and AS1517499 group;(2)compared with M group,the cells in tumor tissue sections of QHF-L group and OXA group were disorganized,some cell boundaries were broken,the cells in QHF-H group and AS1617499 group were loosely arranged,the nuclei gradually shrank and became smaller,and large areas of vacuoles and necrosis appeared;(3)compared with group M,the average fluorescence intensity of CD206 decreased,the average fluorescence intensity of CD80 increased,the M2-TAM decreased and the MI-TAM increased in QHF-H group and AS1517499 group;(4)compared with M group,QHF-H group,OXA group and AS1517499 group can reduce the gene expression of Arg-1,CD206 and IL-10 in tumor tissues,while QHF-H group can increase the gene expression of i NOS,CD86 and TNF-α,which are inhibited by OXA group and AS1517499 group.High dose QHF formula can not only reduce M2 correlation;(5)compared with M group,QHF-H group and AS1517499 group can reduce the expression of IL-4R/STAT6 pathway related genes IL-4R,STAT6 and SOCS1 in tumor tissues,while OXA group can only reduce the expression of STAT6 and SOCS1;(6)compared with M group,all the drug groups can reduce the expression of IL-4R and SOCS1 protein in tumor tissues,while QHF-H group and AS1517499 group can reduce the expression of p-STAT6 protein and the ratio of P-STAT6 to STAT6,but have no effect on the expression of STAT6 protein;(7)the expression of Twist,snail,VEGF and PD-L1 protein in tumor tissues of QHF-H group,AS1517499 group and OXA group was lower than that of M group,but QHF-L group inhibited the expression of VEGF and PD-L1 protein,and compared with OXA group and AS1517499 group,QHF-H group inhibited the expression of PD-L1 and VEGF protein more obviously;(8)compared with group M,each drug group can increase the content of TNF-α and decrease the content of IL-10 in serum of mice,but compared with OXA group and AS1517499 group,the effect of QHF-H group is more significant.Conclusion Traditional Chinese medicine QHF formula can inhibit tumor growth by regulating TAM phenotype and polarizing it from M2 to M1,specifically by inhibiting the expression of IL4R/STAT6 signal pathway in macrophages,down-regulating the expression of Arg-1,CD206 and IL-10 in M2 cells,and up-regulating the expression of M1 markers i NOS,CD80,CD86 and TNF-α,thus inhibiting the expression of invasion and metastasis,vascular survival and immune escape protein of hepatocellular carcinoma. |
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