| Objective:To investigate the effect of Cantharidin(CTD)on the immune function of mice with H22 liver cancer tumor-bearing mice and the effect on the expression of PD-1/PD-L1,and to discuss the possible mechanism of its action,which provided experimental basis for the elucidation of the anti-liver cancer mechanism of CTD.Methods:After the success of tumor grafting in H22 hepatocellular carcinoma-bearing mice,they were randomly divided into model group,5-Fluorouracil positive control group and high,medium and low CTD group,10mice per group,and the blank group were 10 normal mice.The CTD group was injected at a dose of 1 mg/kg,0.5 mg/kg,and 0.25 mg/kg per day,respectively,the 5-Fu group was injected at 25 mg/kg every 2 days,and the model group was injected with normal saline once a day;the dosing cycle was 15 days.1.Observe the general state and behavior of mice,weigh the weight every 2 days apart,and record the weight change.2.Measure the longitudinal and transverse diameter of the tumor twice a week to plot the tumor growth curve,dissect and isolate the tumor tissue,weigh it,and calculate the inhibition rate of the tumor.3.HE staining to observe morphological changes in tumor tissue.4.After separation of the spleen,weigh and calculate the spleen index.5.The ELISA experiment was used to detecte the contents of IL-2,IL-4,IL-10,IFN-γ,TNF-αin tumor suspension.6.The Flow cytometry was used to detect the ratio of CD4~+T,CD8~+T,Treg,B-lymphocyte and CD4~+/CD8~+ratios in peripheral blood.7.Immunohistochemistry(IHC)staining to detect the expression of PD-1 in spleen and PD-L1 in tumor tissues.8.The RT-qPCR was used to detect the expression of PD-1 mRNA in the spleen tissue and PD-L1 mRNA in tumor-bearing mouse.9.The Western Blot method was used to detecte the expression of PD-1 in the spleen and PD-L1,HIF-1αand PI3K/AKT proteins in tumor-bearing mice.Results:1.General behavior:the mice state and diet of the blank group are normal,the mice in the model group have a poor mental state,decreased mobility,dark hair,knots,and the survival status,mobility and diet of the mice in the 5-Fu group and the high-dose group of CTD are better.Weight curve:Compared with the blank group,the mice in each experimental group had significant weight gain,and the weight gain of the model group was more significant(P<0.05);compared with the model group,the 5-Fu group and the high-dose CTD group had the slowest weight gain(P<0.05).2.The tumor growth curve showed that the tumors in the model group grew fast and were large in volume,and the tumor growth of mice in the 5-Fu group and the CTD dose group decreased slowly,and the volume and quality were significantly reduced(P<0.05).The tumor inhibition rate of mice in the 5-Fu group was 45.09%,the tumor suppression rate in the high-dose CTD group was 38.96%,and the tumor suppression effect in the medium and low dose groups was not significant.3.The HE staining results showed that the number of tumor cells in the model group were numerous,tightly arranged,irregular,and deeply stained with nuclei,and the density of tumor cells in the 5-Fu group and the CTD dose group were reduced,loosely arranged,necrosis and apoptosis occurred in different degrees,and the number of vacuoles were increased.4.The spleen index results showed that the spleen index was significantly increased in both the model group and the drug delivery group compared with the blank group(P<0.05),and the spleen index was most significantly reduced in the 5-Fu group and the high-dose CTD group compared with the model group(P<0.05).5.The ELISA experimental results showed that in the tumor suspension,the content of IL-2,IL-4 and IFN-γin the 5-Fu group and the CTD group were increased(P<0.01),and the content of IL-10 and TNF-αwere decreased(P<0.05).6.The FCM test results showed that compared with the blank group,the proportion of CD4~+T,CD8~+T cells and B cells in the model group was significantly reduced(P<0.05),while the expression of Treg was significantly increased(P<0.05),the CD4~+/CD8~+ratio was reduced(P>0.05),and the proportion of CD4~+T,CD8~+T and B cells in the 5-Fu group and CTD group were slightly reduced(P<0.05).Compared with the model group,the proportion of CD4~+T,CD8~+T and B cells in the 5-Fu group and CTD group were increased(P<0.05),the CD4~+/CD8~+ratio of mice in the CTD group was increased(P<0.05),and the expression of Treg in the CTD group was significantly reduced(P<0.01).7.The results of IHC staining showed that the positive expression of PD-1 and PD-L1 in the 5-Fu group and CTD group was significantly reduced compared with the model group(P<0.01).8.The RT-qPCR results showed that compared with the model group,the 5-Fu and CTD could reduce the expression level of PD-1 mRNA in the spleen and PD-L1 mRNA in the tumor tissue(P<0.01).9.The western blot results showed that compared with the model group,the 5-Fu and the CTD could reduce the expression level of PD-1 protein in spleen tissues and PD-L1 protein in tumor tissues(P<0.01 or P<0.05),and downregulate the expression levels of PI3K/AKT and HIF-1αproteins in tumor tissues(P<0.01 or P<0.05).Conclusion:Cantharidin can inhibit the growth of liver cancer tumors in tumor-bearing mice,reduce the expression of PD-1/PD-L1,improve mouse immune function,the mechanism may be to downregulate the expression of HIF-1αto block the PD-1/PD-L1 signaling pathway,thereby inhibiting the development of downstream PI3K/AKT pathway,promoting T and B cell proliferation and differentiation,so that cytokine secretion of IL-2 and IFN-γis increased,thereby enhancing the body’s anti-tumor immune function. |
