Effect Of Periplaneta Americana Extract CⅡ-3 On Expression Of PD-1 And PD-L1 And Preliminary Study On Its Anti-tumor

Objective:In recent years,cancer has become the number one killer threatening the health of residents.In view of the present situation that anti-tumor therapeutic drugs have no specific effects,more researchers continue to explore to find safe,non-toxic,efficient and economical anti-tumor therapeutic drugs.Periplaneta American extract CⅡ-3 is a peptide compound developed by Yunnan Laboratory of Invertebrate Biomedicine Research and Development of Dali University.Many studies have confirmed that it has certain anti-tumor biological activity,but the specific mechanism of anti-tumor action is not fully understood.In this study,the effects of CⅡ-3 on the expression of PD-1 and PD-L1 were analyzed at the cellular and animal levels,and the antitumor mechanism of CⅡ-3 was preliminarily discussed.Methods:1.Expression of PD-1 and PD-L1 in various organs of mice:mice were killed by cervical dislocation,and major organ tissues of mice were extracted under aseptic conditions:Total RNA of heart,liver,spleen,lung,kidney,stomach,small intestine,colon,thymus,bone marrow,lymph node and brain was extracted by Trizol method.With GAPDH as internal reference,PD-1 and PD-L1 primers were used for RT-PCR,and agarose gel electrophoresis was performed on the end products obtained.The expression of PD-1 and PD-L1 in various organs and tissues was analyzed by the images obtained by the gel imaging system.2.Expression of PD-1 and PD-L1 in MFC and L1210 tumor cells:MFC and L1210 cells were resuscitated and transferred to a centrifuge tube after cell culture to a certain number.After washing,the two cells were transferred to RNase-free Eppendorf tube.The total RNA of the two cells was extracted according to the RNA extraction kit provided by QIAGEN,and GAPDH was used as internal reference,PD-1 and PD-L1 primers were used for RT-PCR,and the final products were subjected to agarose gel electrophoresis.The expression levels of PD-1 and PD-L1 in MFC and L1210 cells were analyzed by gel imaging system.3.Effects of Periplanella Americana extract CⅡ-3 on the expression of PD-1 and PD-L1 in MFC,L1210 cells:MFC and L1210 cells were resuspitated after being cultured to a certain number of cells,MFC and L1210 cells were centrifuged respectively,then the supernish was discarded and resuspend cells were counted by trypan blue.Adjust the cell concentration to the experimental requirements,take a certain amount of each into the six-well plate,respectively,divided into negative group and different concentrations of CⅡ-3 drug group,and drug groups of different concentrations were equipped with three rewells,after the completion of the addition of samples,cells were placed in 37℃constant temperature incubator for culture for24h.24h later,the cells in each well in the drawing plate were centrifugally washed and then added into the flow fluorescent antibody staining for incubation.After incubation,the cells were washed twice again,and the cell diluent(scabbard fluid)was added to resuspend.The proportion of MFC and L1210 cells expressing PD-1 and PD-L1 was analyzed by upward flow cytometry analyzer.4.Effects of Periplanella Americana extract CⅡ-3 on PD-1 expression and anti-tumor mechanism of L1210 tumor-bearing mice:L1210 cells were resuscited and cultured to a certain number,then inoculated into the right armpit of BALB/C mice(body weight range:18-20g,6-8 weeks of age).After the establishment of L1210 solid tumor model,mice were randomly divided into 4 groups and treated with drugs:normal group,model group,CⅡ-3 group,and PD-1 antibody group.Normal group and model group were given sterile normal saline(intragastrically,20m L/kg,q.d),CⅡ-3(intragastrically,200mg/kg,20m L/kg,q.d),PD-1 antibody group was given anti-PD-1antibody(intraperitoneal injection,200μg/100μL/time,q.3d).The drug was administered from the day after the model was established and continued for 10days.The next day after the completion of administration,the peripheral blood and spleen of mice were aseptically collected and made into single cell suspension,and the activated T cells,exhausted T cells and regulatory T cells were analyzed by flow cytometry.Results:1.RT-PCR results showed that PD-1 and PD-L1 were expressed in the heart,liver,spleen,lung,kidney,stomach,small intestine,colon,thymus,bone marrow,lymph nodes and brain of mice,and the expression level of PD-1 and PD-L1 in spleen,thymus and bone marrow was higher than that in other tissues.2.RT-PCR results showed that PD-1 and PD-L1 were expressed in both MFC and L1210 cells,PD-1 expression in MFC was higher than that in L1210,and PD-L1expression in L1210 was higher than that in MFC.3.Flow cytometry analysis showed that the expression levels of cell surface molecules PD-1 and PD-L1 of MFC and L1210 cells were decreased after treatment with Periplaneta Americana extract CⅡ-3.The higher the concentration of PD-3,the lower the expression levels of cell surface molecules PD-1 and PD-L1 of MFC and L1210 cells were.4.Flow cytometry analysis results showed that compared with the model group,the proportion of CD3~+PD-1~+T cells and the proportion of CD4~+CD25~+T cells in peripheral blood of CⅡ-3 group decreased.Flow cytometry analysis results showed that compared with the model group,the proportion of CD3~+CD69~+T cells in spleen of CⅡ-3 group was increased,the proportion of CD3~+PD-1~+T cells was decreased,and the proportion of CD4~+CD25~+T cells was decreased.Conclusions:1.Immune checkpoint molecules PD-1 and PD-L1 were expressed to a certain extent in most organs and tissues of normal mice and in tumor cells L1210 and MFC.The expression of PD-1 and PD-L1 in spleen,thymus and bone marrow was higher than that in other tissues,and the expression of PD-L1 in L1210 was higher than that in MFC.2.Periplaneta Americana extract CⅡ-3 could down-regulate the expression of PD-1 and PD-L1 on the surface of L1210 and MFC.3.Periplaneta Americana extract CⅡ-3 can inhibit the expression of PD-1molecule on the surface of T cells in peripheral blood and spleen of tumor-bearing mice of L1210 cells,inhibit the depletion of T cells,enhance the activation of T cells,and reduce the proportion of regulatory T cells.4.Preliminary studies have shown that the effect of Periplaneta Americana extract CⅡ-3 may be to enhance the immune response and play an anti-tumor role by inhibiting the expression of PD-1 molecule in T cells,down-regulating the proportion of PD-1~+T lymphocytes,inhibiting the failure of T cells,reducing the proportion of regulatory T cells and enhancing the activation of T cells.