The Therapeutic Effects Of D-PPA1 In Mouse Endometrial Carcinoma

Objectives To investigate the therapeutic effect of PD-L1 inhibitor D-PPA1 on mouse endometrial carcinoma by detecting the expression of inflammatory cytokines(IL-6,IL-17,TNF-α),matrix metalloproteinases(MMP2,MMP9)and PI3K/Akt/GSK3β signaling pathway proteins.This study provides theoretical basis and effective reference for targeting PD-1/PD-L1 in the treatment of endometrial cancer.Methods 1 The endometrial carcinoma HEC-1-A cells were cultured in vitro to establish the subcutaneous tumor-bearing model of mice.2 Animal grouping: randomly divided into control group,model group,DMSO group(solvent group),paclitaxel group(positive control group),D-PPA1 group(inhibitor group),with 6 animals in each group.3.Drug intervention: D-PPA1 group,paclitaxel group and DMSO group were intraperitoneally injected with D-PPA1(2mg/kg),paclitaxel(7.5mg/kg)and DMSO solution,respectively.Control group and model group were intraperitoneally injected with normal saline,once a day,200μl/ time,continuous administration for 8 days.4 HE staining was used to observe the morphological changes of endometrial cancer in nude mice.5 Real-time fluorescence quantitative PCR: m RNA levels of IL-6,IL-17 and TNF-α gene in tumor tissues of each group were detected.6 Western blotting was used to detecte protein expressions of PD-1,PD-L1,IL-6,IL-17,TNF-α,GSK3β,p-GSK3β,Akt,p-Akt,MMP2,MMP9 in each group.7.ELISA was used to detecte the levels of inflammatory cytokines such as IL-6,IL-17 and TNF-α in serum of each group.Results 1 After 8 days of inoculation,the longest diameter of tumor reached 5mm which indicated the model was successful.After drug intervention,the tumor volume of the DPPA1 group was significantly smaller than that of the model group.2.Expression of PDL1 protein in tumor tissues of mice in each group: D-PPA1 decreased the PD-L1 protein expression in the tumor tissues of mice: the PD-L1 protein expression in the model group,the DMSO group and the paclitaxel group were all higher than that in the control group(P<0.05),and there was no statistical significance between the first three groups.The protein expression of D-PPA1 group was lower than that of paclitaxel group(P<0.05),and slightly higher than that of control group(P>0.05).3 D-PPA1 inhibited the expression of inflammatory factors IL-6,IL-17 and TNF-α: q PCR,Western blotting and ELISA showed the same trend.The m RNA levels and protein expressions of IL-6,IL-17 and TNF-α in model group and DMSO group were higher than those in control group(P<0.05).D-PPA1 group was similar to paclitaxel group(P<0.05),both lower than model group(P<0.05),but still higher than control group(P<0.05).4.D-PPA1 decreased the protein expressions of MMP2 and MMP9 in cancer tissues: the protein expressions of MMP2 and MMP9 in model group and DMSO group were increased compared with the control group(P<0.05),while the protein expressions of MMP9 in paclitaxel group and D-PPA1 group were decreased compared with the model group(P<0.05),and there was no statistical significance between the two groups.MMP2 paclitaxel group was close to model group(P>0.05),higher than control group and D-PPA1 group(P<0.05),and there was no statistical significance between D-PPA1 group and control group.5 D-PPA1 inhibited Akt/GSK3β phosphorylation:(1)The phosphorylation level of Akt protein in the tumor tissues of mice in each group: The p-Akt/Akt ratio in the model group and DMSO group was higher than that in the control group(P<0.05),and the paclitaxel group was lower than that in the model group,but still higher than that in the control group and D-PPA1group(P<0.05).There was no statistical significance between the D-PPA1 group and the control group.(2)The phosphorylation level of GSK3β in the tumor tissues of mice in each group was higher than that in the control group(P<0.05).Paclitaxel group was lower than model group,but higher than control group(P<0.05);D-PPA1 group was lower than model group(P<0.05),and there was no statistical significance between D-PPA1 group and control group and paclitaxel group.Conclusions D-PPA1 inhibited tthe expression of PD-L1 in tumor-bearing mice of endometrial carcinoma,possibly by inhibiting Akt/GSK3β phosphorylation,the expression of inflammatory factors(IL-6,IL-10,TNF-α)and matrix metalloproteinases(MMP2,MMP9)were decreased,and the tumor growth of endometrial carcinoma bearing mice was slowed down.Figure10;Table7;Reference 111...