关键词: 登革热病毒, 内皮细胞, 自噬, 细胞凋亡, 程序性细胞死亡受体-1配体

Abstract: Objective　To investigate the role and mechanism of PD-L1 overexpression in the effect of dengue virus type Ⅱ (DENV-2) on vascular endothelial cell autophagy and apoptosis. Methods　The vascular endothelial cell EAhy926 was used as the research object. The cells were treated with serum-free medium containing DENV-2 (1×108-2×109 pfu/L) for 24 h and 48 h. At the same time, cells were treated with serum-free medium as control group. Add serum-free medium to blank holes as zero adjustment holes. The cell proliferation activity was detected by MTT method. The cells were treated with PD-L1 overexpression lentiviral solution and no-load lentiviral solution, and cells were set as no-load control group and transfection group respectively. Cells treated with DENV-2 (1.2×109 pfu/L) for 12 h, 24 h and 48 h were set as DENV-2 12-h group, DENV-2 24-h group and DENV-2 48-h group, respectively. At the same time, the control group was set (cells treated with serum-free medium). In addition, the no-load control group and the transfection group were treated with DENV-2 for 48 hours, and the protein expression changes of LC3B, Beclin-1 and Bcl-2 were detected by Western blot assay. The cell apoptosis was detected by flow cytometry. Results　1×108-2×109 pfu/L DENV-2 can inhibit the proliferation of EAhy926 cells in a dose-dependent manner, with IC50 of 1.8×109 pfu/L and 1.2×109 pfu/L, respectively. Compared with the control group, the expression leves of LC3Ⅱ/LC3Ⅰ and Beclin-1 protein were up-regulated in the DENV-2 12-h group DENV-2 24-h group and DENV-2 48 h group, the expression of Bcl-2 protein was down-regulated, and the total cell apoptosis rate was increased (P＜0.05). Compared with the empty control group, the expression of PD-L1 protein was up-regulated in the transfection group (P＜0.01). After DENV-2 treatment for 48 hours, compared with the no-load control group, the expressions of LC3Ⅱ/LC3Ⅰ and Beclin-1 protein were down-regulated in the transfection group, the expression of Bcl-2 protein was up-regulated, and the total cell apoptosis rate was reduced (P＜0.01). Conclusion　DENV-2 induces autophagy and apoptosis in EAhy926 cells by up-regulating LC3Ⅱ/LC3Ⅰ, Beclin-1 protein expression and down-regulating Bcl-2 protein expression, while the overexpression of PD-L1 can resist the inducing effect of DENV-2.

Key words: dengue virus, endothelial cells, autophagy, apoptosis, programmed cell death receptor-1 ligand