PD-L1过表达在DENV-2诱导血管内皮细胞自噬和凋亡中的作用机制

目的　探讨PD-L1过表达在Ⅱ型登革病毒（DENV-2）影响血管内皮细胞自噬和凋亡中的作用及机制。方法　以血管内皮细胞EAhy926为研究对象，以含梯度DENV-2（1×108~2×109 pfu/L）的无血清培养基处理细胞24 h和48 h，同时以无血清培养基处理细胞作为对照组，无血清培养基加入空白孔作为调零孔，MTT法检测细胞增殖活力。以PD-L1过表达慢病毒液和空载慢病毒液处理细胞，分别设空载对照组和转染组。DENV-2（1.2×109 pfu/L）处理细胞12、24、48 h作为DENV-2 12 h组、DENV-2 24 h组及DENV-2 48 h组，同时设置对照组（无血清培养基处理）；另以DENV-2处理空载对照组和转染组48 h，以蛋白印迹法检测LC3B、Beclin-1及Bcl-2蛋白表达变化，流式细胞术检测各组细胞凋亡率。结果　1×108~2×109 pfu/L DENV-2可剂量依赖性抑制EAhy926细胞增殖，DENV-2 24 h和48 h的半数抑制浓度（IC50）分别是1.8×109 pfu/L和1.2×109 pfu/L。与对照组比较，DENV-2 12 h组、DENV-2 24 h组及DENV-2 48 h组LC3Ⅱ/LC3Ⅰ和Beclin-1蛋白表达上调，Bcl-2蛋白表达下调，细胞总凋亡率增加（均P＜0.05）。与空载对照组比较，转染组PD-L1蛋白表达上调（P＜0.01）。DENV-2处理48 h，与空载对照组相比，转染组LC3Ⅱ/LC3Ⅰ和Beclin-1蛋白表达下调，Bcl-2蛋白表达上调，细胞总凋亡率降低（均P＜0.01）。结论　DENV-2通过上调LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白表达及下调Bcl-2蛋白表达，从而诱导EAhy926细胞自噬和凋亡，而过表达PD-L1可抵抗DENV-2的诱导效应。

The mechanism of PD-L1 overexpression in DENV-2 induced vascular endothelial cell autophagy and apoptosis

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.