Objective: To investigate the effect of type 2 dengue virus (DENV-2) infection on autophagy in human umbilical vein endothelial cells (HUVECs) and the mechanism mediating the inhibitory effect of baicalin against DENV-2 infection.
Methods: Cultured HUVECs with DENV-2 infection were treated with different concentrations of baicalin, and the changes in autophagy of the cells were detected using transmission electron microscopy. Lyso Tracker Red staining was used to examine pH changes in the lysosomes of the cells, and the expressions of ATG5, beclin-1, LC3, P62, STX17, SNAP29, VAMP8, and PI3K/AKT signaling pathway-related proteins were detected by Western blotting. DENV-2 replication in the cells were evaluated using RT-qPCR. The differentially expressed proteins in DENV-2-infected HUVECs were identified by proteomics screening.
Results: Treatment with baicalin did not significantly affect the viability of cultured HUVECs. Proteomic studies suggested that the PI3K-AKT pathway played an important role in mediating cell injury induced by DENV-2 infection. The results of RT-qPCR demonstrated that baicalin dose-dependently inhibited DENV-2 replication in HUVECs and produced the strongest inhibitory effect at the concentration of 50 μg/mL. Transmission electron microscopy, Lyso Tracker Red staining, RT-qPCR, and Western blotting all showed significant inhibitory effect of baicalin on DENV-2-induced autophagy in HUVECs. DENV-2 infection of HUVECs caused increased cellular expressions of LC3 and P62 proteins, which were significantly lowered by treatment with LY294002 (a PI3K inhibitor).
Conclusion: Baicalin inhibits DENV-2 replication in HUVECs and suppresses DENV-2-induced cell autophagy by inhibiting the PI3K/AKT signaling pathway.
目的: 探究2型登革病毒 （DENV-2）感染人脐静脉内皮细胞（HUVECs）对细胞自噬的影响及黄芩苷（BA）抗DENV-2感染的具体作用机制。
方法: HUVECs体外培养，设置对照组：HUVECs正常培养；DENV-2感染组：HUVECs+DENV-2；黄芩苷组：HUVECs+DENV-2+BA。用DENV-2感染HUVECs通过透射电镜观察自噬；Western blotting检测细胞自噬相关蛋白LC3、P62的表达，采用溶酶体红色荧光探针染色法观察DENV-2感染后HUVECs内溶酶体的pH变化；蛋白质组学筛查DENV-2感染HUVECs后差异蛋白的表达情况；使用CCK-8法测定黄芩苷对HUVECs活性的影响； RT-qPCR检测细胞内病毒RNA的复制情况；检测病毒NS1蛋白的表达情况；通过透射电镜观察细胞自噬；采用Lyso-Tracker Red染色法观察黄芩苷对DENV-2感染后HUVECs内溶酶体酸化的影响；检测细胞自噬相关蛋白ATG5、Beclin-1、LC3、P62的表达，自噬小体和溶酶体融合的关键蛋白STX17、SNAP29、VAMP8表达情况及PI3K/AKT信号通路相关蛋白的表达。
结果: DENV-2感染可诱导细胞自噬小体的形成；DENV-2感染后LC3 Ⅱ/LC3 I表达逐渐增高，48 h达到峰值（P<0.05）；p62表达水平在感染前中期无明显变化，48 h后降低（P<0.05）；Lysotracker red染色结果显示DENV-2感染组红色亮点较多，且染色明亮。CCK-8结果显示50 μg/mL被视为黄芩苷对HUVECs细胞的最大无毒剂量。RT-qPCR结果显示随着黄芩苷浓度增加药物对病毒的抑制作用增强。黄芩苷可降低DENV-2 NS1蛋白的表达（P<0.001）。与DENV-2组相比，经过黄芩苷（50 μg/mL）处理后，自噬减少，加入黄芩苷后抑制溶酶体酸化。黄芩苷组48 h LC3 II/LC3 I比值降低（P<0.05），48 h时p62表达增加（P<0.05）。经过黄芩苷处理后DENV-2诱导的自噬相关蛋白Beclin-1、ATG 5、STX17、SNAP29和VAMP8的表达下调（P<0.01）。蛋白组学结果显示PI3K-AKT通路在DENV的发病机制中可能发挥重要作用。加入PI3K抑制剂（LY294002）后，DENV-2的RNA表达水平降低，各组p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR蛋白相对表达水平的蛋白表达水平降低（P<0.01）。用黄芩苷处理后，DENV-2感染组PI3K、AKT的mRNA表达水平均显著降低（P<0.05），而mTOR mRNA表达无明显变化。DENV-2感染组上调的p-PI3K、p-AKT的蛋白表达水平均降低（P<0.05），而p-mTOR表达无明显变化。DENV-2诱导的LC3、P62蛋白表达降低（P<0.05）；在黄芩苷治疗组中也抑制了病毒诱导的自噬。
结论: DENV-2感染可促进HUVECs细胞自噬的发生，黄芩苷可抑制DENV-2的RNA和NS1蛋白的表达，可能通过抑制自噬发生及自噬体和溶酶体融合，降低DENV-2诱导的自噬，其作用机制可能是通过PI3K/AKT信号通路调控。
Keywords: PI3K/AKT signaling; autophagy; baicalin; dengue virus; human umbilical vein endothelial cells.