黄芩苷通过调控PI3K/AKT信号通路抑制登革病毒感染诱导的人静脉内皮细胞的自噬

doi:10.12122/j.issn.1673-4254.2024.07.07

摘要/Abstract

摘要：

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。

关键词: 登革病毒, 黄芩苷, 自噬, PI3K/AKT, 人脐静脉内皮细胞

Abstract:

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.

Key words: dengue virus, baicalin, autophagy, PI3K/AKT signaling, human umbilical vein endothelial cells

程瑶, 王远迎, 姚飞扬, 胡盼, 陈铭勰, 吴宁. 黄芩苷通过调控PI3K/AKT信号通路抑制登革病毒感染诱导的人静脉内皮细胞的自噬[J]. 南方医科大学学报, 2024, 44(7): 1272-1283.

Yao CHENG, Yuanying WANG, Feiyang YAO, Pan HU, Mingxian CHEN, Ning WU. Baicalin suppresses type 2 dengue virus-induced autophagy of human umbilical vein endothelial cells by inhibiting the PI3K/AKT pathway[J]. Journal of Southern Medical University, 2024, 44(7): 1272-1283.

图/表 13

图1 DENV-2感染C6/36细胞病变图

Fig.1 Pathological changes of C6/36 cells infected by DENV-2 (Original magnification: ×100). A: Blank control cells. B: DENV-2-infected cells.

表1 DENV-2病毒滴度结果统计表

Tab.1 Virulence of DENV-2 virus at different titers in C6/36 cells

Dilution of virus solution	Number of CPE occurrences	No CPE count	Total number		% of CPE holes present
Dilution of virus solution	Number of CPE occurrences	No CPE count	Number of CPE holes	No CPE count	% of CPE holes present
10^-3	8	0	34	0	100
10^-4	8	0	26	0	100
10^-5	8	0	18	0	100
10^-6	8	0	10	2	100
10^-7	2	6	2	6	25
10^-8	0	8	0	14	0
10^-9	0	8	0	22	0
10^-10	0	8	0	30	0

图3 DENV-2感染HUVECs诱导细胞自噬的发生

Fig.3 DENV-2 infection induces autophagy in HUVECs (×15000, n=3). A, B: Control group. C, D: DENV-2 group.

图4 DENV-2感染HUVECs后溶酶体的功能增强

Fig.4 DENV-2 infection enhances lysosomal function in HUVECs (Lyso-tracker Red staining, ×400, n=3).

图5 黄芩苷对HUVECs细胞活性的影响

Fig.5 Effect of Baicalin on viability of HUVECs cells (n=3).

图6 黄芩苷降低DENV-2感染HUVECs的DENV-2 RNA相对表达量

Fig.6 Baicalin reduces relative expression of DENV-2 RNA in infected HUVECs. *P<0.05, ****P<0.001 vs 0 μg/mL group (n=3).

图7 黄芩苷抑制DENV-2 NS1蛋白表达水平

Fig.7 Western blots (A) and quantitative analysis (B) of the protein expressions of DENV-2 NS1 protein in HUVECs infected by DENV-2. ***P<0.001 vs DENV-2 group (n=3).

图8 黄芩苷抑制DENV-2感染HUVEs诱导的自噬

Fig.8 Baicalin inhibits autophagy induced by DENV-2 infection in HUVECs. A-C: Western blotting of protein expressions of LC3 and P62 in HUVECs infected by DENV-2. D: Transmission electron microscopy showing autophagy of HUVECs infected with DENV-2 after baicalin treatment (×15000). **P<0.01, ****P<0.001 vs Contorl group. #P<0.05 vs DENV-2 group (n=3).

图9 黄芩苷抑制DENV-2感染HUVECs后溶酶体的酸化

Fig.9 Baicalin inhibits lysosomal acidification in DENV-2-infected HUVECs (×400, n=3).

图10 黄芩苷抑制DENV-2感染HUVECs后自噬相关蛋白水平

Fig.10 Baicalin inhibits expressions of autophagy-related proteins in HUVECs infected with DENV-2. A: Western blotting of Beclin-1, ATG 5, STX17, SNAP29 and VAMP8 proteins. B-F: Relative expression levels of Beclin-1, ATG 5, STX17, SNAP29 and VAMP8 proteins. **P<0.01, ****P<0.001. #P<0.05, ###P<0.01, ####P<0.001 (n=3).

图11 DENV-2感染后HUEVCs蛋白质组学分析

Fig.11 Proteomic analysis of HUEVCs with DENV-2 infection. A: Heat map of differential proteins in healthy adults and patients with bronchial asthma. B: KEGG signaling pathways enrichment analysis.

图12 LY294002降低DENV-2感染HUVECs的DENV-2 RNA相对表达量

Fig.12 LY294002 reduces relative expression of DENV-2 RNA in HUVECs infected with DENV-2. ****P<0.001 (n=3).

图13 黄芩苷抑制DENV-2诱导的PI3K/AKT信号通路的表达量

Fig.13 Baicalin inhibits the expression level of PI3K/AKT signaling pathway in DENV-2-infected HUVECs. A: Baicalin reduces the relative expressions of PI3K, AKT and mTOR mRNA in DENV-2-infected HUVECs. B: Baicalin inhibits the expression levels of PI3K/AKT signaling pathway-related proteins in DENV-2-infected HUVECs. C: Ratio of p-PI3K/PI3K protein expression. D: Ratio of p-AKT/AKT protein expression. E: Relative expressions of p-mTOR/mTOR protein. *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ##P<0.01, ###P<0.001 (n=3).

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