Baicalin suppresses type 2 dengue virus-induced autophagy of human umbilical vein endothelial cells by inhibiting the PI3K/AKT pathway

doi:10.12122/j.issn.1673-4254.2024.07.07

Abstract

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

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.

Figures/Tables 13

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

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

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

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

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

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).

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).

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).

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

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).

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.

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

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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