Urolithin A alleviates respiratory syncytial virus-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling

doi:10.12122/j.issn.1673-4254.2024.07.17

Abstract

Abstract:

Objective To observe the therapeutic effects of urolithin A (UA) on respiratory syncytial virus (RSV)-induced lung infection in neonatal mice and explore the underlying mechanisms. Methods Babl/c mice (5-7 days old) were subjected to nasal instillation of RSV and received intraperitoneal injection of saline or 2.5, 5 and 10 mg/kg UA 2 h after the infection and then once daily for 2 weeks. Bronchoalveolar lavage fluid (BALF) was then collected for detection of inflammatory cells and mediators, and lung pathology was evaluated with HE staining. RSV-infected BEAS-2B cells were treated with 2.5, 5 or 10 µmol/L UA. Inflammatory factors, cell viability, apoptosis and autophagy were analyzed using ELISA, CCK-8 assay, TUNEL staining, flow cytometry, Western blotting and immunofluorescence staining. The cellular expressions of miR-136 and Sirt1 mRNAs were detected using qRT-PCR. A dual-luciferase reporter system was used to verify the binding between miR-136 and Sirt1. Results In neonatal Babl/c mice, RSV infection caused obvious lung pathologies, promoted pulmonary cell apoptosis and LC3-II/I, Beclin-1 and miR-136 expressions, and increased the total cell number, inflammatory cells and factors in the BALF and decreased p62 and Sirt1 expressions. All these changes were alleviated dose-dependently by UA. In BEAS-2B cells, RSV infection significantly increased cell apoptosis, LC3B-positive cells and miR-136 expression and reduced Sirt1 expression (P<0.01), which were dose-dependently attenuated by UA. Dual-luciferase reporter assay confirmed the binding between miR-136 and Sirt1. In RSV-infected BEAS-2B cells with UA treatment, overexpression of miR-136 and Ex527 treatment both significantly increased the inflammatory factors and cell apoptosis but decreased LC3B expression, and these changes were further enhanced by their combined treatment. Conclusion UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway.

Key words: urolithin A, respiratory syncytial virus, miR-136, Sirt1

Hongzhe WANG, Haitang XIE, Wulan XU, Ming Li. Urolithin A alleviates respiratory syncytial virus-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling[J]. Journal of Southern Medical University, 2024, 44(7): 1370-1381.

Figures/Tables 7

Tab.1 Primer sequences for qRT-PCR

Gene	Primer sequence(5′-3′)
mmu-miR-136	Forward:5'-ACACTCCAGCTGGGACTCCATTTGTTTTGATGA-3'
mmu-miR-136	Reverse:5'-CTCAACTGGTGTCGTGGA-3'
hsa-miR-136	Forward:5'-GCGCACTCCATTTGTTTTGAT-3'
hsa-miR-136	Reverse:5'-GTGCAGGGTCCGAGGT-3'
mmu-U6	Forward:5'-CTCGCTTCGGCAGCACA-3'
mmu-U6	Reverse:5'-AACGCTTCACGAATTTGCGT-3'
hsa-U6	Forward:5'-AAAGCAAATCATCGGACGACC-3'
hsa-U6	Reverse:5'-GTACAACACATTGTTTCCTCGGA-3'
mmu-Sirt1	Forward:5'-GACGCTGTGGCAGATTGTTA-3'
mmu-Sirt1	Reverse:5'-GGAATCCCACAGGAGACAGA-3'
hsa-Sirt1	Forward:5'-TGCCGGAAACAATACCTCCA-3'
hsa-Sirt1	Reverse:5'-AGACACCCCAGCTCCAGTTA-3'
mmu-GAPDH	Forward:5'-GGCCTCCAAGGAGTAAGAAA-3'
mmu-GAPDH	Reverse:5'-GCCCCTCCTGTTATTATGG-3'
hsa-GAPDH	Forward:5'-TGTGGGCATCAATGGATTTGG-3'
hsa-GAPDH	Reverse:5'-ACACCATGTATTCCGGGTCAAT-3'

Fig.1 UA alleviates RSV-induced lung injury in neonatal mice. A: HE staining showing lung tissue injury in the mice (Original magnification: ×200). B: Pathological score based on HE staining results. C: Quantification of the relative copy number of RSV in mouse lung tissues. ****P<0.0001 vs Control group; #P<0.05, ###P<0.001, ####P<0.0001 vs RSV group.

Fig.2 UA reduces RSV-induced lung inflammation in neonatal mice. A-D: Total cell (A), lymphocyte (B), macrophage (C) and neutrophil (D) counts in mouse BALF. E-L: Contents of TNF-α (E), IL-1β (F), IL-4 (G), IL-5 (H), IL-6 (I), IL-13 (J), IFN-γ (K) and CXCL1 (L) in lung tissues of mice detected by ELISA. ****P<0.0001 vs Control group; #P<0.05, ##P<0.01, ####P<0.0001 vs RSV group.

Fig.3 UA treatment inhibits RSV-induced apoptosis in BEAS-2B cells. A: CCK-8 assay for assessing viability of BEAS-2B cells treated with 2.5, 5, 10, 20 and 40 µmol/L UA for 24 or 48 h. B: Flow cytometry for analyzing apoptosis of BEAS-2B cells infected with RSV for 2 h followed by treatment with 2.5, 5 or 10 µmol/L UA for 48 h. C: Quantification of the percentage of apoptotic cells. D: TUNEL assay for detecting cell apoptosis in the lung tissues of the mice (400×). E: Number of TUNEL-positive cells. *P<0.05, **P<0.01, ****P<0.0001 vs Control group; #P<0.05, ###P<0.001, ####P<0.0001 vs RSV group.

Fig.4 UA promotes RSV infection-induced autophagy in BEAS-2B cells. A: Western blotting for detecting LC3-II/I, p62, and Beclin-1 expressions in lung tissues of mice. B-D: Quantification of LC3-II/I (B), p62 (C), and Beclin-1 (D) expressions. E, F: Immunofluorescence assay for detecting LC3B expression in BEAS-2B cells infected with RSV for 2 h followed by treatment with UA (2.5, 5 and 10 µmol/L) for 48 h (×400). ****P<0.0001 vs Control group; #P<0.05, ####P<0.0001 vs RSV group.

Fig.5 UA increases Sirt1 expression by inhibiting miR-136 in RSV-infected BEAS-2B cells. A: Expressions of miR-136 in lung tissues of mice and BEAS-2B cells detected by qRT-PCR. B, C: Sirt1 mRNA (B) and protein (C) levels analyzed by qRT-PCR and Western blotting. D: Prediction of the binding site of miR-136 to 3'-UTR of Sirt1 mRNA using miRanda, targetScan and RNAInter bioinformatics databases. E: Dual-luciferase reporter assay of BEAS-2B cells co-transfected with miR-136 (or miR-NC) and Sirt1-WT or Sirt1-MUT. F-H: Sirt1 mRNA and protein expressions determined by qRT-PCR (F) and Western blotting (G, H) analysis in BEAS-2B cells transfected with miR-136 or miR-NC. I: Western blotting for detecting protein expressions of Sirt1 in RSV (100TCID50)-infected BEAS-2B cells treated with 10 µmol/L UA, transfected with miR-136, or both. ***P<0.001, ****P<0.0001 vs Control or miR-NC group; #P<0.05, ###P<0.001, ####P<0.0001 vs RSV group.

Fig.6 UA inhibits RSV-induced inflammation and apoptosis and promotes autophagy in BEAS-2B cells by activating miR-136-mediated Sirt1 signaling pathway. A-H: ELISA measurement of contents of TNF-α (A), IL-1β (B), IL-4 (C), IL-5 (D), IL-6 (E), IL-13 (F), IFN-γ (G) and CXCL1 (H) in RSV (100TCID50)-infected BEAS-2B cells treated with 10 µmol/L UA, UA+miR-136, UA+Sirt1 inhibitor (Ex527), or UA+miR-136+Ex527. I: Cell apoptosis assessed by flow cytometry. J: Percentage of apoptotic cells. K: Immunofluorescence staining for detecting expression of LC3B (×400). L: Quantification of LC3B-positive cells. **P<0.01, ****P<0.0001 vs Control group; ####P<0.0001 vs RSV group; &P<0.05, &&P<0.01, &&&P<0.001, &&&&P<0.0001 vs RSV+UA group; $P<0.05, $$P<0.01, $$$P<0.001, $$$$P<0.0001 vs RSV+UA+miR-136 or RSV+UA+Ex527 group.

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