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. 2020;53(10):e9183.
doi: 10.1590/1414-431X20209183. Epub 2020 Sep 7.

Lapiferin protects against H1N1 virus-induced pulmonary inflammation by negatively regulating NF-kB signaling

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Lapiferin protects against H1N1 virus-induced pulmonary inflammation by negatively regulating NF-kB signaling

Lishu Pei et al. Braz J Med Biol Res. 2020.

Abstract

H1N1 virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. There is currently no effective strategy against virus infection in lung. The present study evaluated the protective roles of a natural compound, lapiferin, in H1N1 virus-induced pulmonary inflammation in mice and in cultured human bronchial epithelial cells. Initially, Balb/C mice were grouped as Control, H1N1 infection (intranasally infected with 500 plaque-forming units of H1N1 virus), lapiferin (10 mg/kg), and H1N1+lapiferin (n=10/group). Lung histology, expression of inflammatory factors, and survival rates were assessed after 14 days of exposure. Administration of lapiferin significantly alleviated the virus-induced inflammatory infiltrate in lung tissues. Major pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, were decreased at both mRNA and protein levels by lapiferin administration in the lung homogenate. Lapiferin also reduced inflammatory cell numbers in bronchoalveolar fluid. Mechanistically, lapiferin suppressed the transcriptional activity and protein expression of NF-κB p65, causing inhibition on NF-κB signaling. Pre-incubation of human bronchial epithelial cells with an NF-κB signaling specific activator, ceruletide, significantly blunted lapiferin-mediated inhibition of pro-inflammatory cytokines secretion in an air-liquid-interface cell culture experiment. Activation of NF-κB signaling also blunted lapiferin-ameliorated inflammatory infiltrate in lungs. These results suggested that lapiferin was a potent natural compound that served as a therapeutic agent for virus infection in the lung.

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Figures

Figure 1
Figure 1. Lapiferin attenuated H1N1-induced mortality and histopathology in lungs. A, Experimental treatment protocols. B, Fourteen days after H1N1 infection, lung tissues were subjected to paraffin embedding and subsequent HE staining. Representative images from each group are shown (magnification: 400×, scale bar 20 μm). C, Semiquantitative analysis (scores) of inflammatory infiltrate in lungs of each group of mice. D, Mouse mortality from each group (n=10 per group). E, Body weight change from each group of mice. *P<0.05, **P<0.01 as indicated (ANOVA).
Figure 2
Figure 2. Lapiferin treatment decreased expression of pro-inflammatory factors at both mRNA and protein levels in lung. A, B, and C, qRT-PCR analysis of the mRNA levels of major pro-inflammatory factors (IL-1β, IL-6, and TNF-α) in the lung tissues from each group. D, Western blot analysis of the protein levels of IL-1β, IL-6, and TNF-α in the lung tissues of mice. Data are reported as means±SD. **P<0.01; ***P<0.001 as indicated (ANOVA).
Figure 3
Figure 3. Lapiferin treatment decreased the serum levels of pro-inflammatory factors of H1N1 infected mice. A, B, C, and D, ELISA analysis of major pro-inflammatory factors (IL-1β, IL-6, and TNF-α) and fractalkine in the lung homogenates from all groups. Data are reported as means±SD. *P<0.05 and **P<0.01 as indicated (ANOVA).
Figure 4
Figure 4. Lapiferin treatment decreased the number of inflammatory cells in bronchoalveolar fluid. The total inflammatory cell number (A), neutrophil number (B), and macrophage number (C) were counted in the bronchoalveolar fluid. Data are reported as means±SD. *P<0.05 and **P<0.01 as indicated (ANOVA).
Figure 5
Figure 5. Lapiferin suppressed NF-κB signaling. A and B, Western blot analysis and quantitative analysis of NF-κB (p65) in the lung homogenate from each group of mice. C, qRT-PCR analysis of NF-κB in the lung homogenate from each group of mice. D, mRNA levels of ICAM-1 and VCAM-1 in mouse lung tissues. Data are reported as means±SD. *P<0.05; **P<0.01; ***P<0.001 as indicated (ANOVA).
Figure 6
Figure 6. Activation of NF-κB signaling blocked lapiferin-mediated anti-inflammatory effects in vitro and in vivo. A, Western blot assays validated the activation of NF-κB signaling by its specific activator ceruletide. B, ELISA detection of pro-inflammatory cytokines IL-1β and TNF-α in the supernatant of cultured human bronchial epithelial cells. C, Experimental protocol for the in vivo studies. D, Representative histology images after 14 days (magnification: 400×, scale bar: 20 μm). E, Semiquantitative analysis of lung inflammatory infiltrate. F, Total neutrophils levels and (G) total macrophage levels are shown. Data are reported as means±SD. *P<0.05; **P<0.01 as indicated (ANOVA).

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