Naringenin Targets PI3K p85alpha to Suppress PI3K/AKT Signaling Pathway and Ameliorate Disordered MMP-9 Secretion in Cigarette Smoke Extract-Induced Alveolar Macrophages In Vitro

Abstract

Background: Naringenin has demonstrated potential therapeutic effects against cigarette smoke-induced lung injury; however, its underlying mechanisms of regulating matrix metalloproteinase-9 (MMP-9) in alveolar macrophages remain unclear.

Methods: The regulatory mechanisms of naringenin in cigarette smoke extract (CSE)-induced alveolar macrophages were investigated using proteomics, and then, naringenin's targets were further validated by Western blot, molecular docking, molecular dynamics (MD) simulations, cellular thermal shift assay (CETSA), and enzyme activity assay.

Results: The proteomics revealed that the PI3K/AKT signaling pathway might play a crucial role in naringenin's inhibition of MMP-9. Western blot analysis confirmed that naringenin significantly inhibited CSE-upregulated PI3K/AKT signaling pathway and reduced MMP-9 expression in MH-S cells. Notably, the PI3K activator 740Y-P reversed naringenin's effects on MMP-9. Additionally, molecular docking, MD simulations, and CETSA identified PI3K p85alpha as the potential binding site for naringenin, and naringenin markedly inhibited CSE-induced PI3K activity. In in vitro experiments, naringenin inhibiting MMP-9 secretion in alveolar macrophages contributed to alleviating elastin and E-cadherin damage in alveolar epithelial cells. Furthermore, naringenin effectively suppressed CSE-induced MMP-9 secretion in primary mouse alveolar macrophages and human THP-1-differentiated macrophages.

Conclusions: Our findings revealed that naringenin, a potential candidate for treating smoking-induced lung injury, directly targeted PI3K p85alpha, inhibiting PI3K activity and MMP-9 expression in CSE-induced alveolar macrophages via suppressing the PI3K/AKT signaling pathway.

Keywords: MMP-9; PI3K p85alpha; alveolar macrophage; cigarette; naringenin.

Figure 1

Naringenin diminished MMP-9 secretion in CSE-induced MH-S cells. The effects of CSE and naringenin for 24 h treatment on (A) cell viability and (B) level of MMP-9 secreted in MH-S cells. Data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01.

Figure 2

The PI3K/AKT signaling pathway was identified as a significantly altered target through proteomics analysis. (A) Principal component analysis, (B) heat map, (C) differentially expressed proteins, and (D) the top 30 pathway annotations by WikiPathways in identified proteins of the three groups. WikiPathways enrichment analysis of differentially expressed proteins: (E) 2.5% CSE group vs. control group; (F) 2.5% CSE + 100 μM naringenin group vs. 2.5% CSE group. Data are presented with n = 5. The results were filtered by 1% FDR. The differential proteins among groups were accepted when the p-value was less than 0.05 and FC was greater than 1.2 (upregulation) or less than 0.83 (downregulation).

Figure 3

Naringenin inhibited CSE-induced PI3K/AKT/MMP-9 signaling pathway. (A) Representative images and (B–H) relative protein expression levels of (B) p-PI3K p85alpha, (C) PI3K p110beta, (D) p-PI3K p85alpha/PI3K p110beta, (E) p-AKT, (F) AKT, (G) p-AKT/AKT, and (H) MMP-9 in CSE-induced MH-S cells treated with naringenin for 24 h by Western blot. Data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01.

Figure 4

Activation of PI3K phosphorylation neutralized the beneficial effects of naringenin against CSE-induced MMP-9 expression. (A) Representative images and (B–H) quantification for the protein levels of (B) p-PI3K p85alpha, (C) PI3K p110beta, (D) p-PI3K p85alpha/PI3K p110beta, (E) p-AKT, (F) AKT, (G) p-AKT/AKT, and (H) MMP-9 at 24 h treatment by Western blotting. (I) Quantitative protein secretion levels of MMP-9 in MH-S cell supernatants at 24 h treatment by ELISA. Data are expressed as mean ± SD (n = 6). ** p < 0.01.

Figure 5

Molecular docking validation. Ligand–receptor docking revealed that naringenin could bind to (A) PI3K p110beta, (B) PI3K p85alpha, (C) AKT3, (D) AKT2, (E) AKT1, or (F) MMP-9 through amino acid residues. The 3D molecular structure of naringenin is in green, and the 3D protein structure of the corresponding target protein is in blue. The yellow dotted line signifies a potential bond, and the corresponding amino acid residues are in grey.

Figure 6

MD simulations of naringenin and PI3K proteins at a 100 ns trajectory. (A,B) RMSD presentation. (C,D) RMSF presentation. (E) Rg. (F) SASA. (G,H) H-bond. (I,J) FEL. (K,L) Free energy calculations from MMPBSA. (M,N) Energy decomposition analysis of hotspot amino acid residues throughout the trajectory of 100 ns.

Figure 7

Naringenin could bind with PI3K p85alpha and inhibit PI3K activity in MH-S cells. (A) Representative images of PI3K proteins and (B–E) quantification for the protein levels of (B) p-PI3K p85alpha, (C) PI3K p85alpha, (D) PI3K p110beta, and (E) vinculin by CETSA (n = 3). (F) The effects of naringenin on PI3K enzyme activity in CSE-induced MH-S cells after 24 h treatment (n = 6). Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01.

Figure 8

Naringenin protected against ECM injury in alveolar epithelial cells by decreasing the secretion of MMP-9 from alveolar macrophages. (A) Confocal microscope images showing the markers in primary mouse alveolar macrophages. From left to right: white light field of TLD, blue DAPI nuclei, green CD11b marker, red F4/80 marker, and three-color fluorescence overlay field. Scale bar at the bottom right corner: 20 μm. (B,C) The effects of naringenin on the secretion of CSE-induced MMP-9 from mouse primary alveolar macrophages and PMA-induced THP-1 cells with 24 h treatment. (D) MMP-9 levels in CM from drug removal MH-S cells for 12 h. (E–H) The effects of CM and MMP-9-inhibited CM on E-cadherin and elastin at 24 h. Data are expressed as mean ± SD (n = 6). ** p < 0.01.

Figure 9

Schematic diagram illustrating naringenin’s effects on the PI3K/AKT/MMP-9 pathway in CSE-induced alveolar macrophages, ** p < 0.01.