Tongsai Granules inhibit autophagy and macrophage-mediated inflammatory response to improve acute exacerbations of chronic obstructive pulmonary disease in rats

doi:10.12122/j.issn.1673-4254.2024.10.18

Abstract

Abstract:

Objective To investigate the inhibitory effect of Tongsai Granules (TSG) on macrophage-mediated inflammatory response to alleviate acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in rats and explore the underlying mechanism. Methods Twenty-four rats were divided into control group, AECOPD model group, TSG treatment group, and moxifloxacin+salbutamol (MXF+STL) treatment group. In the rat models of COPD, AECOPD was induced by nasal instillation of Klebsiella pneumoniae on day 3 of week 9 after modeling, and saline, TSG or MXF+STL were administered via gavage on days 1 and 2 and days 4 to 7 of week 9. After the treatments, lung tissues were collected for examining for pathologies and expressions of inflammatory markers, MMP2, and MMP9. In cultured macrophage MH-S cells with LPS stimulation, the effect of TSG-medicated serum on IL-1β, IL-6, TNF‑α, COX-2, and iNOS expressions and phosphorylation levels of p38, p-p62, LC3, FoxO3a, and mTOR were evaluated. Results TSG significantly improved lung pathologies and lung function in AECOPD rats by reducing bronchial wall thickness and mean alveolar linear intercept, increasing alveolar numbers, and reducing pulmonary expression of IL-1β, IL-6, TNF-α, MMP2 and MMP9. In MH-S cells, TSG significantly suppressed LPS-induced expressions of inflammatory cytokines, COX-2 and iNOS. Serum pharmacology coupled with network pharmacology identified 10 chemical components in TSG-medicated serum, and functional analysis of their 466 targets suggested that the therapeutic effect of TSG on AECOPD was mediated primarily by luteolin and quercetin, which regulate the MAPK, mTOR, FoxO, and autophagy pathways. In MH-S cells, luteolin significantly inhibited LPS-induced inflammatory responses and expressions of p-p38, FoxO3a, mTOR, p-p62 and LC3. Conclusion TSG reduces macrophage-mediated inflammatory responses to alleviate AECOPD in rats possibly by modulating p38, mTOR, and FoxO3a pathways and inhibiting autophagy.

Key words: Tongsai Granule, acute exacerbation of chronic obstructive pulmonary disease, macrophages, inflammatory response

Mengmeng CHENG, Xinguang LIU, Yanxin WEI, Xiaoxiang XING, Lan LIU, Nan XIN, Peng ZHAO. Tongsai Granules inhibit autophagy and macrophage-mediated inflammatory response to improve acute exacerbations of chronic obstructive pulmonary disease in rats[J]. Journal of Southern Medical University, 2024, 44(10): 1995-2003.

Figures/Tables 5

Fig.1 Tongsai Granules (TSG) alleviates lung pathologies in AECOPD rats. A: Flow chart of animal experiments. B: Histopathological changes of the lung tissues detected in week 10 (HE staining, original magnification: ×200). C: Lung injury scores (BWT, S/Pi, MLI, and MAN). *P<0.05, **P<0.01 vs Control; #P<0.05, ##P<0.01 vs Model. MXF+STL: Moxifloxacin+Salbutamol group. CT: Control group. M: Model group.

Fig.2 TSG inhibits expressions of inflammatory cytokines in the lung tissues of AECOPD rats. A: Expression levels of IL-6, IL-1β and TNF‑α in the lung tissue of the rats analyzed with immunohistochemistry (×200). B: Integral optical density (IOD) of the proteins (Mean±SD). **P<0.01 vs Control, ##P<0.01 vs Model.

Fig.3 TSG inhibits expressions of MMPs in the lung tissues of AECOPD rats. A: Expressions of MMP2 and MMP9 in lung tissue of the rats analyzed by immunohistochemistry (×200). B: IOD of the proteins in each group (Mean±SD). **P<0.01 vs Control, ##P<0.01 vs Model.

Fig.4 Network pharmacological analysis of the mechanisms of TSG for treating AECOPD in rats. A: Serum from TSG-treated rats inhibits expressions of IL-1β, IL-6, TNF-α, COX2, and iNOS in LPS-induced macrophages. B: PPI of 466 targets constructed using STRING. C: Orange circles represent the top 18 nodes ranked by degree value. D: Top 18 nodes analyzed for KEGG pathways. E: Sankey diagram illustrating the representative components of TSG-medicated serum and its target pathways. **P<0.01 vs Control, ##P<0.01 vs Model.

Fig.5 TSG inhibits LPS-induced macrophage inflammation and autophagy. A: Luteolin inhibits expressions of IL-1β, IL-6, TNF-α, COX2, and iNOS in LPS-induced macrophages. B, C: Western blotting of expression levels of p-p38, p38, LC3, p-p62, p-FoxO3a, and p-mTOR (Mean±SD). *P<0.05, **P<0.01 vs Control; #P<0.05, ##P<0.01 vs Model. DEX: Dexamethasone.

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