Integration of serum pharmacochemistry with network pharmacology to reveal the potential mechanism of Yangqing Chenfei formula for the treatment of silicosis.

OBJECTIVE: To explore the mechanisms of Yangqing Chenfei formula (, YCF) in the treatment of silicosis through a comprehensive strategy consisting of serum pharmacochemistry, network pharmacology analysis, and in vitro validation. METHODS: An ultrahigh-performance liquid chroma-tography-tandem mass spectrometry method was used to confirm the active components in YCF-medicated serum. Then, we obtained targets for active components and genes for silicosis from multiple databases. Furthermore, a protein-protein interaction network was constructed, and Kyoto Encyclopedia of Genes and Genomes pathway and biological process analyses were conducted to elucidate the mechanisms of YCF for the treatment of silicosis. Finally, we validated the important components and mechanisms in vitro. RESULTS: Altogether, 19 active components were identified from rat serum after YCF administration. We identified 724 targets for 19 components, which were mainly related to inflammation [phosphatidy linositol 3 kinase/protein kinase B, forkhead box O, hypoxia inducible factor, and T-cell receptor signaling pathway, nitric oxide biosynthetic process], fibrotic processes [vascular endothelial growth factor signaling pathway, extracellular signal regulated kinase (ERK) 1 and ERK2 cascade, smooth muscle cell proliferation], and apoptosis (negative regulation of apoptotic process). In addition, 218 genes for silicosis were identified and were mainly associated with the inflammatory response and immune process [cytokine?cytokine receptor interaction, tumor necrosis factor alpha (TNF-α), toll-like receptor, and nucleotide binding oligomerization domain-like receptor signaling pathway]. Taking an intersection of active component targets and silicosis genes, we obtained 61 common genes that were mainly related to the inflammatory response and apoptosis, such as the phosphatidylinositol-3-kinase/protein kinase B signaling pathway, mitogen activated protein kinases signaling pathway, TNF signaling pathway, toll-like receptor signaling pathway, biosynthesis of nitric oxide, and apoptotic process. In the herb-component-gene-pathway network, paeoniflorin, rutin and nobiletin targeted the most genes. In vitro, paeoniflorin, rutin and nobiletin decreased the mRNA levels of inflammatory factors [interleukin (IL)-6, TNF-α, and IL-1ß], suppressed p-AKT and cleaved caspase-3, and increased B cell lymphoma (Bcl)-2 protein expression in silica-induced macrophages in a concentration-dependent manner. CONCLUSION: YCF could significantly relieve the inflammatory response of silicosis via suppression of the AKT/Bcl-2/Caspase-3 pathway.

Yangqing Chenfei formula alleviates crystalline silica induced pulmonary inflammation and fibrosis by suppressing macrophage polarization.

OBJECTIVE: To explore the underlying mechanisms of the effects of Yangqing Chenfei formula (, YCF) on inflammation and fibrosis in silicosis via inhibition of macrophage polarization. METHODS: A silicotic rat model was established via a single intratracheal instillation of silica particles on the first day of week 0. Subsequently, YCF was administered intragastrically to silicotic rats during weeks 0-2 and 5-8 twice daily. The mouse-derived alveolar macrophage cell line was used to investigate the mechanisms of YCF in M1/M2 polarization. RESULTS: YCF treatment effectively inhibited lung pathological changes, including inflammatory cell infiltration and tissue damage, and increased the forced expiratory volume in the first 0.3 s, functional residual capacity, and maximal mid-expiratory flow in weeks 2 and 8. Furthermore, the treatment improved lung functions by upregulating tidal volume, pause increase, and expiratory flow at 50% tidal volume from weeks 5 to 8. Moreover, YCF could significantly suppressed the progression of inflammation and fibrosis, by reducing the levels of inflammatory cytokines, as well as collagen- I and III. YCF treatment also decreased the numbers of macrophages and M1/M2 macrophages and the level of transforming growth factor-ß (TGF-ß). Additionally, YCF5, the effective substance in YCF, decreased lipopolysaccharide and interferon-γ-induced M1 macrophage polarization in a concentration-dependent manner. The mechanism of anti-M1 polarization might be related to a decrease in extracellular signal-regulated kinase, c-JUN N-terminal kinase, P38, and P65 phosphorylation. Furthermore, YCF5 inhibited interleukin-4-induced M2 macrophages by decreasing the protein and mRNA expressions of arginase-1 and CD206 as well as the levels of profibrotic factors, such as TGF-ß and connective tissue growth factor. The mechanisms underlying the anti-M2 polarization of YCF5 were primarily associated with the inhibition of the nuclear translocation of phosphorylated signal transducer and activator of transcription 6 (p-STAT6). CONCLUSION: YCF significantly inhibits inflammation and fibrosis in silicotic rats probably via the suppression of M1/M2 macrophage polarization mediated by the inhibition of mitogen-activated protein kinase and nuclear factor kappa B signaling pathways and Janus kinase/STAT6 pathways.

Digoxin alleviates pulmonary fibrosis by regulating phosphatidylinositol-3-kinase/Akt signaling through inhibiting the activation of fibroblast: an in vivo and in vitro experiment / 中华危重病急救医学

Objective:To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments. Methods:① In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-Ⅰ and COL-Ⅲ) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-β (TGF-β) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. ② In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-β for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting. Results:① In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-β and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-β and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA ( A value): 5.37±1.10 vs. 9.51±1.66, TGF-β protein (TGF-β/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. ② In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-β was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01]. Conclusion:Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.