Objective To explore the potential mechanism of Polygonum cuspidatum in alleviating silicosis using network pharmacology, molecular docking, and experimental validation. Methods i) The targets of active components of Polygonum cuspidatum and silicosis-related targets were retrieved from multiple databases. The overlapping targets were imported into Venny2.1.0 and STRING platforms. "Polygonum cuspidatum-active components-potential therapeutic targets for silicosis" network was constructed and core targets of Polygonum cuspidatum that can alleviate silicosis were identified using the Cytoscape3.10.2 software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the DAVID database, and molecular docking was conducted with AutoDock software. ii) Human embryonic lung fibroblast MRC-5 cells were divided into three groups. Cells in the control group were cultured in high-glucose Dulbecco's modified Eagle's medium containing 10.00% (V/V)fetal bovine serum. Cells in the silica group were treated with silica suspension at a final concentration of 150 μg·cm² (equivalent to 75 mg/L) for 48 hours. Cells in the silica + β-sitosterol group were first treated with silica suspension for 24 hours, then were cultured in medium containing β-sitosterol at a mass concentration of 50 μg/L for another 24 hours. Immunofluorescence assay and Western blotting were used to detect the effects of β-sitosterol, a core component of Polygonum cuspidatum, on the expression of phosphorylated protein kinase B (p-AKT), B-cell lymphoma-2 (BCL-2), and α-smooth muscle actin (α-SMA) in cells. Results i) A total of ten main active components of Polygonum cuspidatum, 840 targets of the active components, and 231 potential therapeutic targets for silicosis were screened through retrieval. A total of 10 core therapeutic targets were further screened, including tumor protein p53, serine/threonine protein kinase B1 (AKT1), heat shock protein 90-α, interleukin (IL)-6 , tumor necrosis factor, Src tyrosine kinase, estrogen receptor 1, epidermal growth factor receptor, BCL-2, and IL-1β. KEGG enrichment analysis yielded 178 related target pathways, among which, the phosphatidylinositol 3-kinase-protein kinase B (PI3K/AKT) signaling pathway was most relevant to silicosis. Molecular docking results showed that the top five compounds of Polygonum cuspidatum with high degree centrality, including quercetin, luteolin, physovenine, rhein, and β-sitosterol, had strong binding affinities with core targets. Among them, β-sitosterol, a key component of Polygonum cuspidatum, ranked high in binding affinity to all core targets. Molecular docking visualization indicated that β-sitosterol ranked high in binding affinitiy with the core target proteins AKT1 and BCL-2. ii) Both immunofluorescence and Western blotting results showed that, the relative expression of p-AKT, BCL-2, and α-SMA proteins increased in MRC-5 cells of the silica group compared with the control group (all P<0.05), except for p-AKT detected by Western blotting. Compared with the silica group, the relative expression levels of p-AKT, BCL-2, and α-SMA proteins in the silica+β-sitosterol group decreased (all P<0.05). Conclusion β-sitosterol, the core component of Polygonum cuspidatum, may alleviate silicosis fibrosis via the PI3K/AKT signaling pathway.

Background Coal workers' pneumoconiosis (CWP) is a serious occupational disease. Whether ferroptosis, a form of necrotic regulated cell death, is involved in coal dust induced mouse models of CWP needs further survey. Objective This experiment is designed to elucidate the role of ferroptosis in the formation of CWP induced by coal dust in mice. Methods C57BL/6J mice were randomly assigned to a saline group or a CWP group, with eight mice in each group. The mice were treated with 0.1 mL normal saline or 0.1 mL coal dust suspensions (50g·L^-1) via intra-tracheal instillation. HE staining and Masson staining were used to show lung injury and lung fibrosis. Iron concentration in mouse lung tissues was measured using iron assay kit. Lipid peroxidation was estimated in lung tissues by malondialdehyde (MDA) concentration and immunofluorescence intensity, and the ratio of glutathione (GSH) to L-glutathione oxidized (GSSG). Western blotting and real-time fluorescence-based quantitative PCR were used to test protein and mRNA expression levels of glutathione peroxidase 4 (GPX₄) and ferritin in mice. Results Coal dust injured pulmonary structure, thickened alveolar wall, and caused collagen deposition and infiltration of inflammatory cells in the CWP group. The iron concentration in the CWP group [(10.75 ± 5.42) mg·L⁻¹] was higher than that in the saline group [(1.14 ± 0.37) mg·L⁻¹] (P < 0.01). The MDA concentration in the CWP group [(37.32 ± 12.18) μmol·L⁻¹] was higher than that in the saline group [(18.70 ± 8.22) μmol·L⁻¹] (P <0.01). The immunofluorescence intensity of MDA in the CWP group was stronger than that in the saline group. The GSH/GSSG ratio decreased in CWP treated mice (1.50 ± 1.70) compared with the normal saline treated ones (4.95 ± 2.86) (P < 0.01). Compared with the saline group (38.84 ± 15.61 for GPX₄, 225.90 ± 54.34 for ferritin), the relative expression levels of GPX₄ and ferritin mRNA in the CWP group were downregulated (14.29 ± 7.21 for GPX₄, 106.70 ± 36.70 for ferritin) (P < 0.01). Compared with the saline group (1.47 ± 0.54 for GPX₄, 1.73 ± 0.34 for ferritin), the relative expression levels of GPX₄ and ferritin protein in the CWP group were also downregulated (0.92 ± 0.22 for GPX₄, 0.97 ± 0.09 for ferritin) (P < 0.05). Conclusion Ferroptosis may be involved in the formation of coal workers' pneumoconiosis induced by coal dust in mice.