ObjectiveTo explore the distribution of pharyngeal microbiota in coal miners exposed to dust. Methods Eight coal miners who had been engaged in occupational dust exposure for more than 20 years were selected as the dust-exposed group, and four coal miners who were not exposed to dust at work were selected as the control group using the judgment sampling method. Pharyngeal secretions of the coal miners were collected with throat swabs, and its pharyngeal microbiota was analyzed. The diversity, abundance and evenness of the microbiota were analyzed by gene sequencing using the 16sRNA gene high-throughput sequencing technology. Results A total of 254 operational taxonomic units of pharyngeal microbiota were detected in the coal miners in the control group, which was 210 more than that in the dust-exposed group. The Chao1 index, Shannon index, PD-tree index and Pielou index of pharyngeal microbiota in the dust-exposed group decreased compared with the control group (all P<0.01). The abundance of Bacteroidetes and Clostridum, at the phylum level, in the pharynx of coal miners in the dust-exposed group was higher than that in the control group (all P<0.05). The abundance of Prevotella, Neisseria, and Monas, at the genus level, in the pharynx of coal miners in the dust-exposed group was higher than that in the control group(all P<0.05), while the abundance of Lactobacillus decreased (P<0.05). The analysis results of the receiver operating characteristic curve showed that Lactobacillus, Fusobacterium and Rothia may play a role for pharyngeal microbiota imbalance prediction in dust-exposed workers, and the area under the curves were all 1.00±0.00. Conclusion The species diversity and evenness of pharyngeal microbiota in coal miners exposed to dust are decreased, which may be related to the continuous inhalation of coal dust that disrupts the microbial environment of the throat.

Objective To screen mitochondria-targeting differential genes in alveolar macrophages of silicosis pa-tients and explore the role of mitochondrial homeostasis in alveolar macrophages of silicosis patients.Methods High-throughput sequencing dataset GSE174725 was downloaded,and differentially expressed genes were screened with R software and P＜0.05,|LogFC|＞1,and then intermixed with mitochondrial gene bank MitoCarta3.0 to obtain mitochondria-targeted differentially expressed genes.Then enrichment analysis was carried out to obtain the biological processes and pathways involved in differential genes,and the protein-protein interaction network was constructed.In addition,alveolar macrophages from silicosis patients and healthy controls were collected,the ex-pression levels of differential genes were detected by RT-qPCR,and the expressions of mitochondria-related factors mitochondrial fusion protein 1(MFN1),optic atrophy 1(OPA1)and dynamin-related protein 1(DRP1)in alveolar macrophages of silicosis patients were investigated by Western blot.Results A total of 204 differentially expressed genes were screened in silicosis patients,among which 62 differentially expressed genes were up-regulated,142 dif-ferentially expressed genes were down-regulated,and 22 differentially expressed genes were mitochondria-targeted.The concentration analysis of differentially expressed genes targeted by mitochondria showed that the cell compo-nents mainly enriched included mitochondrial membrane,endoplasmic membrane side components,etc.The bio-logical processes mainly enriched included mitochondrial electron transfer from NADH to ubiquinone,inflammatory response,immune response,etc.The main molecular functions enriched included the rotation mechanism of proton transport ATP synthase activity,NADH dehydrogenase activity,chemokine activity,etc.KEGG enrichment analy-sis mainly focused on the involvement in chemical carcinogenesis-ROS,IL-17 signaling pathway,toll-like receptor signaling pathway,chemokine signaling pathway,TNF signaling pathway,etc.In addition,RT-qPCR results showed that the expressions of mitochondrial cytochrome coxidase 1,mitochondrial cytochrome coxidase 2,mito-chondrial cytochrome coxidase 3,mitochondrially encoded NADH dehydrogenase 1,mitochondrially encoded NADH dehydrogenase 3,mitochondrially encoded NADH dehydrogenase 5,superoxide dismutase and mitochondri-ally encoded ATP synthase 6 gene were down-regulated in silicosis patients(P＜0.05).Western blot and RT-qPCR results showed that,in silicosis patients,the expression of MFN1 and OPA1 decreased(P＜0.05),while the expression of DRP1 increased(P＜0.05).Conclusion Bioinformatics analysis and validation,eight mito-chondrial targeted differential genes(MT-CO1,MT-C02,MT-CO3,MT-ND1,MT-ND3,MT-ND5,SOD and MT-ATP6)were finally obtained,which were enriched in mitochondrial respiratory chain and oxidative stress pathways and might play an important role in the process of silicosis.

Objective:To investigate the mechanism of Sulfo-N-succinimidyloleate (SSO) regulating lipid metabolism disorder induced by silicon dioxide (SiO 2) . Methods:In March 2023, Rat alveolar macrophages NR8383 were cultured in vitro and randomly divided into control group (C), SSO exposure group (SSO), SiO 2 exposure group (SiO 2) and SiO 2+SSO exposure group (SiO 2+SSO). NR8383 cells were exposure separately or jointly by SSO and SiO 2 for 36 h to construct cell models. Immunofluorescence and BODIPY 493/ 503 staining were used to detect cluster of differentiation (CD36) and intracellular lipid levels, the protein expression levels of CD36, liver X receptors (LXR), P-mammalian target of rapamycin (P-mTOR) and cholinephosphotransferase 1 (CHPT1) were detected by Western blot, respectively, and lipid metabolomics was used to screen for different lipid metabolites and enrichment pathways. Single-factor ANOVA was used for multi-group comparison, and LSD test was used for pair-to-group comparison. Results:SiO 2 caused the expression of CD36 and P-mTOR to increase ( P=0.012, 0.020), the expression of LXR to decrease ( P=0.005), and the intracellular lipid level to increase. After SSO treatment, CD36 expression decreased ( P=0.023) and LXR expression increased ( P=0.000) in SiO 2+SSO exposure group compared with SiO 2 exposure group. Metabolomics identified 87 different metabolites in the C group and SiO 2 exposure group, 19 different metabolites in the SiO 2 exposure group and SiO 2+SSO group, and 5 overlaps of different metabolites in the two comparison groups, they are PS (22∶1/14∶0), DG (O-16∶0/18∶0/0∶0), PGP (i-13∶0/i-20∶0), PC (18∶3/16∶0), and Sphinganine. In addition, the differential metabolites of the two comparison groups were mainly concentrated in the glycerophospholipid metabolism and sphingolipid metabolism pathways. The differential gene CHPT1 in glycerophospholipid metabolic pathway was verified, and the expression of CHPT1 decreased after SiO 2 exposure. Conclusion:SSO may improve SiO 2-induced lipid metabolism disorders by regulating PS (22∶1/14∶0), DG (O-16∶0/18∶0/0∶0), PGP (i-13∶0/i-20∶0), PC (18∶3/16∶0), SPA, glycerophospholipid metabolism and sphingolipid metabolism pathways.

Objective:To investigate the mechanism of Sulfo-N-succinimidyloleate (SSO) regulating lipid metabolism disorder induced by silicon dioxide (SiO 2) . Methods:In March 2023, Rat alveolar macrophages NR8383 were cultured in vitro and randomly divided into control group (C), SSO exposure group (SSO), SiO 2 exposure group (SiO 2) and SiO 2+SSO exposure group (SiO 2+SSO). NR8383 cells were exposure separately or jointly by SSO and SiO 2 for 36 h to construct cell models. Immunofluorescence and BODIPY 493/ 503 staining were used to detect cluster of differentiation (CD36) and intracellular lipid levels, the protein expression levels of CD36, liver X receptors (LXR), P-mammalian target of rapamycin (P-mTOR) and cholinephosphotransferase 1 (CHPT1) were detected by Western blot, respectively, and lipid metabolomics was used to screen for different lipid metabolites and enrichment pathways. Single-factor ANOVA was used for multi-group comparison, and LSD test was used for pair-to-group comparison. Results:SiO 2 caused the expression of CD36 and P-mTOR to increase ( P=0.012, 0.020), the expression of LXR to decrease ( P=0.005), and the intracellular lipid level to increase. After SSO treatment, CD36 expression decreased ( P=0.023) and LXR expression increased ( P=0.000) in SiO 2+SSO exposure group compared with SiO 2 exposure group. Metabolomics identified 87 different metabolites in the C group and SiO 2 exposure group, 19 different metabolites in the SiO 2 exposure group and SiO 2+SSO group, and 5 overlaps of different metabolites in the two comparison groups, they are PS (22∶1/14∶0), DG (O-16∶0/18∶0/0∶0), PGP (i-13∶0/i-20∶0), PC (18∶3/16∶0), and Sphinganine. In addition, the differential metabolites of the two comparison groups were mainly concentrated in the glycerophospholipid metabolism and sphingolipid metabolism pathways. The differential gene CHPT1 in glycerophospholipid metabolic pathway was verified, and the expression of CHPT1 decreased after SiO 2 exposure. Conclusion:SSO may improve SiO 2-induced lipid metabolism disorders by regulating PS (22∶1/14∶0), DG (O-16∶0/18∶0/0∶0), PGP (i-13∶0/i-20∶0), PC (18∶3/16∶0), SPA, glycerophospholipid metabolism and sphingolipid metabolism pathways.

Background Silicosis is an occupational disease mainly characterized by pulmonary progressive fibrosis induced by the accumulation of free silica (SiO₂) in the lungs due to long-term exposure to SiO₂ dust. It has been shown that neutrophil extracellular traps (NETs) are increased in the lung tissues of silicotic mice after 28 d SiO₂ exposure, but it is unclear how the levels of NETs change throughout entire progression of silicosis in mice. Objective To observe the levels of NETs and pathological changes in the lungs of silicotic mice after different duration of SiO₂ exposure, and to confirm the possible role and significance of NETsin the development of SiO₂-induced pulmonary fibrosis. Methods A total of 28 SPF male C57BL/6J mice were randomly divided into a control group, and a model group, and the model group was subdivided into, a 2 d model group, a 7 d model group, and a 28 d model group, with 7 mice in each group. The mice in the model groups were given intratracheal instillation with 10 mg SiO₂ suspension (50 μL), and the mice in the control group were received same volume of saline. Mice were sacrificed and samples were collected at designed time points. The pathological changes of lung tissues of mice were observed after hematoxylin-eosin (HE) and Van Gieson (VG) staining. Immunofluorescence was used to observe the NETs markers citrullination histone H3 (CitH3) and myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF), and the percentage of NETs-positive cells was calculated. PicoGreen fluorescent dye kit was used to detect the content of extracelluar DNA (ex-DNA) in mouse BALF, and the expression levels of fibrosis-related proteins α-smooth muscle actin (α-SMA) and fibronectin (FN) and NETs marker CitH3 in lung tissues of mice were detected by Western blot (WB). Results Compared with the control group, inflammatory cells accumulation, alveolar wall thickening, and collagen deposition were obviously observed in the lungs of the silicosis model groups, and a large number of silicone nodules were recorded in the lung tissues in the 28 d group. Compared with the control group, the expressions of α-SMA and FN in the lung tissue of the 28 d group were significantly increased (P<0.05). The percentages of NETs in BALF increased significantly in the 2 d and the 7 d model group, then decreased in the 28 d model group (P<0.05). Compared with the control group (7.434±0.258) ng·mL⁻¹, the ex-DNA levels in BALF of mice in the 2 d [(35.110±6.331) ng·mL⁻¹], the 7 d [(39.491±6.948) ng·mL⁻¹], and the 28 d [(23.360±4.809) ng·mL⁻¹] model groups were increased (P<0.05), and the increase of ex-DNA in the 2 d and the 7 d model groups were statistically significant (P<0.05). In comparison with the control group, the protein level of CitH3 was significantly increased in the lung tissues of mice in the 7 d model group (P<0.05). Conclusion The content of NETs increases significantly and reaches a peak in the early inflammatory stage of silicosis, and decreases as the disease progresses to the fibrotic stage, suggesting that NETs may play a role in early stage of silicosis.

Background Pneumoconiosis is the most serious occupational disease in China, and silicosis accounts for about half of it. Any intervention effect of physical exercise as the key and core of lung rehabilitation training on silicosis is still unclear. Objective To explore potential intervention effect of physical exercise on silicotic mice. Methods Forty SPF C57BL/6 male mice were randomly divided into four groups, 10 in each group, including a control group, a physical exercise group, a silicosis model group, and a silicosis model + physical exercise intervention group. Silicotic mouse model was established by using 50 μL SiO₂ suspension (200 mg·mL⁻¹). A treadmill was used to prepare mice receiving physical exercise at 0° inclination, 12.3 m·min⁻¹, 60 min·d⁻¹, 5 d·week⁻¹ for 4 weeks. Pathological morphology of lung tissues was evaluated after hematoxylin-eosin (HE) staining; deposition of collagen in lung tissues was evaluated after Van Gieson (VG) staining; expression of p-protein kinase R-like endoplasmic reticulum kinase (PERK) was detected by immunofluorescence staining; expressions of cyclin dependent kinase inhibitors (p21) and p-p38 mitogen activated protein kinase (p38) were detected by immunohistochemistry. The protein expressions of endoplasmic reticulum stress signal factors [p-inositol-requiring enzyme-1α (p-IRE-1α), p-PERK, and p-eukaryotic initiation factor-2α (p-eIF-2α)], senescence signal factors (p-p53, p21, and p16), mitogen-activated protein kinase (MAPK) signal factors [p-p38, p-extracellular regulated protein kinases (p-ERK), and p-stress-activated protein kinase (p-JNK)] were detected by Western blotting. Results After designed acute SiO₂ exposure, the images of micro computed tomography (CT) showed high density shadows in lung tissues of the silicotic mice and less shadows in lung tissues of the physical exercise intervention mice. After HE staining, the proportions of silicotic nodule area in lung tissues was (18.67±3.89) % in the silicosis model group, and significantly decreased to (8.78±1.05) % in the silicosis model + physical exercise intervention group (P<0.05). After VG staining, the proportion of collagen fiber area of lung tissues was (10.37±2.18) % in the silicosis model group, and significantly decreased to (4.35±0.89) % in the silicosis model + physical exercise intervention group (P<0.05). The results of immunofluorescence staining showed that in the silicosis model group, the expression of p-PERK increased at the location of silicotic nodules, while in the silicotic model + physical exercise intervention group, the expression of p-PERK decreased. The immunohistochemical staining results showed that the expression of p21 and p-p38 increased in the lung tissues of the silicosis model group; the expression of p21 and p-p38 decreased in the lung tissues of the silicosis model + physical exercise intervention group. The results of Western blotting showed that compared with the control group, the expression levels of p-IRE-1α (0.11±0.03), p-PERK (0.95±0.40), p-eIF-2α (3.53±0.91), p-p53 (1.78±0.07), p21 (1.98±0.10), p16 (1.26±0.17), p-p38 (0.41±0.09), p-ERK (0.42±0.05), and p-JNK (3.20±1.23) of the silicosis model group were all upregulated (P<0.05). Compared with the silicosis model group, the expression levels of p-IRE-1α (0.03±0.01), p-PERK (0.31±0.12), p-eIF-2α (0.30±0.06), p-p53 (0.76±0.08), p21 (0.18±0.11), p16 (0.70±0.24), p-p38 (0.03±0.00), p-ERK (0.19±0.03), and p-JNK (0.46±0.21) of the silicosis model + physical exercise intervention group were downregulated (P<0.05). Conclusion Physical exercise may alleviate pulmonary fibrosis in silicotic mice, and inhibit abnormal expressions of endoplasmic reticulum stress signal, MAPK signal, and senescent signal.

Background The pathogenesis of silicosis is complex and treatment methods are limited. SiO₂-induced increase of transforming growth factor-β1 (TGF-β1) can activate fibroblasts to promote collagen deposition, ultimately leading to fibrosis. Previous studies have confirmed that lipid metabolism plays an important role in the progression of silicosis. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) mediates mitochondrial dysfunction and lipid metabolism pathways in diabetic models, but its role in silicosis has not been elucidated. Objective To investigate the effect of PGC1α on lipid metabolism disorder of macrophages induced by SiO₂ and its effect on the progression of silicosis fibrosis. Methods (1) Macrophages were divided into four groups by transfecting and silencing PGC1α and its control sequence in macrophages and followed by SiO₂ stimulation: negative control group (transfected with si-NC for 48 h), si-PGC1α group (transfected with si-PGC1α for 48 h), SiO₂ stimulation group (stimulated with 50 μg·mL⁻¹ SiO₂ for 36 h after transfection with si-NC for 48 h), and si-PGC1α+SiO₂ group (stimulated with 50 μg·mL⁻¹ SiO₂ for 36 h after transfection with si-PGC1α for 48 h). Western blot and cell immunofluorescence were used to test PGC1α expression, 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) and total cholesterol (TC) and free cholesterol (FC) kits were used to test lipid accumulation, and the Oroboros2k-Oxygraph respiratory test system (O2K) was used to assess the effects of PGC1α on mitochondrial respiratory chain. ELISA kits were used to test TGF-β1 expressed in the macrophage supernatant. (2) Lung fibroblasts were divided into the same four groups as above, and stimulated with the supernatant of macrophages in the above groups. The expression of collagen Ι (COL Ι), E-cadherin (Eca), and fibronectin (FN) were detected by cell immunofluorescence and Western blot to further evaluate the effect of silencing PGC1α on fibrosis. Results The protein expression level of PGC1α stimulated by SiO₂ was decreased, and the relative expression level of PGC1α was 0.78 times that of the control group (P<0.05). After transfection with si-PGC1α, the expression of PGC1α was decreased, and the relative protein expression level of the si-PGC1α group was 0.86 times that of the control group (P<0.05). Compared with the SiO₂ stimulation group, the staining area of BODIPY 493/503 in the si-PGC1α+SiO₂ group was enhanced, and the cholesterol-related indexes [TC, FC and cholesterol ester (CE)] were increased to 1.38, 1.10, and 2.26 times those in the SiO₂ stimulation group (P<0.05). The activity of mitochondrial complex Ι was decreased, and the level of complex Ι in the si-PGC1α+SiO₂ group was 0.63 times that in the SiO₂ stimulation group (P<0.05). The secretion of TGF-β1 by macrophages increased, and the level of TGF-β1 in the si-PGC1α+SiO₂ group was 1.15 times that of the SiO₂ stimulation group (P<0.05). In addition, after stimulation of primary lung fibroblasts with macrophage supernatant, silencing PGC1α increased the expression levels of COL Ι and FN, while decreased the expression of Eca. The protein levels of COL Ι, FN, and Eca in the si-PGC1α+SiO₂ group were 1.39, 1.18, and 0.82 times those in the SiO₂ stimulation group, respectively (P<0.05). Conclusion Silencing PGC1α exacerbates SiO₂-induced lipid metabolism disorder, inhibits mitochondrial respiratory chain, and aggravates the fibrosis induced by SiO₂, suggesting that PGC1α may participate silicosis fibrosis by regulating mitochondrial respiratory chain and lipid metabolic disorder induced by SiO₂.

Objective : To explore the regulatory role of microRNA⁃455 ⁃3p ( miR⁃455 ⁃3p) in lymphangiogenesis of rat silicosis model , and to investigate the effect of miR⁃455 ⁃3p targeted regulation of vascular endothelial growth factor C (VEGF⁃C) on the tubular structure formation of human lymphatic endothelial cells ( HLECs) . Methods: The rats were randomly divided into the silicosis model group and the normal control group. The silicosis model group were injected with silicon dioxide (SiO2 )dust suspension , and the control group was injected with the same amount of normal saline. HE , Masson and immunohistochemistry staining were used to observe the pathological changes and lymphangiogenesis of lung tissue. The expression levels of miR⁃455 ⁃3p and VEGF⁃C in lung tissues of rats were detected by Quantitative real⁃time PCR ( RT⁃qPCR) and Western blot; The miR⁃455 ⁃3p inhibitors and negative controls ( NC) were transfected into HLECs , and the expression levels of miR⁃455 ⁃3p and VEGF⁃C in cells were detected by RT⁃qPCR and Western blot. The migration ability of HLECs was detected by scratch test , the ability of tubular structure formation was detected by matrigel tube formation test , and dual luciferase experiments were used to verify the targeting relationship between miR⁃455 ⁃3p and VEGF⁃C. Results : Compared with the normal control group , in the silicosis model group , a large number of inflammatory cells gathered and collagen gradually deposited in the pulmonary interstitium , and there was lymphatic hyperplasia in the lung. The expression of miR⁃455 ⁃3p in the lung tissue was lower than that in the control group , and the expression of VEGF⁃C was higher than that in the control group ; After transfection with HLECs , compared with the NC group , the expression of miR⁃455 ⁃3p in the cells of the Inhibitors group decreased , the expression of VEGF⁃C increased , and the ability of cell migration and tubular structure formation increased(P < 0. 05) ; VEGF⁃C was confirmed as a target gene of miR⁃455 ⁃3p by the dual luciferase experiments. Conclusion miR⁃455 ⁃3p can affect the tubular structure formation ability of HLECs and regulate lymphangiogenesis by targeting the expression of VEGF⁃C.

Objective:To screen the differential metabolites and metabolic pathways in silicosis model by analyzing plasma metabolomics of silicosis rats.Methods:In May 2021, twenty male SD rats were randomly divided into control group (C), 1-week silicosis group (S1W), 2-week silicosis group (S2W) and 4-week silicosis group (S4W), with 5 rats in each group. Rats were intratracheally instillated with 1ml crystalline SiO 2 suspension (50 mg/ml) or normal saline and were sacrificed after 1 week, 2 weeks and 4 weeks, HE staining was used to observe the lung pathology of rats. The plasma samples were analyzed by UPLC-IMS-QTOF mass spectrometer to screen out potential differential metabolites in silicosis models and analyze their lipid enrichment. Results:HE results showed that nodules formed in the silicosis model group, and with the extension of time, nodules gradually increased and alveolar structure was gradually destroyed. Metabolomics screened out 14 differential metabolites in S1W, 24 in S2W, and 28 in S4W, and found that the differential metabolites were mainly enriched in the metabolism of glycerophospholipid metabolism, fatty acid degradation, Glycosylphosphatidylinositol (GPI) -anchor biosynthesis, fatty acid elongation and other metabolic pathways.Conclusion:There are significant changes in plasma lipid metabolites in silicosis rat models.

Objective:To screen the differential metabolites and metabolic pathways in silicosis model by analyzing plasma metabolomics of silicosis rats.Methods:In May 2021, twenty male SD rats were randomly divided into control group (C), 1-week silicosis group (S1W), 2-week silicosis group (S2W) and 4-week silicosis group (S4W), with 5 rats in each group. Rats were intratracheally instillated with 1ml crystalline SiO 2 suspension (50 mg/ml) or normal saline and were sacrificed after 1 week, 2 weeks and 4 weeks, HE staining was used to observe the lung pathology of rats. The plasma samples were analyzed by UPLC-IMS-QTOF mass spectrometer to screen out potential differential metabolites in silicosis models and analyze their lipid enrichment. Results:HE results showed that nodules formed in the silicosis model group, and with the extension of time, nodules gradually increased and alveolar structure was gradually destroyed. Metabolomics screened out 14 differential metabolites in S1W, 24 in S2W, and 28 in S4W, and found that the differential metabolites were mainly enriched in the metabolism of glycerophospholipid metabolism, fatty acid degradation, Glycosylphosphatidylinositol (GPI) -anchor biosynthesis, fatty acid elongation and other metabolic pathways.Conclusion:There are significant changes in plasma lipid metabolites in silicosis rat models.