Objective This study aims to investigate the expression, phenotypic changes, and mechanisms of action of guanylate-binding protein 2 (GBP2) in the process of silica-induced pulmonary fibrosis. Methods The expression and localization of GBP2 in silicotic lung tissue were detected by immunohistochemical staining and immunofluorescence. An in vitro cell model was constructed, and methods such as Western blot and real-time quantitative reverse transcription polymerasechain reaction were utilized to investigate the function of GBP2 in different cell lines following silica stimulation. The mechanism of action of GBP2 in various cell lines was elucidated using Western blot analysis. Results GBP2 was highly expressed in the lung tissue of patients with silicosis. Immunohistochemical staining and immunofluorescence have revealed that GBP2 was localized in macrophages and epithelial cells. In vitro cell experiments demonstrated that silicon dioxide stimulated THP-1 cells to activate the c-Jun pathway through GBP2, promoting the secretion of inflammatory factors and facilitating the occurrence of M2 macrophage polarization. In epithelial cells, GBP2 promoted the occurrence of epithelial to mesenchymal transition (EMT) by upregulating Krueppel-like factor 8 (KLF8). Conclusion GBP2 not only activates c-Jun in macrophages to promote the production of inflammatory factors and the occurrence of M2 macrophage polarization, but also activates the transcription factor KLF8 in epithelial cells to induce EMT, collectively promoting the progression of silicosis.
Humans ; Silicosis/genetics* ; Macrophages/cytology* ; Epithelial Cells/pathology* ; GTP-Binding Proteins/physiology* ; Epithelial-Mesenchymal Transition ; Disease Progression ; Cell Line ; Male

OBJECTIVE: Recent studies have overturned the traditional concept of the lung as a "sterile organ" revealing that pulmonary microbiota dysbiosis and abnormal surfactant proteins (SPs) expression are involved in the progression of silicosis. This study aimed to investigate the relationship between abnormal SPs expression and dysbiosis of lung microbiota in silica-induced lung fibrosis, providing insights into mechanisms of silicosis. METHODS: Lung pathology, SPs expression, and microbiota composition were evaluated in silica-exposed mice. A mouse model of antibiotic-induced microbiota depletion was established, and alveolar structure and SPs expression were assessed. The roles of the lung microbiota and SPs in silicosis progression were further evaluated in mice with antibiotic-induced microbiota depletion, both with and without silica exposure. RESULTS: Silica exposure induced lung inflammation and fibrosis, along with increased expression of SP-A expression. Antibiotics (Abx)-induced microbiota depletion elevated SP-A and SP-D expression. Furthermore, silica exposure altered lung microbiota composition, enriching potentially pathogenic taxa. However, antibiotic-induced microbiota depletion prior to silica exposure reduced silica-mediated lung fibrosis and inflammation. CONCLUSION Lung microbiota is associated with silica-induced lung injury. Overproduction of SP-A and SP-D, induced by Abx-induced microbiota depletion, may enhance the resistance of mouse lung tissue to silica-induced injury.
Animals ; Silicosis/prevention & control* ; Lung/metabolism* ; Mice ; Anti-Bacterial Agents/pharmacology* ; Microbiota/drug effects* ; Silicon Dioxide/toxicity* ; Mice, Inbred C57BL ; Male ; Pulmonary Surfactant-Associated Proteins/genetics*

BACKGROUND: This study aimed to evaluate the correlation between long non-coding RNA (lncRNA)-related single nucleotide polymorphisms (SNPs) and susceptibility to silicosis. METHODS: First, RNA-sequencing (RNA-seq) data were comprehensively analyzed in the peripheral blood lymphocytes of eight participants (four silicosis cases and four healthy controls) exposed to silica dust to identify differentially expressed lncRNAs (DE-lncRNAs). The functional SNPs in the identified DE-lncRNAs were then identified using several databases. Finally, the association between functional SNPs and susceptibility to silicosis was evaluated by a two-stage case-control study. The SNPs of 155 silicosis cases and 141 healthy silica-exposed controls were screened by genome-wide association study (GWAS), and the candidate SNPs of 194 silicosis cases and 235 healthy silica-exposed controls were validated by genotyping using the improved Mutiligase Detection Reaction (iMLDR) system. RESULTS: A total of 76 DE-lncRNAs were identified by RNA-seq data analysis (cut-offs: fold change > 2 or fold change < 0.5, P < 0.05), while 127 functional SNPs among those 76 DE-lncRNAs were identified through multiple public databases. Furthermore, five SNPs were found to be significantly correlated with the risk of silicosis by GWAS screening (P < 0.05), while the results of GWAS and iMLDR validation indicated that the variant A allele of rs1814521 was associated with a reduced risk of silicosis (OR = 0.76, 95% CI = 0.62-0.94, P = 0.011). CONCLUSION The presence of the SNP rs1814521 in the lncRNA ADGRG3 is associated with susceptibility to silicosis. Moreover, ADGRG3 was found to be lowly expressed in silicosis cases. The underlying biological mechanisms by which lncRNA ADGRG3 and rs1814521 regulate the development of silicosis need further study.
Case-Control Studies ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; Polymorphism, Single Nucleotide ; RNA, Long Noncoding/genetics* ; Silicosis/genetics*

OBJECTIVETo investigate the relationship between polymorphisms of surfactant protein D (rs3088308 and rs721917) and the susceptibility to silicosis.

METHODSThis case-control study included 125 silicosis patients and 125 individuals exposed to industrial dust but without silicosis (control group), who were strictly matched with the case group for age, gender, work type and cumulative length of dust exposure. The rs3088308 and rs721917 polymorphisms of surfactant protein-D were detected in all the participants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

RESULTSThe frequencies of T/T, T/A and A/A genotypes of surfactant protein-D rs3088308 locus were 22.2%, 71.2% and 5.6% in the case group, significantly different from the frequencies of 17.6%, 58.4% and 24.0% in the control group, respectively (P<0.05). The frequencies of C/C, C/T and T/T genotypes of rs721917 locus were 17.6%, 56.8% and 25.6% in the case group, similar to the frequencies of 15.2%, 60.0% and 24.8% in the control group, respectively (P>0.05).

CONCLUSIONSurfactant protein-D rs3088308 polymorphism is significantly associated with silicosis, and the T allele may be a risk factor for silicosis in individuals exposed to industrial dust.

Alleles ; Case-Control Studies ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Pulmonary Surfactant-Associated Protein D ; genetics ; Risk Factors ; Silicosis ; genetics

Silicosis is one of the most serious occupational diseases in China and dates back to centuries ago. In this study, we successfully established a rat model of silicosis by intratracheal silica injection for 28 days and determined hydroxyproline levels to evaluate collagen metabolism in lung homogenates. Oxidative stress status was evaluated by detecting catalase and glutathione peroxidase activities. Expression levels of peroxiredoxins (Prx I and Prx VI) were detected by Western blotting. Pulmonary surfactant protein A (SP-A) levels in rat serum and lung tissue were analyzed by ELISA, and SP-A and Prx expression levels in lung tissues were detected by immunohistochemistry. The results suggest that Prx proteins may be involved in pulmonary fibrosis induced by silica. Downregulation of SP-A expression caused due to silica is an important factor in the occurrence and development of silicosis.
Animals ; Disease Models, Animal ; Humans ; Lung ; enzymology ; metabolism ; Male ; Oxidative Stress ; Peroxiredoxin VI ; genetics ; metabolism ; Peroxiredoxins ; genetics ; metabolism ; Pulmonary Surfactant-Associated Protein A ; genetics ; metabolism ; Rats ; Silicon Dioxide ; toxicity ; Silicosis ; genetics ; metabolism

OBJECTIVETo investigate the differential gene expression profile of the lung tissues in experimental silicosis rats and to screen for and identify the key signal transduction pathways in pulmonary silicotic fibrosis.

METHODSA total of 80 rats were randomly divided into control group (n = 40) and silica-instilled group (n = 40). Each group was equally divided into five subgroups, and each subgroup was treated at 1, 7, 14, 21, or 28 d. Intratracheal instillation was used to give 1 ml of silica suspension (50 mg/ml) in the silica-instilled group and normal saline in the control group. Silicotic nodules and type I and III collagen were observed through hematoxylin and eosin staining and Sirius red staining, respectively. Differentially expressed genes in pulmonary silicotic fibrosis were selected by the rat whole-genome gene expression RatRef-12 BeadChip (Illumina, USA), and a fold change cutoff was applied. Quantitative real-time polymerase chain reaction (qRT-PCR) was also used to verify differentially expressed genes. Through bioinformatics databases such as Visualization and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG), preliminary research was performed on the biological pathways of differential genes, key biological signal transduction pathways were identified, and key differentially expressed genes in each pathway at different time points were searched for.

RESULTSA total of 2694 genes were differentially expressed and changed dynamically. The KEGG pathway analysis showed that 141 signal transduction pathways were involved in the development and progression of pulmonary silicotic fibrosis, among which 48 pathways were more significant than others (P < 0.01), with the mitogen-activated protein kinase (MAPK) pathway exceptionally significant. The differentially expressed genes interleukin-1 receptor (IL-1R), tumor necrosis factor receptor (TNFR), and transforming growth factor beta (TGF-β) in the MAPK pathway were up-regulated at different time points after silica instillation. The results of real-time PCR showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) was over-expressed at 4 time points and under-expressed at 1 time point compared with the control group.

CONCLUSIONThe MAPK signal transduction pathway plays a very important role in the development of pulmonary silicotic fibrosis. Both IL-1R and TNFR may play major roles during inflammation phase through the P38/Jun N-terminal kinase (JNK) pathway, and TGF-β may have important function through the extracellular-signal-regulated kinase (ERK) pathway in the formation of fibrosis.

Animals ; Gene Expression ; Lung ; metabolism ; pathology ; Male ; Pulmonary Fibrosis ; genetics ; metabolism ; pathology ; Rats ; Rats, Wistar ; Signal Transduction ; Silicosis ; genetics ; metabolism ; pathology

OBJECTIVETo investigate the relationship between the genetic polymorphisms of matrix metalloproteinase-2 (MMP-2) (-735) and matrix metalloproteinase-3 (MMP-3) (-1171) and the susceptibility to silicosis.

METHODSA case-control study was conducted in 113 patients diagnosed with stage I silicosis (case group) and 115 dust-exposed workers without silicosis (control group); the two groups had the same sex, ethnic group, and type of dust and similar age and cumulative exposure time. Polymerase chain reaction-restriction fragment length polymorphism was used to determine the genotypes of MMP-2 (-735) and MMP-3 (-1171).

RESULTSNo significant difference was observed in age, cumulative exposure time, or smoking rate between cases and controls (P > 0.05). The frequencies of genotypes C/C, C/T, and T/T at MMP-2 C-735T in the case group were 57.5% (65/113), 31.0% (35/113), and 11.5% (13/113), respectively, which were significantly different from those of the control group (69.6% (80/115), 26.9% (31/115), and 3.5% (4/115)), χ² = 6.542, P < 0.05). The frequencies of T allele in cases and controls were 27.0% and 17.0%, respectively, which were significantly different from each other χ² = 6.704, P < 0.05). Carriage of T allele at MMP-2 C-735T increased the risk of silicosis (OR = 1.811, 95% CI: 1.151-2.847). The frequencies of genotypes 6A/6A, 5A/6A, and 5A/5A at MMP-3 A-1171A were 67.2% (76/113), 24.8% (28/113), and 8.0% (9/113), respectively, in the case group, versus 59.1% (68/115), 37.4% (43/115), and 3.5% (4/115) in the control group (χ² = 5.519, P > 0.05).

CONCLUSIONGenetic polymorphism at MMP-2 C-735T is significantly associated with the development of silicosis. Carriage of T allele increases the risk of silicosis among workers exposed to dust. No significant association was found between MMP-3 A-1171A polymorphism and silicosis in this study.

Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Genetic Predisposition to Disease ; Genotype ; Humans ; Matrix Metalloproteinase 2 ; genetics ; Matrix Metalloproteinase 3 ; genetics ; Middle Aged ; Polymorphism, Genetic ; Silicosis ; genetics

OBJECTIVETo investigate the relationship between epidermal growth factor (EGF) gene polymorphisms at G-61A, R431K, and D784V and susceptibility to silicosis.

METHODSIn a case-control study, 116 patients diagnosed with stage I silicosis were included in the case group, and 149 workers without silicosis of the same gender and nationality, exposed to the same nature of dust, and with similar age and cumulative time of dust exposure were included in the control group. Peripheral venous blood was collected, DNA was extracted by salting out, polymerase chain reaction-restriction fragment length polymorphism was used to identify the genotypes at three polymorphic loci of EGF and the allele frequencies, and their distributions in the case group and control group were analyzed.

RESULTSThe genotype frequencies of G-61A GG, GA, and AA in the case group were 50.9%, 34.5%, and 14.7%, respectively, and significant differences were found when comparing the data with those in the control group (35.6%,44.3%, and 20.1%), (χ(2) = 6.283, P = 0.048). The distribution frequencies of allele A in the case group and control group were 31.9%and 42.3%, respectively, and the difference between the two groups was statistically significant (χ2 = 5.554, P = 0.018). The risk of silicosis in workers carrying allele G at G-61A was increased by 1.564 times (OR = 1.564, 95%CI: 1.092∼2.024). The genotype frequencies of D784V AA, AT, and TT in the case group were 58.6%, 34.5%, and 6.9%, respectively, versus 65.1%, 31.5%, and 3.4% in the control group, and the differences between the two groups were not statistically significant (χ(2) = 2.278, P = 0.320). The genotype frequencies of R431K GG, GA, and AA in the case group were 56.9%, 39.7%, and 3.4%, respectively, versus 55.0%, 39.6%, and 5.4% in the control group, and the differences between the two groups were not statistically significant (χ(2) = 0.572, P = 0.751).

CONCLUSIONThe EGF gene polymorphism at G-61A is associated with susceptibility to silicosis, and the risk of silicosis in dust-exposed workers carrying GG genotype is relatively high. No relationship between EGF gene polymorphisms at D784V and R431K and silicosis is found.

Aged ; Aged, 80 and over ; Alleles ; Case-Control Studies ; Epidermal Growth Factor ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Middle Aged ; Polymorphism, Single Nucleotide ; Silicosis ; epidemiology ; genetics

OBJECTIVETo investigate the inhibitory effects of CD36-targeting RNA interference on the latent transforming growth factor β1 (L-TGF-β1) activation and silicotic fibrosis in rat silicosis model.

METHODSWistar rats were divided into four groups: saline control group (n=24), SiO2 model group (10 mg SiO2 per rat) (n=24), SiO2+Lv-shCD36 group (lentiviral vector expressing specific shRNA against CD36) (n=24), and SiO2+Lv-shCD36-NC group (non-silence control lentivirus) (n=24). At 7, 21, and 28 d after instillation, the rats were sacrificed. The activity of TGF-β1 in bronchoalveolar lavage fluid (BALF) was measured by evaluating its inhibitory effect on the proliferation of mink lung epithelial cells. The pathological changes of lung tissue were observed by HE staining and van Gieson staining. The hydroxyproline content in the lungs was determined by alkaline lysis method.

RESULTSAt 7 d after instillation, the expression of CD36 mRNA in alveolar macrophages was significantly lower in the SiO2+Lv-shCD36 group than in the saline control group, SiO2 model group, and SiO2+Lv-shCD36-NC group (P < 0.05); the quantity and percentage of active TGF-β1 in BALF were significantly lower in the SiO2+Lv-shCD36 group than in the SiO2 model group and SiO2+Lv-shCD36-NC group (P < 0.05). At 28 d after instillation, there were cellular silicotic nodules in the lungs of rats in SiO2+Lv-shCD36 group and fibrotic cellular silicotic nodules in the lungs of rats in SiO2 model group and SiO2+Lv-shCD36-NC group. At 21 and 28 d after instillation, the hydroxyproline content was significantly lower in the SiO2+Lv-shCD36 group than in the SiO2 model group and SiO2+Lv-sh CD36-NC group (P < 0.05).

CONCLUSIONCD36-targeting RNA interference has inhibitory effects on the L-TGF-β1 activation and silicotic fibrosis in rat silicosis model.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; CD36 Antigens ; genetics ; metabolism ; Disease Models, Animal ; Female ; Hydroxyproline ; chemistry ; Macrophages, Alveolar ; metabolism ; Male ; RNA Interference ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Silicosis ; metabolism ; prevention & control ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo screen the differentially expressing genes between silicotic lung tissue and normal lung tissue, to identify the differentially expressing genes of matrix metalloproteinase-12 (MMP-12) and Cathepsin E and to explore the roles of those genes in silicosis development.

METHODSThirty male SD rats were divided randomly into two groups: control group (6 rats) and exposure group (24 rats) which was exposed to SiO2 by intra-tracheal perfusion. On the 30 th, 60 th and 90 th days after exposure, 8 rats in model group and 2 rats in control group were executed and the lung tissues were obtained. The morphologic changes of lung tissues were observed with HE staining and VG staining under a light microscope. The gene microarrays were used to identify differentially expressing genes of lung tissues in rats exposed to SiO2 for 60 days. Two significantly up-regulated genes, MMP-12 and Cathepsin E, were validated using RT-PCR, immunohistochemistry and Western Blot assay.

RESULTSA total of 338 differentially expressing genes were identified from the 26 962 genes between silicotic rats and normal rats, including 267 up-regulated genes and 71 down-regulated genes. The results of RT-PCR showed that in the lung tissues of exposure group on the 30 th, 60 th and 90 th days, the mRNA expression levels of MMP-12 were 4.306, 5.338, 6.713 times higher than those in the control group, the mRNA expression levels of Cathepsin E were 1.434, 2.974, 3.889 times higher than those in the control group, respectively. The results of immunohistochemical showed that in the lung tissues of exposure group on the 30th, 60th and 90th days, the mRNA expression levels of MMP-12 were 1.435, 1.746, 2.069 times higher than those in the control group, the mRNA expression levels of Cathepsin E were 1.372, 1.663, 2.103 times higher than those in the control group, respectively. The results of immunohistochemical showed that in the lung tissues of exposure group on the 30th, 60th and 90th days, the expression levels of MMP-12 protein were 1.214, 1.531, 1.959 times higher than those in the control group, the expression levels of Cathepsin E protein were 1.262, 1.828, 1.907 times higher than those in the control group, respectively. Compared with the control group, the mRNA and protein expression levels of MMP-12 and Cathepsin E in lung tissues of exposure group were significantly up-regulated (P < 0.05).

CONCLUSIONThe differentially expressing genes in rat lung tissues screened by gene chip were validated, which suggested that a complex gene regulatory network may be contributed to occurrence of silicosis. MMP-12 and Cathepsin E genes may be involved in the development of silicotic pulmonary fibrosis by degrading the basement membrane of alveolar wall and participating in the immune response.

Animals ; Cathepsin E ; genetics ; metabolism ; Gene Expression ; Lung ; metabolism ; Male ; Matrix Metalloproteinase 12 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Silicosis ; genetics ; metabolism