Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

Abstract

Pneumoconiosis is the most common and serious disease among coal miners. In earlier work on this subject, we documented that coal dust (CD) nanoparticles (CD-NPs) induced pulmonary fibrosis (PF) more profoundly than did CD micron particles (CD-MPs), but the mechanism has not been thoroughly studied. Based on the GEO database, jveen, STRING, and Cytoscape tools were used to screen hub genes regulating PF. Particle size distribution of CD were analyzed with Malvern nanoparticle size potentiometer. Combining 8 computational methods, we found that IGF1, POSTN, MMP7, ASPN, and CXCL14 may act as hub genes regulating PF. Based on the high score of IGF1 and its important regulatory role in various tissue fibrosis, we selected it as the target gene in this study. Activation of the IGF1/IGF1R axis promoted CD-NPs-induced PF, and inhibition of the axis activation had the opposite effect in vitro and in vivo. Furthermore, activation of the IGF1/IGF1R axis induced generation of reactive oxygen species (ROS) to promote epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs) to accelerate PF. High-throughput gene sequencing based on lung tissue suggested that cytokine-cytokine receptor interaction and the NF-kB signaling pathway play a key role in PF. Also, ROS induced inflammation and EMT by the activation of the NF-kB/NLRP3 axis to accelerate PF. ROS can induce the activation of AKT/GSK3β signaling, and inhibition of it can inhibit ROS-induced inflammation and EMT by the NF-kB/NLRP3 axis, thereby inhibiting PF. CD-NPs induced PF by promoting inflammation and EMT via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals. This study provides a valuable experimental basis for the prevention and treatment of coal workers' pneumoconiosis. Illustration of the overall research idea of this study: IGF1 stimulates coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and EMT via the NF-κB/NLRP3 pathway driven by ROS-mediated AKT/GSK3β signals.

Illustration of the overall research idea of this study: IGF1 stimulates coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and EMT via the NF-κB/NLRP3 pathway driven by ROS-mediated AKT/GSK3β signals.

Fig. 1. IGF1 identified as a hub gene in PF.

A, B The up- and down-regulated intersection genes among the 5 datasets were selected by jvenn tool. C The protein–protein interaction (PPI) network analysis of the up- and down-regulated intersection genes was performed by STRING and Cytoscape software. D The hub genes were predicted by the plug-in cytoHubba of Cytoscape software and the top three nodes ranked by MCC, DMNC, MNC, Degree, EPC, BottleNeck, EcCentricity and Closeness.

Fig. 2. IGF1/IGF1R signaling is significantly activated in models of pre-fibrosis in vitro and in vivo.

A, B The size distribution and polydispersity index (PDI) of CD-MPs and CD-NPs were measured with a Malvern Nanoparticle Size Potentiometer. C, D IGF1/IGF1R signaling was detected by western blot and qRT-PCR in models of pre-fibrosis in vitro. E Cell morphology in an in vitro pre-fibrosis model was examined by inverted microscope. F, G IGF1/IGF1R signaling was detected by immunohistochemistry and western blot in models of pre-fibrosis in vivo.

Fig. 3. Activation of IGF1/IGF1R signaling positively regulates CD-NPs-induced PF.

A, C After inhibition of IGF1R in an in vitro models of pre-fibrosis, IGF1/IGF1R signaling was detected by western blot. B, D After inhibition of IGF1R in an in vitro models of pre-fibrosis, cell viability was examined by CCK-8 assay. E, F IGF1/IGF1R signaling was detected by western blot and immunohistochemistry in an in vivo models of pre-fibrosis. G Lung coefficients in an in vivo pre-fibrotic model. Data were expressed as the mean ± SD, n = 3. *P < 0.05 and ***P < 0.001.

Fig. 4. Activation of IGF1/IGF1R signaling mediates EMT in in vitro and in vivo models by promoting the generation of ROS.

A, C ROS levels were detected by ROS fluorescent probe DCFH-DA in vitro. B, D The expression level of HO-1 was detected by western blot. E ROS levels were detected by ROS fluorescent probe DCFH-DA in vivo. Data were expressed as the mean ± SD, n = 3. ***P < 0.001. F, G The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1) was detected by western blot and immunohistochemistry. H, I The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Fig. 5. High-throughput gene sequencing analysis of lung tissue based on in vivo pre-fibrosis model mice.

A Clustering heatmap of differential gene expression in normal mouse lung tissue and pre-fibrotic model mouse lung tissue. In the figure, each row represents a gene, and each column represents a sample. The color represents the expression level of the gene in the sample. Red represents the gene with high expression level in the sample, and green represents the low expression level. B Volcano plot of differential gene expression in normal mouse lung tissue and pre-fibrotic model mouse lung tissue. The horizontal axis is the fold-change value of the gene expression difference between samples in different groups, and the vertical axis is the statistically significant p Value representing the change in gene expression. The smaller the pValue, the larger the difference in −log₁₀(pValue), the more obvious the difference. Each dot represents a gene, where red indicates upregulated genes, green indicates down-regulated genes, and black indicates non-differential genes. C GO enrichment analysis of differentially expressed genes. The vertical axis represents the functional annotation information, the horizontal axis represents the Rich factor corresponding to the function (the number of differential genes annotated to the function divided by the number of genes annotated to the function), and the red box marks the two functions with the most significant enrichment of differential genes. D KEGG-enrichment analysis of differentially expressed genes. The vertical axis represents the KEGG pathway information, the horizontal axis represents the Rich factor corresponding to the KEGG pathway (the number of differential genes annotated to the KEGG pathway divided by the number of genes annotated to the KEGG pathway), and the red boxes mark the three signaling pathways that are significantly enriched for the differential genes focused on in this study.

Fig. 6. Inhibition of IGF1/IGF1R signaling can alleviate CD-NPs-induced inflammation by inhibiting ROS-driven NF-κB/NLRP3 pathway.

A, B The activation level of NF-κB/NLRP3 pathway was detected by western blot in vitro. C Inflammatory factor IL-1β and inflammatory chemokine CXCL2 in the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ***P < 0.001. D, E The activation level of NF-κB/NLRP3 pathway was detected by western blot in vivo. F Inflammatory factor IL-1β and inflammatory chemokine CXCL2 in mouse bronchoalveolar lavage fluid (BALF) were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ***P < 0.001.

Fig. 7. Inhibition of NLRP3 reverses the effects of CD-NPs on inflammation and EMT in vitro.

A The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1) and inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) were detected by western blot. B The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay. C Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 8. ROS-mediated AKT/GSK3β signals drive NF-κB/NLRP3 pathway to promote inflammation and EMT.

A, B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C, D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. **P < 0.01 and ***P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.