Exploring the key pathways of tetrandrine in the treatment of early silicosis based on bioinformatics and in vitro experiments

LIANG Chao, ZHOU Jia-wei, LIU Ya-feng, GUO Jian-qiang, WANG Qing-sen, SU Yixin, XING Ying-ru, HU Chun-xiao, XIE Jun, WU Jing,2,3?, HU Dong,2,3?

1. School of Medicine,Anhui University of Science and Technology, Huainan 232001,China

2. Anhui Occupational Health and Safety Engineering Laboratory, Huainan 232001,China

3. Key Laboratory of Advanced Industrial Dust Purification and Occupational Health and Safety of Education Department,Huainan 232001,China

4. Department of Clinical Laboratory, Gengjiu Hospital of Anhui Zhongke, Hefei 232001,China

5. Affiliated Cancer Hospital of Anhui University of Science and Technology/Huainan Oriental Hospital Group Cancer Hospital, Huainan 232001,China

Keywords:

ABSTRACT Objective: Exploring the key pathways affecting the development of early silicosis based on bioinformatics and in vitro experiments. Method: Collecting differentially expressed genes in silicosis patients through literature mining; Collecting differentially expressed genes in silicon dioxide infusion mice by using a high-throughput gene expression database (GEO);Obtaining disease targets related to silicosis by means of online human Mendelian genetic database (OMIM), GeneCards and comparative toxicgenomics database (CTD); differentially expressed genes and disease targets were subjected to gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis via R-package and Metascape platforms, respectively. The Schr?dinger and Pymol software were used for molecular docking and modification. Silicon dioxide-stimulated macrophages and epithelial cells were modeled and analyzed by PCR and western blot (WB). Result: 2065 differentially expressed genes in silicosis patients, 2 291 differentially expressed genes in rat infused with silicon dioxide, and 803 targets for silicosis-related diseases were screened out. GO enrichment analysis mainly involves G protein-coupled receptor binding, the regulation of inflammatory response, and participation in immune response. The enrichment analysis of KEGG pathway mainly included ECM-receptor interaction, TNF signaling pathway, and IL-17 signaling pathway. IL-17 signaling pathway was screened out from different genes and disease targets,indicating that IL-17 signaling pathway might be the key pathway for the development of silicosis. Molecular docking results showed that the silicosis drug tetrandrine had good binding effect with the RAF/MEK/ERK pathway in the IL-17 signaling pathway. Cellular experiments showed that tetrandrine reduced the expression of inflammatory factors such as IL-6 and TGF-β in macrophages by regulating the RAF/MEK/ERKpathway, and inhibited the epithelialmesenchymal transition and expression of inflammatory factors in epithelial cells. Conclusion:Tetrandrine regulates the inflammatory response and epithelial-mesenchymal transition (EMT)through the RAF/MEK/ERK pathway and thus affects the early progression of silicosis.

1. Introduction

Silicosis is a disease of lung fibrosis due to inhalation of silica or stimulated by silica and is one of the most common occupational diseases. China has the largest number of silicosis patients, with an average of more than 5000 confirmed cases and more than 20 000 deaths per year. More than 3 million people with silicosis worldwide are in Europe and more than 2 million in the United States [1]. Silicosis is often associated with macrophage receptor activation, inflammation of small bodies, silica is inhaled into the lungs and engulfed by macrophages, macrophages release inflammatory factors to stimulate epithelial cells to transform into interstitial cells and the recruitment of fibroblasts, fibroblasts are stimulated to differentiate into myofibroblasts, producing collagen fibers [2, 3]. Silicon dioxide has been shown to stimulate PI3K protein kinase to regulate autophagosome accumulation to promote silicosis development[4], as well as silicosis progression by inhibiting NFκB or TGF-β [5]. However, the key pathways of early silicosis are not clear, and there is no effective means to identify, diagnose, and treat early silicosis [6], so the in-depth study of early silicosis has important clinical and social significance.

According to the characteristics of silicosis syndrome, ancient Chinese medicine practitioners used traditional Chinese medicine to supplement as a method, to use general, and to treat silicosis with general supplementation, and Chinese medicine self-defense was one of them [7]. Self-prevention has a long history of medicinal use, which has been recorded in ancient books such as "Shennong Materia Medica", "Treatise on Typhoid Fever and Miscellaneous Diseases", "Theory of Medicinal Properties", etc., and self-defense has recorded bitter cold in the "Shennong Materia Medica", which has the effect of treating rheumatism and edema and eczema pulse floating [7]. In modern medical treatment, it has anti-tumor,anti-rheumatoid arthritis, cardiovascular protection and other effects. Studies have shown that the main chemical component of Tetrandrine is Tetrandrine (tetrandrine), which is a quinoline alkaloid, which has anti-tumor, anti-inflammatory, and anti-fibrosis.Pharmacological effects such as antisilicosis[7]. Clinical studies have shown that powder antihexanin has a significant clinical effect on silicosis and can improve the level of lung function in patients [8].It was also found that powdered hexane can reduce the outbreak of lipopolysaccharide-induced macrophages[9], reduce the production of oxygen radicals and tumor necrosis factor-α (TGF-α). The accumulation of inflammatory factors such as interleukin-6 (IL-6)delays the inflammatory phase in the pre-silicosis period, reduces the production of collagen fibers, and improves the formation of pulmonary fibrosis [10]. Based on the research results of traditional Chinese medicine in the previous research group, this study used three datasets of silicosis patient genes, silica instillation mouse genes and network pharmacology for screening and prediction, and explored the key pathways of powder hexane in the treatment of early silicosis through molecular docking and in vitro experimental verification, providing a theoretical basis for the treatment of early silicosis.

2. Information and methods

2.1 Chip and target genes

The relevant chip data of patients with silicosis are derived from the literature, and this study collected lung tissue RNA sequencing data from 10 patients with silicosis and 7 patients with non-silicosis For analysis, the lung tissue of 10 silicosis patients and 7 non-silicosis patients were all from Wuxi People's Hospital, the lung tissue was obtained at the time of lung transplantation, lymph nodes and organs were avoided during sampling, and all participants gave written informed consent and obtained the approval of the Institutional Review Committee of Nanjing Medical University and the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences [11]. Chip data for silica instillation rats comes from the Gene Expression Omnibus, GEO, https://www.ncbi.nlm.nih.gov/geo/geo/ ). Search for the keyword "silicosis" in the GEO database, and the original data of the chip GSE188520 and the platform annotation file GPL2 5290 are filtered The sample in GSE188520 consisted of 6 common rats and 6 silica instillation rats. The above chip data were analyzed by using the limma package[13], and the filter conditions for the differential genes were P＜0.05, log(fold change, FC) ＞1 Or ＜-1,the screened genes are sorted according to the absolute size of log FC, and the top 100 genes are up-regulated and down-regulated for subsequent analysis.

2.2 Collection of silicosis targets

In the OMIM database[14], the CTD database[15], the GeneCard database[16]. The key word "silicon lung" was used to search, and the intersection target was extracted and constructed using the target intersection visualization tool [17].

2.3 Functional enrichment analysis

To illustrate the signaling pathways and biological functions of the selected key genes and targets, R studio software was used and R/ggplot2, limma, pheatmap, and ggsci were used DPLYR packages performed gene ontologic analysis and signaling pathway enrichment analysis of key differential genes[18].

2.4 Protein-protein interaction diagram

The pathways screened by different gene sets intersect to obtain key pathways, and the relevant targets of key pathways are imported into the STRING database[19], the species is set to "Home sapiens", and the download file is saved in tsv format. Key proteins were obtained after beautification of cytoscape 3.8.0 [20].

2.5 Molecular docking

The format chemistry of key proteins in the PDB[21] database,downloaded in PDB format. Download the chemical structure of the Tetrandrine in PubChem [22] and save it in SDF format. Use the Protein Preparation Wizard in Schrodinger software for protein structure optimization, select retention or deletion of chains, waters and ligands, repair and fill lost side chains, and minimize energy.Ligand, water position, and charge correction in the Review and Modify options, and final protein structure optimization in the Refine option. Ligand preparation by LigPrep, hydrogenation,desalting, neutralization of charged groups, protonization state and finally tautomer structure. The software's Ligand Docking module is used for molecular docking, evaluating the affinity of the drug and protein structures by binding energies, and finally modifying the performance with pymol.

2.6 MTS detects apoptosis rate

After washing the Dish twice with PBS, digest the cells with pancreatin, select the appropriate pancreatic digestion time according to the different cell states, and after pancreatic enzyme digestion,pipette into a 15mL centrifuge tube, centrifuge for 5 min, and the speed is 5000 per minute Turn, centrifuge after the supernatant is poured out, wash with PBS and centrifuge again for 5 min at a speed of 5000 rpm. Add 1 mL of culture medium after pouring the supernatant to prepare the cell suspension, prepare for counting,seed the cells into a 96-well plate, adjust the cell concentration at around 5×103, and add medium 1 per well 100 μL, repeat 3-5 wells per sample, add different concentrations of the drug after about 6 h in the 37 degree incubator, After 24 h into the incubator, add 20μL of MTS solution per well and absorb absorbance is determined after four h of placement in the incubator again Value.

2.7 Q-PCR determines cellular inflammatory factor expression

Wash the cell samples with PBS and add 1 ml of Trizol, leave on ice for 5 min, pipett and mix well and put in 1.5 mL without R Na enzyme EP tube, add chloroform 200 μL, shake mix well and let stand for 10 min. Centrifuge at 4 degrees 12000 rpm for 15 min and aspirate the supernatant with an R NA-free tip and add to 500 μL isopropanol is shaken to stand for 10 min at 4 ℃12000 rpm Collect the RNA pellet at 10 min, wash with 75% absolute ethanol and centrifuge for five min to supernatant, add an appropriate amount of DEPC after natural air drying Water dissolves the precipitation.Then the RNA concentration was measured on the machine, and the amount of RNA in each group was adjusted to the same level,and the reverse transcription system was configured. After reverse transcription is complete, cDNA, fluorescent dyes, and different primers are configured into different mixed systems on the machine.

2.8 Western blot detects protein expression

After washing the cell sample with PBS, according to the amount of cells, add the RIPA lysate with protease inhibitors, leave it in the refrigerator at 4 degrees for 30 minutes, take the cell mixture and centrifuge to remove the supernatant to take the pellet. Measure cell concentrations in different groups and adjust to the same concentration. Each histone protein was isolated with 8% SDSPAGE, electrokined in ice water for 80 min, and the protein was molded into a PVDF membrane with BSA After the blocking solution was blocked for one hour, it was combined with the primary antibody, placed in a 4-degree refrigerator overnight, combined with the secondary antibody for 60 minutes after washing the film,washed the film again, and finally obtained the electrophoresis band diagram [23].

2.9 Statistical analysis

Experimental data are expressed in±s. The experimental results were statistically analyzed using GraphPad Prism 8 software, and the multi-component comparison used one-way variance (One-Way ANOVA In the analysis, P＜0.05 was statistically significant for the difference.

3. Results

3.1 Silicosis differential gene expression screening

In order to reveal the expression of silicosis differential genes,the single-cell sequencing of lung parenchyma in 7 normal and 10 patients with silicosis was first analyzed [11], and a total of 2 065 differential genes were obtained, and the data were normalized accordingly Analysis of limma packets screened out 100 up-anddown-regulated genes, respectively. Secondly, the expression spectrum chip data G SE188520 was downloaded from the GEO database, which included 6 normal mice and 6 silica induced mice 2 291 differential genes were screened by differential genes in the R package, and 100 up-and-down-regulated genes were screened out. Finally, using network pharmacology, it was retrieved through The Genes, CTD, OMIM databases, and a total of 803 genes related to silicosis disease were obtained. 1 of 29 intersecting genes were obtained, which are thought to be genes closely linked to silicosis.See Figure 1

3.2 Analysis of KEGG pathway enrichment of key difference genes in silicosis

Firstly, the differential genes of normal people and silicosis patients were analyzed for KEGG pathway enrichment, and a total of 13 were screened out based on P＜0.05 The KEGG pathway,in which extracellular matrix-receptor interactions are associated with the formation of fibrosis. Human papillomavirus infection is associated with cell cycle and apoptosis, IL-17 signaling pathway(IL-17 Signaling pathway) is related to cell growth, differentiation,inflammation, etc. Secondly, after enriching the intersecting gene pathways of the network pharmacology database, a total of 66 pathways were screened, and the main signaling pathways were AGE-RAGE signaling pathway and TNF Signaling pathways, IL-17 signaling pathways, receptor factor interaction signaling pathways,etc. Finally, 22 pathways were screened out after the differential genes of silicosis rats were enriched, mainly related to gastric acid secretion, IL-17 signaling pathway, malaria, amoeba disease and other signaling pathways, and the results showed IL-17 Signaling pathways may be key pathways that affect silicosis. See Figure 2.

Fig 1 Screen of key silicosis differential genes

3.3 GO enrichment analysis

GO analysis of differential genes in silicosis patients by R language showed a total of 282 biological processes, 33 cellular components,and 35 molecular function. The main results include ciliary motility,extracellular matrix tissue, activation of T cells, participation in the activation of neutrophils in the immune response and other biological processes, G protein-coupled receptor binding, collagen binding, growth factor binding, Wnt protein binding and other molecular functions, active cilia, collagen-containing extracellular matrix, basement membrane and other cellular components.Secondly, A GO analysis of the network pharmacology database showed 2576 biological processes, 6cellular components, and 142 Molecular functions, biological processes include responses to lipopolysaccharides, cellular responses to biological stimuli, reactive oxygen metabolism processes, regulation of inflammatory responses,etc., molecular functions include cytokine receptor binding, protein serine / threonine kinase activity, receptor ligand activity, G Proteincoupled receptor binding, etc., cell components including vesicle cavity, transcriptional regulator complex, etc., and finally GO analysis of differential genes in silicosis mice, including Toll-like receptor signaling pathways, cell response to tumor necrosis factor,Molecular functions such as cell-to-biological stimulus response,glycosaminoglycan binding, G protein-coupled receptor binding, and chemokine activity are roughly the same as the above two groups of cell components. See Figure 3.

3.4 PPI Network Diagram

Fig 2 Analysis of differential gene KEGG pathway

In the PPI network diagram, the target gene is represented by nodes, and the connections between the targets represent the interaction between genes, and the lowest interaction score is set to the highest confidence. The target genes contained in the IL-17 signaling pathway were introduced into the STRING database, and the interaction scores between nodes were arranged from high to low, and most of the target genes with higher scores were related to MAPK Signaling pathways are related, and most of these targets are related to the inflammatory process, indicating that the MAPK signaling pathway may play a key role in the IL-17 signaling pathway, thereby promoting high expression of inflammatory factors to promote the development of silicosis. See Figure 4.

3.5 Molecular docking results

The key protein of the IL-17 signaling pathway was molecularly docked with the small molecule compound Tetrandrine, and the binding position of the two was determined, that is, the size of the active pocket, and the hydrogen bonding position between the small molecule and the key protein was shown. The docking affinity fraction is calculated by Schr?dinger. The molecular docking results show that there is a good binding force between the Tetrandrine and the key targets RAF and ERK in the MAPK pathway. The above results indirectly prove that the Tetrandrine may regulate the RAF/MEK/ERK pathway in the MAPK signaling pathway Plays a key role in influencing early silicosis. See Figure 5.

3.6 Cell experiments

3.6.1 Effect of Tetrandrine on the apoptosis rate of macrophages and epithelial cells

In the macrophage line THP-1, as the drug concentration increases,the ability of Tetrandrine to inhibit the proliferation of macrophages gradually increases, and when the drug concentration is 5 μmol/L,the apoptosis rate of the cells is 20 %, when the drug concentration is 10 μmol/L, the apoptosis rate of the cells is 83%, and the data show that when the dosing concentration is greater than 5 μmol/L, the apoptosis rate is significantly increased, not suitable for cell experiments, and the dosing concentration in macrophages is set to 5 μmol/L is the most appropriate concentration. In the epithelial cell line BEAS-2B, the apoptosis rate of epithelial cells increases linearly as the drug concentration increases, and at 5 μmol/L, the apoptosis rate of cells is 5%. At 10 μmol/L, the apoptosis rate of cells was 16%. According to the calculation of the apoptosis rate at different dosing concentrations, the concentration is the most effective when the dosing concentration is 20 μmol/L.

Fig 3 GO enrichment analysis of differential gene

3.6.2 Effects of Tetrandrine on TNF-α, TGF-β, IL-1β, IL-1αlevels in macrophages and epithelial cells

TNF-α is mainly released by macrophages and activated T cells,which can not only stimulate other fibrogels, but also stimulate TGF-β promote collagen formation. IL-1β, IL-1α can induce adsorption of inflammatory cells, regulate other inflammatory factors, and promote the proliferation of fibroblasts. P CR was used to detect the expression of inflammatory factors in the plus silicon group and the dosing group. Compared with the normal group, macrophage plus silicon group of TNF-α, TGF-β, IL-1β, ILExpression of 1α was elevated (P＜0.05), of which the expression of TNF-α and TGF-β increased significantly ( P＜0.01), compared with the silicon group, the Tetrandrine base group of TNF-α, TGF-β,IL-1β, I The expression of L-1 α decreased significantly (P＜0.05),of which the expression of TNF-α decreased most significantly P＜0.001), see Figure 7A. Compared with the normal group, epithelial cells plus silicon group of TNF-α, TGF-β, IL-1β, IL- The expression of 1 α was increased (P＜0.05), and the expression of TNF-α was significantly increased (P＜0.001 Compared with the silicon group,the TNF-α, TGF-β, IL-1β, IL-1 α of the dosing group the expression of the ＜a0＞P＜0.05) decreased significantly.

Fig 4 IL-17 signaling pathway protein interaction

Fig 5 Docking of tetrandrine and pathway protein pattern diagram

Tab 1 Effect of tetrandrine on apoptosis of macrophages and epithelial cells(n=3,±s)

Tab 1 Effect of tetrandrine on apoptosis of macrophages and epithelial cells(n=3,±s)

Cell Grouping(μmol/L) Apoptosis(%) F P 5 22.01±1.53 Macrophages 10 82.98±3.8 50 87.05±1.2 100 83.27±0.88 358.30 ＜0.000 1 5 4.5±0.82 Epithelial cells 10 15.68±0.64 50 72.51±2.6 433.70 ＜0.000 1 100 78.18±0.8

3.6.3 Effect of Tetrandrine on E-Cadherin, N-Cadherin and Vimentin levels in epithelial cells

Epithelial mesenchymal transformation plays an important role in the progression of silicosis, epithelial cell damage can lead to transformation and thus induce pulmonary fibrosis, in the process of transformation, the expression of epithelial cell marker E-cadherin decreases, while mesenchymal cell markers N-cadherin and vimentin are gradually elevated, so that epithelial cells transform into fibroblasts. The expression of epithelial interstitial conversion-related proteins was detected by WB, and the expression of E-Cadherin in the silicon-plus group decreased significantly (P＜0.01)compared with the normal group N-Cadherin expression increased significantly (P＜0.000 1) and Vimentin expression was elevated but not significant. Compared with the silicon group, the expression of E-Cadherin in the dosing group increased significantly (P＜0.01) and the expression of N-Cadherin decreased significantly (P＜0.000 1),Vimentin expression decreased but was not statistically significant.The results proved that Tetrandrine plays a role in inhibiting epithelial cells for epithelial interstitial conversion. See Figure 6.

Tab 2 Expression of TNF-α,TGF-β,IL-1β,IL-1α in macrophages and epithelial cells (n=3,±s)

Tab 2 Expression of TNF-α,TGF-β,IL-1β,IL-1α in macrophages and epithelial cells (n=3,±s)

Cell index Grouping Apoptosis F P Control 1 Silicon 2.92±0.3792 Tet 1.19±0.4712 TNF-β TGF-α 27.78 ＜0.001 Control 1 65.94 ＜0.000 1 Macrophages Silicon 2.49±0.2955 Tet 1.47±0.2512 IL-1α Control 1 Silicon 1.41±0.2628 Tet 0.35±0.2511 19.45 ＜0.01 Control 1 IL-1β Silicon 1.37±0.2458 Tet 0.49±0.0600 27.83 ＜0.01 Control 1 TGF-α Silicon 2.47±0.21 Tet 0.78±0.4106 35.02 ＜0.001 Control 1 TNF-β 24.91 ＜0.01 Epithelial cells Silicon 4.43±1.2536 Tet 0.49±0.2888 IL-1α Control 1 Silicon 2.333±0.4343 Tet 0.39±0.060 IL-1β 46.10 ＜0.001 Control 1 Silicon 2.74±0.5892 Tet 0.52±0.1877 31.97 ＜0.001

Fig 6 Expression of E-Cadherin、N-Cadherin、Vimentin protein in BEAS-2B epithelial cells in each group

3.6.4 Effect of powdered antihexane on the level of phosphorylated RAF and ERK protein in macrophages

The RAF protein is a protein kinase encoded by the RAF gene that plays an important role in the MAPK signaling pathway, and the RAF protein regulates downstream MEK Protein and ERK protein activity, thereby regulating the proliferation, apoptosis,inflammatory response and other functions of cells. The expression of MAPK-related proteins was detected using WB, adding the RAF proteins and E of the silicon group compared to the normal group The phosphorylation level of RK protein was significantly increased (P＜0.01). Compared with the silicon-added group, the phosphorylation levels of RAF protein and ERK protein in the powder antihexane group were significantly reduced (P＜0.000 1),it is shown that powder antihexane can inhibit the phosphorylation levels of RAF protein and ERK, thereby participating in the functional regulation of cells. See Figure 7.

Figure7 Expression of Phospho-c-Raf、Phospho-ERK1/2 in THP-1 macrophage cells in each group(n=3,±s)

Tab 3 Expression of E-Cadherin、N-Cadherin、Vimentin protein in BEAS-2B epithelial cells in each group(n=3,±s)

Tab 3 Expression of E-Cadherin、N-Cadherin、Vimentin protein in BEAS-2B epithelial cells in each group(n=3,±s)

Index Grouping Expression F P E-Cadherin Control 1.000 0±0.000 0 Silicon 0.490 0±0.105 3 Tet 1.070 0±0.147 3 N-Cadherin 27.5 ＜0.001 Control 1.000 0±0.000 0 Silicon 1.350 0±0.053 4 Tet 0.366 6±0.030 5 Vimentin 651.2 ＜0.000 1 Control 1.000 0±0.000.0 Silicon 1.100 0±0.245 8 Tet 0.700 0±0.185 9 4.875 NS

Tab 4Expression of Phospho-c-Raf、Phospho-ERK1/2 in THP-1 macrophage cells in each group(n=3,±s)

Tab 4Expression of Phospho-c-Raf、Phospho-ERK1/2 in THP-1 macrophage cells in each group(n=3,±s)

Index Grouping Expression F P Phospho-c-Raf Control 1±0 64.04 ＜0.000 1 Control 1±0 Silicon 1.556±0.088 8 Tet 0.8312±0.029 6 147.7 ＜0.000 1

4. Discussion

Silicosis is a common occupational disease, mainly inflammation and fibrosis of the lungs, which can lead to heart failure in severe cases and is irreversible. Most patients with silicosis present at an advanced stage, which seriously affects the quality of life and has a poor prognosis, and the current treatment of silicosis is not optimistic [24, 25]. In recent years, the treatment and pathogenesis of silicosis have gradually become a hot topic, and related articles have focused on the pathogenesis of silicosis, and even a small number of methods have been successfully carried out in silicosis models [3],but the relevant research on the development mechanism of silicosis is still insufficient. Powder antihexane is a drug extracted from the Chinese medicine Han anti-hexane plant, which is a clinical drug used to treat silicosis [26], and is well known for its effects on treating cancer and tumors [27] [28]. Studies have reported that powdered antihexane has a significant role in inhibiting the inflammatory response and regulating oxidative stress [10]. Therefore, from the perspective of bioinformatics and network pharmacology, this study further explores the mechanism of action of powder antihexane in the treatment of early silicosis, which provides a theoretical basis for subsequent clinical research.

Bioinformatics and network pharmacology have become an important means to study disease pathways and key proteins, this study for the first time silicosis patients, silica instillation mice RNA sequencing data and network database related to silicosis related targets for synchronous analysis, from different species, different sources of data to analyze the signaling pathways affecting early silicosis. Studies have shown that the development of silicosis is closely related to a variety of signaling pathways. Zhang et al.inhibited the NF-κB signaling pathway by using the autophagy inhibitor 3-methyladenine (3-MA). Mediated development of early lung inflammation in silicosis [29]. Elhameed AG found that aspirin and krill oil can downregulate NF-κ B and the inflammatory factor TGF-β1 in silica mouse lung tissue, thereby reducing silica-induced lung damage [5]. Yan et al. demonstrated that the PI3K/Akt pathway plays a key role in silica-induced pulmonary fibrosis [30]. Helal et al. reduced P-AKT and mTOR levels in mice with intrapulmonary tissue disease by using the β receptor blocker carvedilol This alleviates silica-induced pulmonary fibrosis [31]. Wu et al. found that rhubarb stinging pill (DHZCP) can be used by adjusting the p38 MAPK/TGF-β 1/Smad pathway, Reduces stimulation by inflammatory factors and thereby inhibits silicosis progression [32].Wang et al. discovered P38 MAPK using the p38 MAPK-specific inhibitor SB203580 Pathways are key to silicosis and EMT in rats[33]. Related articles mention that inflammatory factor IL-6 induces EMT in gastric cancer cells through JAK2/STAT3, while IL-6 The occurrence of FMT can also be induced by the STAT3 signal[34]. CHeng et al. found that metformin inhibits the inflammatory response of macrophages by inhibiting the activation of fibroblasts by inhibiting the TGF-β1 signal [35]. Reports indicate that there is more than one pathway that affects the development of silicosis, but it is still inconclusive which pathway is the most critical pathway to silicosis. From the analysis of GO and KEGG enrichment, it was found that the IL-17 signal path existed in three different data sets[36], indicating that the IL-17 signaling pathway plays an extremely important role in the early development of silicosis. The relevant literature confirms that interleukin-17 is an important inflammatory cytokine of the interleukin family, and high levels of interleukin-17 lead to worsening lung diseases, including pneumonia, asthma and other diseases, and pirfenidone can inhibit interleukin 1 The secretion of 7A to improve silica-induced lung inflammation in mice is consistent with the conclusions of this study, confirming that theIL-17 signaling pathway plays an important role in the development of silicosis.

The PPI network diagram shows the interaction between different targets, and its results confirm that the MAPK pathway plays an important role in the IL-17 signal path , and the targets such as IL-6,TNF-α, and CCL2 are in the PPI The network diagram has a high impact score, and Shambhoo Sharan et al. demonstrated that IL-6 expression is closely related to silica-induced pulmonary fibrosis[37], cheng et al. demonstrated Decreased expression of TNF-α can reduce the activation of fibroblasts [35]. CCL2 is a chemotaxi factor that mediates inflammation and can promote the early progression of silicosis by promoting the release of inflammatory factors by macrophages, all of which indicate that the screened IL-17 signaling pathway plays an important role in early silicosis.

Powder antihexane was approved by the Food and Drug Administration as a new drug for the treatment of silicosis, extracted from Materia Medica powder anti-self, and plays an important role in anti-inflammatory, anti-fibrosis, immune response, and antioxidant,but its anti-fibrosis therapeutic effect is still controversial, and the mechanism of the influence of powder antihexane in the progression of silicosis has not been determined [38]. In this study,it was experimentally found that powder antihexane can inhibit the phosphorylation level and E MT of the key pathways RAF and ERK proteins Occurrence, in which epithelial cell markers E-Cadherin are upregulated, mesenchymal cell markers N-Cadherin and Vimentin are expressed Downgraded. PCR experiments have found that powder antihexane significantly inhibits the expression of inflammatory factors IL-6, TNF-α, TGF-β in macrophages and epithelial cells.

In summary, powder antihexane has a significant inhibitory effect on the development of early silicosis, and the predicted mechanism is to reduce phosphorylation of the RAF/MEK/ERK pathway, inhibit the conversion of epithelial cells to interstitial cells in early silicosis lung tissue, reduce the release level of macrophage and epithelial inflammatory factors, thereby affecting the progression of early silicosis. The research provides a theoretical basis and experimental basis for the treatment of silicosis, and lays a foundation for the research and development of silicosis drugs and subsequent further development. This study still has limitations in terms of data adoption, most of the data comes from the database, there may be incomplete data, the key pathway verification only stays at the cellular level, it has not been verified in animals, and the mechanism of powder antihexane in the treatment of early silicosis needs to be further studied.

Conflict of Interest Description: All authors do not have any conflict of interest

Author Contribution: Liang Chao, as the first author, conducted the article conception, experimental design, completed the analysis of bioinformatics and network pharmacology, and carried out the operation of cell experiments. Zhou Jiawei and Liu Yafeng are responsible for the guidance and adjustment of bioinformatics analysis. Guo Jianqiang and Wang Qingsen gave help in the operation of the experiment. Su Yixin and Hu Chunxiao contributed to the analysis of experimental data. Xie Jun, Xing Yingru, Hu Dong,and Wu Jing participated in the writing and revision of the paper. All authors were involved in the design and revision of the paper. All authors read and approved the final version of the manuscript.