Inhibitory Effects of 3-Cyclopropylmethoxy-4-(difluoromethoxy) Benzoic Acid on TGF-β1-Induced Epithelial–Mesenchymal Transformation of In Vitro and Bleomycin-Induced Pulmonary Fibrosis In Vivo

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by lung inflammation and excessive deposition of extracellular matrix components. Transforming growth factor-β1 (TGF-β1) induced epithelial–mesenchymal transformation of type 2 lung epithelial cells leads to excessive extracellular matrix deposition, which plays an important role in fibrosis. Our objective was to evaluate the effects of 3-cyclopropylmethoxy-4-(difluoromethoxy) benzoic acid (DGM) on pulmonary fibrosis and aimed to determine whether EMT plays a key role in the pathogenesis of pulmonary fibrosis and whether EMT can be used as a therapeutic target for DGM therapy to reduce IPF. Firstly, stimulation of in vitro cultured A549 cells to construct EMTs with TGF-β1. DGM treatment inhibited the expression of proteins such as α-SMA, vimentin, and collagen Ⅰ and increased the expression of E-cadherin. Accordingly, Smad2/3 phosphorylation levels were significantly reduced by DGM treatment. Secondly, models of tracheal instillation of bleomycin and DGM were used to treat rats to demonstrate their therapeutic effects, such as improving lung function, reducing lung inflammation and fibrosis, reducing collagen deposition, and reducing the expression of E-cadherin. In conclusion, DGM attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in rats.


Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease that progresses to loss of lung function [1]. IPF is characterized by structural remodeling of lung tissue, inflammatory responses in the interstitium of the lung, destruction of the alveolar structure, and massive deposition of the extracellular matrix (ECM) components in the interstitium and in the basement membrane, resulting in progressive destruction of the lung structure and irreversible loss of lung function [2][3][4]. The pathogenesis of IPF involves several risk factors, including environmental exposure, smoking, chronic viral infections, and certain co-morbidities, among which genetic risk factors are the most critical [5]. IPF has a median survival time of 2 to 4 years [6]. The incidence of IPF in North America and Europe is higher than in South America and East Asia, and is more prevalent among older patients [4,7]. Nintedanib and pirfenidone (PFD), which were approved by the United States Food and Drug Administration in 2014 for the treatment of IPF, can restore vital capacity of the lung, but are not associated with a reduction in mortality [8]. With the high

Effects of DGM on TGF-β1 Induced Viability of A549 and H1299 Cells
To simulate the process of EMT of lung epithelial cells in vivo, as shown in Figure 1B,C, we selected two types of lung epithelial cells, A549 and H1299, and treated them with 2.5-20 ng/mL TGF-β1 for 24 h, 36 h, 48 h, and 72 h. We found that after 24 h, 36 h, and 72 h of stimulation, the two types of cells did not proliferate, but when TGF-β1 of various concentrations was used after 48 h of stimulation, the two types of cells could proliferate in a concentration-dependent manner; thus, 5 ng/mL TGF-β1 was selected for subsequent experiments. The incubation condition at 48 h mimics the process of EMT in vivo. To detect the cytotoxicity of DGM ( Figure 1A) for subsequent experiments, we treated two types of epithelial cells with 50-200 µM DGM, as shown in Figure 1D,E. The MTT assay was used to detect the concentration of DGM within 200 µM, which had no toxic effect on the two cell lines after 48 h of treatment. Next, both cell lines were treated with TGF-β1 (5 ng/mL) for 48 h in combination with DGM. We found that DGM was effective in inhibiting TGF-β1 induced cell proliferation ( Figure 1F,G). These results indicated that DGM did not exhibit toxicity in the two types of epithelial cells within our experimental dose range and also that DGM could inhibit the proliferation of epithelial cells stimulated by TGF-β1. treated two types of epithelial cells with 50-200 μM DGM, as shown in Figure 1D,E MTT assay was used to detect the concentration of DGM within 200 μM, which ha toxic effect on the two cell lines after 48 h of treatment. Next, both cell lines were tre with TGF-β1 (5 ng/mL) for 48 h in combination with DGM. We found that DGM wa fective in inhibiting TGF-β1 induced cell proliferation ( Figure 1F,G). These results cated that DGM did not exhibit toxicity in the two types of epithelial cells within ou perimental dose range and also that DGM could inhibit the proliferation of epithelial stimulated by TGF-β1. Values are pres as mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group. ## 0.0001 vs. Control group; * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001 vs. TGF-β1 group.

DGM Inhibited the Activation of the Smad Signaling Pathway Stimulated by TGF-β1
To further investigate the mechanism of inhibition by DGM of TGF-β1 induced EMT of A549, we investigated the effects of DGM on activation of the Smad signaling pathway. As shown in Figure 3A, after stimulation of A549 cells with TGF-β1, the protein expression of Smad 2/3 changed hardly, and the protein level of phosphorylated Smad2/3 was obviously increased. After DGM treatment, the expression of phosphorylated Smad2/3 decreased markedly, especially at the concentration of 200 µM. The ratio of p-Smad2/3 to Smad2/3 for different groups is shown in Figure 3B. The above results suggest that DGM inhibition of EMT-related protein expression could be achieved by inhibiting activation of the Smad signaling pathway. of Smad 2/3 changed hardly, and the protein level of phosphorylated Smad2/3 was obviously increased. After DGM treatment, the expression of phosphorylated Smad2/3 decreased markedly, especially at the concentration of 200 μM. The ratio of p-Smad2/3 to Smad2/3 for different groups is shown in Figure 3B. The above results suggest that DGM inhibition of EMT-related protein expression could be achieved by inhibiting activation of the Smad signaling pathway.

DGM Improved Bleomycin-Induced Weight Loss and Decreased Lung Function in Rats
To study the anti-IPF activity of DGM in vivo, we treated rats with bleomycin (BLM) to establish a model of pulmonary fibrosis. As shown in Figure 4A, BLM could lead to a continuous loss of body weight in the first 7 days when compared to the control group Compared to the BLM-induced group, the weight of rats in the DGM treatment groups improved markedly. Next, we used lung function indicators (PFT Animal Lung Function Test System) to assess the severity of pulmonary fibrosis in each treatment group. Forced vital capacity (FVC), deep inspiratory volume (IC), forced expiratory volume (FEV), maximum mid-expiratory flow (MMEF), supplementary expiratory volume (ERV), and other indices in the BLM group were significantly lower than in the control group, while lung function was significantly improved by DGM administration. The DGM-treated group showed a stronger effect than PFD treatment at the same dose, with 60 mg/kg DGM achieving a greater effect than 30 mg/kg DGM ( Figure 4B-G). When comparing the contro group with the BLM-induced group, the lung coefficient of the model group was much higher than that of the control group, and after DGM treatment, the lung coefficient decreased significantly, with the high-dose treatment achieving a better effect than that of lower doses ( Figure 4H).

DGM Improved Bleomycin-Induced Weight Loss and Decreased Lung Function in Rats
To study the anti-IPF activity of DGM in vivo, we treated rats with bleomycin (BLM) to establish a model of pulmonary fibrosis. As shown in Figure 4A, BLM could lead to a continuous loss of body weight in the first 7 days when compared to the control group. Compared to the BLM-induced group, the weight of rats in the DGM treatment groups improved markedly. Next, we used lung function indicators (PFT Animal Lung Function Test System) to assess the severity of pulmonary fibrosis in each treatment group. Forced vital capacity (FVC), deep inspiratory volume (IC), forced expiratory volume (FEV), maximum mid-expiratory flow (MMEF), supplementary expiratory volume (ERV), and other indices in the BLM group were significantly lower than in the control group, while lung function was significantly improved by DGM administration. The DGM-treated group showed a stronger effect than PFD treatment at the same dose, with 60 mg/kg DGM achieving a greater effect than 30 mg/kg DGM ( Figure 4B-G). When comparing the control group with the BLM-induced group, the lung coefficient of the model group was much higher than that of the control group, and after DGM treatment, the lung coefficient decreased significantly, with the high-dose treatment achieving a better effect than that of lower doses ( Figure 4H).

DGM Reduced BLM-Induced Lung Inflammation and Fibrosis
To investigate the effects of DGM on lung fibrosis in rats, we used a BLM-induced rat model of pulmonary fibrosis. On Day 0, the BLM group, the DGM group, and the PFD group were subjected to a tracheal drip BLM solution, and animals were euthanized around Day 28. During this period, the control group and the BLM group were injected with normal saline, and the administration group was injected with different drugs for treatment. As shown in Figure 5A, the lung tissue of the control group by H&E staining exhibited an intact alveolar wall structure, an intact bronchial structure, and no shedding or hyperplasia of the bronchial epithelial cells, while the alveolar wall structure of the lung tissue of rats in the BLM group was destroyed, pulmonary consolidation occurred, bronchial epithelial cells exhibited sloughing and hyperplasia, and secretions could be seen in the lumen. The lung consolidation of the PFD group was significantly reduced, although

DGM Reduced BLM-Induced Lung Inflammation and Fibrosis
To investigate the effects of DGM on lung fibrosis in rats, we used a BLM-induced rat model of pulmonary fibrosis. On Day 0, the BLM group, the DGM group, and the PFD group were subjected to a tracheal drip BLM solution, and animals were euthanized around Day 28. During this period, the control group and the BLM group were injected with normal saline, and the administration group was injected with different drugs for treatment. As shown in Figure 5A, the lung tissue of the control group by H&E staining exhibited an intact alveolar wall structure, an intact bronchial structure, and no shedding or hyperplasia of the bronchial epithelial cells, while the alveolar wall structure of the lung tissue of rats in the BLM group was destroyed, pulmonary consolidation occurred, bronchial epithelial cells exhibited sloughing and hyperplasia, and secretions could be seen in the lumen. The lung consolidation of the PFD group was significantly reduced, although pathological changes, such as thickening of the alveolar wall, hyperemia and edema of lung tissue, and cell infiltration of pulmonary interstitial cells, could still be observed. Lung inflammation was obviously reduced in the DGM group, and the pathological score of the high-dose DGM group had a better effect than that of the low-dose group. Masson staining was used to evaluate the fibrosis and collagen deposition of rat lung tissue. Compared to the control group, the area of lung tissue fibrosis of the BLM group was significantly increased, the thickness of the alveolar membrane was significantly thicker, and the lung tissue was significantly destroyed. Compared to the BLM group, the degree of collagen deposition and fibrosis in the DGM group and the PFD group improved significantly, collagen deposition in the low-dose DGM group was less than that of the PFD group, and the effect of the high-dose DGM group was better than that of the low-dose group. These results suggest that DGM reduces the degree of lung damage and fibrosis caused by BLM at doses of 30 mg/kg and 60 mg/kg ( Figure 5B). pathological changes, such as thickening of the alveolar wall, hyperemia and edema of lung tissue, and cell infiltration of pulmonary interstitial cells, could still be observed. Lung inflammation was obviously reduced in the DGM group, and the pathological score of the high-dose DGM group had a better effect than that of the low-dose group. Masson staining was used to evaluate the fibrosis and collagen deposition of rat lung tissue. Compared to the control group, the area of lung tissue fibrosis of the BLM group was significantly increased, the thickness of the alveolar membrane was significantly thicker, and the lung tissue was significantly destroyed. Compared to the BLM group, the degree of collagen deposition and fibrosis in the DGM group and the PFD group improved significantly, collagen deposition in the low-dose DGM group was less than that of the PFD group, and the effect of the high-dose DGM group was better than that of the low-dose group. These results suggest that DGM reduces the degree of lung damage and fibrosis caused by BLM at doses of 30 mg/kg and 60 mg/kg ( Figure 5B).

DGM Reduced the Expression of α-SMA, Hydroxyproline, and Total Collagen in Lung Tissue
In the pulmonary fibrosis process, fibroblast differentiation into myofibroblasts is a very critical stage, and the expression of α-SMA was characteristic of myofibroblasts. Hydroxyproline is a main component of collagen, and overexpression of collagen is also considered a characteristic of the ECM. Next, the expression of α-SMA, HYP, and total collagen in lung tissue was detected by immunohistochemical staining and enzyme-linked immunosorbent assays. As shown in Figure 6A, compared to the control group, the expression of α-SMA in the BLM group increased significantly, and its expression was significantly reduced after DGM and PFD treatment. As shown in Figure 6B,C, the expression of HYP and total collagen increased significantly in the BLM group, and treatment with DGM (30 mg/kg and 60 mg/kg) treatment significantly improved their expression.

DGM Reduced the Expression of α-SMA, Hydroxyproline, and Total Collagen in Lung Tis sue
In the pulmonary fibrosis process, fibroblast differentiation into myofibroblasts is very critical stage, and the expression of α-SMA was characteristic of myofibroblasts. H droxyproline is a main component of collagen, and overexpression of collagen is also co sidered a characteristic of the ECM. Next, the expression of α-SMA, HYP, and total coll gen in lung tissue was detected by immunohistochemical staining and enzyme-linked im munosorbent assays. As shown in Figure 6A, compared to the control group, the expre sion of α-SMA in the BLM group increased significantly, and its expression was signi cantly reduced after DGM and PFD treatment. As shown in Figure 6B,C, the expressio of HYP and total collagen increased significantly in the BLM group, and treatment wi DGM (30 mg/kg and 60 mg/kg) treatment significantly improved their expression.

DGM Reduced the Content of Inflammatory Cells and Inflammatory Cytokines in Rat Bron choalveolar Lavage Fluid
We examined the effects of DGM on the inflammatory cell infiltration in the bro choalveolar lavage fluid (BALF) of rats. As shown in Figure 7A, compared to the contr group, the total number of inflammatory cells, neutrophils, lymphocytes, and eosinoph in the BALF of rats in the BLM group increased significantly. At the same time, compare to rats in the BLM group, DGM administration significantly reduced the number of tot inflammatory cells, neutrophils, lymphocytes, and eosinophils in BALF, and the effect 60 mg/kg was better than 30 mg/kg treatment.
To assess whether DGM influenced TNF-α, TGF-β1, IL-6, and other fibrotic facto in the fibrosis process of rats, we used the ELISA assay to detect cytokine levels in th BALF of rats. Compared to the control group, BLM significantly increased the productio of TNF-α, TGF-β1, IL-6, MCP-1, and MMP-7 production. Compared to the BLM grou DGM administration inhibited the increase of TNF-α, TGF-β1, IL-6, MCP-1, and MMP

DGM Reduced the Content of Inflammatory Cells and Inflammatory Cytokines in Rat Bronchoalveolar Lavage Fluid
We examined the effects of DGM on the inflammatory cell infiltration in the bronchoalveolar lavage fluid (BALF) of rats. As shown in Figure 7A, compared to the control group, the total number of inflammatory cells, neutrophils, lymphocytes, and eosinophils in the BALF of rats in the BLM group increased significantly. At the same time, compared to rats in the BLM group, DGM administration significantly reduced the number of total inflammatory cells, neutrophils, lymphocytes, and eosinophils in BALF, and the effect of 60 mg/kg was better than 30 mg/kg treatment. accumulation of inflammatory cells in rat lung tissue and secreted several cytokines to accelerate the fibrosis process, while the intervention with DGM reduced the number of inflammatory cells in the lungs of rats and attenuated the secretion of these cytokines.

DGM Repressed the BLM-Induced EMT and Fibroblast Activation in Rat Lung Tissue
To determine whether DGM could alleviate BLM-induced pulmonary fibrosis, the expression of the EMT marker proteins and fibroblast activation marker protein: vimentin, E-cadherin, α-SMA and collagen I, collagen III, as well as the enzyme MMP-2 related to the degradation of the ECM of lung tissue, were detected. As shown in Figure  8A, the expression of lung tissue vimentin, α-SMA, collagen I, collagen III, and MMP-2 was significantly elevated, and the expression of E-cadherin was significantly reduced compared to the control group. The DGM 30 mg/kg and 60 mg/kg groups were able to significantly down-regulate the expression of the vimentin, α-SMA, collagen I, collagen III, and MMP-2 proteins in lung tissue and up-regulate the expression of E-cadherin. Statistical analysis of protein levels showed that DGM could inhibit vimentin, α-SMA, collagen I, collagen III, and MMP-2 expression and showed a dose-dependent relationship and higher E-cadherin expression in a dose-dependent manner ( Figure 8B-G). The findings showed that DGM could inhibit the EMT of rat lung tissue induced by BLM, thus inhibiting the fibroblast activation to alleviate pulmonary fibrosis. To assess whether DGM influenced TNF-α, TGF-β1, IL-6, and other fibrotic factors in the fibrosis process of rats, we used the ELISA assay to detect cytokine levels in the BALF of rats. Compared to the control group, BLM significantly increased the production of TNF-α, TGF-β1, IL-6, MCP-1, and MMP-7 production. Compared to the BLM group, DGM administration inhibited the increase of TNF-α, TGF-β1, IL-6, MCP-1, and MMP-7 in a dose-dependent manner ( Figure 7B-F). These results showed that BLM induced the accumulation of inflammatory cells in rat lung tissue and secreted several cytokines to accelerate the fibrosis process, while the intervention with DGM reduced the number of inflammatory cells in the lungs of rats and attenuated the secretion of these cytokines.

DGM Repressed the BLM-Induced EMT and Fibroblast Activation in Rat Lung Tissue
To determine whether DGM could alleviate BLM-induced pulmonary fibrosis, the expression of the EMT marker proteins and fibroblast activation marker protein: vimentin, E-cadherin, α-SMA and collagen I, collagen III, as well as the enzyme MMP-2 related to the degradation of the ECM of lung tissue, were detected. As shown in Figure 8A, the expression of lung tissue vimentin, α-SMA, collagen I, collagen III, and MMP-2 was significantly elevated, and the expression of E-cadherin was significantly reduced compared to the control group. The DGM 30 mg/kg and 60 mg/kg groups were able to significantly down-regulate the expression of the vimentin, α-SMA, collagen I, collagen III, and MMP-2 proteins in lung tissue and up-regulate the expression of E-cadherin. Statistical analysis of protein levels showed that DGM could inhibit vimentin, α-SMA, collagen I, collagen III, and MMP-2 expression and showed a dose-dependent relationship and higher E-cadherin expression in a dose-dependent manner ( Figure 8B-G). The findings showed that DGM could inhibit the EMT of rat lung tissue induced by BLM, thus inhibiting the fibroblast activation to alleviate pulmonary fibrosis.

Discussion
Alveolar epithelial cell injury and subsequent repair are the main factors involved the irreversible pathogenesis of IPF [28,29]. Alveolar epithelial cells are the main play in the composition of alveolar structures and play a critical role in lung tissue homeosta [30]. Type II alveolar epithelial cells are the most important cells on the alveolar surfa and when stimulated by environmental pathogens, in order to protect the alveolar surfa type II alveolar epithelial cells repair epithelial tissue through proliferation and differe tiation [31]. In this study, DGM inhibited cell proliferation induced by TGF-β1 in A5 cells and H1299 cells, suggesting that DGM could slow the development of fibrosis improving the proliferation of type II alveolar epithelial cells. To rule out that the inhi tory effects of DGM in the proliferation of type II alveolar epithelial cells induced by TG β1 may be related to toxicity, we evaluated cell viability using the MTT assay, and t results showed that DGM had no significant toxic effect on both cell lines within the in cated concentration range. In this study, we demonstrated that DGM could inhibit TG β1-induced proliferation of A549 cells, suggesting that DGM could slow down the dev opment of pulmonary fibrosis at some level by inhibiting the proliferation of type II e thelial cells in the presence of injury, thereby reducing the occurrence of the EMT proce TGF-β1 is considered to be a cellular regulator of fibrosis in a variety of organs, pecially in the lung, and has a certain regulatory effect on ECM protein deposition,

Discussion
Alveolar epithelial cell injury and subsequent repair are the main factors involved in the irreversible pathogenesis of IPF [28,29]. Alveolar epithelial cells are the main players in the composition of alveolar structures and play a critical role in lung tissue homeostasis [30]. Type II alveolar epithelial cells are the most important cells on the alveolar surface, and when stimulated by environmental pathogens, in order to protect the alveolar surface, type II alveolar epithelial cells repair epithelial tissue through proliferation and differentiation [31]. In this study, DGM inhibited cell proliferation induced by TGF-β1 in A549 cells and H1299 cells, suggesting that DGM could slow the development of fibrosis by improving the proliferation of type II alveolar epithelial cells. To rule out that the inhibitory effects of DGM in the proliferation of type II alveolar epithelial cells induced by TGF-β1 may be related to toxicity, we evaluated cell viability using the MTT assay, and the results showed that DGM had no significant toxic effect on both cell lines within the indicated concentration range. In this study, we demonstrated that DGM could inhibit TGF-β1-induced proliferation of A549 cells, suggesting that DGM could slow down the development of pulmonary fibrosis at some level by inhibiting the proliferation of type II epithelial cells in the presence of injury, thereby reducing the occurrence of the EMT process.
TGF-β1 is considered to be a cellular regulator of fibrosis in a variety of organs, especially in the lung, and has a certain regulatory effect on ECM protein deposition, inflammation, and tumorigenesis [32]. Under stimulation of TGF-β1, type II alveolar epithelial cells undergo EMT, epithelial cells lose epithelial cell characteristics, acquire the fibroblast phenotype, and develop fibroblast foci and cause excessive protein deposition in the ECM [33]. EMT is involved in the development of many diseases and plays a very important role in pulmonary fibrosis. On treating A549 cells with TGF-β1 in vitro to mimic the EMT process, we observed an up-regulation in the expression of vimentin, α-SMA and collagen, EMT-related biomarkers, and the main components of the ECM. Further, DGM could attenuate this up-regulation. E-cadherin, the most critical indicator of EMT, was down-regulated on exposure to TGF-β1 stimulation [34], and DGM attenuated this change. These results suggest that DGM inhibits excessive ECM deposition by inhibiting the occurrence of EMT. At the same time, we found that DGM could reduce the upregulation of MMP-2 induced by TGF-β1, suggesting that this reversal effect by DGM was related to the degradation of ECM components. Fibroblasts are also very critical cells in the development and progression of lung fibrosis. In this study, we have mainly demonstrated that DGM could inhibit the occurrence of EMT in epithelial cells, but whether there is an effect on the activation of fibroblasts needs to be explored more. Many signaling pathways are involved in the biological processes of EMT, such as Wnt/β-catenin, NF-κB, and TGF-β1/Smad [12]. The TGF-β1/Smad signaling pathway is the most cited EMT-related signaling pathway. TGF-β1 binds to its receptor on the membrane, which subsequently leads to phosphorylation of Smad2/3, which in turn binds to Smad2/3 to form a heterologous complex, which translocates to the nucleus where it regulates the expression of target genes [35][36][37]. DGM decreased the phosphorylation levels of Smad2 and Smad3 stimulated by transforming growth factor-β1, suggesting that DGM has the ability to inhibit transforming growth factor-β1/Smad-dependent signaling pathways. DGM may inhibit the stimulation of the type II alveolar epithelial transformation process by inhibiting Smad2/3 phosphorylation and stimulating the EMT process of type II alveolar epithelial cells. The specific regulatory mechanism for inhibition needs to be further studied.
Pulmonary function testing (PFT) is an important clinical examination and diagnostic indicator of pulmonary fibrosis, and the lung function of patients with IPF will continue to decline. In this study, a PFT animal lung function assay was used to measure lung function. The results showed that DGM reversed the changes in various indicators of lung function in rat models of IPF, and the effect was better than that of the positive drug PFD at high doses. Combined with the comprehensive analysis of the above lung function indicators, our findings strongly suggest that DGM improved lung function of IPF rats.
Inflammation is observed throughout the course of pulmonary fibrosis. In the early stage of the BLM-induced pulmonary fibrosis model in rats, damage to alveolar epithelial cells led to a large release of crude pro-inflammatory fibrosis biomarkers and increased neutrophil infiltration, which caused a strong inflammatory response in the lungs. With the progression of the disease, chronic inflammation dominated by lymphocyte infiltration will occur in the later stage [38], therefore inhibiting the inflammatory response is conducive to blocking the further development of pulmonary fibrosis. The experimental results showed that after BLM stimulation, the overall number of inflammatory cells in the BALF increased significantly, especially neutrophils, lymphocytes, and eosinophils. DGM significantly and dose-dependently reduced the total number of inflammatory cells, with the 60 mg/kg dose being more effective than that of PFD treatment. TNF-α, TGF-β1, IL-6, MCP-1, and MMP-7 are inflammatory mediators and profibrotic factors involved in lung tissue damage and play a significant role in the initiation and progression of pulmonary fibrosis [39][40][41][42][43]. Therefore, we measured the levels of these cytokines in the BALF supernatant. The results showed that DGM could reduce the levels of inflammatory factors. Pathomorphological H&E staining also showed a decrease in structural alveolar lesions and a decrease in inflammatory cell infiltration. These results indicated that DGM could reduce lung inflammation and protect alveolar epithelial cells, which requires further experimental verification. The above results showed that DGM could slow the progression of pulmonary fibrosis by inhibiting lung inflammation.
Pulmonary fibroblast activation is an important factor in the development of pulmonary fibrosis, and fibroblasts are activated by processes such as EMT and endothelial-mesenchymal transformation, and a large amount of ECM is deposited in lung tissue, resulting in changes in lung structure [44]. Integrins activate downstream pathways to promote the transformation of fibroblasts into myofibroblasts [45]. Myofibroblasts produce large amounts of collagen, characterized by the presence of α-SMA stress fibers. The HYP content in lung tissue is often used as a fibrosis marker to characterize the degree of collagen deposition in fibrotic tissue. Immunohistochemistry, WB, and ELISA experiments have shown that DGM could reduce ECM deposition and reduce lung fibroblast activation, thus exerting antifibrotic effects.
Previously, our laboratory studied the activity of the marine derivative MBH-212 in the treatment of COPD, and its intermediate metabolite, DGM, in pulmonary fibrosis. In summary, DGM inhibits the alveolar epithelial cell EMT process by inhibiting the TGF-β1/Smad signaling pathway in vitro, and slows the development of pulmonary fibrosis in vivo by reducing lung inflammation, improving lung function, and reducing remodeling of the ECM. However, the effects of DGM on non-Smad-dependent signaling pathways are unclear and require further study. This current study actually provides another piece of the puzzle on the overall picture of the benefits and effectiveness of DGM in acute and chronic lung disease, although the detailed mechanism of action urgently needs to be elucidated. This is the first study of the effect of DGM on pulmonary fibrosis, and this paper explores the selectivity of benzoic acid compounds in IPF, but it is unclear whether DGM can improve pulmonary fibrosis by modulating other factors, and further research is needed. Whether the same therapeutic effect is achieved when administered after 14 days of tracheal drip requires further study.

Cell Culture and Treatment
The A549 cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in RPMI.1640 culture medium with 10% inactivated fetal bovine serum in an incubator with 5% CO 2 at 37 • C. A549 cells were seeded in a 96-well plate for 12 h, then underwent serum starvation for 12 h. Treatment with 5 ng/mL TGF-β1 for 48 h was used to induce the in vitro EMT model.

MTT Assay
After A549 cells were treated with different concentrations (50, 100, 200 µM) of the test drug for 48 h, cell viability was detected by the MTT assay. A 5 mg/mL MTT solution was added to a 96-well plate treated with drugs, incubated in a 37 • C incubator for 4 h, then dissolved with DMSO to form crystals. Finally, absorbance was measured at 490 nm with a microplate reader.

Western Blotting Analysis
A549 cells were seeded in 6-well plates at a density of 5 × 10 5 , cultured for 12 h, serum starved for 12 h, and then stimulated with TGF-β1 for 24 h. Cells were then lysed to extract protein by adding a 150 µL volume of lysis buffer (containing protease inhibitors) to each well of the 6-well plate. Cells were lysed on ice for 25 min, and lysates were collected in a centrifuge tube by cell scraping. The lysate was centrifuged at 14,000 rpm at 4 • C for 5 min, the supernatant was removed, and the pellet was discarded. The protein concentration of the extracted supernatant was measured using the BCA assay. For electrophoresis, 25 g of protein was added to each well, and different separation gels were selected according to the different molecular weights of the target protein. Proteins were then transferred to NC membrane, blocked with 5% BSA for 2 h, and then exposed to the corresponding primary antibody and incubated overnight at 4 • C. The next day, the primary antibody solution was recovered. The film was washed 6 times with 1 × TBST for 6 min each time. The corresponding secondary antibody solution was added after washing and incubated at room temperature for 1 h. The film was washed 6 times, and then the strips were visualized with the ECL luminescence kit. Finally, ImageJ software was used for grayscale densitometric analysis.

Animal Models
Male Sprague-Dawley rats (aged 6-8 weeks) were obtained from Jinan Pengyue Laboratory Animal Co., Ltd. (Jinan, China). The living environment of the rats provided adequate water and food, an ambient temperature of 25 • C, and a light/dark cycle of 12 h. Before the beginning of the experiment, rats were placed in an experimental environment and reared adaptively for one week. All animal experiments were conducted according to the guidelines of the Animal Experiment Ethics Committee of Ocean University of China (OUC-SMP-2022-06-01).

Mold Making and Treatment Scheme
The 60 rats were randomly divided into five groups of 12 rats, namely: (1) control group, (2) BLM group, (3) BLM + PFD (60 mg/kg) group, (4) BLM + DGM (30 mg/kg) group, and the (5) BLM + DGM (60 mg/kg) group. PFD was used as the positive test drug. Prior to tracheal drip, all rats were injected with 4% chloral hydrate solution for anesthesia. To establish the pulmonary fibrosis (PF) model, rats received BLM (5 mg/kg, volume 100 µL/200 g of body weight) dissolved in sterile PBS by intratracheal instillation. Subsequently, the BLM + PFD group, BLM + DGM (30 mg/kg) group, and BLM + DGM (60 mg/kg) group were treated every two days; the BLM group and the control group were given the same amount of normal saline, and the rats were euthanized on Day 28 and sampled for testing.

Pulmonary Function Tests
The rats were anesthetized by injection of 4% chloral hydrate, cutting the skin at the midline of the neck, bluntly peeling off the exposed trachea, cutting a small opening in the trachea with small scissors, inserting the head of the pulmonary function tester cannula into the trachea, and starting running a PFT Animal Lung Function Test System, which was purchased from Shanghai Tawang Intelligent Technology Co. (Shanghai, China),to test the lung function of the rats.

Lung Coefficient Detection
After the rat was euthanized, lung tissue was sampled. All residual blood stains on the tissue were washed with PBS. Excess connective tissue was removed, and then the water was removed with filter paper. The lung tissue was weighed, and the weight was divided by the weight of the rat before being euthanized and then multiplied by 100%, which defined the lung coefficient.

H&E and Masson's Staining
After sacrifice of the rat, the lung tissue was removed and immersed in 4% paraformaldehyde. It was then dehydrated, embedded, sliced, and stained with H&E and Masson, respectively. Finally, pathological changes such as lung tissue inflammatory infiltration and collagen deposition were observed under a light microscope, and three random fields were randomly selected for each sample and taken at magnifications of 200× and 400×.

Immunohistochemical Staining
Detection of α-SMA expression in rat lung tissue by immunohistochemistry. Briefly, tissue sections were pretreated in a microwave oven to inactivate enzymes, blocked, and reacted with primary and secondary antibodies. The samples were chromogenized, counterstained, decolorized, sealed, and finally scanned with a digital pathology slide scanner.

Determination of Hydroproline and Collagen
Rat lung tissue was stored at −80 • C and once thawed, RIPA lysis buffer was added, placed in a Xianou-24 homogenizer for homogenization, and allocated to the corresponding wells of 96-well culture plates, as determined by ELISA kits, which were purchased from FANKEI Industrial Co., Ltd. (Shanghai, China).

BALF Collection and Inflammatory Cell Count
After anesthetizing the rat, the trachea was exposed, and the rat was then intubated. The lung tissue was rinsed twice with 1.5 mL of pre-cooled PBS, and BALF was collected in a 4 mL centrifuge tube, followed by centrifugation at 2000 rpm for 10 min. The supernatant was collected and stored at −80 • C for subsequent cytokine detection. The BALF precipitate was suspended in 200 µL of PBS, and the total numbers of white blood cells, neutrophils, lymphocytes, and eosinophils in the BALF were determined with ProCyte Dx ® Hematology Analyzer (IDEXX). Laboratories Inc., Westbrook, ME, USA).

ELISA Method for the Detection of Cytokines in BALF Supernatant
The levels of TGF-β1, TNF-α, IL-6, MCP-1, and MMP-7 in the BALF were determined by ELISA, as indicated by the corresponding kit instructions.

Statistical Analysis
Data are expressed as the mean ± SEM. GraphPad Prism 9.3.1 software (San Diego, CA, USA) was used for statistical analysis, and one-way analysis of variance (ANOVA) followed by multiple comparison tests was performed. Statistical significance was accepted at p < 0.05.  Institutional Review Board Statement: The animal experimental protocol was performed following the ethical standards of ARRIVE guidelines, and efforts were made to minimize animal suffering. Animal experiments were approved by the Ethics and Animal Welfare Committee of the Ocean University of China (OUC-SMP-2022-06-01).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.