Background

Silicosis, which exhibits high morbidity and mortality rates in develo** countries, is an irreversible occupational respiratory disease caused by long-term inhalation of crystalline silica dust [1, 2]. Silicosis is characterized by chronic inflammation and progressive interstitial fibrosis [3]. The pathogenesis of silicosis is unclear, thus, no effective clinical treatments to retard the progression of silicosis are available [4]. Pulmonary alveolar macrophages are the predominant cells involved in the development of silicosis [5]. When silica enters the lung, it can stimulate macrophages to engulf silica particles, resulting in M1 polarization and the induction of inflammation and tissue damage due to the release of large amounts of pro-inflammatory mediators, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) [25]. However, no specific drug is available to treat silicosis, and lung transplantation is the only effective treatment. Thus, novel drugs are urgently needed to suppress the progression of silicosis. YCF is a Chinese medicine for pneumoconiosis patients with yin deficiency and dryness heat syndrome or dryness invading lung syndrome. Previous studies demonstrated that YCF effectively improves lung function and alleviates clinical symptoms, such as coughing, shortness of breath, and dyspnea. The present study suggests that YCF significantly improves lung tissue damage and inhibits inflammation and fibrosis in rats with silicosis. Moreover, YCF can significantly inhibit macrophage M1 polarization in vivo and in vitro. By integrating transcriptomic analysis and network pharmacology, we found that YCF has a variety of active components, including ginsenoside Rg3, platycoside D, cucurbitacin D, which can bind to multiple targets and regulate various signaling pathways, including the mTOR, PI3K/AKT, JAK/STAT, MAPK, and NF-κB signaling pathway. These pathways may be involved in the mechanisms by which YCF inhibits inflammation and suppresses the progression of silicosis.

Although the pathogenesis of silicosis remains unclear, increasing evidence suggests that silica-induces persistent pulmonary inflammation, causing tissue damage and fibrosis [26, 27]. In response to silica exposure, activated macrophages polarize into M1 macrophages, which release large amounts of pro-inflammatory mediators, such as IL-1β, IL-6, and TNF-α, that further trigger the inflammatory cascade, leading to tissue damage and fibrosis [28,29,30]. Thus, inhibition of the macrophage-induced inflammatory response may effectively ameliorate silicosis. A previous study demonstrated that bone marrow mesenchymal stem cells exert therapeutic effect in rats with silicosis by ameliorating inflammation and reducing the release of inflammatory cytokines [31]. Moreover, dioscin protects against crystalline silica-induced lung inflammation by suppressing the production of inflammatory factors and inhibiting the activation of macrophages [32]. In our study, YCF treatment significantly inhibited pulmonary inflammation in silica-exposed rats, by reducing inflammatory cell infiltration, and decreasing the secretion of inflammatory chemokines, including TNF-α, IL-1β, and IL-6. YCF also exhibited remarkable anti-fibrotic effects by inhibiting collagen deposition. Moreover, YCF decreased the amount of M1 macrophages in the lung tissue of rats with silicosis. Therefore, we speculate that YCF may exert anti-inflammatory properties via inhibiting M1 macrophage polarization.

To clarify the therapeutic mechanisms of YCF, we separated YCF into five fractions using macroporous resins, and examined the anti-inflammatory effects of these fractions on LPS and IFN-γ-induced M1 macrophages. YCF5 substantially inhibited the production of pro-inflammatory cytokines. These results indicate that YCF5 may be the essential fraction that exerts anti-inflammatory effects on M1 macrophages to attenuate inflammation and delay the progression of silicosis. However, the underlying mechanism of YCF5 attenuation of inflammation via altered macrophage polarization needs to be explored. Thus, we obtained the main active components of YCF5 by MS, network pharmacology and transcriptomics revealed the potential anti-inflammatory mechanisms of YCF5.

Traditional Chinese formulas exert curative effects through multiple compounds and targets with synergistic effects. The holistic and systematic characteristics of network pharmacology are consistent with the “holism concept” in traditional Chinese medicine. Network pharmacology can be used to construct a “compound-target-pathway” network to understand the overall perspective of effective substances and their mechanisms. In recent years, the combination of transcriptomics and network pharmacology has proven to be an effective approach for exploring the therapeutic mechanisms of traditional Chinese prescriptions. Using network pharmacology analysis, we identified the core targets of YCF5, including AKT1, JAK2, MAPK3, STAT3, MYC, EGFR, mTOR, VEGFR, and TNF. These signaling pathways and biological processes, including Toll-like receptor, TNF, and IL-17 signaling pathways, are strongly linked to immunological responses and inflammation.

AKT1, a serine/threonine-protein kinase, is activated through the PI3-kinase pathway to regulate cellular survival signals in response to growth factors and cytokines [33]. The activation of AKT1 accelerates the degradation of IκB and leads to the phosphorylation of NF-κB p65, which promotes the transcriptional activity of NF-κB [34]. Mitogen-activated protein kinase 3 (MAPK3 or ERK) is involved in cell proliferation, growth, migration, metabolism, and transcription [35]. MAPK3 (ERK1) levels are dramatically elevated in the peripheral blood mononuclear cells of patients with silicosis, and crystalline silica may accelerate the release of ROS [36]. ROS further activate the inflammasome through MAPK3 and phosphorylate Ser276 of p65 NF-κB and Ser641 and Ser643 of HIF-1α, to promote the development of silicosis [36,37,38]. JAK2 and STAT3, crucial proteins in the JAK/STAT signaling pathway, play a significant role in regulating macrophage polarization in silicosis; inhibiting the expression of these proteins can delay the progression of silicosis [39]. PI3K-Akt targets mTOR, a crucial component of the rapamycin (mTOR) signaling pathway [40, 41]. Furthermore, mTOR increases autophagy and aggravates the progression of pulmonary fibrosis in silicosis under the regulation of AMP-activated protein kinase (AMPK) [42]. VEGFA, a ligand for the VEGF receptor, plays an important role in angiogenesis and inflammation [43]. Manipulating VEGFA inhibits fibrosis factor release, suppressing the expression of TGF-β and α-SMA in the lung tissue of rats with silicosis [44]. Thus, the targets of YCF5 play vital roles in the inflammatory response, and modulating these targets can delay the progression of silicosis.

Using transcriptomic analysis, we identified 117 reversal genes for YCF5 inhibition of M1 macrophage polarization. These reversal genes are primarily involved in immunological and inflammatory responses, including the regulation of interleukins and tumor necrosis factor production. By integrating network pharmacology and transcriptomic analysis, we constructed a PPI network of YCF5 targets and reversal genes. The anti-inflammatory mechanisms of YCF5 were associated with various targets, including AKT1, TNF, TRP53, IL6, MAPK3, and JUN, and signaling pathways, including the including PI3K-Akt, MAPK, TNF, JAK-STAT, mTOR, NF-κB, and AMPK signaling pathways. These signaling pathways play critical roles in regulating inflammation. For example, MAPKs and NF-κB regulate the expression of pro-inflammatory factors [22]. The activation of mTORC1 also influences the inflammatory response [23]. The targets enriched in these pathways are related to 81 active components, which may be the main active components of YCF. In vitro, we confirmed the activity of YCF5 and the active components of YCF, including ginsenoside Rg3, Ginsenoside Rd, and Cucurbitacin D, which suppressed the activation of these signaling pathways.

Conclusions

Our study demonstrated that YCF treatment improved the pathological changes, inflammatory response, and fibrosis in rats with silicosis, probably by inhibiting M1 macrophage polarization. Moreover, network pharmacology, transcriptomics, molecular docking, and in vitro experiments showed that YCF contains multiple effective compounds with various targets that exert anti-inflammatory effects by inhibiting the pathway networks, such as mTOR, MAPK, and NF-κB signaling pathways. Although this study provides an explanation for the anti-silicosis effects of YCF, there are several limitations to this study. For instance, 101 active compounds identified in YCF5 exert anti-inflammatory effects; however, the identity of the specific active substance of YCF with beneficial effects on silicosis is not known. In future work, we will perform the effective-constituent compatibility-based analysis to identify the critical ingredients from YCF and then form the effective-constituent compatibility (ECC) of JCF, which has the potential bioactive equivalent of JCF [45]. Moreover, we will explore the anti-silicosis mechanisms and potential targets of the ECC of YCF.