Emodin attenuates silica-induced lung injury by inhibition of inflammation, apoptosis and epithelial-mesenchymal transition

https://doi.org/10.1016/j.intimp.2020.107277Get rights and content

Highlights

Abstract

Silicosis is a fatal pulmonary disease caused by the inhalation of silica dust, and characterized by inflammation and fibrosis of the lung, with no effective treatment to date. Here we investigate the effect of emodin, an anthraquinone derivative isolated from rhubarb using a mouse silicosis model and in vitro cultured human macrophages and alveolar epithelial cells. Results from histological examination indicated that emodin reduced the degree of alveolitis and fibrosis in the lungs of mice exposed to silica particles. We also demonstrated that emodin effectively inhibited the phosphorylation of Smad3 and NF-κB and reduced the levels of inflammatory factors in the lung tissue of mice treated with silica particles. In addition, we found that emodin inhibited apoptosis and demonstrated an anti-fibrotic effect by down-regulating the pro-apoptotic protein Bax and up-regulating the anti-apoptotic protein Bcl-2. Furthermore, emodin increased E-cadherin levels, reduced the expression of Vimentin, α-SMA and Col-I, as well as pro-inflammatory factors TGF-β1, TNF-α and IL-1β in vivo and in vitro. These results suggested that emodin can regulate epithelial–mesenchymal transition (EMT) through the inhibition of the TGF-β1/Smad3 signaling pathway and the NF-κB signaling pathway to prevent alveolar inflammation and apoptotic process. Overall, this study showed that emodin can alleviate pulmonary fibrosis in silicosis through regulating the inflammatory response and fibrotic process at multiple levels.

Introduction

Silicosis is an occupational lung disease caused by inhalation of free silica particles. Characterized by persistent inflammation and pulmonary fibrosis, silicosis is an incurable disease with a high mortality rate. Occupational exposure to silica dust is an important risk factor for pulmonary fibrosis that perpetuates even after cessation of occupational exposure [1]. Pulmonary fibrosis includes a heterogeneous group of lung disorders characterized by progressive and irreversible destruction of lung architecture caused by scar formation that ultimately leads to organ malfunction, disruption of gas exchange and death from respiratory failure [2], [3]. Although numerous studies on silicosis have been carried out in recent years, the exact mechanism of disease remains unclear and there is currently no effective treatment except lung transplantation.

The pathogenesis of silicosis is a complex process involving different cell types, cytokines and pathways [4]. When silica dust enters the lungs, macrophages are the first line of defense and engulf silica particles causing DNA damage, cell injuries and death, which triggers the innate immune system to release inflammatory cytokines that including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) [5]. These inflammatory factors induce multiple signaling pathways, such as the TGF-β/Smad signaling and NF-κB signaling pathway. Meanwhile more macrophages are recruited from circulation leading to more inflammation responses [6]. As alveolar epithelial cells are damaged, they undergo autophagy and apoptosis [7]. Repeated inflammations and cell death promote the formation of fibroblasts, myofibroblasts and the production of large quantities of extracellular matrix, accelerating the release of fibrogenic factors and the process of epithelial mesenchymal transformation (EMT), resulting in alveolar structure destruction and fibrosis [8]. In recent years, EMT has been shown to play an important role in fibrosis, wound healing and tumor formation [9] (see Fig. 1).

Emodin is an anthraquinone derivative isolated and purified from rhubarb that possesses a variety of biological activities. Pharmacological research showed that emodin has anti-tumor, anti-inflammation, anti-fibrosis, and other protective effects [10], [11]. These pharmacological properties suggest that emodin might be a valuable therapeutic option for the prophylaxis and treatment of various diseases. Emodin has been reported to decrease collagen deposition in pulmonary injuries and down-regulate TGF-β signaling pathway which is upregulated in many human cancers [12], [13], [14]. In addition, emodin has been used in the treatment of rheumatic arthritis, on which it exerts its anti-inflammatory effects by inhibiting the NF-κB pathway [12]. Furthermore, emodin demonstrated its immunosuppressive and antitumor effects by inducing apoptosis in human T cells and A549 cells via the downregulation of Bcl-2 and upregulation of Bax [15], [16]. Emodin has been shown to have protective effects against ovalbumin-induced airway inflammation in mouse model [17]. There were sporadic studies investigating the effects of emodin on silicosis. A recent study showed that emodin can suppress the silica-induced lung fibrosis by promoting Sirt1 signaling [18]. Silicosis is a multiple process involving cell death, inflammatory response and fibrotic reactions. Understand the effects of emodin on these key processes of silicosis will not only help to elucidate the molecular pathogenesis of silicosis but also provide clues to identify the better ways for prevention and treatment of the disease.

In this study, we developed an in vivo model of silicosis in mouse model, in which silica suspension were injected into the lung once with a non-exposed tracheal tube and investigated the effects of emodin on pulmonary fibrosis. The molecular mechanisms underlying the anti-silicotic effects further explored in vitro using a co-culture system with THP-1 and A549 cells. Results showed that Emodin have anti-inflammatory and fibrogentic effect in vitro and in vivo models.

Section snippets

Reagents

Emodin (99.39%) was purchased from MedChemExpress Co.,Ltd (Shanghai, China). Stock solutions of emodin were prepared by dissolving emodin powder and diluted to create doses of 25 and 50 mg/kg of Emodin. Pirfenidone (PFD) was purchased from Beijing Continent Pharmaceutical Co., Ltd (Beijing, China). PFD is an oral anti-fibrosis drug that has been approved for the clinical treatment of idiopathic pulmonary fibrosis (IPF) [19]. Silica powder was purchased from Sigma Chemical Co. (St. Louis, MO,

Emodin recovered the lung histological changes induced by silica particles

The results of HE staining showed normal alveolar structure of mice from the negative control group and the emodin only control group. The alveolar wall is thin, clear and complete, without obvious inflammatory cell infiltration or granuloma. Lung of C57BL/6 mice exposed to silica particles showed clear pathological alterations. As shown in Fig. 2A, there is severe pulmonary inflammation and damaged alveolar structures occurred in the lungs after silica instillation. However, compared with the

Discussion

Occupational exposure to silica particles can lead to silicosis, which is characterized by inflammation and abnormal tissue repair. Lung epithelial cells responding to repeated injury experience persistent inflammation and sustained EMT, leading to progressive lung fibrosis [9]. So far, silicosis is incurable but preventable disease. The purpose of this study was to investigate the potential anti-inflammatory and anti-fibrosis effects of emodin on silicosis caused by silica.

The aim of the study

Conclusion

In summary, our findings demonstrated that emodin can significantly alleviate the degree of pulmonary alveolitis and fibrosis in a dose–response manner in silica-induced silicotic mice in a dose–response manner. The mechanism underlying the anti-silicotic effects may involve multiple processes of silicosis. We demonstrated that emodin can inhibit EMT, and reduce the production of collagen, which may be attributed to its regulation of the NF-κB and TGF-β1/Smad3 signaling pathway and inhibition

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81872603), Natural Science Foundation of Shandong Province (ZR2019MH102), Science and Technology Development Plan of Shandong Province and Jinan City (2016GSF201047, 201907061), the Innovation Project of Shandong Academy of Medical Sciences, and Academic Promotion Programme of Shandong First Medical University (2019QL001), and Tsung Cho Chang Education Foundation, Taiwan (UQ: 023142).

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