Exposure to free silica induces silicosis and myofibroblasts are regarded as primary effector cells. Fibrocytes can differentiate into myofibroblast. Therefore, the present study was designed to investigate whether fibrocytes participate in silicosis. The rat model of silicosis was established. Hematoxylin-eosin stainings and Masson stainings were used to evaluate the histopathology and collagen deposition. Flow cytometry and immunofluorescence were performed to detect the number of fibrocytes and their contribution to myofibroblasts. Results showed that fibrocytes participate in silicosis. Trend analysis of different sources of myofibroblasts during silicosis indicated that fibrocytes and lung type II epithelial cell-derived myofibroblasts play an important role in the early stage of silicosis, while resident lung fibroblast-derived myofibroblasts play a predominant role during the fibrosis formative period.

Chronic inhalation of crystalline silica (SiO2) is associated with the development of silicosis, which is characterized by tissue remodelling, fibroproliferation, and deposition of extracellular matrix (ECM). Myofibroblasts, as key effector cells, are believed to play a major role in the pathogenesis of fibrotic diseases. The source of these cells has been intensively debated. Resident lung fibroblasts were conventionally thought to be the main source of myofibroblasts. However, recent studies have demonstrated that epithelial-mesenchymal differentiation or recruitment of fibrocytes also contribute to myofibroblasts in lung fibrosis. Little attention has been focused on fibrocytes and the contribution of different sources of myofibroblasts in silicosis.

Among the possible sources of lung myofibroblasts, fibrocytes are bone marrow-derived cells that co-express CD45 and collagen type I (CD45+ Col I+)[1]. Fibrocytes participate in both tissue fibrosis and tissue remodelling by producing ECM and matrix metalloproteinases[2]. Recent studies have reported that the number of fibrocytes is obviously increased in patients with idiopathic pulmonary fibrosis (IPF) and that higher numbers of them are related to early mortality[3]. Taken together, a growing body of evidence indicates that fibrocytes participate in lung. In our present study, we sought to evaluate the contribution of fibrocytes to silicosis. We compared lung tissue and peripheral blood from rats with silicosis and control rats for the presence and number of fibrocytes and contribution of the three sources of myofibroblasts during the process of experimental silicosis. Our findings support the notion that fibrocytes may participate in silicosis and that different sources of myofibroblasts may play roles at different stages of silicosis.

The animal experiments were approved by the Experimental Animal Ethics Committee of Zhengzhou University, and our study was conducted in accordance with the guidelines of the Chinese Association of Laboratory Animal Care. The SD rats were randomly divided into the model group and control group (n = 36 per group). The rats in the model group were administered a single intratracheal instillation of SiO2 solution (100 mg/0.5 mL per rat) and those of control group were instilled an equal amount of 0.9% saline. All of the rats were anaesthetized with chloral hydrate and then sacrificed by cervical dislocation for further study at 1, 2, 3, 6, 9, and 12 weeks (n = 12 per week) after silica/saline instillation.

Hematoxylin-eosin (HE) and Masson stainings were used to evaluate the histopathology and collagen deposition. The extent of lung fibrosis was evaluated by the measurement of the area of fibrosis (%) using Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA). Double-colour immunofluorescence was performed to identify fibrocytes or different sources of myofibroblasts in the lung tissue. The fibrocytes were labelled by CD45+ Collagen I+. The fibrocytes-derived myofibroblasts were labelled by CD45+ α-smooth muscle actin+ (SMA). The lung type II epithelial cells (AT II)-derived myofibroblasts were labelled by human pulmonary surfactant associatedprotein (SPC+ α-SMA+). The nuclei was stained by 4’, 6’-diamidino-2-phenylindole dihydrochloride (DAPI). The sections were observed by a Nikon Eclipse Ti microscope (Nikon, Totyo, Japan). For the counting of fibrocyte-derived collagen I-secreting cells or fibrocyte-derived and AT II-derived myofibroblasts in the lungs, two observers assessed the number of double-positive cells. Ten fields were randomly selected to be examined, and the numbers of double positive cells were counted and calculated as the mean number of positively stained cells per total number of collagen I or α-SMA positive cells at sections of the lungs at 200× magnification.

The statistical analysis was performed using SPSS 21.0 (IBM, NC, USA). Comparisons were evaluated by Student’s t test. P values that were less than 0.05 were used to define statistically significant differences.

In the present study, the rat silicosis model was successfully established (Figure S1 available in ).

文章来源：《农业环境科学学报》 网址: http://www.nyhjkxxb.cn/qikandaodu/2021/0201/483.html

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