Objectives The animal model of silicosis of non-exposed tracheal instillation induced by SiO₂ in rats lung fibrosis were studied. To investigate:

1. whether SiO₂ can induce fibrosis in lung tissue by induction of epithelial interstitial transformation (EMT);

2. EMT in poly guanine nucleotide (PolyG) molecular biology mechanism of silicosis fibrosis reversal.

3. the effects of PolyG preventive and therapeutic interventions on EMT in rats exposed to silica dust and the effect of intervention at different time points.

Methods Ninety-six healthy male Sprague-Dawley (SD) rats weighing 180~220 g were randomly divided into normal saline group (n=32), silicosis model group (n=32), PolyG prevention group (n=16) and PolyG (28 days) treatment group (n=16). After the rats were anaesthetised with ether, the control group was injected with 1 ml normal saline by bronchial instillation. The rats in other groups were treated with non-exposed tracheal infusion of a one-time infusion of 50 ml/L silica suspension 1 ml. The rats in the PolyG preventive group were treated with PolyG 2.5 mg/kg body weight (the weight of each rat was determined 28 days after modelling) at the same time by tail vein injection. The rats in the PolyG treatment group were treated with intravenous injection of PolyG 2.5 mg/Kg body weight (by modelling 28 days after each rat weight to determine the appropriate dose). The rats in the polyG prevention group and the treatment group were sacrificed on the 28th day and the 56th day after the corresponding administration. Silicosis model group and saline control group also have 8 rats were sacrificed at the same time point.(Macrophage receptor with collagenous structure, MARCO), E-cadherin (E-cadherin) were detected by Western blotting. The macrophage receptor (MARCO) Cadherin, E-cadherin, α-smooth muscle actin (α-SMA), vimentin and type I and type III collagen; the right amount of lung tissue, The relative expression levels of E-cadherin, α-SMA, vimentin and type I and type III procollagen mRNA were detected by Real-time PCR. The right middle lobe tissue was fixed with 4% paraformaldehyde, paraffin section, the histopathological changes of lung tissue were observed by HE staining and Masson staining. Immunohistochemical staining was used to detect the localization and expression of E-cadherin, α-SMA and vimentin in lung tissue.

Results

1. Histopathological changes of lung tissue in different groups: The lung tissue structure of the rats in the control group was normal, and the infiltration of inflammatory cells was observed in the surrounding area, and the lung tissue structure of the control group did not change significantly over time; There were a large number of inflammatory cell infiltration in the lung tissue of the rats in the silicosis model, and the typical fibrous nodules were formed. The nodules were composed of macrophages and fibroblasts. Some areas of the alveolar structure still existed, showing different alveolar walls and small blood vessel wall thickening; with the dying time, the number of fibrous silicon nodules increased, some of the silicon nodules have a trend of integration and becoming bigger, in particular, is completely fibrous tissue, or even fibrotic changes; In PolyG prevention group and treatment group, the number of cell nodules or silicon nodules was lower than that of the model group; the degree of lesion in the rats was significantly lower than that in the control group. The results of immunohistochemistry showed that α-SMA and vimentin positive cells were significantly increased in the silicosis model group, and the number of E-cadherin positive cells was significantly decreased. After administration of PolyG, the number of E-cadherin-positive cells and the number of α-SMA and vimentin-positive cells were significantly increased.

2. The expression of MARCO, E-cadherin, α-SMA and vimentin in the lung tissue of the control group were not significantly different (p<0.05). There was no significant difference in the expression of MARCO and EMT-related proteins in rat lung tissue at different time points (p<0.05). The levels of E-cadherin in the lung tissue of the silicosis model group were increased with the prolongation of observation time (p<0.05). The expression levels of MARCO, α-SMA and vimentin protein in the lung tissue of the PolyG intervention group (prophylactic and therapeutic) were lower than those in the control group, but the E-cadherin was higher than that of the silicosis group model group (p<0.05).

3. The comparison of mRNA expression levels of E-cadherin, α-SMA and vimentin in lung tissue of different groups: the expression of E-cadherin, α-SMA and vimentin mRNA in lung tissue of all groups were the same as those of corresponding protein.

4. The expression of collagen I and III in the lung tissue of different groups were significantly higher than those in the control group (p<0.05). There was no significant difference in the expression of collagen I and III in rat lung tissue of control groups at different time points (p<0.05). The expression level of collagen I and III in the lung tissue of silicosis model group increased with the prolongation of dying time (p<0.05). The protein content of collagen I and III in PolyG intervention group was significantly higher than that in the control group (p<0.05). The mRNA expression of type I and III procollagen were the same as that of I and III collagen.

Conclusions

• The protein content and mRNA expression of E–cadherin were decreased, while α–SMA and vimentin in silicosis model group were increased, which indicated that there was epithelial–mesenchymal transition in the development of silicosis.

• After inhibition of MARCO combined with SiO₂, the protein content and mRNA expression of α–SMA and vimentin were significantly down–regulated, and the protein content and mRNA expression of E–cadherin were significantly up–regulated; I, III collagen content and I, III type procollagen mRNA expression levels were decreased and lung tissue pathological changes were reduced in vary degrees.

• PolyG intervention (prophylactic and therapeutic) can effectively inhibit the progression of EMT, and further delay the formation of pulmonary fibrosis, and the effect of early to give preventive intervention is better.