The Role And Mechanism Of EMT In MARCO-mediated Silicosis In Rats

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;（2）The molecular biological mechanism of EMT reversing silicosis in polyglycine nucleotides（Poly G）（3）the effects of preventive and therapeutic interventions of Poly G 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）,Poly G prevention group（n=16）and Poly G（28 days）treatment group（n=16）.After the rats were anesthetized with ether,the control group was injected with 1ml normal saline by bronchial instillation.The rats in other groups were treated with non-exposed tracheal infusion of a one-time infusion of 50ml/L silica suspension 1ml.The rats in the Poly G preventive group were treated with Poly G 2.5mg/kg body weight（the corresponding dose was determined by the weight of each rat in the day of modeling）at the same time by tail vein injection.The rats in the Poly G treatment group were treated with intravenous injection of Poly G 2.5mg/Kg body weight（the corresponding dose was determined by weight of each rat after 28 days of modeling）.The rats in the poly G prevention group and the treatment group were sacrificed on the 28 th day and the 56 th day after the corresponding administration.Silicosis model group and saline control group also have 8 rats were sacrificed at the same time point.After taking appropriate amount of left lung tissue and homogenating,Macrophage receptor with collagenous structure,E-cadherin,α-smooth muscle actin,vimentin,collagen Ⅰand collagen Ⅲwere detected by Western blotting;the relative expression levels of mRNA of E-cadherin,α-SMA,vimentin,procollagen Ⅰ and procollagen Ⅲ 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 Poly G 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 Poly G,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 Poly G 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 comparation of mRNA expression levels of E-cadherin,α-SMA and vimentin in lung tissue of different groups: the expression of Ecadherin,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 Poly G 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 1 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.2 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.3 Poly G intervention（preventive 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.