Evaluation Of The Effect Of Endoplasmic Reticulum Transmembrane Protein Inhibitors On Silicotic Fibrosis

BackgroundSilicosis is the most serious occupational disease in pneumoconiosis.There is no effective radical cure at present.It mainly adopts the comprehensive prevention and control strategy which is the main prevention and the combination of prevention and treatment.The main pathological changes are the formation of silicosis and diffuse interstitial fibrosis in the lung tissue.A variety of hypotheses have been proposed for the pathogenesis of silicosis fibrosis,The relationship between endoplasmic reticulum stress and pulmonary fibrosis is a new research direction in recent years,and many mechanisms are still unclear.Previous studies have found that the endoplasmic reticulum transmembrane protein inhibitors have a therapeutic effect on silicosis in rats,but their therapeutic effects are not very satisfactory.The aim of this study was to evaluate the preventive effect of prophylacticadministration of endoplasmic reticulum transmembrane protein inhibitors on silicotic fibrosis in rats.ObjectiveThree kinds of endoplasmic reticulum transmembrane protein inhibitors were used to block the unfolded protein response pathway in endoplasmic reticulum stress.Endoplasmic reticulum stress-mediated cell apoptosis and lung fibrosis in rats exposed to silica dust were studied.The present study was aimed to evaluate the role of three transmembrane protein inhibitors in the preventive intervention of pulmonary fibrosis in rats.MethodsAfter one week of adaptive feeding,100 SPF level of healthy adult male SD rats,were randomly divided into saline control group,silicosis model group,IRE1 inhibitor group,ATF6 inhibitor group and PERK inhibitor group according to body weight,20 rats in each group.In addition to the saline control group,other groups of rats were all inhaled 1.0 ml of silica suspension with 50.0 g/L mass concentration by inhalation tracheal instillation.The control group was given an equal volume 0.9%saline of instillation.On the day of modeling,the rats of three inhibitor groups began to be given different intervention treatments,and 10 rats were respectively sacrificed on the 28th and the 56th day after the dust was stained.HE staining and Masson staining were used to observe the pathological changes of lung tissue.According to the Ashcroft scoring method,the degree of pulmonary fibrosis in each group was determined by double-blind scoring.Relative expression levels of the related proteins GRP78,ATF6,IRE1,PERK,CHOP,Bcl2,Bax,Caspase12 and Caspase3 of Endoplasmic reticulum stress and apoptosis were measured by Western Blotting（WB）.Results1 The pathological changes of rat lung tissueUnder the microscope observation,it was found that after 28 days of dusting,the structure of lung tissue was clear and a small number of inflammatory cells infiltrated in the interstitial tissue of rats in normal saline control group;In the lung tissue of model group rats,a lot of dust cells were infiltrated and typical fibrous nodules were appeared.The nodules were composed of fibroblasts and collagen fibers,and the alveolar structure in some regions was fine.The alveolar wall and small vessel wall were thickened in different degree,and there were a large number of inflammatory cell infiltration and diffuse fibrosis changes in lung tissue in the three kinds of inhibitor groups,and there were scattered cell nodules and no obvious fibrous nodules.At 56 days after dusting,comparing with the 28th day,there was no significant change in the lung tissue structure of the control rats;in the silicosis model group,the number of fibrous nodular nodules in the lungs gradually increased,and some of the nodules showed the tendency of fusion,and in the severe cases the vitreous changes were observed.There were a large number of inflammatory cell infiltration and diffuse fibrosis changes in lung tissues in the three inhibitor groups,and scattered cellular nodules were observed,but no obvious fibrous nodules compared with the 28th day in each group.2 Ashcroft score of pulmonary fibrosis in ratsCompared with normal saline control group,28 days after dusting,the Ashcroft scores of lung fibrosis in the model group and the three inhibitor groups were increased（P<0.05）;The Ashcroft score of pulmonary fibrosis in the three inhibitor groups was lower than that in the silicosis model group,but the difference was not statistically significant;56 days after the dust was stained,the Ashcroft scores of lung fibrosis in the model group and the three inhibitor groups were increased（P<0.05）;The Ashcroft scores of pulmonary fibrosis in the three inhibitor groups were significantly lower than that in the silicosis model group（P<0.05）.The Ashcroft score of pulmonary fibrosis in ATF6 inhibitor group was lower than that of 28 days（P<0.05）.The Ashcroft score of pulmonary fibrosis in other groups was not statistically significant.3 Comparison of UPR signal Pathway-related proteins in different groups28 days after dusting,the contents of PERK,GRP78,CHOP,ATF6,Caspase12,Caspase3,Bcl2 and Bax in the lungs of the model group were increased compared with the saline group（P<0.05）,compared with the saline group,the levels of CHOP in PERK inhibitor group and IRE1 inhibitor group were all increased（P<0.05）;compared with the same time period,the contents of PERK,ATF6,GRP78,Bax and Bcl2 in lung tissue of ATF6 inhibitor group were decreased（P<0.05）,The contents of GRP78,Bax,Caspase12and Caspase3 in lung tissue of PERK inhibitor group were decreased（P<0.05）,IRE1inhibitor group GRP78 content decreased,Caspase3 content increased（P<0.05）.At 56days after dusting,compared with the saline group at the same time,the contents of PERK,IRE1,GRP78,CHOP,ATF6,Caspase12,Caspase3,Bcl2 and Bax in the lung tissue of the silicosis model group were all increased（P<0.05）.Compared with the silicosis model group at the same time,the contents of PERK,ATF6,GRP78 and Bax in lung tissue of ATF6 inhibitor group were decreased（P<0.05）,and the contents of PERK,ATF6,GRP78,Bax and Caspase3 in lung tissue of PERK inhibitor group were all increased.The level of PERK,ATF6,GRP78 and Caspase3 in lung tissue of IRE1 inhibitor group were decreased（P<0.05）.Comparison with dust exposure on the 28th day,and with the extension of observation time,the contents of PERK,ATF6,GRP78,Caspase12 and CHOP in lung tissue of the model group were increased（P<0.05）.The content of PERK in lungs of rats with PERK inhibitors was decreased（P<0.05）.The content of GRP78 were increased and the content of Caspase3 were decreased in lung tissue of IRE1 inhibitor group（P<0.05）.The contents of ATF6,GRP78 and Caspase12 in lung tissue of ATF6 inhibitor group were increased（P<0.05）.GRP78 and Caspase12 of rat lung tissue were found interaction between time and intervention agent in PERK inhibitor group（F=5.625,P=0.007;F=3.486,P=0.039,respectively）.GRP78 of rat lung tissue was found interaction between time and intervention agent in IRE1 inhibitor group（F=5.092,P=0.010）.PERK,GRP78 and Bax of rat lung tissue were found interaction between time and intervention agent in ATF6inhibitor group（F=3.315,P=0.045;F=5.526,P=0.007;F=4.619,P=0.015,respectively）.ConclusionApoptosis was mediated by UPR signaling pathway which induced silicosis fibrosis by SiO₂,and the UPR signal transduction pathway was blocked by three endoplasmic reticulum transmembrane protein inhibitors,respectively,which can down-regulate apoptosis and improve the degree of pulmonary fibrosis in rats exposed to silica dust.