| Objectives Study of the role of inhibitors of endoplasmic reticulum transmembrane proteins including inositol requiring enzyme 1(IRE1), activating transcription factor 6(ATF6) and double stranded RNA dependent protein kinase like endoplasmic reticulum kinase(PERK) inhibitor in the apoptosis pathway of endoplasmic reticulum stress(ERS) in pulmonary fibrosis rats by silica, and the intervention effect evaluation of three kinds of inhibitors on silicosis fibrosis rats.Methods Specific pathogen free(SPF) healthy adult male SD 100 rats, Rats were randomly divided into 5 groups: normal saline control group, silicosis model group, IRE1 inhibitor group, ATF6 inhibitor group, PERK inhibitor group, with 20 rats in each group. Except for the saline control group, the other rats were injected with non tracheal exposure concentration of 50.0g/L silica suspension 1 ml modelled, the physiological saline control group injected with the same volume of sterilized 0.9% Sodium Chloride Physiological Solution. 28 d after modeling, Each group of 2 randomly selected rats. The three inhibitors group rats began to give different intervention treatment, rats in each group were fifty-sixth days, eighty-fourth days after the dusts rats were sacrificed 8 rats in each. Alveolar macrophages(AM) from lung lavage fluid were collected, separated, purifi and culture, the mitochondrial depolarization ratio and reactive oxygen species(ROS) positive rate of AM were detected by flow cytometry. The right lung was appropriate, real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) detected the relative expression level of C/EBP homologous protein(CHOP), ATF6, PPERK, type I and type III procollagen m RNA; homogenated a suitable amount of lung tissue, after cell lysised, determinated the relative expression level of glucose regulated protein-78(GRP78),IRE1α,B cell lymphoma/leukemia protein-2(Bcl-2), Bcl-2 associated X protein(Bax) and aspartate specific cysteine protease(Caspase-12) in lung tissue by Western blot. The right middle lobe of lung tissue by 10% formalin fixed, paraffin section, hematoxylin eosin staining, observing the pathological changes of lung tissue.Results 1 different groups of rat lung tissue pathological changes: lung tissue structure of the physiological saline control group rats was normal, and no inflammatory cell infiltrated, with the extension of exposure, physiological saline control group rats lung tissue without structure change tendency; silicosis model group within a large number ofinflammatory cell infiltration, appearing typical fibrous nodules, nodules formed by macrophages and fibroblasts, the alveolar structure in some areas still exist, the alveolar walls and the vessel wall thickened in different degrees, it was visible; along with the extension of exposure, the number of fibrous silicotic nodule increased, There was trend of becoming bigger and bigger among some silicotic nodule fusion, serious silicotic nodule became fibrous tissue, and became even glassy degeneration; among three inhibitor groups, lung tissue of rats infiltrated a large number of inflammatory cell and exist different degree fibrosis, and there are scattered cells nodules, without obvious fibrous nodules. With the extension of time for observating; There were not obvious the trend between IRE1 inhibitor group and PERK inhibitor group, the rats lung tissue fibrosised seriously in ATF6 inhibitor group. 2 Comparison of relevant protein content in different groups of ERS apoptosis pathway: The physiological saline control group at the two time points lung tissue proteins of rat GRP78, IRE1α,Caspase-12, Bax and Bcl-2 expression levels were no statistical significance(P>0.05); after exposure to dust levels 56 d and 84 d, IRE1α, Caspase-12 and Bax expression of rats lung tissue protein in three inhibitor groups were lower than the same period of silicosis model group, were higher than that of the physiological saline control group, but the Bcl-2 were higher than that of the silicosis model group and inhibitor groups(P<0.05); In the rats lung tissue of silicosis modelgroup, the content of Bcl-2 decreased with prolonged time, besides GRP78 other protein levels were increased with observation time(P<0.05). As the dust exposure time prolong,there was not statistically significant among expression of GRP78, IRE1α, Caspase-12, Bax and Bcl-2 in IRE1 inhibitor group and PERK inhibitor group(P>0.05); Caspase-12 and Bax of ATF6 inhibitor group with the time were significantly increased(P<0.05), the other indexes showed no statistical significance(P>0.05). 3 Different groups of rats AM mitochondrial depolarization rate and the positive rate of ROS: after exposure to dust levels 56 d and 84 d, AM mitochondrial depolarization rate and the positive rate of ROS of three inhibitors group rats were lower than silicosis model group, were significantly higher than those in the physiological saline control group(P<0.05); along with the prolonging of the exposure, AM mitochondrial depolarization rate and the positive rate of ROS of the silicosis model group and ATF6 inhibitor group rats were elevated(P<0.05), AM mitochondrial depolarization rate and the positive rate of ROS of the physiological saline control group and AM mitochondrial depolarization rate of twoother inhibitors showed no significant difference(P>0.05), the positive rate of ROS IRE1 inhibitor group and PERK inhibitor increased(P<0.05). 4 Analysis of different groups of rat lung tissue cells of CHOP, ATF6, P-PERK, type I and type III procollagen m RNA expression:After exposure to dust 56 d and 84 d, CHOP, ATF6, P-PERK, type I and type III procollagen m RNA expression levels of rats lung tissue in three inhibitors group were higher than those in the physiologycal saline control group, lower than the same period in the silicosis model group(P<0.05), the 5 indexes of rat lung tissue in the silicosis model group and ATF6 inhibitor group with dust exposure time prolonged increased(P<0.05); with dust exposure time prolonged, ATF6 m RNA expression levels the IRE1 inhibitor group and PERK inhibitor group of increased(P<0.05); while other indexes were no significant between the two groups(P>0.05).Conclusions 1 The pathological changes of lung tissue in rats with silicosis become serious, changes in the levels of the mitochondrial membrane potential and ROS, the relative expression levels of protein and m RNA in lung tissue were increased to varying degrees, indicating that ERS apoptosis pathway of IRE1, ATF6 and PERK may play an important role in the occurrence and development of silicosis fibrosis. 2 Three kinds of transmembrane protein inhibitors block the UPR signaling pathways, the inhibitors reduce rat lung tissue fibrosis degree, and expression level of lung tissue protein and m RNA become lower, that inhibitors antagonism the effect of pulmonary fibrosis. 3 Three kinds of transmembrane protein inhibitors block the UPR signaling pathways, mitochondrial depolarization ratio and ROS of rats AM bacome lower, and relative expression levels of lung tissue poptosis-promoting protein are lower and relative expression levels of lung tissue anti-apoptotic protein are higher, among ERS apoptosis pathway and mitochondrial apoptosis pathway and oxidative stress induced apoptosis pathway is closely related. |
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