The Role Of Autophagy In The Development Of Silicosis Pulmonary Fibrosis

?Objective?To observe the changes of pulmonary autophagy in rats exposed to silica dust at different stages,and to explore the role and mechanism of autophagy in silicosis.?Methods?By establishing silicosis rat model,the changes of autophagy in silicosis at different time points were observed.72 SPF wistar male rats were randomly divided into SiO₂ model group and solvent control group.Rats in model group were given SiO₂ suspension to establish silicosis model at one time by non-exposed tracheal drip method.Rats in solvent control group were given 0.9%Nacl solution.The rats were observed daily and their weight was recorded.Six rats were sacrificed 1,7,14,21,28 and 60 days after the establishment of the model,weighing the wet lung weight and calculating the lung organ coefficient.Histopathological changes and fibrosis were observed by hematoxylin-eosin?HE?staining and Masson staining.Transforming growth factor-??TGF-??and interleukin-1?IL-1?and Tumor Necrosis Factor-??TNF-??in lung homogenate were detected by enzyme linked immunosorbent assay?ELISA?.Levels of microtubules associated protein 1 light chain 3?LC3?and yeast autophagy associated gene 6?Beclin1?in lung tissue were detected by Western blot,and the number of autophages in lung tissue was observed by transmission electron microscopy.To further clarify the role of autophagy in silicosis,autophagy inhibitor 3-MA and autophagy agonist Rapamycin were used to intervene to observe the progress of fibrosis in silicosis rats.168 SPF Wistar male rats were randomly divided into four groups:SiO₂ model group?S group?,SiO₂+Rapa group?R group?,SiO₂+3-MA group?M group?and solvent control group?C group?.The method of modeling is the same as the above.Rats in the group M and group R were injected 3-Methyladenine?2.5mg/kg?and Rapamycin?1mg/kg?once every two days starting from two days before the silica injection expriment.Six rats were sacrificed 1,7,14,21,28 and 60 days after the establishment of the model.Lung tissues were taken and the detection methods were the same as the above.And detecting the Epithelial-mesenchymal transition?EMT?-related proteins Vimentin and?-SMA by Western blot.Human macrophages and human alveolar epithelial cells?A549?were co-cultured and stimulated by SiO₂ to explore the possible mechanism of EMT transformation in alveolar epithelial cells.THP-1 cells were induced into macrophages by 100ng/ml PMA.Rapa?100nM?and 3-MA?5mM?were used to pretreat group R and group M cells for 2 hours,and SiO₂?100?g/ml?was used to treat group S,M and R cells for 24 hours.Group C was not treated.After 24 hours,3×10⁴/ml A549 cells were inoculated into 0.4um Transwell chamber and cultured for 24 hours.CCK-8 was used to detect the survival rate of macrophages and A549 cells.The supernatant of macrophages was collected and the concentration of TGF-?was detected by ELISA.Western blot was used to detect the expression levels of autophagy-related proteins LC3,Beclin1,P62 and EMT-related proteins Vimentin,?-SMA and E-cad in A549 cells.?Results?1.General Situation and Weight of Rats.The rats in the control group?group C?were in good condition during the experiment.Model group?group S?rats began to have rough coat,poor mobility,shortness of breath and moist rale on the 14th day of the experiment.The general condition of rats in group M was similar to that in group S,and the lung moist rale was more severe than that in group S.Rats in group R also had rough coat at the beginning of 14 days,and had worse activity than those in group S.Rats in group R still had shortness of breath but less moist rale than those in group S.The body weight of rats in S,M and R groups was significantly lower than that in C group after 7 days of experiment?P<0.05?.The body weight of rats in group R was significantly lower than that in group S on day 45?P<0.05?.2.Pulmonary organ coefficient in rats.The pulmonary organ coefficients in S,M and R groups were significantly higher than those in C group from 7 to 60 days?P<0.05?.M group was significantly higher than S group and R group from 7 to 60 days?P<0.05?.Group R was significantly lower than group S on the60th day of the experiment?P<0.05?.3.Gross anatomical observation of rat lungs.The lungs of group C showed pink,smooth surface and good elasticity at each time point;the lungs of group S showed pink on the first day after the experiment,with occasional bleeding spots on the surface and soft texture.As time progressed,the lungs of group S gradually showed pink white on the 14th day,with scattered gray-white spot nodules and hard texture on the surface.And White spots of group M were more widely distributed and hard;the lungs of rats in group R had fewer gray-white spots at 14 days,less than those in group S.4.Pathological examination of rat lung.The results of HE staining showed that mild inflammatory cell infiltration was observed in group C on the 1st day,and returned to normal on the 7th day,and the lung tissue structure was normal.In group S,the alveolar wall became thicker and inflammatory cells infiltrated in pulmonary interstitium on day 1-14,which was moderate alveolitis.And on the 21st day,a few inflammatory nodules were seen in lung tissue,and nodular fusion occurred on the 60th day.And the inflammatory score at each time point was significantly higher than that in group C?P<0.05?.The inflammatory score of group M at each time point was significantly higher than that of group S?P<0.05?.The inflammatory score of group R was significantly lower than that of group S?P<0.05?.Masson staining showed that there was no obvious proliferation of collagen fibers in lung tissue of rats in group C.In group S,a small amount of collagen fibers began to proliferate on the 14th day of the experiment,which was mild fibrosis,and medium fibrosis was achieved on the 28th day.At the 60th day,a large number of collagen fibers were observed,and the fibrosis score was close to 3 points.The scores of 14-60 days in group S were significantly higher than those in group C?P<0.05?.The 14-60 day scores of group M were significantly higher than those of group S?P<0.05?.The scores of 14-60 days in group R were significantly lower than those in group S?P<0.05?.5.Changes of IL-1,TNF-?and TGF-?levels in rat lung tissue.ELISA results showed that the expression levels of IL-1,TNF-?and TGF-?in lung homogenate of rats in group S were significantly higher than those in group C?P<0.05?.The expression levels of three inflammatory factors in group M were significantly higher than those in group S?P<0.05?.The expression levels of three inflammatory factors in group R were significantly lower than those in group S?P<0.05?.6.Changes of autophagy-related proteins LC3 and Beclin1 in rat lung.Western blot results showed that the expression of LC3 protein in lung tissues of rats in group S showed a general downward trend,and it was significantly higher than that in group C on the 1st to 60th day?P<0.05?.LC3 protein in group R was significantly higher than that in group S at 14,28 and 60 days?P<0.05?.LC3 protein in group M was significantly higher than that in group C at 14,28 and 60 days,and lower than that in group S?P<0.05?.And the expression level of Beclin1 protein in lung tissue of rats in group S also showed a downward trend,and it was significantly higher than that in group C on the 7th to 60th day?P<0.05?.Beclin1 protein in group R was significantly higher than that in group S at 14,28 and 60 days?P<0.05?.Beclin1 protein in group M was significantly higher than that in group C at 14,28and 60 days,and lower than that in group S?P<0.05?.7.Changes of EMT-related proteins Vimentin and?-SMA in rat lung.Western blot results showed that the expression levels of Vimentin and?-SMA protein in lung tissue of rats in group S showed an overall upward trend.The expression levels of Vimentin and?-SMA protein in group S were significantly higher than those in group C from14 to 60 days?P<0.05?.Group M was significantly higher than S group from 14 to 60 days?P<0.05?.Group R was significantly lower than group S from 14 to 60 days?P<0.05?.8.Survival of macrophages and A549 cellsAfter 24 hours of co-culture of macrophages and A549 cells,CCK-8 results showed that the survival rates of both cells were above 90%.9.Levels of autophagy-related proteins LC3 and Beclin1 in Macrophages.Western blot results showed that the expression levels of LC3 and Beclin1 in SiO₂ group were significantly higher than those in control group?P<0.05?.3-MA significantly inhibited the expression of LC3 and Beclin1 in SiO₂ group?P<0.05?.Rapa intervention significantly promoted the expression of LC3 and Beclin1 in SiO₂ group?P<0.05?.10.The expression of TGF-?in supernatant of MacrophagesThe level of TGF-?in cell supernatant of SiO₂ group was significantly higher than that of control group?P<0.05?.The intervention of 3-MA significantly promoted the increase of SiO₂-induced TGF-?level?P<0.05?,while Rapa significantly reversed the increase of SiO₂-induced TGF-?level?P<0.05?.11.Expressions of EMT-related proteins E-Cad,Vimentin and?-SMA in A549 cellsSimilar to the changes of Vimentin and?-SMA in lung tissue,the levels of Vimentin and?-SMA in SiO₂ group were significantly higher than those in control group,and the levels of E-Cad were significantly lower than those in control group?P<0.05?.The elevation of TGF-?level in group M significantly promoted the elevation of Vimentin and?-SMA levels and the decrease of E-Cad levels?P<0.05?,while the decrease of TGF-?level in group R significantly reversed the increase of Vimentin and?-SMA proteins and increased the expression of E-Cad?P<0.05?.?Conclusion?1.The level of autophagy in lung tissue of rats exposed to silicosis varies with the onset stage of silicosis.The level of autophagy increases in the early inflammatory reaction of silicosis and decreases gradually with the aggravation of silicosis fibrosis.2.Enhanced autophagy with Rapamycin can inhibit the inflammatory reaction of lung tissue and reduce the degree of fibrosis in silicosis rats,but long-term use of Rapamycin has some toxic side effects.3.Rapamycin combined with SiO₂ could decrea the expression of TGF-?after enhancing the autophagy level of macrophages,and inhibit the EMT process of A549 cells.