Study On The Effect Of 3-methyladenine On Lps-interfered Silicosis Fibrosis Through Inhibiting Autophagy In Mice

Objective:To investigate the effects of 3-methyladenine（3-MA）on autophagy,apoptosis and inflammation by constructing a silicosis mice model with lipopolysaccharide（LPS）interference,and explore the effects and possible mechanisms of autophagy inhibitor 3-MA on silicotic fibrosis.Method:1.64 C57BL/6 mice were randomly divided into four groups:Ctrl group,Silica group,Silica+LPS group,and Silica+LPS+3-MA group,with 16 mice in each group.In the Silica group,50u L 3 mg/50μL Si O₂ suspension was injected into the lungs of the mice without tracheal exposure.In the Ctrl group,non-tracheal exposure was used and the same amount of saline was injected into the lungs.The mice in the Silica+LPS group was injected with 50u L 3 mg/50μL Si O₂ suspension containing 100μg/m L LPS into the lungs without tracheal exposure.The Silica+LPS+3-MA group was treated with an intraperitoneal injection of 30 mg/kg 3-MA for five consecutive days.The mice were sacrificed on the 7th day and the28th day after perfusion.2.To analyze the effect of 3-MA on the inflammatory response of LPS-interfered silicosis mice model,the expression levels of inflammatory factors IL-1β,IL-6 and TNF-αwere detected by ELISA,and the expression level of nucleoprotein NF-κB p65 was detected by Western blot on the 7th day and the 28th day.3.On the 7th day and the 28th day,the expression levels of autophagy related protein LC3,autophagy substrate protein p62,lysosomal membrane associated protein LAMP1 and apoptosis related protein cleaved caspase-3 were detected by Western blot to analyze the effects of 3-MA on autophagy and apoptosis of LPS-interfered silicosis mice model.4.On the 28th day,the lung tissues of the four groups were stained with HE and Masson.The content of HYP in lung tissue was measured.Western blot was used to detect the expression of fibrosis-associated proteinα-SMA and Col-1 in lung tissues of each group.Results:1.The Elisa experiments indicated that on the 7th or 28th day,the expression levels of the inflammatory factors IL-1β,IL-6,and TNF-αin the AMs culture supernatant of the LPS+silica group were significantly higher than those in the silica group（P<0.05）;on the 7th day,compared with the LPS+silica group,the expression levels of these factors in the silica+LPS+3-MA group were significantly decreased（P<0.05）;and on the 28th day,comparing the expression level of these factors between the silica+LPS+3-MA group and LPS+silica group,there was no statistically significant difference（P>0.05）.Western Blot revealed that the expression level of AMs nuclear protein NF-κB p65 in the LPS+silica dust group was significantly higher than its expression level in the silica group（P<0.05）;the expression level of it in the silica+LPS+3-MA group was significantly reduced on the 7th day compared to the LPS+silica group（P>0.05）,but there was no statistically significant difference between these two groups on the 28th day（P<0.05）.2.The levels of LC3-II/LC3-I and p62 were detected by Western Blot.On the 7th day,the expression levels of these factors in the LPS+silica group was significantly higher than those in the silica group（P<0.05）;compared with the silica+LPS+3-MA group.the expression levels,in the LPS+silica group,were significantly reduced（P<0.05）;on the 28th day,there was no significant difference in expression levels between the silica+LPS+3-MA group and the LPS+silica group（P>0.05）.Western Blot further revealed that on the 7th or 28th day,there was no significant difference in the expression level of the LAMP1 among the silica group,the LPS+silica group and the silica+LPS+3-MA group（P>0.05）.Western Blot also indicated that on the 7th or 28th day,the expression level of the cleaved caspase-3 in the LPS+silica group was significantly higher than that in the silica group（P<0.05）.On the 7th day,its expression level in the silica+LPS+3-MA group was significantly lower than that in the LPS+silica group（P<0.05）,and on the 28th day,the difference between these two groups was not statistically significantly different（P>0.05）.3.HE staining showed that compared with the control group,the alveolar structure of the silica group was disordered,the alveolar septum was thickened,and there was a large number of inflammatory cell infiltrations,and cellular nodules appeared;while the alveolar structure of the LPS+silica group was more disordered,the alveolar septum was further thickened,the inflammatory cell infiltration was more serious,and the cellular nodules were more obvious.However,there was no significant difference in these characteristics between the silica+LPS+3-MA group and the LPS+silica group.Masson staining revealed that the area of fibrosis in the LPS+silica group was significantly larger than that in the silica group（P<0.05）;while the silica+LPS+3-MA group showed no significant difference in the area of fibrosis compared with the LPS+silica group（P>0.05）;compared with the silica group,the level of the lung tissue HYP in the LPS+silica group was significantly increased（P<0.05）,while compared with the silica+LPS+3-MA group,the lung tissue HYP level was not statistically significantly different（P>0.05）.Furthermore,compared with the silica group,the levels of Col-1 andα-SMA in the lung tissue of the LPS+silica group were significantly increased（P<0.05）,while compared with the silica+LPS+3-MA group,the levels of Col-1 andα-SMA in lung tissue were not statistically significant different（P>0.05）.Conclusion:1.In the early stage of silicosis,3-MA may reduce the inflammatory response of AMs induced by LPS;2.In the early stage of the development of silicosis,3-MA may inhibit the formation of autophagosomes,thereby alleviating LPS-induced lung tissue apoptosis in mice with silicosis;3.3-MA has no obvious inhibitory effect on fibrosis in silicosis mice with LPS intervention.