| Long-term inhalation of silica dust can cause silicosis,a serious occupational lung disease characterized by persistent inflammation and progressive fibrillation of lung tissue.Silicosis is one of the high-incidence occupational diseases in China,it’s pathogenesis is not completely clear and there is a lack of effective treatment.Previous studies have suggested that the inhaled silica dust was first engulfed by alveolar macrophages,causing a variety of cell infiltrations and the release of pro-inflammatory cytokines to expand the inflammatory response.However,the mechanisms of macrophage death and inflammatory response caused by silica dust is still unclear.Our previous research found that growth arrest specific protein 6（Gas6）was involved in the regulation of pulmonary inflammation in mice and down-regulated macrophage activity caused by silica dust.Combined with recent reports on the key role of macrophage autophagy in silica-induced pulmonary inflammation and collagen deposition.We propose a hypothesis:Does Gas6 participate in regulating the inflammatory response induced by silica through autophagy?In this study,we first observed the localization and effect of Gas6 on autophagy in lungs of mice exposed to silica,and then analyzed the effect of Gas6 in regulating autophagy on the inflammatory response by macrophages after silica exposure.The research was divided into the following two parts.Part Ⅰ.The role of Gas6 in silica-induced autophagy in mice lungObjective:Silica staining was performed on Wild Type（WT）mice and Gas6 gene knockout(Gas6-/-)mice to observe the autophagy level of mice lung at different time points,so as to explore the effect of Gas6 on silica-induced pulmonary autophagy in mice.Methods:A single instillation of silica dust suspension into the trachea was used to establish a dust-stained mouse model.According to their body weight,48 male WT C57BL/6 mice and 48 Gas6-/-mice of the same species were randomly divided into WT saline group,WT silica group,Gas6-/-saline group,Gas6-/-silica group,24 mice per group.The mice in saline group were instilled with 50μL sterile saline,and the silica group mice were instilled with 50μL of silica dust suspension with a concentration of50 mg/m L.At the 7th,28th,and 84th day after silica exposure,8 mice in each group were sacrificed and sampled.Immunofluorescence was used to detect autophagy protein LC3 and macrophage marker F/480 levels,and the ultrastructure of autophagosomes in mouse lung tissue was detected by transmission electron microscope.The level of Gas6in BALF was detected by enzyme-linked immunosorbent assay（ELISA）,quantitative real-time quantitative PCR（q RT-PCR）was used to detect m RNA expressions of Gas6and autophagy-related factors Atg5,Beclin1,P62 and lysosome marker LAMP1,and western blotting（WB）was used to determine the protein expressions of autophagy-related factors LC3,p-m TOR,Atg5,Beclin1,P62 and lysosomal membrane protein LAMP1.Results:（1）On the 7th,28th,84th days after silica exposure,the number of autophagosomes in the lungs of WT mice increased,the autophagy protein LC3increased,and the m RNA expressions of autophagy-related genes Atg5,Beclin1,P62were also significant increased,but the number of lysosomes decreased,and the m RNA expression of lysosome marker LAMP1 decreased（P<0.05）.Immunofluorescence results showed that autophagy protein LC3 could be co-labeled with F4/80,indicating that mouse lung autophagy occurred in the lung macrophages.At the same time,the level of Gas6 protein in BALF and the expression of Gas6 m RNA in silica group were higher than those in saline group（P<0.01）.The results suggested that silica increased Gas6 level and autophagy in the lungs of mice and damaged lysosomes.（2）Compared with WT silica group,the number of autophagosomes and the LC3and P62 protein expressions in Gas6-/-silica group significantly decreased at three exposure time（P<0.05）.The protein expression of Atg5 significantly reduced on the 7th day after silica exposure（P<0.05）,but there was no significant difference at the other two time points.The p-m TOR protein increased significantly at all three exposure time（P<0.05）.Compared with the WT silica group,the number of lysosomes and the protein expression of lysosomal membrane LAMP1 increased significantly at all three exposure time（P<0.05）.These results indicated that Gas6 gene knockout alleviated autophagosome accumulation caused by silica and restored lysosomal damage to some extent.Conclusions:silica exposure increased autophagy in mouse lung macrophages and destroyed lysosomes,leading to obstruction of autophagolysosome degradation,which jointly promoted autophagosome accumulation.However,Gas6 gene knockout could significantly inhibit autophagy,alleviate the accumulation of autophagosomes,and promote the degradation of autophagosomes.Part Ⅱ.Study on Gas6 regulating autophagy to promote the inflammatory response of macrophages induced by silicaObjective:To explore the role of Gas6 in regulating autophagy on silica-induced macrophage inflammatory response.Methods:Raw264.7 macrophages were exposed to respiratory standard silica dust（a particle size of 1μm）at different concentrations（0,37.5,75,150,300μg/m L）for 24h.Additionally,silica-activated（300μg/m L）macrophages were treated with Rapa,3-MA and si RNA-Gas6 respectively to get autophagy promotion group,autophagy inhibition group,Gas6 gene silencing group,Gas6 gene silencing+autophagy promotion group,Gas6 gene silencing+autophagy inhibition group.CCK8 assay was used to detect the cell vitality,lact dehydrogenase kit to detect LDH activity,expressions of Gas6 and IL-6/TNF-αin supernatant were detected by ELISA,LC3 and P62 levels were determined by immunofluorescence assay,m RNA expressions of autophagy related factors LC3/Beclin1/P62 were detected by q RT-PCR,protein levels of Gas6 and autophagy related factors LC3/Beclin1/P62 were determined by WB.Results:1.Silica dust induced autophagy enhancement of macrophagesWith the increase of silica concentration,the activity of macrophages decreased and LDH activity increased.The m RNA and protein expressions of autophagy-related factors LC3,Beclin1 and P62 in the silica exposure groups increased with the increase of silica concentration（P<0.05）.The immunofluorescence assay results showed that the protein expressions of LC3 and P62 were increased（P<0.05）.The results showed that silica induced autophagy of macrophages.2.Autophagy was involved in the inflammatory response of macrophages induced by silica 24 hours after macrophages were exposed to silica,the expressions of inflammatory factors IL-6 and TNF-αincreased in a concentration-dependent manner（P<0.05）.Compared with silica group,the cell viability of the autophagy promotion group（Rapa+Silica）decreased（P<0.01）,the m RNA expressions of autophagy related factors Beclin1 and P62 increased（P<0.05）,and the expressions of IL-6 and TNF-αin supernatant decreased（P<0.01）.Compared with the silica group,the cell viability of the autophagy inhibition group（3-MA+Silica）increased,the m RNA expressions of Beclin1 and P62 decreased（P<0.01）,and the expressions of IL-6 and TNF-αincreased（P<0.01）.The results suggested that autophagy activation could significantly down-regulate the inflammatory response,and autophagy inhibition could further promote the inflammatory response caused by silica in macrophages.3.Silencing Gas6 inhibited autophagy of macrophages caused by silicaCompared with control group,the expression of Gas6 in silica groups increased（P<0.05）,and in Gas6 silencing group（si RNA-Gas6+silica）decreased（P<0.01）.Compared with silica group,the fluorescence intensity of LC3 and the protein expressions of autophagy-related factors LC3,Beclin1 and P62 decreased in the Gas6silencing group（P<0.05）,suggesting that Gas6 silencing inhibited macrophage autophagy.4.Gas6 gene silencing promoted macrophages to secrete inflammatory factors by inhibiting autophagyCompared with silica group,the expressions of IL-6、TNF-αin Gas6 silencing group significantly increased（P<0.05）.However,RAPA treatment could reverse the effect of Gas6 gene silencing.Compared with Gas6 silencing group,RAPA treatment reduced the secretion of IL-6 and TNF-α（P<0.05）,while 3-MA treatment further promoted this effect of Gas6 and increased the secretion of IL-6 and TNF-α（P<0.05）.The results suggested that Gas6 participated in the secretion of macrophages inflammatory factors through autophagy.Conclusions:The inflammatory response of macrophages induced by silica was influenced by Gas6 and autophagy.Gas6 gene silencing could aggravate the stimulation of silica on the inflammatory factors of macrophages by inhibiting autophagy,thus promoting the occurrence of inflammatory response. |
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