Effect Of Silica Exposure On LAP And STING Signaling Pathways In Lung Tissues Of Mice

Objective: Silicosis is one of the most common and serious occupational diseases,still no effective treatment for this disease.Alveolar macrophages play a very important role in the pathogenesis and development of silicosis,they are the main target cells of silica.At the same time,due to the toxic effect of silica,some alveolar macrophages disintegrate and die,releasing phagocytic silica and cell content double-stranded DNA(ds DNA)combines with auto-antibodies to form immune complexes,aggravating pulmonary inflammation.LC3-associated phagocytosis(LAP),a new non-classical form of autophagy,is an important mechanism for the body to clear dead cells.Studies have shown that LAP defects,under the condition of apoptosis cells can’t degradation of fracture and release the cell contents ds DNA interferon genes stimulating factor(Stimulator of interferon genes,STING)pathway,induction of Interferons(IFNs)Interferons secretion,Increase the body’s inflammatory response.In this study,the animal model of mice exposed to silica was established and treated with different concentrations of silica to explore the effects of silica on LAP and STING signal pathways in the lungs of mice.Methods: In this study,the model of silica exposure in C57BL/6 mice was established by endotracheal instillation of silica suspension.Firstly,three silica exposure time points were set up on the 7,14 and 56 day.According to the concentration of silica,they were divided into normal saline control group,low silica(1.25mg/50μl volume),medium(2.5mg/50μl volume)and high(5mg/ 50μl volume)dose group.The lung tissues of mice in each group and at each time point were taken to carry out our experimental study.The expression levels of key node proteins and genes of LAP and STING signal were detected by Western blot and RT-PCR methods,and the effect of silica exposure on this pathway was explored.The pathological changes of the lungs were observed and detected by HE and Masson staining.Western blot and RT-PCR techniques were used to detect STING and its downstream signal molecules,and to detect the activation of the pathway induced by silica exposure.The apoptosis of lung tissue was observed by DAB Tunel staining.The content of ds DNA in bronchoalveolar lavage fluid(BALF)was detected by DNA detection kit.Results:1.Different concentrations of silica exposure causes lung inflammation and fibrosis.H&E results showed that there was no or only slight inflammation in the lung tissue of mice in normal saline control group at 7,14 and 56 days after silica exposure,and the lung tissue structure was intact.The alveolar wall of mice exposed to silica was damaged,and a large number of inflammatory cells gathered around the damaged tissue.With the increase of silica concentration,the alveolar wall was obviously thickened,and the alveolar cavity was narrowed.At 56 days,a large number of inflammatory cells still gathered.In the high-dose silica group,obvious nodules appeared,and lung tissue repair was abnormal.Masson results showed that there was no significant blue collagen deposition in normal saline control group and silica exposure group at 7 and 14 days.At 56 days,there was no change in lung tissue of saline control group,but blue collagen deposition was found in lung tissue of silica exposure group.With the increase of silica concentration,the proportion of collagen deposition increased.2.Different concentrations of silica exposure caused increase in apoptotic cells in mice lung tissue.DAB results showed that there was no apoptotic cells in the mice in the saline control group in Silica exposed to 7,14 and 56 days.Compared with the saline control group,there were a small amount of apoptotic cells in 7 and 14 days;the proportion of apoptotic cells in each of the 56 days of time is increased.3.Exposure to silica can injure LC3-associated phagocytosis in lung tissue of mice.Western blot results showed that the protein expression levels of Rubicon,NOX2 and TIM4 in the lungs of mice in some silica exposure groups decreased with the increase of silica concentration compared with the saline control group at 7,14,56 after silica exposure(P <0.05).Meanwhile,Realtime-PCR results showed that Rubicon and TIM4 gene transcription levels were decreased at each time point in some silica exposed groups(P <0.05).The transcription level of NOX2 gene was increased at three time points in silica exposure groups(P <0.05).4.Exposure to different concentrations of silica resulted in the increase of ds DNA content in BALF of mice.The ds DNA results showed that,compared with the normal saline control group,the ds DNA content in BALF of the silica dust exposure groups increased at 7,14 and 56days(P <0.05).5.Exposure to silica at different concentrations can activate STING signaling pathway.Western blot showed that compared with saline control group,the expression level of STING protein in lung of some mice exposed to silica increased with the increase of silica concentration at 7,14 and 56 days(P<0.05).Meanwhile,the results of Realtime-PCR showed that the transcription level of STING gene in the lungs of some mice exposed to silica increased at various time points(P<0.05).Conclusion:1.Silica exposure could increase apoptosis and decrease the expression of LAP specific protein in lung tissue of mice.2.Silicon exposure could activate STING signaling pathway in lung tissue of mice.