Study On The Effect Of Silica Exposure On Macrophage LC3-associated Phagocytosis(LAP) And The Protective Effect Of Dioscin

Objective: Long-term exposure to silica can cause lung diseases and seriously affect one's health.Silica can enter the lung tissue and cause silicosis,which is a type of pneumoconiosis.Studies showed that the occurrence of silicosis promoted the occurrence of autoimmune diseases.Studies also indicated that deficiencies in LC3-associated phagocytosis(LAP)function can cause autoimmune diseases.In this study,J774 A.1 mononuclear macrophages were used to investigate the effect of silica exposure on the LAP function of macrophages and the protective effect of dioscin on it in vitro.Methods: In our study,J774 A.1 mononuclear macrophages was used for vitro experiments.After treatment of macrophages with different concentrations of silica suspension,the phagocytic ability of macrophages was detected by flow cytometry.Western Blot and RT-PCR were used to detect the expression level of LAP-specific proteins Rubicon,NOX2 and genes RUBCN and CYBB.Different concentrations of dioscin were used to treat silica-exposed macrophages,and flow cytometry was used to detect the phagocytic ability of macrophages.Western Blot and RT-PCR methods were used to detect LAP-specific proteins Rubicon and NOX2 at the protein and gene expression levels,the ELISA was used to detect the secretion level of inflammatory factor TNF-?.Results:1.The silica exposure treatment does not affect the phagocytic ability of J774 A.1macrophages.J774A.1 macrophages were treated with different concentrations of silica suspensions for 6,24 and 48 hours.The phagocytic ability was detected by flow cytometry.Compared with the blank control group,different concentrations of silica suspension treatment had no effect on the phagocytic ability of the cells.2.Silica exposure reduces the expression of Rubicon and NOX2 in J774 A.1macrophages.After treating cells with silica suspension for 6,24 and 48 hours,RT-PCR andWestern Blot were used to detect the expression of Rubicon and NOX2 at the m RNA and protein levels.The results showed that the expression levels of Rubicon and NOX2 decreased with the concentration of silica increasing,and there was a certain statistical significance between the partial treatment groups at 6,24 and 48 hours(P<0.05).3.Different doses of dioscin do not affect the phagocytic ability of silica-exposure J774 A.1 macrophages.After treatment of silica-exposure macrophages with different concentrations of dioscin for 6,24 and 48 hours,flow cytometry was used to detect the phagocytic ability of the cells.Compared with the blank control group,different doses of dioscin treatment had no effect on the phagocytic ability of the silica-exposure macrophages.4.Dioscin up-regulates the reduction of Rubicon and NOX2 expression in J774 A.1macrophages caused by silica exposure.The results of Western Blot showed that compared with the blank control group,the Rubicon and NOX2 protein expression was reduced after treatment with silica suspension.After treatment with dioscin,compared with the blank control group,Rubicon,NOX2 expression levels were significantly increased with the concentration of dioscin increasing.The results of RT-PCR experiments showed that compared with the blank control group,the transcription levels of RUBCN and CYBB in the silica exposure group also decreased to varying degrees.Compared with the silica exposure group,the transcription level of the two genes in the dioscin treatment group with different concentrations also showed a certain increase.There was statistical significance between these treatment groups(P<0.05).5.Dioscin inhibits the level of inflammation of J774 A.1 macrophages caused by silica exposure.The results of ELISA experiments showed that the secretion of tumor necrosis factor(TNF-?)was increased at 6,24 and 48 hours after exposure to silica,and the secretion was significantly reduced after treatment with dioscin at 6,24 and 48 hours.At different time points,the difference between the low-,medium-,and high-dosedioscin treatment group was significantly different from that of the silica exposure group(P<0.05).Conclusion: 1.Silica exposure and dioscin treatment could not affect the phagocytic ability of J774 A.1 macrophages separately or in combination.2.Silica exposure could damage the LAP function of J774 A.1 macrophages,and dioscin could have a protective effect on the damage of LAP caused by silica exposure.