Study On The Mechanism Of Dioscin In Inhibiting The Occurrence And Development Of Systemic Lupus Erythematosus Accelerated By Silica In The Lupus-prone MRL/lpr Mice

Objective: So far,it's estimated that millions of people around the world have been exposed to crystalline silica dust.Crystalline silica in lung tissue can lead to silicosis,which is a type of pneumoconiosis.It is a progressive occupational lung disease,which characterized as chronic lung inflammation and irreversible pulmonary fibrosis.However,it is not to be ignored that exposure to crystalline silica dust can also induce to autoimmune diseases.Systemic lupus erythematosus(SLE),caplan syndrome and rheumatoid arthritis(RA)had been reported frequently in silicosis patients.However,the pathogenesis of autoimmune diseases induced by crystalline silica dust haven't been fully elucidated.Recent studies have found that the apoptosis and the LC3-associated phagocytosis play an important role in the pathogenesis of autoimmune diseases.This study used MRL/lpr mice to study the role of apoptosis and LAP in inducing autoimmune diseases and the inhibition mechanism of dioscin,which will provide new ideas for the prevention and treatment of autoimmune diseases caused by silica.Methods: In this study,experiment mice model was induced by intratracheal instillation of crystalline silica suspension and saline,and then 80 mg/kg dose of dioscin and CMC-Na were given to the mice through gastric irrigation.The experimental animals were divided into four groups: saline+CMC-Na group,saline+dioscin group,silica+CMC-Na group,silica+dioscin group.In 7 days,28 days and 56 days,mice were killed.We used H&E staining and the inflammatory cell count in the alveolar lavage to detect the changes in the pulmonary inflammatory condition.ELISA was applied to detect anti-body(anti-ds-DNA,ANA)in serum.IF was used to illuminate C3 and Ig G depositing in the kidney.Serum creatinine and urine creatinine was determined by creatine oxidase method.The urine protein contents of mouse was determin by the Bradford method.Flow cytometry analysis was used to detect the apoptosis percentage of lymphocytes in the spleen.Besides,we used Western blot experimental technique to detect the expression of mitochondrial-mediated-apoptosis related protein in spleen tissue.We adopted Western blot and RT-PCR techniques to detect the protein expression levels of Rubicon,NOX2,Beclin1,LC3 and genes of Rubcon,CYBB,BECN and LC3 in kidney tissue.Results:1.Dioscin could reduce the pulmonary inflammation caused by silica in the lupusprone MRL/lpr mice.HE result found that the inflammation in saline+CMC-Na and saline+dioscin groups were slight and alveolar structure basic was complete without damage.However,the lungs of silica+CMC-Na group in 7,28 day appeared obvious inflammation,gathering a large number of inflammatory cells in the alveolar interval and lung parenchyma.Besides,normal alveolar structure has been destruction.In 56 days,the inflammation decreased,but the tissue still infiltrated with inflammatory cells and obvious cell nodules were present.What's worse,the alveolar structure was damaged.The phenomenon of silica+dioscin group was relieved.In addition,Giemsa staining showed that in 7 day and 28 day,the cells of macrophages in BALF of silica+CMC-Na group were higher than the other three groups,with the statistical significant.There was no statistical significance with silica+dioscin group in 56 day.The rates of Lymphocytes and neutrophils in silica+CMC-Na group in 7day were higher than saline+CMC-Na group and silica+dioscin group.In 28 day and 56 day,lymphocytes and neutrophils in silica+CMC-Na group and silica+ dioscin group gradually increased,and the proportions of them in silica+ dioscin group were higher than silica+CMC-Na group and saline+dioscin group,with statistical significant.2.Dioscin could reduce the levels of autoantibodies in the silica-exposed lupusprone MRL/lpr mice.Compared with saline+CMC-Na group and saline+dioscin group,the levels of these two antibodies in the serum in silica+CMC-Na group and silica+dioscin group were also increased,which was also statistically significant.At the same time,compared with silica+CMC-Na group,the serum autoantibodies of the group of silica+ dioscin group were significantly decreased with statistical significant.3.Dioscin could improve the function of kidney,and relieve the renal pulmonary inflammation and the deposition of immune complexes in the silica-exposed lupus-prone MRL/lpr mice.Compared with the saline+CMC-Na group and the saline+dioscin group,the ratio of urinary protein creatinine and serum creatinine in silica+ CMC-Na group and silica+ dioscin group were significantly increased.Compared with the silica+ CMC-Na group,the urine protein creatinine ratio and serum creatinine were decreased silica+ dioscin group.Compared with the saline+CMC-Na group and the saline+dioscin group,the results of H&E staining in silica+ CMC-Na group and silica+ dioscin group showed that the renal inflammation was serious.Compared with silica+ CMC-Na group,the saline+dioscin group showed lighter inflammation of the kidney: gather around a small amount of inflammatory cells in glomeruli,glomerular mesangial mild hyperplasia and a small amount of sample transparent thrombus formation.Immunofluorescence results show that deposition of the Ig G and C3 in silica+ CMC-Na group and silica+ dioscin group were higher than the saline+CMC-Na group and the saline+dioscin group,and the immune complex deposition in silica+dioscin group was lower than that in silica+ CMC-Na group.4.Dioscin could reduce the level of lymphocyte apoptosis,and promote the LAP function of renal cells in the silica-exposed lupus-prone MRL/lpr mice.The results of flow cytometry showed that there were only a less degree changes in the saline+CMC-Na group and the saline+dioscin group,and there was no statistical significance.However,compared with the silica+ CMC-Na group,the proportion of cell apoptosis in silica+dioscin group significantly reduced,and both of the early and late apoptosis were statistically significant.Compared with the saline+CMC-Na group and the saline+dioscin group,Caspase9,Caspase3,Bax and Cytc also increased in silica+ CMC-Na group and silica+dioscin group were significantly increased.And the antiapoptotic proteins,the Bcl-2 was significantly decreased,also with statistically significant.The expression of Caspase9,Caspase3,Bax and Cytc in the silica+ dioscin decreased,while the expression of bcl-2 protein increased compared with silica+CMC-Na,both of which were statistically significant.With the saline+CMC-Na group and the saline+dioscin group,protein expression levels of Rubicon,NOX2 reduced,while the level of Beclin1 and LC3 increased,with statistical significance in silica+CMC-Na group and silica+dioscin group.Compared with the silica+CMC-Na group,the expression of Rubicon,NOX2,Beclin1 and LC3 protein in the drug group was increased in silica+dioscin group,all of which were statistically significant.Compared with the saline+CMC-Na group and the saline+dioscin group,the transcriptional levels of RUBCN,CYBB also reduced in silica+CMC-Na group and silica+dioscin group,while BECN and LC3 were increased.Compared with the s silica+CMC-Na group,the transcriptional levels of also increased in silica+dioscin group.Conclusion: 1.Dioscin could reduce the pulmonary inflammation caused by silica in the lupus-prone MRL/lpr mice.2.Dioscin could reduce the level of autoantibodies in the serum in the silica-exposed lupus-prone MRL/lpr mice.3.Dioscin could reduce the ratio of urinary protein creatinine and serum creatinine content to improve renal function,and reduce renal inflammation and the accumulation of Ig G and C3 in the silica-exposed lupus-prone MRL/lpr mice.4.Dioscin could inhibit the occurrence and development of SLE by inhibiting the mitochondrial apoptosis and promoting the LAP function of renal cell in the silica-exposed lupus-prone MRL/lpr mice.