Molecular Mechanism Of MiR-155 Promoting Podocyte Pyrodeath By Inhibiting IL-18BP In A Mouse Model Of Lupus Nephritis

Objective:To establish the mouse model of miR-155^-/-Fas^lpr/lprgenotype,and investigate the pathogenesis of lupus nephritis（LN）induced by micro RNA-155（miR-155）and promoting cell pyrosis in mice.It provides a new idea for diagnosis and treatment of lupus nephritis.Methods:（1）Grouping:The mir155 knockout mouse model was constructed,the miR-155 knockout mice were hybridized with the LN mice,and the miR-155^-/-Fas^lpr/lprgenotypic mice were identified by Polymerase Chain Reaction（PCR）and agarose gel electrophoresis.The experimental animals were divided into three groups:blank group（C57group）,model group（LN group）and miR-155^-/-group(miR-155^-/-Fas^lpr/lprknockout group),with 8 females in each group.（2）Detection indexes and methods:The urine of mice in each group was collected for 24 hours every two weeks from the 14th week to detect the protein content of mice in urine.After 20 weeks,the animals were sacrificed.Kidney tissue and serum were collected from mice in each group.enzyme-linked immunosorbent assay（ELISA）was used to measure the contents of anti-Smith antibody and anti-ds DNA antibody.real-time quantitative polymerse chain reaction,real-time quantitative polymerse chain reaction,RT-q PCR)were used to detect the expression of miR-155 and caspase-1,IL-1β,IL-18BP,IL-18,GSDMD（Gasdermin D）,P-cadherin and Nephrin at the gene level in kidney tissues of each group.The expressions of caspase-1,IL-1β,IL-18BP,IL-18,GSDMD（Gasdermin D）,P-cadherin and Nephrin at the protein level were detected by Western blot.hematoxylin-eosin staining（HE）,Periodic Acid-Schiff stain,hematoxylin-Eosin staining（HE）,periodic acid-Schiff stain,PAS),periodic acid-silver metheramine（PASM）and Masson staining were used to observe the damage of kidney tissue in mice.The integrity of glomerular podocytes and basement membrane was observed by transmission electron microscopy.The expression and distribution of Caspase-1 and Nephrin in glomeruli were analyzed by immunofluorescence.（3）Bioinformatics analysis predicts the binding site of the 3’Untranslated Region（UTR）of the target gene that miR-155 may bind,and double luciferase reporter gene detects the activity of IL-18BP gene.Results:（1）Compared with the blank group,urine protein content,lupus-specific antibody anti-smith antibody and anti-ds DNA antibody of mice in the model group and miR-155^-/-group are increased,and caspase-1,IL-1β,IL-18,GSDMD,and NLRP3 are increased.The expressions of IL-18BP,P-cadherin and Nephrin were decreased,and the differences were statistically significant（P<0.05）.The results of HE,PAS,PASM,MASSON staining and electron microscopy also showed increased inflammatory infiltration,excessive activation of inflammatory cells,renal fiber hyperplasia and serious pathological injury.（2）Compared with the model group,the urine protein content and lupus specific antibody of mice in miR-155^-/-group were decreased,the expressions of caspase-1,IL-1β,IL-18,GSDMD and NLRP3 were decreased,and the expressions of IL-18BP,P-cadherin and Nephrin were increased.The difference was statistically significant（P<0.05）,and the pathological staining results also showed that the kidney injury was significantly relieved.（3）Bioinformatics analysis predicted that combination of dual luciferase reporter genes further verified that miR-155 could bind to IL-18BP 3’UTR.Conclusion:（1）The expression of miR-155 was up-regulated in the mouse model of lupus nephritis,and the expression of pyroptosis related factors was positively correlated with the disease.Specific knockout of miR-155 could reduce pyroptosis of foot cells and thus improve the symptoms of lupus nephritis.（2）Mi R-155 may act by binding and inhibiting the IL-18BP gene.