Mechanism Research Of Adropin Deficiency Trigerring ANCA Associated Vasculitis

Objective: Antineutrophil cytoplasmic antibodies(ANCA)associated vasculitis is a systemic autoimmune disease characterized by necrotizing vasculitis,the mortality rate was significantly higher than that of other autoimmune diseases,which target antigen for serine protease 3(PR3)and myeloperoxidase(MPO).The pathogenic mechanism of ANCA is unclear.Adropin is the product of energy homeostasis associated(ENHO),which is related to insulin resistance and endothelial cell function.We found that MPO-ANCA associated lung injury patients carry with ENHO mutations.We hypothesized that ENHO gene can regulate vascular endothelial function,and ENHO gene mutation may lead to vascular endothelial dysfunction and vascular inflammation.In order to confirm our conjecture,the ENHO knockout C57BL/6J mouse model was used to verify the relationship between MPO-AAV and adropin associated vasculitis,and to explore its possible mechanism.Methods:(1)Extraction of blood DNA from 152 cases ANCA associated vasculitis patients and 220 cases control group,gene sequencing and localization of gene mutation through BLAST on PUBMED.(2)ELISA analysis of serum adropin,ET-1 and SLPI in ANCA patients and healthy individuals.(3)ENHO knockout mice(Adr KO)were obtained by Crisp-cas9 technology,and then DNA was extracted and sequenced to verify whether the ENHO gene was successfully knocked out.(4)Biomarkers were analyzed by ELISA,tissue H&E staining and immunohistochemistry to observe the phenotypic differences among Adr KO mice,Adr HET mice and WT mice serum.(5)We selected 3 Adr KO mice and 3 WT 8-week old littermates,then carried out experiments of extraction of lung tissue RNA for RNA-seq,and the possible pathway protein was verified by immunohistochemical staining and Westernblot.Finally,the protein interaction was analyzed by laser confocal.Results:(1)We found 6 cases p.Ser43 Thr mutation,5 cases p.Cys56 Trp mutation,1 case p.Tyr72 Tyr,and other 15 cases intron mutation(contain:c.365G>A,c.718G>A,c.895G>A,c.994G>A)in all the patients with MPO-ANCA,while no mutations were found in the corresponding region of the ENHO gene in the control group.(2)The level of serum adropin in patients with ANCA associated pneumonia(423.030 + 178.302 pg/ml)was significantly higher than the normal control group(91.670 + 66.091 pg/ml)(P<0.001),and serum ET-1 level in patients with ANCA pneumonia(56.723 + 12.503 pg/ml)was significantly higher than the normal control group(48.991 + 7.067 pg/ml).(3)Adr KO 627 bp fragment was deleted in the ENHO transcription region,and the ENHO knockout mice were successfully constructed.The ENHO knockout mice and the same strain wild type mice were reared in the common animal breeding condition,and the offspring mice(F1-F6)grew well.The three genetypes(homozygous,heterozygous and wild genotypes)were identified in the offspring mice.(4)The serum levels of CRP,TNF-alpha,AECA,OPN,ET-1 and INS were negative correlated with the level of adropin in mice with different genotypes(CRP:P=0.0169,r=-0.5034;TNF-?:P=0.0389,r=-0.4430;AECA:P=0.0956,r=-0.3643;OPN:P=0.0772,r=-0.3846;ET-1:P=0.0003,r=-0.6988;INS:P=0.064,r=-0.4015)?(5)In the study of lung tissue H&E staining,we found that pulmonary vascular of Adr KO mice have inflammatory infiltration,vascular wall destruction,extravasation of red blood cells,while the wild-type mice pulmonary vessels intact.(6)In the immunohistochemical staining of lung tissue,we found that immune cells CD3,CD20 and CD38 positive cells were significantly increased in Adr KO mice.(7)The results of RNA-seq analysis showed that the expression of adropin and other cytokines induced by interferon in lung tissue was decreased.The cell proliferation related genes in two groups of mice also showed significant differences in expression levels.The down regulated gene expression in Adr KO mice was mainly concentrated in the development of cardiac function and blood circulation.(8)Westernblot results showed that p Akt1(T450),Akt1,e NOS and Mapk1 were not affected by the level of Adropin.The p-AKT1(Ser473)and p-e NOS(1177)in lung tissue of Ad KO mice were lower than those of the littermates' wild type.(9) Immunofluorescence staining showed that the expression of CD31 and ICAM-1 in lung tissue of Adr KO mice was significantly higher than that in WT mice,and the expression of Akt1 was significantly lower than that of WT mice.We found that CD31 and ICAM-1 were co-expressed in vascular endothelial cells and vascular smooth muscle cells by CD31 and ICAM-1 co-localization.In Akt1,VEGFR2,PI3 K immunofluorescence co-localization,we found that in Adr KO mice,VEGFR2,PI3 K co-expression was lower than WT mice.Conclusion:(1)MPO-ANCA associated lung injury may be associated with the occurrence of ENHO gene mutation or adropin disorder.(2)Adr KO mice have similar phenotypes of chronic pulmonary hemorrhage with human MPO-AAV,and ENHO knockout may induce MPO-ANCA related lung injury.(3)The decreased expression of adropin resulted in the decline of phosphorylation level of Akt1,and then led to the decreased expression of p-e NOS through PI3K-Akt1 pathway.After that,the loss of protection from vascular endothelial increased the expression of vascular endothelial growth factor(VEGF)enhancing the proliferation of vascular endothelial cells,inflammation.Finally,secretion of IL-1 and TNF-? would induce aggregation of neutrophil autoimmunity on vascular endothelium,at this point MPO-ANCA associated lung injury easily occurred in the susceptible constitution.