| Background:Takayasu Arteritis(TAK)is a chronic granulomatous disease,mainly involving the aorta and its branches.It is manifested as vascular stenosis and occlusion,arterial dilation or aneurysm formation,resulting in insufficient blood supply to limbs,internal organs and brain.At present,the pathogenesis of Takayasu Arteritis is not clear.Genetic factors play an important role in the pathogenesis of arteritis.Single nucleotide polymorphism in the LILRA3 gene was identified by genome Wide Association study(GWAS).SNP(rs103294)locus is a susceptibility gene for TAK.Studies have shown that the polymorphism of LILRA3 gene is associated with a variety of autoimmune diseases,but there is almost no research on TAK at present.We speculated that LILRA3 is involved in the pathogenesis of TAK.Therefore,this study preliminarily explored the polymorphism and protein expression of rs103294 in LILRA3 gene and its correlation with disease.Objective:To investigate the gene polymorphism and protein expression of the single nucleotide polymorphism rs103294 of leukocyte immunoglobulin like receptor A3 in peripheral blood of Takayasu arteritis,and LILRA3 correlation with vascular typing and monocytes.Methods:We enrolled 52 TAK patients and 79 healthy individuals to analyze polymorphism of rs103294 of LILRA3 gene with Sanger sequencing and detected expression of LILRA3 with ELISA.Chi-square test and correlation analysis were used to analyze the correlation between genotyping,vascular typing and monocytes.Results:It was no significant difference in genotype frequency and allele frequency of SNP RS103294 in LILRA3 between TAK patients and healthy controls(P>0.05);There was no significant difference in LILRA3 protein expression between TAK patients and healthy controls(P>0.05).There was no significant difference in LILRA3 protein expression in all TT,CT and CC genotypes(P>0.05).TAK genotype and allele frequency were not correlated with vascular typing(P>0.05).The number of monocytes in TAK patients was higher than that in healthy controls,but there was no correlation with LILRA3 protein expression(P>0.05).The expression of LILRA3 protein in TAK group was correlated with CRP(P<0.05),but not with ESR(P>0.05).Conclusion:The SNP rs103294 polymorphism and protein level of LILRA3 showed no differences in peripheral blood of TAK.The genotype had not related to vascular typing of TAK;The expression of LILRA3 was not correlation with increase of monocytes.The expression of LILRA3 protein in peripheral blood of TAK patients was related to CRP.Background:Takayasu arteritis is a kind of granulomatous inflammation involving large blood vessels.A variety of inflammatory cell infiltration can be seen in the early stage.With the progress of the disease,vascular lumen stenosis and occlusion,vascular dilatation to form aneurysms.In recent years,adventitia fibroblasts have been paid more and more attention as the main component of vascular adventitia.During vascular injury,adventitia fibroblasts can be activated and transformed into myofibroblasts with stronger contractile ability,and then migrate to the intima of vascular wall to promote the inflammatory response of vascular wall,resulting in vascular remodeling and lumen stenosis.As the main symptom of Takayasu arteritis is vascular disease,the adventitia of mouse aorta was selected as the research object in this study.Lipopolysaccharide(Lipopolysaccharide,LPS)was often used as a stimulant to induce cell injury.In this study,LPS was selected to establish an in vivo inflammation model.In the early stage,our research team adopted a two-stage research strategy including 20240 human proteome high-throughput chip(Hu Prot chip)small sample screening and large sample verification to find a new autoantibody of TAK-anti-IL-17 RB antibody.IL-17 RB belongs to the IL-17 family of receptors,and its ligands include IL-17 B and IL-25(IL-17E),but the affinity of IL-25 and IL-17 RB is much higher than that of IL-17 B,IL-25 and IL-17 RB in Takayasu’s arteritis.IL-25 plays its role through IL-17 RB and IL-17 RA,IL-17 through IL-17 RA and IL-17 RC,IL-25 and IL-17 share IL-17 RA subunits,but the relationship between IL-25 and IL-17 is not clear.Therefore,we studied the expression of IL-25 and IL-17 RB in peripheral blood of Takayasu arteritis,and the effects of IL-25,IL-17 and IL-17 RB on LPS-induced mouse aortic adventitia fibroblasts.Objective:To study the expression of IL-25 and IL-17 RB in peripheral blood of patients with Takayasu arteritis,and to study the effects of IL-25/IL-17 RB on the proliferation,migration and apoptosis of mouse adventitia fibroblasts induced by LPS,as well as its effects on inflammatory factors.Methods:(1)Detection of IL-25 and IL-17 RB protein levels in peripheral blood: peripheral blood samples were collected from 52 patients with TAK,14 patients with ANCA-associated vasculitis,16 patients with BD and 55 healthy controls.The levels of IL-25 and IL-17 RB protein in serum of TAK patients and HC patients were detected by ELISA.The correlations between IL-25,IL-17 RB and vascular classification and inflammatory indexes(ESR,CRP)of TAK were analyzed.(2)Establishment of inflammatory model: Setting up blank group and different LPS concentration(0.01μg/m L,0.1μg/m L,1μg/m L,5μg/m L,10μg/m L,100μg/m L,200μg/m L).The activity of AF was detected by CCK-8 method,and the best intervention concentration was determined.The m RNA level of IL-6 was detected by PCR to determine whether the modeling was successful.(3)In vitro experiment:Purchase of mouse aortic adventitia fibroblasts(primary cells).The experimental groups were divided into normal group,different intervention groups(LPS group,LPS+IL-25 group,LPS+IL-17 group,LPS+IL-25+IL-17 group),IL-17RB-si RNA transfection intervention group(si RNA+LPS group,si RNA+LPS+IL-25group).(4)Si RNA transfection experiment: small interference RNA(Smallinterfering RNA,si RNA)of IL-17 RB was designed and synthesized,and si RNA was transferred into AF by si RNA-mate transfection reagent.In this experiment,healthy control group and IL-17 RB interference group(si RNA group)were set up.RCR was used to detect the inhibition of IL-17 RB,and the si RNA with the highest interference rate was selected for follow-up experiments.(5)Detection of cell proliferation,migration and apoptosis: The proliferative activity of cells in the experimental group and control group was detected by CCK8 method,apoptosis was detected by flow cytometry,and the migration ability of adventitia fibroblasts in each group was detected by cell scratch method.ELISA was used to detect the expression of MMP2 and 9,MCP-1,TGF-β1 inflammatory factors.The expression ofα-SMA inflammatory factors was detected by q PCR.Results:1.Analysis of IL-17 RB and IL-25 protein levels in peripheral blood of patients with arteritis: compared with healthy controls and vasculitis disease controls,the level of IL-17 RB protein in peripheral blood of patients with arteritis increased(P<0.05).Compared with healthy controls,IL-25 protein levels were increased in Takayasu arteritis,ANCA-associated vasculitis and Behcet’s disease(P<0.05).2.The correlation between the levels of IL-17 RB and IL-25 protein in peripheral blood and vascular classification in patients with Takayasu arteritis: the level of IL-17 RB protein in peripheral blood of patients with arteritis was related to the vascular classification of Takayasu arteritis(P<0.05),but the increase of IL-25 protein level was not related to the vascular classification of Takayasu arteritis(P>0.05).3.Correlation analysis of IL-17 RB and IL-25 protein levels in peripheral blood with ESR and CRP in patients with arteritis: there was a correlation between IL-17 RB protein level and ESR(P<0.05).There was no correlation between IL-17 RB protein level and CRP(P>0.05).There was no correlation between IL-25 protein level and ESR,CRP(P>0.05).4.Establishment of inflammatory model: the OD values of different LPS concentration intervention groups were detected by CCK8,the results showed that LPS concentration was the best concentration of 5ug/m L,and could significantly increase the expression of IL-6m RNA.5.Effects of IL-25 and IL-17 on LPS-induced of AF function(1)Proliferation: Compared with the normal group,LPS can significantly promote the proliferation of AF(P<0.05).Both IL-25 and IL-17 can promote the proliferation of AF induced by LPS(P<0.05).Compared with IL-25 alone,IL-25 and IL-17 co-stimulation inhibited the proliferation of AF(P<0.05).Compared with IL-17 group,IL-25 group had a stronger effect on the proliferation of LPS-induced AF(P<0.05).(2)Apoptosis: Compared with the normal group,LPS can not promote the apoptosis of AF;IL-25,IL-17 and IL-25+IL-17 could not promote LPS-induced AF cell apoptosis,and the difference was not statistically significant(P>0.05)(3)Migration: Compared with the normal group,LPS group promoted the migration of AF;IL-25,IL-17 and IL-25+IL-17 groups can promote the migration of AF induced by LPS.IL-25 has a stronger effect on the migration of AF induced by LPS than IL-17.The migration effect of IL-25 and IL-17 co-stimulation group on LPS-induced AF was stronger than that of single stimulation group,and the difference was statistically significant(P<0.05)(4)Effects on inflammatory factors: 1.IL-25 and IL-17 increase the expression level of α-SMAm RNA.2.Compared with other intervention groups,IL-25 and IL-17co-stimulation group decreased the protein levels of MCP-1,MMP2,MMP9 and TGF-β1secreted by LPS-induced AF,and increased the expression level of α-SMAm RNA.6.Comparison of function of IL-25/IL-17 RB on LPS-induced AF before and after transfection Transfection of IL-17RB-si RNA knocked down the expression of IL-17 RB to study the effect of IL-25/IL-17 RB on the function of LPS-induced AF.(1)Proliferation: Compared with the non-transfected IL-17RB-si RNA group,the transfected LPS group significantly increased the proliferation rate of AF(P<0.05);After transfection,IL-25 group reduced the increment rate of AF(P<0.05).(2)Apoptosis: Compared with the non-transfected IL-17RB-si RNA group,LPS group and LPS+IL-25 stimulation group can not reduce the apoptosis rate of AF(P>0.05).(3)Migration: Compared with the non-transfected IL-17RB-si RNA group,the cell migration rate of LPS group and LPS+IL-25 group decreased(P<0.05).(4)Effects on inflammatory factors: Compared with the non-transfected IL-17RB-si RNA group,the protein levels of MCP-1,TGF-β1,MMP9 and α-SMAm RNA in the transfected LPS+IL-25 group decreased,and there was no significant difference in the protein level of MMP2.Conclusion:1.The levels of IL-25 and IL-17 RB protein in peripheral blood of patients with Takayasu’s arteritis increased.2.The levels of IL-17 RB protein in peripheral blood of patients with arteritis were correlated with ESR and vascular typing.3.IL-25 and IL-17 synergistically promote LPS-induced AF migration,and IL-25 and IL-17 may play an antagonistic role in proliferation and inflammatory factors.4.Il-25/IL-17 RB promotes LPS-induced AF proliferation,migration and up-regulation of inflammatory cytokines. |
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