| Background:Ankylosing spondylitis(AS)is a chronic autoimmune inflammatory disease that mainly affects the axial joints and large peripheral joints.In severe cases,it can lead to joint deformity and even disability.A large number of studies have confirmed that the Janus protein tyrosine kinase/signal transducer and activator of transcription(JAK/STAT)signaling pathway is closely related to a variety of rheumatic immune diseases.The JAK/STAT signaling pathway is involved in the inflammatory response of AS,but less research has been done on the JAK3/STAT5 signaling pathway in AS,and its specific mechanism in the pathogenesis of AS is still unclear.At present,the JAK inhibitor tofacitinib has been approved for the treatment of AS and its effect is clear,but its specific treatment mechanism is not fully understood.Objective:To detect the expression of JAK3/STAT5 signal pathway in peripheral blood of patients with AS and to explore its role in the occurrence and development of AS as well as to explore the therapeutic effect and mechanism of tofacitinib on AS.Materials and methods:1.Clinical data,peripheral blood and laboratory tests were collected from 30 patients with ankylosing spondylitis activity(ASA),30 patients with ankylosing spondylitis stability(ASS)and 50 healthy controls(HC).Quantitative real-time-polymerase chain reaction(RT-qPCR)was used to detect the relative expression levels of JAK3 mRNA,STAT5a mRNA and STAT5b mRNA in peripheral blood mononuclear cells(PBMCs)of all subjects.The protein expression levels of JAK3,STAT5a,STAT5b,P-JAK3,P-STAT5a and P-STAT5b were detected by Western-blot.Plasma interleukin-2(IL-2)expression was detected by enzyme-linked immunosorbent assay(ELISA).Spearman was used to analyze the correlation between variables.Receiver operating characteristic curve(ROC)was used to evaluate the value of JAK3 mRNA,STAT5a mRNA and STAT5b mRNA in monitoring AS activity.2.20 AS patients were treated with tofacitinib for 3 months.Clinical data,peripheral blood and laboratory tests of the patients before and after treatment were collected.The relative expression levels of JAK3 mRNA,STAT5a mRNA and STAT5b mRNA in PBMCs before and after treatment were detected by RT-qPCR.The protein expression levels of JAK3,STAT5a,STAT5b,P-JAK3,P-STAT5a and P-STAT5b were detected by Western-blot.The bath ankylosing spondylitis disease activity index(BASDAI)and bath ankylosing spondylitis functional index(BASFI),laboratory tests and JAK3,STAT5a,STAT5b gene expression levels and protein phosphorylation levels were compared before and after treatment.Results:1.Compared with the ASA group,the BASDAI,BASFI,erythrocyte sedimentation rate(ESR),hypersensitivity C-reactive protein(hsCRP)and monocyte(MO)in the ASS group were significantly decreased(all P<0.05).Compared with the HC group,the white blood cell(WBC),neutrophil(GR),MO,platelet(PLT),creatinine(Crea)were significantly increased and the glomerular filtration rate(eGFR)was significantly decreased in the ASA group(all P<0.05)while the WBC,GR and MO were significantly increased in the ASS group(all P<0.05).2.The expression levels of JAK3 mRNA,STAT5a mRNA and STAT5b mRNA in the AS group were higher than those in HC group(all P<0.05).The differences of JAK3 mRNA,STAT5a mRNA and STAT5b mRNA among the ASA group,ASS group and HC group were statistically significant(F=65.98,21.148,13.666;all P<0.001),which were higher in the ASA group than in the ASS group and HC group(all P<0.001).The expression level of JAK3 mRNA in the human leukocyte antigen B27(HLA-B27)positive group was higher than that in the HLA-B27 negative group(P<0.05).The protein ratios of P-JAK3/JAK3,P-STAT5a/STAT5a and P-STAT5b/STAT5b were significantly different among the ASA group,ASS group and HC group(F=91.557,25.15,178.592;all P<0.001),in the ASA group were higher than ASS group and HC group(all P<0.001).The expression level of IL-2 was significantly different among the ASA group,ASS group and HC group(F=3.317,P<0.05),which was higher in the ASA group than ASS group and HC group(both P<0.05).3.Spearman correlation analysis showed that JAK3 mRNA,STAT5a mRNA and STAT5b mRNA in the AS group were positively correlated with BASDAI(all P<0.05),JAK3 mRNA and STAT5a mRNA were positively correlated with BASFI(both P<0.05),and STAT5a mRNA was positively correlated with PLT(P<0.05).In the ASA group,JAK3 mRNA was positively correlated with IL-2(P<0.001),negatively correlated with eGFR(P<0.05),STAT5a mRNA was positively correlated with ESR(P<0.05),STAT5b mRNA was positively correlated with hsCRP(P<0.05).4.The ROC curve showed that JAK3 mRNA predicted the area under ROC curve(AUC)of ASA with a 95%CI of 0.92(0.853,0.987),sensitivity and specificity of 86.7%and 90%,respectively.STAT5a mRNA predicted the AUC 95%CI of ASA was 0.874(0.787,0.961),and the sensitivity and specificity were 96.7%and 66.7%,respectively.STAT5b mRNA predicted the AUC 95%CI of ASA was 0.749(0.617,0.881),and the sensitivity and specificity were 73.3%and 80%,respectively.5.After tofacitinib treatment for 3 months,the expression levels of STAT5a mRNA and STAT5b mRNA in PBMCs of 20 AS patients were significantly lower than before treatment(both P<0.05),and there was no significant change in JAK3 mRNA(P>0.05).The protein ratios of P-JAK3/JAK3,P-STAT5a/STAT5a and P-STAT5b/STAT5b were significantly lower than before treatment(all P<0.05),BASDAI,BADFI,ESR and hsCRP were significantly lower than before treatment(all P<0.05).Conclusions:1.In addition to elevated inflammatory indexes,AS patients can also cause renal damage.2.AS patients have abnormal expression of JAK3/STAT5-related genes and proteins,and JAK3/STAT5-related genes are correlated with some clinical and laboratory tests,suggesting that JAK3/STAT5 pathway may play a role in the pathogenesis of AS.3.The abnormal expression of IL-2 in AS patients is correlated with JAK3 gene,suggesting that IL-2 may be involved in the pathogenesis of AS through the JAK3/STAT5 pathway.4.The expression level of JAK3 gene has a certain value in the monitoring of AS disease activity.5.After tofacitinib treatment,BASDAI,BASFI and inflammatory indexes of AS patients were significantly reduced,which proved that tofacitinib can effectively inhibit the inflammatory response and improve clinical symptoms of AS patients.The JAK3/STAT5-related genes and protein phosphorylation levels were significantly reduced after treatment,suggesting that tofacitinib may act by inhibiting the JAK3/STAT5 pathway. |
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