| Research background:Ankylosing spondylitis(AS)is a chronic immune-mediated inflammatory arthritis that mainly affects axial joints and peripheral large joints.In severe cases,spinal deformities and ankylosing can occur.A large number of studies have confirmed that autophagy is related to the progress of various rheumatic diseases,including systemic lupus erythematosus,rheumatoid arthritis,osteoarthritis,and systemic sclerosis.Autophagy is an important mechanism by which cells decompose their components through lysosomes or vacuoles to maintain the normal physiological activity of cells and homeostasis.They can be divided into three categories:macroautophagy,microautophagy,and partner-mediated autophagy.Autophagy is a multi-step,multi-path process,which is regulated by a variety of autophagy-related genes(ATG).However,autophagy is rarely studied in the pathogenesis of ankylosing spondylitis,which is also a rheumatic disease.The current research mainly focuses on the association between ATG gene polymorphism and AS and the expression of ATG in different tissues.Whether the expression level of ATG is related to inflammation in AS patients and Whether ATG can be used as an AS disease activity evaluation has not been reported.Studies have found that tumor necrosis factor alpha(TNF-α)can stimulate and induce autophagy in a variety of cells,and TNF-α inhibitors(TNFi),which play a role in antagonistic TNF-α,have a clear therapeutic effect on AS,but the specific mechanism is not fully understood.Whether TNFi can exert its anti-inflammatory effect by affecting the autophagy of AS patients is currently unknown.Objective:To study the expression of autophagy-related genes in peripheral blood mononuclear cells(PBMCs)of patients with AS and different disease activity stages,and to observe the changes of autophagy-related gene expression levels in AS patients before and after treatment with tumor necrosis factor inhibitors.To investigate whether autophagy is involved in the pathogenesis of AS,the relationship between autophagy-related genes and AS disease activity,and the effect of tumor necrosis factor inhibitor treatment on autophagy in patients with AS.Methods:1.Collect clinical data and peripheral blood of 30 patients with active AS(AAS:BASDAI≥ 6,or 6>BASDAI>4 and ESR>22 mm/h,or 6>BASDAI>4 and hsCRP>9 mg/L),and 30 inactive AS patients(IAS:BASDAI≤4)and 30 healthy controls(HC),isolation and extraction of mononuclear cells(PBMCs),total RNA extraction,real-time quantitative PCR(RT-qPCR)detection the expression of LC3,Beclinl,ATG5,ATG12,ATG16L1 of autophagy-related genes in three groups of PBMCs.The correlations between LC3,Beclinl,ATG3,ATG5,ATG12,and ATG16L1 levels and different disease activity levels and clinical laboratory indicators in AS were analyzed by Spearman correlation test.2.Eighteen AS patients were treated with TNFi for 3 consecutive months.Clinical data and peripheral blood were collected before and after treatment.Total RNA was extracted after PBMCs were isolated.RT-qPCR was used to detect the expression levels of autophagy-related genes LC3,Beclinl,ATG3,ATG5,ATG12,and ATG16L1 in PBMCs of the study subjects,and then the expression levels of autophagy related genes LC3,Beclinl,ATG3,ATG5,ATG12 and ATG16L1 in PBMCs before and after treatment were compared.Results:1.(1)The expressions of LC3,ATG5,ATG12,and ATG16L1 in PBMC of AS patients were significantly lower than those in HC group(P<0.05,respectively)[LC3:4.25(3.74)×10-4 vs 6.92(4.42)×10-4;ATG5:5.65(2.57)×10-3 vs 7.78(3.77)×10-3;ATG12:6.00(3.74)×10-3 vs 10.30(4.27)×10-3;ATG16L1:2.14(1.36)×10-3 vs 3.13(2.30)×10-3],while Beclinl and ATG3 expression was no significant difference between the two groups(P>0.05)[Beclin1:5.63(3.19)×10-3 vs 5.41(2.62)×10-3;ATG3:11.57(5.22)×10-3 vs 9.65(4.96)×10-3].(2)The expression levels of LC3 in AAS group and IAS group were significantly lower than those in HC group(P<0.05,respectively),but there was no significant difference between AAS and IAS groups(P>0.05)[LC3 AAS:4.51(2.90)×10-4 vs IAS:4.00(4.01)×10-4 vs HC:6.92(4.42)×10-4].The expression levels of Beclinl were not significantly different between the three groups of AAS,IAS and HC(P>0.05,respectively)[Beclinl:5.10(2.45)×10-3 vs 6.67(3.91)×10-3 vs 5.42(2.62)×10-3].The expression level of ATG3 was significantly higher in the IAS group than in the HC group(P<0.05),while the AAS group was not significantly different from the IAS group and the HC group(P>0.05,respectively)[ATG3:10.22(4.98)×10-3 vs 12.64(5.66)×10-3 vs 9.65(4.96)×10-3].The expression level of ATG5 in the AAS group was significantly lower than that in the IAS group and the HC group(P<0.05,respectively),and there was no significant difference between the IAS group and the HC group(P>0.05)[ATG5:5.05(1.94)×10-3 vs 6.21(3.69)×10-3 vs 7.78(3.77)×10-3].The expression level of ATG12 was significantly lower in the AAS and IAS groups than in the HC group(P<0.05,respectively),and the AAS group was also significantly lower than in the IAS group(P<0.05)[ATG12:5.91(2.31)×10-3 vs 7.94(4.63)×10-3 vs 10.30(4.27)×10-3].The expression level of ATG16L1 in AAS group was significantly lower than that in HC group(P<0.05),but there was no significant difference between IAS group and AAS group and HC group(P>0.05,respectively)[ATG16L1:2.06(0.81)×10-3 vs 2.60(1.89)×10-3 vs 3.13(2.30)×10-3].(3)ATG5 and ATG12 expression levels were negatively correlated with BASDAI,ESR,and hsCRP(P<0.05,respectively),ATG3 expression levels were negatively correlated with BASDAI,BASFI and ESR(P<0.05,respectively).However,there was no significant correlation between LC3,Beclinl,ATG16L1 and BASDAI,BASFI,ESR,hsCRP(P>0.05).2.(1)The BASDAI,BASFI,and serum ESR and hsCRP of AS patients were significantly lower than those before treatment(P<0.05,respectively).(2)LC3,Beclinl,ATG3,ATG5,ATG12,ATG16L1 expression levels in PBMCs of AS patients before and after treatment were not statistically different(P>0.05,respectively)[LC3:5.05(4.27)×10-4 vs 4.83(1.99)×10-4;Beclin1:7.36(3.73)×10-3 vs 5.63(3.65)×10-3;ATG3:9.97(7.21)×10-3vs 11.38(10.48)×10-3;ATG5:5.62(4.67)×10-3 vs 5.17(6.05)×10-3;ATG12:5.31(3.55)×10-3 vs 4.81(4.89)×10-3;ATG16L1:3.48(3.55)×10-3 vs 2.47(1.72)×10-3]。Conclusion:1.The decreased expression of autophagy-related genes in AS patients suggests that autophagy dysfunction exists in AS patients.Autophagy may act as a "stabilizer" to regulate the occurrence and development of AS.2.The expression of ATG5 and ATG12 in AAS patients is significantly lower than that in IAS patients,and it is negatively correlated with disease activity index and inflammation indicators,suggesting that they may be involved in AS inflammation regulation,and are expected to be used as biomarkers to assess AS disease activity.3.After TNFi treatment,the disease activity index of AS patients was significantly reduced,and the expression level of autophagy-related genes was not significantly different from that before treatment,suggesting that TNFi treatment effectively inhibited inflammation in AS patients,but it may not directly participate in this inflammation regulation process through autophagy. |
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