抗IL-17A单抗调控痛风自噬及痛风炎症的机制

Mechanism of monoclonal antibody against interleukin IL-17A regulating autophagy and inflammation in gout

目的探讨抗IL-17A单抗(司库奇尤单抗,secukinumab)调节痛风自噬及痛风炎症的机制。方法收集57份急性期痛风(acute gout,AG)、57份间隙期痛风(intermittent gout,IG)患者和82份健康志愿者外周静脉血,RT-qPCR法检测自噬相关基因(autophagy-related gene,ATG)ATG4B、ATG7、ATG16L1、Beclin-1和微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3B,LC3B)mRNA转录水平。在健康志愿者外周静脉血中加入单钠尿酸盐(monosodium urate,MSU)晶体,建立急性痛风炎症模型,RT-qPCR和ELISA法检测在0、1、2、4、6、8 h及不同浓度(0、100、200、400μmol/L)司库奇尤单抗作用下IL-1β转录及蛋白表达水平。将健康志愿者外周血分为对照(未经MSU处理)、MSU(100μg/mL)、MSU+秋水仙碱(100μg/mL+30μg/mL)、MSU+司库奇尤单抗(100μg/mL+400μmol/L)组,RT-qPCR和Western blot法检测IL-1β、ATG mRNA转录及蛋白表达水平,ELISA法检测IL-1β、IL-12、IL-35表达水平。结果AG、IG和健康对照组间ATG4B、Beclin-1和LC3B mRNA转录水平差异均有统计学意义(F分别为3.896、11.78、3.856,P均<0.05),其中AG组均低于IG及HC组(t分别为2.692、3.234、2.231和2.085、4.795、2.748,P均<0.05);ATG16L1 mRNA转录水平在3组间差异有统计学意义(F=7.949,P<0.001),其中AG组显著低于HC组(t=3.860,P<0.001)。在急性痛风炎症模型中,IL-1βmRNA转录及蛋白表达水平在2~4 h达到高峰,司库奇尤单抗浓度在400μmol/L时抗炎作用最强。与MSU组相比,MSU+司库奇尤单抗组ATG16L1、LC3B mRNA转录水平均显著降低(t分别为2.343、2.916,P均<0.05),ATG4B、ATG7、Beclin-1、ATG16L1、LC3B-Ⅱ蛋白表达水平均显著降低(t分别为28.84、11.6、8.402、4.124、2.458,P均<0.05);对照、MSU、MSU+秋水仙碱、MSU+司库奇尤单抗组间IL-12和IL-35蛋白表达水平差异均有统计学意义(F分别为7.009、6.518,P均<0.01),与MSU组相比,MSU+司库奇尤单抗组中IL-12蛋白表达水平显著降低(t=2.604,P<0.05),IL-35蛋白表达水平也降低,但差异无统计学意义(t=1.928,P>0.05)。结论司库奇尤单抗可调控自噬相关基因mRNA及蛋白表达,降低促炎细胞因子水平,抑制痛风炎症,为痛风治疗提供了参考。

Objective To investigate the mechanism of anti-IL-17A monoclonal antibody(secukinumab)regulating autophagy and inflammation in gout.Methods The peripheral venous blood samples from 57 patients with acute gout(AG),57patients with intermittent gout(IG)and 82 healthy volunteers were collected and measured for the mRNA transcription levels of autophagy-related genes(ATGs)ATG4B,ATG7,A TG16L1,Beclin-1 and LC3B by RT-qPCR.The model of AG inflammation was established by adding monosodium urate(MSU)crystals into the peripheral venous blood samples of healthy volunteers,and the transcription and protein expression of IL-1βwere detected by RT-qPCR and ELISA at 0,1,2,4,6 and8 h and different concentrations(0,100,200 and 400μmol/L)of secukinumab.The peripheral blood samples of healthy volunteers were divided into control(without MSU treatment),MSU(100μg/mL),MSU+colchicine(100μg/mL+30μg/mL)and MSU+secukinumab(100μg/mL+400μmol/L)groups,which were detected for the mRNA transcription and protein expression of IL-1βand ATGs by RT-qPCR and Western blot,and for the expression of IL-1β,IL-12 and IL-35 by ELISA.Results The mRNA expression levels of ATG4B,Beclin-1 and LC3B in AG,IG and healthy control groups were significantly different(F=3.896,11.78 and 3.856,respectively,each P<0.05),among which the mRNA levels in AG were lower than those in IG and HC groups(t=2.692,3.234,2.231 and 2.085,4.795,2.748,respectively,each P<0.05);the expression levels of ATG16L1 mRNA were significantly different in the three groups(F=7.949,P<0.001),and was significantly lower in AG group than HC group(t=3.860,P<0.001).In AG inflammation model,the mRNA and protein expression of IL-1βreached their peak in 2—4 h,and the anti-inflammation effect of secukinumab was the strongest at the concentration of 400μmol/L.Compared with MSU group,the mRNA levels of ATG16L1 and LC3B(t=2.343 and 2.916,respectively,each P<0.05)as well as the expression levels of ATG4B,ATG7,Beclin-1,ATG16L1 and LC3B-Ⅱproteins(t=28.84,11.6,8.402,4.124 and 2.458,respectively,each P<0.05)in MSU+secukinumab group decreased significantly.The expression levels of IL-12 and IL-35 in the control,MSU,MSU+colchicine and MSU+secukinumab groups showed significant difference(F=7.009 and 6.518,respectively,each P<0.01).Compared with MSU group,the expression level of IL-12 significantly decreased(t=2.604,P<0.05)in MSU+secukinumab group,and the expression level of IL-35 also decreased,while with no significant difference(t=1.928,P>0.05).Conclusion Secukinumab can regulate the mRNA and protein expression of ATGs,reduce the levels of pro-inflammatory cytokines,and inhibit gout inflammation,which provides a reference for the treatment of gout.