自噬晚期阶段在原发性痛风性关节炎中的研究

Study on the late stage of autophagy in primary gouty arthritis

目的探索自噬晚期阶段在原发性痛风性关节炎(GA)患者PBMCs中的表达及临床意义。方法收集30例急性期痛风患者(AG)、30例间歇期痛风患者(IG)和50名健康对照组的外周血、临床资料及实验室检查指标;采用实时荧光定量PCR(RT-qPCR)检测自噬基因(ATG5、ATG12、ATG16、ATG3、ATG7、ATG10、ATG4B、LC3-2/LC3B)mRNA表达水平。计量资料符合正态分布采用t检验或方差分析(ANOVA),非正态分布用Mann-Whitney检验或Kruskal-Wallis H检验,3组间两两比较采用SNK;变量间的相关关系采用Spearman相关分析。结果①ATG5 mRNA、ATG12 mRNA、ATG16 mRNA、ATG10 mRNA、LC3-2 mRNA在AG组的表达水平低于IG组及健康对照组,IG组表达水平低于健康对照组[9.16×10^(-3)(6.04×10^(-3),15.00×10^(-3))、14.48×10^(-3)(9.95×10^(-3),21.38×10^(-3))和20.08×10^(-3)(12.21×10^(-3),42.79×10^(-3)),H=19.377,P<0.01;18.89×10^(-3)(13.85×10^(-3),24.92×10^(-3))、21.13×10^(-3)(12.11×10^(-3),28.06×10^(-3))和33.57×10^(-3)(13.11×10^(-3),49.89×10^(-3)),H=7.545,P=0.023;8.72×10^(-3)(4.96×10^(-3),13.74×10^(-3))、10.62×10^(-3)(7.48×10^(-3),24.71×10^(-3))和20.07×10^(-3)(11.99×10^(-3),39.56×10^(-3)),H=20.962,P<0.01;1.05×10^(-3)(0.73×10^(-3),1.84×10^(-3))、1.60×10^(-3)(0.93×10^(-3),2.58×10^(-3))和1.69×10^(-3)(1.05×10^(-3),3.54×10^(-3)),H=8.193,P=0.017;2.31×10^(-3)(1.22×10^(-3),3.53×10^(-3))、2.78×10^(-3)(1.68×10^(-3),5.96×10^(-3))和3.68×10^(-3)(2.00×10^(-3),5.67×10^(-3)),H=7.135,P=0.028]。ATG4B mRNA在AG、IG组的表达水平高于健康对照组[9.95×10^(-3)(6.32×10^(-3),12.23×10^(-3))、10.86×10^(-3)(8.80×10^(-3),17.03×10^(-3))和8.07×10^(-3)(5.52×10^(-3),11.63×10^(-3)),H=8.531,P=0.014];ATG3 mRNA、ATG7 mRNA组间差异无统计学意义(H=0.539,P>0.05;H=3.739,P>0.05)。②急性期痛风患者ATG3与PDW、MPV呈负相关(r=-0.499,P=0.006;r=-0.463,P=0.011);ATG4B与HDL-C呈正相关(r=0.408,P=0.048);ATG7与球蛋白呈负相关(r=-0.554,P=0.001);ATG10与白蛋白呈正相关(r=0.412,P=0.024),与Crea、hsCRP呈负相关(r=-0.459,P=0.011;r=-0.375,P=0.045);ATG12与MO呈负相关(r=-0.434,P=0.017);ATG16与ALT、AST呈负相关(r=-0.389,P=0.034;r=-0.366,P=0.047);LC3-2与血尿酸呈正相关(r=0.381,P=0.041),与MPV、PDW呈负相关(r=-0.413,P=0.026;r=-0.449,P=0.015)。间歇期痛风患者ATG3、ATG4B与apoB100呈负相关(r=-0.555,P=0.011;r=-0.462,P=0.040);ATG5与Crea呈负相关(r=-0.456,P=0.011);ATG10与TC、LDL-C、apoB100呈负相关(r=-0.526,P=0.017;r=-0.556,P=0.011;r=-0.515,P=0.020)。结论自噬参与了痛风的发生发展,且与痛风患者的炎性指标及代谢性等指标相关,提示自噬是GA发病机制中的一个重要特征。

Objective To explore the expression and clinical significance of late autophagy in per-ipheral blood mononuclear cells(PBMCs)of patients with primary gouty arthritis(GA).Methods Peripheral blood,clinical data,and laboratory tests were collected from 30 patients with acute gout(AG),30 patients with intermittent gout(IG),and 50 healthy controls(HC).Quantitative polymerase chain reaction(RT-qPCR)was used to detect mRNA expression levels of autophagy-related genes(ATG5,ATG12,ATG16,ATG3,ATG7,ATG10,ATG4B,LC3-2/LC3B).Measurement data conformed to normal distribution were tested using t test or analysis of variance(ANOVA),and non-normal distribution data were tested using Mann-Whitney test or Kruskal-Wallis H test.SNK was used for pairwise comparison among the three groups.Correlation between variables was tested by Spearman correlation analysis.Results①The expression level of ATG5 mRNA,ATG12 mRNA,ATG16 mRNA,ATG10 mRNA and LC3-2 mRNA in the AG group was lower than that of the IG group and the HC group,and the expression level of the IG group was lower than that of the HC group[9.16×10^(-3)(6.04×10^(-3),15.00×10^(-3))vs 14.48×10^(-3)(9.95×10^(-3),21.38×10^(-3))vs 0.08×10^(-3)(12.21×10^(-3),42.79×10^(-3)),H=19.377,P<0.001;18.89×10^(-3)(13.85×10^(-3),24.92×10^(-3))vs 21.13×10^(-3)(12.11×10^(-3),28.06×10^(-3))vs 33.57×10^(-3)(13.11×10^(-3),49.89×10^(-3)),H=7.545,P=0.023;8.72×10^(-3)(4.96×10^(-3),13.74×10^(-3))vs 10.62×10^(-3)(7.48×10^(-3),24.71×10^(-3))vs 20.07×10^(-3)(11.99×10^(-3),39.56×10^(-3)),H=20.962,P<0.001;1.05×10^(-3)(0.73×10^(-3),1.84×10^(-3))vs 1.60×10^(-3)(0.93×10^(-3),2.58×10^(-3))vs 1.69×10^(-3)(1.05×10^(-3),3.54×10^(-3)),H=8.193,P=0.017;2.31×10^(-3)(1.22×10^(-3),3.53×10^(-3))vs 2.78×10^(-3)(1.68×10^(-3),5.96×10^(-3))vs 3.68×10^(-3)(2.00×10^(-3),5.67×10^(-3)),H=7.135,P=0.028].The expression level of ATG4B mRNA in the AG and IG group was higher than that in HC group,and there was significant difference between IG group and AG group,IG group and HC group[9.95×10^(-3)(6.32×10^(-3),12.23×10^(-3))vs 10.86×10^(-3)(8.80×10^(-3),17.03×10^(-3))vs 8.07×10^(-3)(5.52×10^(-3),11.63×10^(-3)),H=8.531,P=0.014].There was no significant difference between the ATG3 mRNA and ATG7 mRNA groups(H=0.539,3.739,bothall P values>0.05).②The results of Spearman correlation analysis suggested that in patients with acute gout,ATG3 was negatively correlated with PDW and MPV(r=-0.499,P=0.006;r=-0.463,P=0.011);ATG4B was positively correlated with HDL-C(r=0.408,P=0.048);ATG7 was negatively correlated with GLOB(r=-0.554,P=0.001);ATG10 was positively correlated with ALB(r=0.412,P=0.024)and negatively correlated with Crea and hsCRP(r=-0.459,P=0.011;r=-0.375,P=0.045);ATG12 was negatively correlated with MO(r=-0.434,P=0.017);ATG16 was negatively correlated with ALT and AST(r=-0.389,P=0.034;r=-0.366,P=0.047);LC3-2 was positively correlated with UA(r=0.381,P=0.041)and negatively correlated with MPV and PDW(r=-0.413,P=0.026;r=-0.449,P=0.015).In patients with intermittent gout,ATG3 and ATG4B were negatively correlated with apoB100(r=-0.555,P=0.011;r=-0.462,P=0.040);ATG5 was negatively correlated with Crea(r=-0.456,P=0.011);ATG10 was negatively correlated with TC,LDL-C,and apoB100(r=-0.526,P=0.017;r=-0.556,P=0.011;r=-0.515,P=0.020).Conclusion Autophagy is involved in the development of gout,and is correlated with ibflammatory and metabolic indicators,suggesting that autophagy is an important feature in the pathogenesis of GA.