细胞焦亡相关分子在原发性痛风性关节炎中的表达及其临床意义

Expression of pyroptosis related molecules in primary gouty arthritis and its clinical value

目的探讨原发性痛风性关节炎患者PBMCs中细胞焦亡的分子机制,为治疗痛风提供新思路。方法采集30例急性期痛风(AG)患者(AG组)、30例间歇期痛风(IG)患者(IG组)和40名健康患者(HC组)的外周血样本及临床资料;实时荧光定量检测细胞焦亡相关分子,包括核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶-1/4/5(caspase-1/4/5)、消皮素A/B/C/D/E(GSDMA/B/C/D/E)的mRNA表达水平。蛋白质免疫印迹法检测了NLRP3、前体caspase-1(pro-caspase-1)、剪切的caspase-1(caspase-1+p10)、GSDMD、N段GSDMD(GSDMD-N)、前体IL-1β(pro-IL-1β)、成熟IL-1β(cleced IL-1β)。正态或近似正态分布的计量资料采用独立样本t检验或单因素方差分析(ANOVA),非正态分布的计量资料采用Mann-WhitneyU检验或Kruskal-WallisH检验,计数资料使用χ^(2)检验进行比较。正态分布的连续性变量间采用Pearson相关分析,非正态分布的连续性变量间采用Spearman相关分析。Logistic回归分析用于评估危险因素。结果①一般资料比较:AG和IG组之间的药物持有率(MPR)、BMI差异无统计学意义(χ^(2)=0.64,P=0.426;t=0.04,P=0.972),病程差异具有统计学意义([25.0(9.8,63.0)月,54.0(33.0,102.0)月,Z=2.01,P=0.044)]。实验室指标比较:AG和IG组之间的ESR差异具有统计学意义(t=5.24,P<0.001),肾小球滤过率(eGFR)、中性粒细胞(GR)、淋巴细胞(LY)、红细胞(RBC)、红细胞压积(HCT)、尿酸、肌酐、ALT、AST 3组差异之间具有统计学意义。②3组间caspase-1、GSDMC、GSDMD、GSDME、NLRP3 mRNA表达水平差异有统计学意义。其中,在AG、IG组中caspase-1[(1.55±0.62、1.58±0.62)、GSDMD(4.7±1.4、3.5±1.5)]、NLRP3[(2.63(2.03,4.10)、2.39(1.57,3.49)]mRNA表达水平高于HC组[1.24±0.59,P=0.037,P=0.023;1.16±0.71,P<0.001,P<0.001;1.16(0.52,2.34),P<0.001,P<0.001],在IG组中GSDMD(3.5±1.5)mRNA表达水平相比较AG组(4.7±1.4)降低(P<0.001),而在AG组GSDMC、GSDME[(0.57(0.33,0.78)、(0.32±0.15)mRNA表达水平低于HC组[0.80(0.47,1.86),P=0.004;1.06±0.36,P<0.001],同时IG组中GSDME(0.62±0.29)mRNA表达水平也降低(P<0.05),但在IG组中GSDMC、GSDME(0.87(0.51,1.53)、(0.62±0.29)高于AG组[0.57(0.33,0.78),P=0.003;(0.32±0.15),P<0.001]。③3组间的NLRP3、pro-caspase-1、caspase-1+p10、GSDMD、GSDMD-N、pro IL-1β、cleved IL-1β蛋白表达水平差异具有统计学意义(F=50.04,P<0.001;F=9.65,P=0.013;F=30.71,P=0.001;F=7.38,P=0.024;F=23.66,P=0.001;F=30.11,P=0.001;F=6.01,P=0.036)。其中,NLRP3蛋白表达水平在AG组(1.14±0.12)显著高于IG、HC组(0.35±0.18)、(0.17±0.03)(P均<0.001),pro-caspase-1、caspase-1+p10蛋白表达水平在AG组(1.11±0.15)、(0.93±0.38)和IG组(0.98±0.14)、(1.14±0.17)高于HC组(0.42±0.28)、(0.29±0.16)(P=0.006,P=0.015),GSDMD蛋白表达水平在AG组(1.04±0.16)高于IG、HC组(0.53±0.26)、(0.39±0.22)(P=0.029,P=0.011),GSDMD-N蛋白表达水平在AG、IG组(0.97±0.06、0.90±0.04)高于HC组(0.27±0.23)(P=0.001,P=0.001),pro IL-1β蛋白表达水平在AG组(1.01±0.06)显著高于IG、HC组(0.32±0.14)、(0.64±0.11)(P<0.001,P=0.006),而在IG组(0.32±0.14)低于HC组(0.64±0.11)(P=0.011)。Cleved IL-1β蛋白表达水平在AG组(1.08±0.20)高于HC组(0.33±0.24)(P=0.014)。相关性分析:NLRP3、GSDMD与LY负相关(r=-0.32,P=0.001;r=-0.24,P=0.017);GSDMD与WBC、GR正相关(r=0.43,P<0.001;r=0.23,P=0.019)。Logistic回归分析:在AG组比HC组中,GSDMD和NLRP3是AG的危险因素[OR值(95%CI)=11.29(3.92,32.48),P<0.001;OR值(95%CI)=2.21(1.00,4.85),P=0.049];在IG组比HC组中,GSDMD是IG的危险因素[OR值(95%CI)=6.84(2.52,18.53),P<0.001];而在IG组比AC组中GSDMD是IG的保护因素[OR值(95%CI)=0.61(0.41,0.30),P=0.013]。结论细胞焦亡相关分子NLRP3、caspase-1、GSDMD在AG患者PBMCs中表达增加,GSDMC、GSDME在AG患者PBMCs中表达水平降低。其中,细胞焦亡经典途径NLRP3-caspase-1/GSDMD可能与急性痛风性关节炎的发作密切相关。

Objective To explore the molecular mechanism of cell death of the peripheral blood mononuclear cells(PBMCs)of patients with primary gouty arthritis,and provide new idea for the treatment of gout.Methods Peripheral blood samples and clinical data were collected from 30 patients with acute gout(AG),30 patients with intermittent gout(IG)and 40 healthy controls(HC).Real-time fluorescence quantitative detection of cell apoptosis related molecules,including the mRNA expression level of nucleotide binding oligomerization domain like domain like receptor protein 3(NLRP3),cysteine aspartic proteinase-1/4/5(caspase-1/4/5),Gasdermin A/B/C/E.And NLRP3,precursor caspase-1(pro-caspase-1),clipped caspase-1(caspase-1+p10),Gasdermin D(GSDMD),N segment GSDMD(GSDMD-N),precursor IL-1β(pro IL-1β),mature IL-1β(clevated IL-1β)were detected by western blot.The measurement data of normal or approximate normal distribution were analyzed by independent sample t test or one-way variance analysis(ANOVA),the measurement data of non-normal distribution were analyzed by Mann-Whitney U test or Kruskal-Wallis H test,and the counting data was compared by Chi-square test.Pearson's correlation analysis was used for the continuous variables with normal distribution,and Spearman's correlation analysis was used for the continuous variables with non-normal distribution.The logistic regression analysis was used to assess risk factors.Results①There were no significant differences in MPR and BMI between AG and IG(χ^(2)=0.64,P=0.426;t=0.04,P=0.972),and there was significant difference in disease course[25.0(9.8,63.0),54.0(33.0,102.0)mouth,Z=2.01,P=0.044].Comparison of labora-tory parameters:there were statistical significant differences in ESR between AG and IG(t=5.24,P<0.001),eGFR,GR,LY,RBC,HCT,UA,Creatinin,ALT and AST.②In the three groups,the expression lev-els of caspase-1,GSDMC,GSDMD,GSDME,NLRP3 mRNA were statistically significantly different.In AG and IG groups,mRNA expression levels of caspase-1(1.55±0.62),(1.58±0.62),GSDMD(4.7±1.4),(3.5±1.53),NLRP3[2.63(2.03,4.10),2.39(1.57,3.49)]were higher than those of the HC group[(1.24±0.59),1.16±0.71,1.16(0.50,2.34)](P=0.037,P=0.023,P<0.001,P<0.001,P<0.001,P<0.001).In IG group,mRNA expression levels of GS-DMD(3.53±1.53)were lower than those of AG group(4.68±1.43)(P<0.001).The mRNA expression levels of GS-DMC and GSDME[0.57(0.33,0.78),(0.32±0.15)]were lower than those of the HC group[0.80(0.47,1.86),(1.06±0.36)(P=0.004,P<0.001),and the mRNA expression levels of GSDME(0.62±0.29)in the IG group were lower(P=0.004,P<0.001),However,in the IG group,GSDMC and GSDME[0.87(0.51,1.53),(0.62±0.29)]were higher than those in the AG group[0.57(0.33,0.78),(0.32±0.15)](P=0.003,P<0.001).③The expression levels of NLRP3,pro-caspase-1,caspase-1+p10,GSDMD,GSDMD-N,pro-IL-1β,clevated IL-1βprotein were statistically different among the three groups[(F=50.04,P<0.001;F=9.65,P=0.013;F=30.71,P=0.001;F=7.38,P=0.024;F=23.66,P=0.001;F=30.11,P=0.001;F=6.01,P=0.036].The expression of NLRP3 protein in the AG group(1.14±0.12)was significantly higher than that in the IG and HC group(0.35±0.18),(0.17±0.03)(all P=0.001),the expression levels of Pro caspase-1,caspase-1+p10 protein in the AG(1.11±0.15),(0.93±0.38)and IG(0.98±0.14),(1.14±0.17)group were higher than those in the HC(0.42±0.28),(0.29±0.16)(P=0.006,P=0.015).The expression levels of GSDMD protein in the AG group(1.04±0.16)were higher than those in the IG and HC group(0.53±0.26),(0.39±0.22)(P=0.029,P=0.011).The expression level of GSDMD-N protein in the AG and IG group(0.97±0.06),(0.90±0.04)was higher than that in the HC(0.27±0.23)(P=0.001,P=0.001).The expression level of pro-IL-1βprotein in the AG group(1.01±0.06)was significantly higher than that in the IG and HC group(0.32±0.14),(0.64±0.11)(P<0.001,P=0.006),but lower than that in the HC(0.64±0.11)(P=0.011).The expression of clevated IL-1βprotein was higher in the AG group(1.08±0.20)than in the HC group(0.33±0.24)(P=0.014).④Negative correlation between NLRP3,GSDMD and LY(r=-0.32,P=0.001;r=-0.24,P=0.017)and positive correlation between GSDMD and WBC,GR(r=0.43,P<0.001;r=0.23,P=0.019)were found and Logistic regression analysis showed that the GSDMD and NLRP3 were risk factors for AG[OR(95%CI)=11.29(3.92,32.48),P<0.001;OR(95%CI)=2.21(1.00,4.85),P=0.049].GSDMD was risk factor for IG[OR(95%CI)=6.84(2.52,18.53),P<0.001];While GSDMD was the protective factor for IG[OR(95%CI)=0.61(0.41,0.30),P=0.013].Conclusion The expression's of NLRP3,Caspase-1 and GSDMD are increased in PBMCs of AG patients,while the expression's of GSDMC and GSDME are decreased.NLRP3/Caspase-1/GSDMD may be associated with the onset of acute gouty arthritis.