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Objective:To investigate the expression and clinical significance of decoy receptor 3 (DcR3) and its signal pathway-related molecules in PBMCs of patients with ankylosing spondylitis (AS).Methods:Peripheral blood samples, clinical data and laboratory test results were collected from 100 patients with ankylosing spondylitis [50 patients with AS activity (ASA), 50 patients with AS stability (ASS)], 30 patients with osteoarthritis and 30 patients with gouty arthritis (as disease control group), and 60 healthy controls (HC). The mRNA expression levels of DcR3 and its signal pathway related genes (DR3, TL1A, Fas, FasL, LIGHT, LIGHTR, LTβR) were measured by real-time fluorescence quantitative polymerase chain reaction. Measurement data among the three groups in normal distribution were analyzed by t test or one-way analysis of variance, pairwise comparisons using LSD- t test, non-normal distribution data were analyzed by Mann-Whitney test or Kruskal-Wallis H test, χ2 test was used for correlation analysis of categorical variables. Correlation analysis between variables were analyzed using Spearman correlation analysis. Results:① By comparing the AS group, disease control group and HC group, the expression levels of DcR3 mRNA and DR3 mRNA in the AS group were lower than those in disease control group and HC group, and DcR3 mRNA and DR3 mRNA in disease control group were lower than those in the HC group {DcR3mRNA: [6.21 (3.89, 10.70)]×10 -4vs [9.51 (5.89, 16.65)]×10 -4vs [17.81 (11.27, 24.20)]×10 -4, H=55.28, P<0.001; DR3 mRNA: [41.05 (24.09, 66.95)]×10 -4vs [58.28 (28.41, 94.38)]×10 -4vs [94.79 (54.07, 144.51)]×10 -4, H=37.10, P<0.001}. The expression level of TL1A mRNA in the AS group was higher than that in disease control group {[14.71(4.91, 42.22)]×10 -4vs [4.00(1.07, 16.60)]×10 -4vs [7.70 (3.52, 27.83)]×10 -4, H=17.71, P<0.001}; The expression level of Fas mRNA in AS group and disease control group was lower than that in HC group {[20.99(4.63, 62.89)]×10 -4vs [23.97(15.82, 38.99)]×10 -4vs [78.45 (27.32, 146.46)]×10 -4, H=31.17, P<0.001}. The expression level of FasL mRNA in AS group was higher than that in disease control group and HC group {[42.87(6.57, 91.21)]×10 -4vs [5.45(2.83, 10.32)]×10 -4vs [6.88 (4.57, 23.79)]×10 -4, H=46.42, P<0.001}. The expression level of LIGHTR mRNA in AS group was lower than that in disease control group {[52.66 (7.20, 143.21)]×10 -4vs [98.80 (53.11, 166.24)]×10 -4vs [63.47(40.85, 138.07)]×10 -4, H=11.96, P<0.001}. There were no significant differences in LIGHT mRNA and LTβR mRNA among all groups ( H=0.86, P>0.05; H=3.18, P>0.05). ②The expression levels of DcR3 mRNA, DR3 mRNA and Fas mRNA in ASA group and ASS group were lower than those in HC group. DcR3 mRNA in ASA group was higher than that in ASS group, and DR3 mRNA in ASA group was lower than that in ASS group {DcR3 mRNA: [7.28 (4.92, 16.56)]×10 -4vs [4.59 (2.49, 7.03)]×10 -4vs [17.81 (11.27, 24.20)]×10 -4, H=62.63, P<0.001; DR3 mRNA: [30.93(16.18, 66.66)]×10 -4vs [47.17(29.91, 67.40)]×10 -4vs [94.79(54.07, 144.51)]×10 -4, H=41.48, P<0.001; Fas mRNA: [20.04(3.29, 62.30)]×10 -4vs [22.49(5.63, 64.79)]×10 -4vs [78.45(27.32, 146.46)]×10 -4, H=23.54, P<0.001}. The expression levels of TL1A mRNA and LTβR mRNA in the ASA group were higher than those in the ASS group and the HC group {TL1A mRNA: [32.36(10.09, 97.84)]×10 -4vs [9.98(1.29, 21.63)]×10 -4vs [7.70(3.52,27.83)]×10 -4, H=21.14, P<0.001; LTβR mRNA: [6.13(2.16,20.06)×10 -4vs [2.13(0.53,8.04)]×10 -4vs [2.72 (1.24,5.73)]×10 -4, H=12.86, P<0.001}. The expression level of FasL mRNA in the ASA group and the ASS group was higher than that in the HC group {[60.70 (8.16, 106.16)]×10 -4vs [30.14 (5.37, 78.40)]×10 -4vs [6.88 (4.57, 23.79)]×10 -4, H=18.99, P<0.001}. The expression level of LIGHTR mRNA in ASS group was lower than that in HC group {[49.79(10.75, 168.48)]×10 -4vs [15.92(3.27, 105.91)]×10 -4vs [63.47(40.85, 138.07)]×10 -4, H=11.80, P<0.001]. There was no significant difference in LIGHT mRNA among all groups ( H=4.15, P>0.05). ③Spearman correlation analysis showed that DcR3 level was positively correlated with BASDAI score and hsCRP in AS patients ( r=0.52, P<0.001; r=0.35, P<0.01), and DR3 level was negatively correlated with BASDAI score, ESR and hsCRP level ( r=-0.28, P<0.001; r=-0.25, P<0.001; r=-0.31, P<0.001). TL1A was positively correlated with BASDAI score, ESR and hsCRP level ( r=0.23, P=0.046; r=0.26, P=0.015; r=0.25, P=0.017). Conclusion:DcR3 and its signal pathway-related molecules are differentially expressed in PBMCs of patients with AS, suggesting that they may participate in the occurrence and development of AS.
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Objective:To This study was to investigate the expression and possible clinical significance of microRNA-146b (miR-146b) and signal transducer and transcriptional activator 1 and 3 (STAT1/3) in peripheral blood mononuclear cells (PBMCs) of patients with primary gouty arthritis (GA).Methods:The peripheral blood samples, clinical data and laboratory indexes of 120 male cases of GA [including 57 cases of acute (AG group) and 63 cases of intermittent (IG group)]and 66 healthy subjects (HC group) were collected. The expression levels of miR-146b and STAT1/3 in PBMCs were detected by real-time fluorescence quantitative PCR (RT-qPCR). The differences among the three groups were compared and the correlation between them and clinical indexes was analyzed. The receiver operating characteristic curve (ROC) was constructed to evaluate its diagnostic value in GA. After the PBMCs of 18 healthy subjects were stimulated by 100 μg/ml MSU for 3 hours to simulate acute gout inflammatory environment, the transcriptional changes of IL-1β, miR-146b and STAT1/3 were detected by RT-qPCR, and the expressions of IL-1β, STAT1/3 protein and phosphorylated protein were detected by Western blotting. T test or one-way ANOVA and LSD- t test were used in accordance with the normal measurement data, Kruskal-Wallis H and Mann-Whitney U test were used in the non-normal data, Spearman correlation analysis was used in the correlation between variables, and the diagnostic value was evaluated by the receiver working characteristic curve ROC. Results:①There were statistical differences in the expression of miR-146b, STAT1 and STAT3 among the three groups ( F=7.02、19.52、17.07, all P<0.001). The expression of miR-146b in gout group [(0.32±0.28)] was significantly higher than that in HC group (0.19±0.18)( t=2.96, P=0.003), while STAT1(0.019±0.012) and STAT3(0.014±0.010) were significantly lower than those in HC group (0.038±0.029),(0.025±0.016)( t=6.26, 5.56, both P<0.001). Further subgroup analysis showed that the expression of miR-146b in AG and IG groups was higher than that in HC group[(0.27±0.17), (0.38±0.35), (0.19±0.18), t=2.09, 3.30, both P<0.05], but that in AG group was lower than that in IG group ( t=2.02, P<0.05). The expression of STAT1 mRNA in AG and IG groups was lower than that in HC group [(0.020±0.012), (0.019±0.012), (0.038±0.029), t=4.89, 4.56, both P<0.001], but there was no statistical significance between AG and IG groups ( t=0.24, P>0.05). The expression of STAT3 mRNA in AG and IG groups was lower than that in HC group [(0.016±0.012), (0.012±0.008), (0.025±0.016), t=5.64, 3.33, both P<0.01], and the expression of STAT3 mRNA in AG group was higher than that in IG group ( t=2.12, P<0.05). ② Spearman correlation analysis showed that the expression of miR-146b in GA was negatively correlated with HCY ( r=-0.37, P=0.014), STAT1 was negatively correlated with Crea ( r=-0.29, P=0.019), positively correlated with eGFR ( r=0.25, P=0.047), and STAT3 was negatively correlated with HDL-C ( r=-0.27, P=0.033). ③ROC curve showed that the AUC (95% CI) of miR-146b, STAT1 and STAT3 were 0.679(0.582, 0.776), 0.710(0.629, 0.791) and 0.705(0.626, 0.783), and the combined AUC(95% CI) of the three was 0.836 (0.765, 0.907). ④Compared with blank control group and negative control group, the expression of miR-146b in PBMCs of 18 cases of HC was significantly decreased ( H=14.44, P=0.003), while the expression of IL-1β, STAT1 and STAT3 mRNA was significantly increased after 3 h of MSU stimulation ( H=26.44、27.26、15.90, all P<0.001). The expression of IL-1β, STAT1 and STAT3 protein and phosphorylated protein in the model group were significantly higher than those in the blank control group, and the differences were statistically significant ( t=9.97、6.63、7.48、11.25、6.28, all P<0.01). Conclusion:The abnormal expression of miR-146b and STAT1/3 in GA is related to some clinical indicators, suggesting that it may be involved in the regulation of gout immune inflammatory response and metabolism, and the specific mechanism is worth further study.
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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. Results:① There were no significant differences in MPR and BMI between AG and IG ( χ2=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. ② In 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). ③ The expression levels of NLRP3, pro-caspase-1, caspase-1 + p10, GSDMD, GSDMD-N, pro-IL-1β, clevated IL-1β protein 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-1β protein 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-1β protein was higher in the AG group (1.08±0.20) than in the HC group (0.33±0.24) ( P=0.014). ④ Negative 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.
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Objective:To explore the three long non-coding RNA (long non-coding ribonucleic acid, the expression of lncRNA NR_002578, NR_038264 and NR_046252) in peripheral blood mononuclear cells (PBMCs) of patients with primary gout arthritis (GA) and their clinical value.Methods:Peripheral venous blood, clinical data and laboratory data were collected from 60 gout patients (including 30 AG patients in acute stage and 30 IG patients in intermittent stage) and 50 healthy subjects (HC group). Quantitative reverse transcription PCR (RT-qPCR) was used to detect the expression levels of PBMCs of 3 lncRNAs in GA and HC groups, and the differences of 3 lncRNAs expression levels in different groups were compared and the correlation analysis was conducted with clinical indicators. Receiver operating characteristic curve (ROC) was constructed to evaluate the possible efficacy of lncRNAs in gout diagnosis. The measurement data conforming to normal distribution were tested by t test or variance analysis, and non-normal distribution were tested by Mann-Whitney U test or Kruskal-Wallis H test. The comparison among the three groups was conducted by SNK. Results:① The expression of NR_002578 in GA was significantly lower than that in HC [60.2(16.8, 100.1)×10 -3vs 149.5 (92.6, 221.8)×10 -3, Z=-5.75, P<0.001], subgroup analysis showed that the expression of NR_002578 in AG was significantly lower than that in IG and HC [34.3(8.6, 72.8)×10 -3vs 88.3(47.7, 109.6)×10 -3vs 149.5(92.6, 221.8)×10 -3, H=40.12, P<0.001], and lower in IG than that in HC ( P<0.001). The expression of NR_046252 in GA was significantly higher than that in HC [6.5(2.1, 21.5)×10 -3vs 2.1(1.2, 3.5)×10 -3, Z=-4.21, P<0.001]. The expression of NR_046252 in AG and IG were higher than that of the HC group [6.3(2.0, 12.0)×10 -3vs 7.2(2.4, 30.6)×10 -3vs 2.1(1.2, 3.5)×10 -3, H=21.33, P<0.001], but there was no significant difference between the AG and IG group ( P>0.05). ② Spearman correlation analysis showed that NR_002578 expression was negatively correlated with erythrocyte sedimentation rate (ESR) ( r=-0.29, P=0.024)and hypersensitive C-reactive protein (hs-CRP) ( r=-0.35, P=0.006) in gout patients. ③ The areas under ROC curve of NR_002578 and NR_046252 for diagnosing gout were 0.819 and 0.750, respectively. Conclusion:The abnormal expression of NR_002578 and NR_046252 in gout patients suggests that NR_002578 may be involved in the pathogenesis of gout.