Clinical data and laboratory test indices
The general data and clinical characteristics of the subjects are shown in Table 3. Age, gender, LY, HDL, and LDLC did not differ statistically between the three groups (P > 0.05). Compared with the HC group: IL−6, sUA, Crea, eGFR, Cysc, GLU, Globulin, WBC, GR, MO, TG, TC, VLDL, apoA1, apoB100 were significantly higher in the AG group than in the HC group; IL−6, sUA, Crea, eGFR, Cysc, GLU, Globulin in the IG group, WBC, GR, LY, MO, TG, TC, VLDL, apoA1, and apoB100 were higher than those in the HC group (all P < 0.05). IL−6, sUA, Cysc, Globulin, ESR, hsCRP, WBC, GR, MO, and apoA1 were higher in the AG group than in the IG group (all P < 0.05).
Multifactorial Logistic Regression of Acute Gouty Attacks
In order to make the model more stable, the IG and HC groups were combined, and then all the clinical data and serum IL−6 concentrations of the AG and combined groups were analysed using univariate regression, and the variables in the univariate analyses with a P < 0.05 and a VIF < 10 were included in the logistic regression as shown in Table 4. According to the forward stepwise regression method, the entry SLE was set to 0.05 and the stay was set to 0.1, and the final results of the logistic regression analyses found that IL−6 was considered a risk factor for acute gouty attacks.
Table 3
Comparison of clinical data and laboratory indicators between groups
Items
|
Gout group(n = 111)
|
AG group(n = 55)
|
IG group(n = 56)
|
HC group(n = 57)
|
F/H value
|
P value
|
Age(years)(‾x ± SD)
|
40.09 ± 10.02
|
39.73 ± 10.34
|
40.45 ± 9.78
|
39.11 ± 11.86
|
0.22
|
0.801
|
Gender F/M
|
0/111
|
0/55
|
0/56
|
0/57
|
-
|
-
|
sUA(umol/L)(‾x ± SD)
|
502.0 ± 125.0a
|
529.3 ± 138.9ab
|
475.2 ± 103.9a
|
347.8 ± 42.4
|
46.36
|
< 0.001
|
Crea(mmol/L)(‾x ± SD)
|
86.05 ± 14.15a
|
84.99 ± 14.54a
|
87.09 ± 13.82a
|
70.99 ± 10.49
|
7.55
|
< 0.001
|
eGFR(ml·min− 1·1.73min − 2)
(‾x ± SD)
|
89.20 ± 13.83a
|
90.51 ± 14.95a
|
87.91 ± 12.64a
|
100.10 ± 15.81
|
11.02
|
< 0.001
|
Cysc(mg/L)(‾x ± SD)
|
1.11 ± 0.31a
|
1.04 ± 0.22ab
|
1.18 ± 0.36a
|
0.82 ± 0.13
|
28.56
|
< 0.001
|
GLU(mmol/L)(‾x ± SD)
|
5.62 ± 0.62a
|
5.57 ± 0.67a
|
5.68 ± 0.57a
|
4.75 ± 0.70
|
34.35
|
< 0.001
|
Globulin(g/L)(‾x ± SD)
|
32.76 ± 3.48a
|
33.53 ± 2.54ab
|
32.00 ± 4.09a
|
29.14 ± 2.93
|
26.22
|
< 0.001
|
ESR(mm/1 h)[M(Q1,Q3)]
|
14.00(8.00, 17.00)
|
17.00(14.00, 22.00)b
|
10.00(6.00, 13.00)
|
-
|
6.39
|
< 0.001
|
hsCRP(mg/L)[M(Q1,Q3)]
|
8.39(2.29, 28.39)
|
28.39(16.64, 41.54)b
|
2.33(1.15, 4.21)
|
-
|
9.05
|
< 0.001
|
WBC(×109/L)(‾x ± SD)
|
7.62 ± 1.98a
|
8.76 ± 1.87ab
|
6.51 ± 1.36a
|
6.02 ± 1.10
|
54.76
|
< 0.001
|
GR(×109/L)(‾x ± SD)
|
4.66 ± 1.14a
|
5.26 ± 0.80ab
|
4.07 ± 1.12a
|
3.52 ± 0.82
|
51.70
|
< 0.001
|
LY(×109/L)(‾x ± SD)
|
2.07 ± 0.57a
|
2.06 ± 0.64
|
2.07 ± 0.50a
|
1.89 ± 0.57
|
2.27
|
0.106
|
Mo(×109/L)(‾x ± SD)
|
0.48 ± 0.14a
|
0.54 ± 0.13ab
|
0.41 ± 0.12a
|
0.33 ± 0.10
|
47.66
|
< 0.001
|
TG(mmol/L)(‾x ± SD)
|
2.05 ± 0.81a
|
1.94 ± 0.85a
|
2.15 ± 0.77a
|
1.15 ± 0.44
|
31.41
|
< 0.001
|
TC(mmol/L)(‾x ± SD)
|
4.77 ± 1.05a
|
4.61 ± 0.94a
|
4.92 ± 1.13a
|
4.32 ± 0.54
|
6.17
|
0.003
|
HDL(mmol/L)(‾x ± SD)
|
1.16 ± 0.28
|
1.14 ± 0.26
|
1.18 ± 0.29
|
1.23 ± 0.30
|
1.37
|
0.257
|
LDLC(mmol/L)(‾x ± SD)
|
2.59 ± 0.63
|
2.50 ± 0.66
|
2.68 ± 0.60
|
2.52 ± 0.46
|
1.72
|
0.182
|
VLDL(mmol/L)(‾x ± SD)
|
0.88 ± 0.28a
|
0.88 ± 0.28a
|
0.97 ± 0.42a
|
0.63 ± 0.21
|
18.06
|
< 0.001
|
apoA1(mmol/L)(‾x ± SD)
|
1.14 ± 0.22a
|
1.10 ± 0.22ab
|
1.18 ± 0.20a
|
1.34 ± 0.20
|
20.05
|
< 0.001
|
apoB100(mmol/L)(‾x ± SD)
|
0.89 ± 0.20a
|
0.86 ± 0.19a
|
0.91 ± 0.23a
|
0.75 ± 0.12
|
12.62
|
< 0.001
|
IL-6(pg/ml)
|
16.61(7.57, 40.98)a
|
40.98(26.71, 61.68)ab
|
7.67(6.04, 9.55)a
|
4.02(2.12, 5.63)
|
199.90
|
< 0.001
|
Note: a P < 0.05 vs HC group; b P < 0.05 vs IG group. |
Table 4
Univariate and multivariate logistic regression analysis of acute gouty attacks
Variables
|
AG group
(n = 55)
|
Combined group
(n = 113)
|
Univariate analysis
|
Multivariate analysis
|
P1-value
|
P2-value
|
OR(95%CI)
|
IL-6(pg/ml)
|
40.98(26.71, 61.68)
|
5.72(4.00, 7.86)
|
0.009
|
0.009
|
2.175(1.219, 3.881)
|
sUA(umol/L)
|
529.3 ± 138.9
|
410.9 ± 101.5
|
< 0.001
|
0.179
|
|
Globulin(g/L)
|
33.53 ± 2.54
|
30.56 ± 3.82
|
< 0.001
|
0.171
|
|
ESR(mm/1 h)
|
17.00(14.00, 22.00)
|
10.00(6.00, 13.00)
|
< 0.001
|
0.765
|
|
WBC(×109/L)
|
8.76 ± 1.87
|
6.26 ± 1.25
|
< 0.001
|
0.492
|
|
GR(×109/L)
|
5.26 ± 0.80
|
3.79 ± 1.02
|
< 0.001
|
0.564
|
|
Mo(×109/L)
|
0.54 ± 0.13
|
0.37 ± 0.11
|
< 0.001
|
0.088
|
|
TG(mmol/L)
|
1.94 ± 0.85
|
1.65 ± 0.80
|
0.035
|
0.134
|
|
apoA1(mmol/L)
|
1.10 ± 0.22
|
1.26 ± 0.21
|
< 0.001
|
0.165
|
|
Note: Multifactorial Logistic Regression for Acute Gouty Attacks. combined group=(IG + HC)group; P1 values were obtained by unifactorial logistic regression, P2 and OR values were obtained by multifactorial logistic regression. |
Transcriptional and translational expression of IL−1β, IL−6, JAK2, STAT1/3 in PBMCs from gout patients and healthy controls
The expression of IL−1β, IL−6, JAK2, and STAT1/3 mRNA was significantly lower in the gout group than in the HC group (all P < 0.001), and further subgroup analysis revealed statistically significant differences in expression among the three groups (all P < 0.001). The expression of IL−6 mRNA was significantly lower in both AG and IG groups than in the HC group, and was lower in both AG than IG groups (all P < 0.05) ; The expression of JAK2, STAT3 and IL−1β mRNA was significantly lower than that of HC group in both AG and IG groups, and AG group was higher than IG group (P < 0.05); the expression of STAT1 mRNA was significantly lower than that of HC group in both AG and IG groups (P < 0.001), but the difference between AG and IG groups was not statistically significant (P > 0.05) (Fig. 1a). The differences in protein levels of IL−6, JAK2, STAT1/3, p-JAK2, p-STAT1/3, and IL−1β among the three groups were statistically significant (P < 0.001). Specifically, the AG group exhibited significantly higher levels compared to the IG and HC groups. Compared to the HC group, the IG group showed significant increases in IL−6, JAK2, STAT3, and IL−1β protein levels, while STAT1, p-JAK2, and p-STAT1/3 protein levels were significantly decreased (P < 0.05) (Fig. 1b). These findings indicate elevated serum IL−6 levels and dysregulated expression of IL−6/JAK2/STAT1/3 signaling pathway-related genes in gout patients.
Correlation analysis and ROC curves of IL−6 and JAK2 mRNA or protein expression with inflammatory markers in gout patients
ESR, CRP, WBC, GR, Mo and LY, as inflammation-related indicators, are important experimental indicators for evaluating the disease activity of gouty arthritis.Spearman correlation analysis showed that ESR, CRP, WBC, GR and Mo were significantly positively correlated with serum IL−6 protein expression (all P < 0.05), and that CRP, WBC, GR, Mo and JAK2 mRNA expression were significantly positively correlated (P < 0.05) (Fig. 2a); the AUC (95% CI) of IL−6 and JAK2 mRNA expression in GA was 0.709 (0.632, 0.786) and 0.711 (0.631 0.791), respectively, while the AUC (95% CI) of IL−6 mRNA expression in AG was 0.781 (0.697 0.865) (Fig. 2b). The data suggest that IL−6 and JAK2 are associated with clinical and laboratory activities in GA, and have an adjunctive value in the diagnosis of gout.
Changes in the expression levels of IL-1β, IL-6, JAK2 and STAT1/3 in an in vitro gouty inflammation model in human blood
Peripheral blood PBMCs from healthy individuals were stimulated in MSU to establish an in vitro gout model in human blood, and the expression of relevant genes at different time points was detected. Compared with 0h, the expression of IL-1β and IL-6 proteins was elevated after 1h (both P < 0.05), and the peak of inflammation appeared at 4-6h, suggesting that the acute gout inflammation model was successfully established (Fig. 3a). And in the 2h human blood in vitro gout inflammation model, IL-1β, IL-6, JAK2 mRNA expression and IL-1β, IL-6, JAK2, STAT1/3, p-JAK2, p-STAT1/3 protein expression in the model group were significantly higher than that in the blank control group and the PBS-negative control group and the differences were statistically significant (all P < 0.05); while the the differences between the blank control group and the PBS-negative control group were not statistically significant (both P > 0.05) (Fig. 3b and 3c). These findings suggest that the IL-6-JAK2-STAT1/3 signaling pathway may be involved in the activation of acute gout inflammation or its pathogenesis.
The THP-1 gout 6-hour inflammation model, combined with IL-6 agonist, can amplify the inflammatory response through the JAK2-STAT1/3 signaling pathway.
THP1 macrophages were stimulated in MSU to establish an acute gout cell model, and the expression of related genes at different time points was detected. Compared with 0h, the expression of IL-1β and IL-6 proteins gradually increased, and both were statistically significant after 3h (both P < 0.05), and from the figure, we can see that the peak of inflammation may be after 12h (Fig. 4a). In the 6h cellular model of acute gout, the expression of IL-1β, IL-6, JAK2, STAT1/3 mRNA and proteins and their phosphorylated proteins in the model group was significantly higher than that in the blank control group, and the differences were all statistically significant (all P < 0.05); whereas, in the model group with the addition of IL-6 agonist, it was found that the expression of IL-1β, IL-6, JAK2, STAT1/3 mRNA and IL-1β, IL-6, JAK2, STAT1/3, p-JAK2, p-STAT1/3 protein expression was significantly higher than that in the model group, and the differences were all statistically significant (all P < 0.05) (Fig. 4b and 4c). These results suggest that IL-6 agonists make the inflammatory response more intense and can amplify inflammation through the JAK2-STAT1/3 signalling pathway.
IL-6 knockout mice (IL-6 KO) exhibit milder arthritis compared to wild-type B6 mice (WT).
MSU crystals were injected into the footpads of both IL-6 knockout mice (IL-6 KO) and wild-type B6 mice (WT) to establish an acute gouty arthritis mouse model. Compared to baseline (0h), the swelling index of the footpads in WT mice and IL-6 KO mice showed statistically significant differences at 6h, 12h, and 24h (P < 0.05), indicating successful establishment of the acute gouty arthritis mouse model (Fig. 5a). At 6h and 12h post-injection, the swelling index of footpads in IL-6 KO mice was significantly lower than that in the WT control group (P < 0.05), consistent with observed increases in footpad swelling, with the most significant difference observed at 12h, hence selected for subsequent experimental observations (Fig. 5b). HE staining revealed more severe inflammatory cell infiltration in WT mice at 12h and 24h compared to IL-6 KO mice, with no significant difference observed at 0h (Fig. 5c). These results indicate that IL-6 knockout attenuates MSU-induced inflammation and arthritis.
IL-6 KO mice avoid developing more severe gouty arthritis by impairing the JAK2-STAT1/3 signalling pathway
In IL-6 KO mice, mRNA and protein expression of IL-6 were detected to be significantly lower compared with untreated WT mice (both P < 0.05), whereas other genes were transcribed and translated at similar levels (Fig. 6a). In the acute gout mouse model, IL-1β, IL-6, JAK2, STAT1/3 mRNA and IL-1β, IL-6, JAK2, STAT1/3, p-JAK2, p-STAT1/3 protein expression were significantly down-regulated in IL-6 KO mice as compared to 12h WT mice (both P < 0.05) (Figs. 6a and 6b); also IHC staining showed decreased positive expression of p-JAK2, p-STAT1/3 (both P < 0.05) (Fig. 6c). Compared with WT mice at 24h, IL-6 mRNA and protein expression were similar (P > 0.05), IL-1β mRNA and protein expression as well as JAK2 and STAT3 mRNA expression were down-regulated (both P < 0.05), while STAT1 mRNA expression was similar (P > 0.05) (Fig. 6a and 6b). These results suggest that IL-6 KO can downregulate inflammation and avoid the development of more severe gouty arthritis by impairing the JAK2-STAT1/3 signalling pathway.