重症溃疡性结肠炎患者血清NOD样受体蛋白3、半胱氨酸天冬氨酸特异性蛋白酶1和核因子κB mRNA表达水平及临床意义

doi:10.3877/cma.j.issn.1674-6880.2019.01.006

目的

探讨重症溃疡性结肠炎（UC）患者血清NOD样受体蛋白3（NLRP3）、半胱氨酸天冬氨酸特异性蛋白酶1（CASP1）和核因子κB（NF-κB）mRNA表达水平及临床意义。

方法

选取温州市中西医结合医院消化内科收治的重症UC患者116例作为重症UC组，同期健康体检者50例作为对照组，采用实时荧光定量PCR（qRT-PCR）法检测受试者血清NLRP3、CASP1和NF-κB mRNA表达水平，采用酶联免疫吸附剂测定（ELISA）法检测所有受试者血清白细胞介素1β（IL-1β）、IL-18表达水平，采用Pearson相关分析重症UC组患者血清NLRP3、CASP1、NF-κB mRNA与IL-1β、IL-18表达的相关性。

结果

重症UC组患者血清NLRP3 [（6.3 ± 1.1）vs.（1.2 ± 0.3）]、CASP1 [（5.4 ± 1.1）vs.（1.1 ± 0.3）]和NF-κB [（6.73 ± 1.13）vs.（0.51 ± 0.15）mRNA表达水平均显著高于对照组（t = 32.016、27.873、38.710，P均< 0.001）。重症UC组Ⅰ、Ⅱ、Ⅲ级患者NLRP3 [（2.4 ± 0.6）、（4.5 ± 1.1）、（6.8 ± 1.2）]、CASP1 [（2.2 ± 0.4）、（3.9 ± 1.0）、（5.9 ± 1.1）]及NF-κB [（2.5 ±0.6）、（5.4 ± 1.2）、（7.2 ± 1.4）] mRNA表达水平比较，差异均有统计学意义（F= 49.852、43.224、33.293，P均< 0.001）。进一步两两比较发现，Ⅱ级、Ⅲ级重症UC患者NLRP3、CASP1和NF-κB mRNA的表达水平均显著高于Ⅰ级患者（P均< 0.05），Ⅲ级重症UC患者NLRP3、CASP1和NF-κB mRNA的表达水平均显著高于Ⅱ级患者（P均< 0.05）。重症UC组患者血清IL-1β [（90 ± 7）ng/L vs.（69 ± 7）ng/L]、IL-18 [（121 ± 4）ng/L vs.（102 ± 6）ng/L]表达水平较对照组均显著升高（t= 16.555、24.249，P均< 0.001）。重症UC患者血清NLRP3 mRNA与CASP1、及NF-κB mRNA表达均呈正相关（r= 0.767、0.676，P均< 0.001），而CASP1 mRNA与NF-κB mRNA表达无相关性（r= 0.263，P= 0.175）。重症UC患者血清NLRP3、CASP1、NF-κB mRNA与IL-1β表达均呈正相关（r= 3.372、3.724、3.424，P= 0.035、0.011、0.026），与IL-18表达亦均呈正相关（r= 2.963、3.513、3.312，P= 0.043、0.018、0.023）。

结论

重症UC患者血清NLRP3、CASP1和NF-κB mRNA表达水平均显著升高，且NLRP3 mRNA与CASP1、NF-κB mRNA表达呈正相关。NLRP3、CASP1和NF-κB mRNA参与重症UC病理过程，临床上可通过检测NLRP3、CASP1和NF-κB表达水平判断UC病程进展。

关键词: 结肠炎，溃疡性, NOD样受体蛋白3, 半胱氨酸天冬氨酸特异性蛋白酶1, 核因子κB, 病理分级, 相关性

Objective

To investigate expressions of serum NOD-like receptor protein-3 (NLRP3), cysteiny aspartate specific protease-1 (CASP1) and nuclear factor-kappa B (NF-κB) mRNA in patients with severe ulcerative colitis (UC) and its clinical significance.

Methods

A total of 116 patients with severe UC were selected as the severe UC group and 50 healthy subjects were used as the control group in the Department of Gastroenterology, Wenzhou Integrated Chinese and Western Medicine Hospital. The expressions of NLRP3, CASP1 and NF-κB mRNA in serum were detected by quantitative real-time PCR (qRT-PCR), and expressions of interleukin 1 beta (IL-1β) and IL-18 in serum were detected by enzyme linked immunosorbent assay (ELISA). Correlation between serum NLRP3, CASP1, NF-κB mRNA and IL-1β, IL-18 expressions in patients with severe UC was detected by Pearson correlation analysis.

Results

The mRNA expressions of NLRP3 [(6.3 ± 1.1) vs. (1.2 ± 0.3)], CASP1 [(5.4 ± 1.1) vs. (1.1 ± 0.3)] and NF-κB [(6.73 ± 1.13) vs. (0.51 ± 0.15)] in the severe UC group were significantly higher than those in the control group (t= 32.016, 27.873, 38.710; all P < 0.001). The mRNA expressions of NLRP3 [(2.4 ± 0.6), (4.5 ± 1.1), (6.8 ± 1.2)], CASP1 [(2.2 ± 0.4), (3.9 ± 1.0), (5.9 ± 1.1)] and NF-κB [(2.5 ± 0.6), (5.4 ± 1.2), (7.2 ± 1.4)] in grade Ⅰ, Ⅱ and Ⅲ patients with severe UC were significantly different (F= 49.852, 43.224, 33.293; all P < 0.001). Further comparison showed that expressions of NLRP3, CASP1 and NF-κB mRNA in grade Ⅱ and Ⅲ UC patients were significantly higher than those in grade Ⅰ patients (all P < 0.05), and the expression levels of NLRP3, CASP1 and NF-κB mRNA in grade Ⅲ patients were significantly higher than those in grade Ⅱ patients (all P < 0.05). The levels of serum IL-1β [(90 ± 7) ng/L vs. (69 ± 7) ng/L] and IL-18 [(121 ± 4) ng/L vs.(102 ± 6) ng/L] in the severe UC group were significantly higher than those in control group (t= 16.555, 24.249; both P < 0.001). There were positive correlations between expressions of NLRP3 and CASP1, NF-κB mRNA in patients with severe UC (r= 0.767, 0.676; both P < 0.001), but no correlation between expressions of CASP1 and NF-κB mRNA(r= 0.263, P= 0.175). The levels of NLRP3, CASP1, NF-κB mRNA were positively correlated with IL-1β (r= 3.372, 3.724, 3.424; P= 0.035, 0.011, 0.026) and IL-18 (r= 2.963, 3.513, 3.312; P= 0.043, 0.018, 0.023).

Conclusions

The levels of serum NLRP3, CASP1 and NF-κB significantly increased in severe UC patients, and the expression of NLRP3 was positively correlated with CASP1 and NF-κB. Clinical progress of UC can be judged by expression levels of NLRP3, CASP1 and NF-κB as they are involved in the pathological process of severe UC.

Key words: Colitis, ulcerative, NOD-like receptor protein-3, Cysteiny aspartate specific protease-1, Nuclear factor-kappa B, Pathological grading, Relevance

表1 qRT-PCR扩增引物序列

基因	正向引物5 ’-3 ’	反向引物5 ’-3 ’
NLRP3	AAAGAGATGAGCCGAAGTGGG	TCAATGCTGTCTTCCTGGCA
CASP1	CTCAGGCTCAGAAGGGAATG	CGCTGTACCCCAGATTTTGT
NF-κB	TCAATGGCTACACAGGACCA	CACTGTCACCTGGAAGCAGA
β-actin	CTGGAACGGTGAAGGTGACA	AGGGACTTCCTGTAACAATGCA

表1 qRT-PCR扩增引物序列

基因	正向引物5 ’-3 ’	反向引物5 ’-3 ’
NLRP3	AAAGAGATGAGCCGAAGTGGG	TCAATGCTGTCTTCCTGGCA
CASP1	CTCAGGCTCAGAAGGGAATG	CGCTGTACCCCAGATTTTGT
NF-κB	TCAATGGCTACACAGGACCA	CACTGTCACCTGGAAGCAGA
β-actin	CTGGAACGGTGAAGGTGACA	AGGGACTTCCTGTAACAATGCA

表2 两组患者血清NLRP3、CASP1和NF-κB mRNA表达水平比较（±s）

组别	例数	NLRP3 mRNA	CASP1 mRNA	NF-κB mRNA
对照组	50	1.2 ± 0.3	1.1 ± 0.3	0.51 ± 0.15
重症UC组	116	6.3 ± 1.1	5.4 ± 1.1	6.73 ± 1.13
t值		32.016	27.873	38.710
P值		< 0.001	< 0.001	< 0.001

表2 两组患者血清NLRP3、CASP1和NF-κB mRNA表达水平比较（±s）

组别	例数	NLRP3 mRNA	CASP1 mRNA	NF-κB mRNA
对照组	50	1.2 ± 0.3	1.1 ± 0.3	0.51 ± 0.15
重症UC组	116	6.3 ± 1.1	5.4 ± 1.1	6.73 ± 1.13
t值		32.016	27.873	38.710
P值		< 0.001	< 0.001	< 0.001

表3 不同病理分级血清重症UC患者NLRP3、CASP1和NF-κB mRNA表达水平比较（±s）

组别	例数	NLRP3 mRNA	CASP1 mRNA	NF-κB mRNA
Ⅰ级	4	2.4 ± 0.6	2.2 ± 0.4	2.5 ± 0.6
Ⅱ级	18	4.5 ± 1.1^a	3.9 ± 1.0^a	5.4 ± 1.2^a
Ⅲ级	94	6.8 ± 1.2^ab	5.9 ± 1.1^ab	7.2 ± 1.4^ab
F值		49.852	43.224	33.293
P值		< 0.001	< 0.001	< 0.001

表3 不同病理分级血清重症UC患者NLRP3、CASP1和NF-κB mRNA表达水平比较（±s）

组别	例数	NLRP3 mRNA	CASP1 mRNA	NF-κB mRNA
Ⅰ级	4	2.4 ± 0.6	2.2 ± 0.4	2.5 ± 0.6
Ⅱ级	18	4.5 ± 1.1^a	3.9 ± 1.0^a	5.4 ± 1.2^a
Ⅲ级	94	6.8 ± 1.2^ab	5.9 ± 1.1^ab	7.2 ± 1.4^ab
F值		49.852	43.224	33.293
P值		< 0.001	< 0.001	< 0.001

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Expressions and clinical significance of serum NOD-like receptor protein-3, cysteiny aspartate-specific protease-1 and nuclear factor-kappa B in patients with severe ulcerative colitis

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Expressions and clinical significance of serum NOD-like receptor protein-3, cysteiny aspartate-specific protease-1 and nuclear factor-kappa B in patients with severe ulcerative colitis