白细胞介素-1β参与curdlan诱导的慢性肉芽肿性疾病高炎症反应的初步研究

Role of interleukin-1β in hyperinflammation of chronic granuiomatous disease

目的探讨curdlan诱导慢性肉芽肿性疾病（CGD）高炎症性反应的分子机制，初步探讨NOX2（NADPH oxidase 2）缺失与高炎症反应之间的关系及可能机制。方法β-葡聚糖（β-glucan）是CGD感染并发症的主要刺激物，以NOX2基因缺失的小鼠耳背注射curdlan（1，3-β-slucan，200μg／50μL）／PBS 50μL建立CGD高炎症反应动物模型，观察其炎症反应并分析炎症因子的表达变化。在注射后的第2、7天提取鼠耳蛋白检测其中炎症因子白细胞介素（IL）-1β（interleukin-1β）的表达变化。Westernblot检测对照组及NOX2缺失组caspase-1的表达情况。体外实验中，用10、50、100、200μg／mL curdlan处理从上述小鼠中分离培养的巨噬细胞，建立CGD细胞模型。在处理后第1、2天和第5天收取细胞上清用酶联免疫吸附试验（ELISA）检测相关的炎症因子IL-1β表达变化。结果NOX2缺失的小鼠注射curdlan后表现出高炎症性反应，与对照组相比，炎症因子IL-1β显著高表达（第2天：P=0．0189，第7天：P=0．0450）；NOX2缺失小鼠caspase-1的活性形式caspase-1 p10表达显著增加（第2天：P=0．3980，第7天：P=0．0001）。在CGD细胞模型中，我们发现在注射后第5天与正常对照组相比，炎症因子IL-1β水平显著升高（P=0．0011），且局限于NOX2缺失的骨髓来源的巨噬细胞（BMDM）中，其水平与curdlan浓度呈剂量依赖性。结论Curdlan通过激活巨噬细胞中的caspase-1导致炎症因子IL-1β显著高表达，炎症因子IL-1β参与了NOX2缺失导致的慢性肉芽肿病高炎症性反应。

Objective To clarify the molecular mechanism underlying hyperinflammation in CGD（ chron- ic granulomatous disease）induced by curdlan and demonstrates the potential mechanism involved in the link be- tween NOX2 and hyperirinflammation. Methods β-glucan is considered to be the main stimulus of the CGD in- flammatory complication. In our study, mice model was established by 200 μg/50 μL/PBS 50 μL of curdlan （ 1, 3-β-glucan） injection into the ears of NOX2/ mice. IL-1β protein in ears was detected using ELISA kit at day 2 and day 7. Caspase-1 was also detected by Western blot. In addition, bone marrow-derived macrophages （ BM- DM） isolated from these mice were treated with 10,50,100 and 200 μg/mL of curdlan. Supernatant of curdlan- treated macrophages was collected from day 1,2 and 5 to detect level of IL-1β. Results NOX2 deficient mice （NOX2 KO） displayed hyperinflammation,increased ear thickness and elevated IL-1β level after curdlan injec- tion （ Day2 ：P =0.0189 ,Day 7 ：P =0.0450）. The expression of active form of caspase-1 （ caspase-1 pl0） also increased （ Day2 ：P =0. 3980, Day 7 ： P = 0. 0001 ）. In cell model, macrophages isolated from these mice were stimulated with 10,50,100,and 200 μg/mL of curdlan. IL-1β was up-regulated in BMDM （P =0. 0011 at Day 5） and the level of IL-1 increased with a dose-dependent manner. Conclusions Curdlan led to high level of IL-1β production in macrophages through caspase-1 activation. IL-1β was involved in the hyperinflammation in chronic granulomatous disease induced by NOX2 deficiency.