张宏男, 陈振辉, 范宏英, 朱嘉伟, 郭进强, 覃旻, 王致, 孟晓静. NLRP3炎症小体在小鼠热应激损伤中的作用[J]. 环境与职业医学, 2020, 37(5): 453-460. DOI: 10.13213/j.cnki.jeom.2020.19899
引用本文: 张宏男, 陈振辉, 范宏英, 朱嘉伟, 郭进强, 覃旻, 王致, 孟晓静. NLRP3炎症小体在小鼠热应激损伤中的作用[J]. 环境与职业医学, 2020, 37(5): 453-460. DOI: 10.13213/j.cnki.jeom.2020.19899
ZHANG Hong-nan, CHEN Zhen-hui, FAN Hong-ying, ZHU Jia-wei, GUO Jin-qiang, QING Min, WANG Zhi, MENG Xiao-jing. Effects of NLRP3 inflammasome on heat stress-induced damage in mice[J]. Journal of Environmental and Occupational Medicine, 2020, 37(5): 453-460. DOI: 10.13213/j.cnki.jeom.2020.19899
Citation: ZHANG Hong-nan, CHEN Zhen-hui, FAN Hong-ying, ZHU Jia-wei, GUO Jin-qiang, QING Min, WANG Zhi, MENG Xiao-jing. Effects of NLRP3 inflammasome on heat stress-induced damage in mice[J]. Journal of Environmental and Occupational Medicine, 2020, 37(5): 453-460. DOI: 10.13213/j.cnki.jeom.2020.19899

NLRP3炎症小体在小鼠热应激损伤中的作用

Effects of NLRP3 inflammasome on heat stress-induced damage in mice

  • 摘要: 背景

    热应激(HS)可导致机体重要脏器损伤甚至引起死亡,全身性炎症反应是其主要原因之一。NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体在介导组织损伤引起的无菌性炎症中起重要作用,但其在HS损伤中的作用及机制尚不清楚。

    目的

    探讨NLRP3炎症小体在HS小鼠系统性炎症反应和多器官损伤中的作用。

    方法

    设立野生型C57BL/6雄性小鼠对照组、HS组,以及NLRP3敲除HS组(Nlrp3-/-HS组),每组8只。利用温度(41.0±0.5)℃、湿度(65±5)%的高温环境模拟舱对HS组和Nlrp3-/-HS组小鼠进行HS。每隔15~20 min监测肛温以及记录一般精神身体状况。当HS组小鼠肛温均超过42.0℃,立即将两组小鼠移至正常室温环境,于室温恢复4 h,解剖三组小鼠。HE染色观察小鼠肝、肾、脾、脑和肠组织的病理改变;采用ELISA试剂盒检测各组的血清肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β和IL-6的水平;采用内毒素检测试剂盒检测血清内毒素水平;采用免疫印记法检测肠闭锁蛋白(Occludin)、闭锁小带蛋白-1(ZO-1)的表达水平,以及脑、肝脏和肠道组织NLRP3、半胱天冬酶(caspase)-1、IL-1β的蛋白表达水平。

    结果

    受热90min时,HS组小鼠的肛温(42.1±0.2)℃高于对照组(36.6±0.2)℃,Nlrp3-/-HS组小鼠肛温(40.1±0.7)℃和体重减轻量(1.3±0.2)g低于HS组(P < 0.05)。HE染色显示HS组小鼠的肝、肾、脾、脑和肠组织有不同程度的损伤,而Nlrp3-/-HS组小鼠的各个组织病理损伤程度减轻。与对照组相比,HS组小鼠血清的TNF-α、IL-1β和内毒素水平均升高,肠道Occludin、ZO-1表达量降低,Nlrp3-/-HS组小鼠的IL-1β分泌量较HS组减少,肠道Occludin、ZO-1表达量增多(P < 0.05);HS组小鼠脑、肝脏和肠道组织的NLRP3、caspase-1、IL-1β蛋白水平与对照组相比升高(P < 0.05),Nlrp3-/-HS组小鼠的NLRP3、caspase-1和IL-1β表达量较HS组降低(P < 0.05)。

    结论

    NLRP3炎症小体活化参与了急性HS所致的系统性炎症反应以及多器官功能损伤。

     

    Abstract: Background

    Heat stress (HS)-caused systemic inflammation response can lead to injuries to vital organs and even death in patients. NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) plays an important role in mediating aseptic inflammation caused by tissue injury, but its role in HS-induced damage is not clear.

    Objective

    This experiment explores the role of NLRP3 inflammasome in systemic inflammatory response and multiple organ damage in mice after HS.

    Methods

    This experiment included three groups:control group (wild type C56BL/6 male mice without HS), HS group (wild type C57BL/6 male mice with HS), and Nlrp3-/-HS group (NLRP3 knockout C57BL/6 male mice with HS), with eight mice in each group. HS was induced by exposure to an ambient temperature of (41.0±0.5)℃ with relative humidity of (65±5)%. Anal temperature and general mental and physical conditions were recorded every 15-20min. When the anal temperature of the mice in the HS group rose above 42.0℃, the HS group and the Nlrp3-/-HS group were removed to room temperature and then anesthetized and dissected after 4 h. Selected organs of the mice, such as liver, kidney, spleen, brain, and intestine, were stained with HE for histopathological examination. Levels of serum tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6 were measured with ELISA kit; level of endotoxin was detected with endotoxin kit; expression levels of intestinal Occludin and zonula occludens-1 (ZO-1), as well as NLRP3, caspase-1, and IL-1β in brain, liver, and intestine tissues were analyzed by Western blotting.

    Results

    After the heat exposure for 90min, the HS group showed higher anal temperature(42.1±0.2)℃ than the control group(36.6±0.2)℃, and the Nlrp3-/-HS group showed lower anal temperature(40.1±0.7)℃ and less body weight loss(1.3±0.2) g than the HS group (P < 0.05). The pathological results after HE staining showed different degrees of injury to liver, kidney, spleen, brain, and intestine in the HS group, while only mild histopathological changes in the Nlrp3-/-HS group. The levels of serum TNF-α, IL-1β, and endotoxin were higher and the expression levels of intestinal Occludin and ZO-1 were lower in the HS group than those in the control group, and the level of IL-1β was lower and the expression levels of intestinal Occludin and ZO-1 were higher in the Nlrp3-/-HS group than those in the HS group (P < 0.05). The expression levels of NLRP3, caspase-1, and IL-1β in brain, liver, and intestine tissues of mice in the HS group were higher than those in the control group (P < 0.05), while the levels in the Nlrp3-/-HS group were lower than those in the HS group (P < 0.05).

    Conclusion

    The activation of NLRP3 inflammasome plays a key role in systemic inflammatory response and multiple organ function damage during acute HS exposure.

     

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