骨骼肌纤维NLRP3炎症小体激活、亚细胞定位及3D重建

doi:10.13418/j.issn.1001-165x.2025.2.10

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中国临床解剖学杂志 ›› 2025, Vol. 43 ›› Issue (2) : 168-174. DOI: 10.13418/j.issn.1001-165x.2025.2.10

实验研究

骨骼肌纤维NLRP3炎症小体激活、亚细胞定位及3D重建

厉洋洋¹，菅晓婷¹，黄静雯¹，王琪森¹，桂炜超²
张小龙²，赵子炜¹，胡稷杰²*，廖华¹*

作者信息 +

Activation, subcellular localization, and 3D reconstruction of NLRP3 inflammasome in skeletal muscle fibers

Li Yangyang¹, Jian Xiaoting¹, Huang Jingwen¹, Wang Qisen¹, Gui Weichao², Zhang Xiaolong², Zhao Ziwei¹, Hu Jijie²*, Liao Hua¹*

Author information +

文章历史 +

摘要

目的探讨体外炎性环境中肌纤维内NLRP3炎症小体的激活、亚细胞定位及其3D重建。方法 C2C12细胞体外培养、马血清分化；脂多糖（Lipopolysaccharide/LPS）及尼日利亚菌素（Nigericin/Nig）刺激C2C12肌管内NLRP3炎症小体激活；qPCR及Western blot分析NLRP3、ASC、Caspase-1基因及蛋白水平；免疫荧光分析NLRP3、ASC的聚集与共定位；共聚焦观察、Imaris软件重建显示聚合态NLRP3、ASC与特定细胞器（高尔基体、线粒体、内质网）的关联。结果体外条件下，LPS/Nig联合刺激可致肌纤维内NLRP3、ASC、Caspase-1基因、蛋白水平上调，胞浆内可见NLRP3、ASC聚集；肌纤维内线粒体功能分子TOM20、高尔基体分子TGN38显著上调，聚集态NLRP3、ASC与线粒体、高尔基体存在共定位。结论 LPS/Nig刺激可诱导骨骼肌纤维内NLRP3炎症小体激活。活化的NLRP3炎症小体组分与线粒体及高尔基体密切关联。

Abstract

Objective To investigate the activation, subcellular localization, and 3D reconstruction of the NLRP3 inflammasome within muscle fibers under in vitro inflammatory conditions. Methods C2C12 cells were cultured in vitro and differentiated with horse serum. Lipopolysaccharide (LPS) and Nigericin (Nig) were used to stimulate NLRP3 inflammasome activation in C2C12 myotubes. qPCR and Western blot were employed to analyze the gene and protein levels of NLRP3, ASC, and Caspase-1. Immunofluorescence analysis was performed to assess NLRP3 and ASC aggregation and co-localization. Confocal microscopy and Imaris software were used to visualize and reconstruct the associations of aggregated NLRP3 and ASC with specific organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum. Results In vitro, LPS/Nig co-stimulation increased the gene and protein expression of NLRP3, ASC, and Caspase-1 in muscle fibers, with cytoplasmic aggregation of NLRP3 and ASC observed. The mitochondrial functional molecule TOM20 and Golgi marker TGN38 were significantly upregulated, and co-localization was detected between aggregated NLRP3, ASC, and both mitochondria and the Golgi apparatus. Conclusions LPS/Nig stimulation induces activation of the NLRP3 inflammasome in skeletal muscle fibers. The activated components of the NLRP3 inflammasome are closely associated with mitochondria and the Golgi apparatus.

导出引用

厉洋洋, 菅晓婷, 黄静雯, 王琪森, 桂炜超, 张小龙, 赵子炜, 胡稷杰, 廖华. 骨骼肌纤维NLRP3炎症小体激活、亚细胞定位及3D重建[J]. 中国临床解剖学杂志. 2025, 43(2): 168-174 https://doi.org/10.13418/j.issn.1001-165x.2025.2.10

Li Yangyang, Jian Xiaoting, Huang Jingwen, Wang Qisen, Gui Weichao, Zhang Xiaolong, Zhao Ziwei, Hu Jijie, Liao Hua. Activation, subcellular localization, and 3D reconstruction of NLRP3 inflammasome in skeletal muscle fibers [J]. Chinese Journal of Clinical Anatomy. 2025, 43(2): 168-174 https://doi.org/10.13418/j.issn.1001-165x.2025.2.10

中图分类号： R392.31

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