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.
Key words
NLRP3 Inflammasome /
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C2C12 /
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Imaris /
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3D Reconstruction /
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Organelles
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