Circadian disruption by simulated shift work aggravates periodontitis via orchestrating BMAL1 and GSDMD-mediated pyroptosis

Abstract

Approximately 20% to 30% of the global workforce is engaged in shift work. As a significant cause of circadian disruption, shift work is closely associated with an increased risk for periodontitis. Nevertheless, how shift work-related circadian disruption functions in periodontitis remains unknown. Herein, we employed a simulated shift work model constructed by controlling the environmental light-dark cycles and revealed that shift work-related circadian disruption exacerbated the progression of experimental periodontitis. RNA sequencing and in vitro experiments indicated that downregulation of the core circadian protein brain and muscle ARNT-like protein 1 (BMAL1) and activation of the Gasdermin D (GSDMD)-mediated pyroptosis were involved in the pathogenesis of that. Mechanically, BMAL1 regulated GSDMD-mediated pyroptosis by suppressing NOD-like receptor protein 3 (NLRP3) inflammasome signaling through modulating nuclear receptor subfamily 1 group D member 1 (NR1D1), and inhibiting Gsdmd transcription via directly binding to the E-box elements in its promoter. GSDMD-mediated pyroptosis accelerated periodontitis progression, whereas downregulated BMAL1 under circadian disruption further aggravated periodontal destruction by increasing GSDMD activity. And restoring the level of BMAL1 by circadian recovery and SR8278 injection alleviated simulated shift work-exacerbated periodontitis via lessening GSDMD-mediated pyroptosis. These findings provide new evidence and potential interventional targets for circadian disruption-accelerated periodontitis.

Fig. 1

Circadian disruption by shift work simulation aggravates experimental periodontitis in mice. a The intensity and diurnal oscillation of physical activity in mice under normal or simulated shift work conditions measured by voluntary running wheel assay. b Relative mRNA expressions of indicated circadian clock genes in gingiva of mice under normal or simulated shift work conditions (n = 3). c The representative sagittal tridimensional and bidimensional views of the maxillary molars scanned by micro-CT (scale bar: 500 μm). d Quantification of the BMD, BV/TV, and the distance from the CEJ to the ABC in indicated groups (n = 7). e H&E staining of paraffin-embedded sections (scale bar: 500 μm; 75 μm). Data are represented as the mean ± SD. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: ZT zeitgeber time, NN mice under normal light-dark cycles without ligature, NL mice under normal light-dark cycles with ligature, DN mice under disturbed light-dark cycles without ligature, DL mice under disturbed light-dark cycles with ligature, BMD bone mineral density, BV/TV bone volume fraction, CEJ cement-enamel junction, ABC alveolar bone crest, H&E hematoxylin and eosin

Fig. 2

Circadian entrainment and pyroptosis are involved in the development of circadian disruption-accelerated periodontitis. a Heatmap of the expressions of genes related to circadian entrainment and pyroptosis (n = 3). b, c The diurnal rhythms of Bmal1, Nr1d1, and Gsdmd measured by qRT-PCR in the gingiva of mice at indicated times (n = 3). d, e Quantification of the diurnal oscillation of BMAL1, NR1D1, as well as Fl-GSDMD and N-GSDMD at indicated times by western blot (n = 3). Data are represented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: NN mice under normal light-dark cycles without ligature, NL mice under normal light-dark cycles with ligature; DL mice under disturbed light-dark cycles with ligature; Fl-GSDMD full-length of GSDMD, N-GSDMD N-terminal cleavage fragment of GSDMD

Fig. 3

BMAL1 functions in the regulation of GSDMD-mediated pyroptosis. a, f The representative images of cell morphology in indicated groups examined by SEM (scale bar: Low magnification = 50 μm; High magnification = 10 μm) b, g Quantification of LDH activity in mGFs treated with P. gingivalis LPS and Bmal1 siRNA (n = 5) or overexpression plasmid (n = 4). c, h The representative images of PI staining in indicated groups were examined by LCFM (scale bar: 100 μm). d, i Relative mRNA expressions of genes related to GSDMD-mediated pyroptosis in indicated groups measured by qRT-PCR (n = 3). e, j Relative levels of proteins related to NLRP3 inflammasome signaling in indicated groups detected by western blot (n = 3). Data are represented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: LPS lipopolysaccharide, LDH lactate dehydrogenase, PI propidium iodide, SEM scanning electron microscope, LCFM laser confocal fluorescence microscopy

Fig. 4

BMAL1 regulates Gsdmd transcription independent of NR1D1. a Relative mRNA expressions of Bmal1, Nr1d1, and genes related to NLRP3 inflammasome signaling in indicated groups measured by qRT-PCR (n = 3). b Relative expressions of BMAL1, NR1D1, and proteins related to NLRP3 inflammasome signaling in indicated groups detected by western blot (n = 3). c The representative images of PI staining in indicated groups examined by LCFM (scale bar: 100 μm). d Quantification of LDH activity in indicated groups (n = 4). e Sequence logos of consensus DNA binding sites for genes regulated by BMAL1. f Verification of the binding between BMAL1 and the Gsdmd promoter region by ChIP assay. g The binding sites of BMAL1 and Gsdmd, and the design for luciferase reporters. h Normalized luciferase activity after co-transfection of pcDNA or Bmal1 plasmid together with GSDMD-WT or GSDMD-MUT luciferase reporters measured by dual luciferase assay (n = 3). Data are represented as the mean ± SD. ns, no significance; **P < 0.01; ***P < 0.001. Abbreviations: LPS lipopolysaccharide, LCFM laser confocal fluorescence microscopy, PI propidium iodide, LDH lactate dehydrogenase; WT wild type, MUT mutant type

Fig. 5

Inhibition of GSDMD-mediated pyroptosis improves the progression of periodontitis. a Relative expressions of proteins related to GSDMD-mediated pyroptosis in the gingiva of mice with periodontal ligature and periodontal local injection of YVAD. b, c Representative images (scale bar: 100 μm) and quantitative analysis of GSDMD expression in indicated groups by immunofluorescence staining (n = 6). d The representative sagittal tridimensional and bidimensional views of the maxillary molars in indicated groups scanned by micro-CT (scale bar: 500 μm). e Quantification of the distance from CEJ to the ABC as well as the index of BV/TV and BMD in indicated groups (n = 7). f Representative images of H&E staining of the gingiva in indicated groups (scale bar: 200 μm; 50 μm). Data are represented as the mean ± SD. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: BMD bone mineral density, BV/TV bone volume fraction, CEJ cement-enamel junction, ABC alveolar bone crest, H&E hematoxylin and eosin

Fig. 6

BMAL1-upregulation alleviates circadian disruption-accelerated periodontitis via lessening GSDMD-mediated pyroptosis. a Representative images and quantitative analysis of BMAL1, NR1D1, and proteins related to NLRP3 inflammasome signaling in indicated groups detected by western blot (n = 3). b, c Representative images (scale bar: 100 μm) and quantification of GSDMD expression in indicated groups by immunofluorescence staining (n = 3). d The representative sagittal tridimensional and bidimensional views of the maxillary molars in indicated groups scanned by micro-CT (scale bar: 500 μm). e Quantification of the BMD, the BV/TV, and the distance from the CEJ to the ABC in indicated groups (n = 6). f Representative images of H&E staining of the gingiva in indicated groups (scale bar: 500 μm; 75 μm). Data are represented as the mean ± SD. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: BMD bone mineral density, BV/TV bone volume fraction, CEJ cement-enamel junction, ABC alveolar bone crest, H&E hematoxylin and eosin

Fig. 7

Circadian disruption caused by shift work simulation aggravates periodontitis via orchestrating GSDMD-mediated pyroptosis through BMAL1 in gingival fibroblasts