| [Background] Periodontitis manifests as a chronic and progressive inflammation state of the periodontal supporting tissues,initiated by microbial plaque infection,which is the leading cause of tooth loss in adults.Furthermore,periodontitis is strongly correlated with various systemic diseases and an elevated risk of mortality,posing a significant threat to overall health.The current high prevalence of periodontitis,coupled with an aging population and longer tooth retention,will greatly increase the medical and economic burden on society.Researching the pathogenesis of periodontitis and controlling its occurrence and development is an urgent social problem.Circadian rhythm refers to the phenomenon that the life activities of organisms change periodically with the circadian cycle.The circadian rhythm operates on a 24-hour cycle,which is regulated by external environmental signals,internal autonomous timing system,and the coordination between them.And the negative feedback loop of transcription and translation,which is composed by core circadian proteins including BMAL1 and NR1 D,regulates its normal output.Circadian rhythm disruption is a disordered state that negatively affects the function of the circadian rhythm.It is mainly caused by the asynchrony between the sleep-wake cycle and the environmental light-dark cycle,manifested by abnormal changes in periods,phases and amplitudes of the circadian rhythm,which can cause serious adverse effects on physical and mental health.Due to the demands of an evolving economy and service culture,shift work,which refers to the work schedules outside of conventional daytime hours,is becoming increasingly common in contemporary society.However,light is the most influential timing factor.Delayed sleep time and excessive light exposure at night can disrupt circadian rhythm physiology,resulting in severe circadian rhythm disruptions.Numerous epidemiological studies indicate that prolonged participation in shift work increases the risk of various diseases,including cardiovascular and cerebrovascular diseases,cancer,obesity,diabetes,and mental disorders.These conditions can have serious detrimental effects on the body.It is worth noting that previous studies have shown a close relationship between circadian sleep disturbances caused by shift work and an increased risk of periodontitis.However,there are no experimental studies to clarify whether shift work-related circadian rhythm disruption plays a role in the progression of periodontitis and its possible molecular mechanisms.Answering the above questions is of great practical significance for the health construction of the current society.Pyroptosis is a form of programmed cell death triggered by pro-inflammatory signals during microbial infections.It relies on the multimerization of N-terminal domain fragments resulting from the cleavage of GSDMD or other Gasdermin family proteins.This process leads to the perforation of the cell membrane,causing the formation of numerous pores on the surface of the cell membrane,the disintegration and rupture of cells,and the release of a large number of intracellular components.Pyroptosis is closely related to periodontitis.Proper pyroptosis can be involved in the activation and recruitment of immune cells to help eliminate and prevent the spread of pathogens,but overactive and prolonged pyroptosis can accelerate the progression of periodontitis by increasing cell death and upregulating inflammatory factor levels.The NLRP3 inflammasome signaling pathway is a crucial regulator of GSDMD-mediated pyroptosis.NLRP3 assembles with CASPASE-1,ASC,and NEK7 to form NLRP3 inflammasome,activates the self-cleavage of CASPASE-1 and forms its active protein fragments,which is the molecular basis for the cleavage of IL-1β precursor,IL-18 precursor,and GSDMD to form functional proteins.It is worth noting that the circadian proteins are closely related to the NLRP3 inflammasome signaling pathway.However,the expression of circadian proteins is abnormal in the condition of circadian disruption.Nevertheless,whether it can cause the abnormal regulation of pyroptosis and further lead to the circadian disruption-related periodontitis requires further investigation.In summary,this study proposes the hypothesis that the circadian rhythm disruption caused by shift work triggers abnormal expression of circadian proteins,further affecting the regulation of pyroptosis and accelerating the progression of periodontitis.[Aim] The aim of this study was to investigate the impact of circadian rhythm disruption caused by shift work on the development of periodontitis,to further elucidate the regulatory effect of the core circadian protein BMAL1 on GSDMD-mediated pyroptosis and its underlying molecular mechanism,as well to clarify the role of BMAL1/GSDMD axis in the circadian disruption-aggravated periodontitis.The specific objectives of the study are as follows:(1)To construct a mouse model of circadian disruption by shift work combined with periodontitis,and evaluate the impact of shift work on the progression of periodontitis;(2)To measure the gene expression profiles of the inflammatory gingival tissue derived from the circadian disruption mice,and identify the key molecules that affect the progression of periodontitis due to circadian disruption;(3)To clarify the modulatory pathway of BMAL1 on GSDMD-mediated pyroptosis;(4)To determine the therapeutic role of BMAL1/GSDMD axis in the circadian disruption-aggravated periodontitis.[Methods] 1.A mouse model of circadian disruption by simulating shift work was constructed by controlling the light-dark cycles;To verify the effect of model construction,voluntary running wheel assay was used to monitor the intensity and diurnal oscillation of physical activity in mice;RT-q PCR and Western Blot experiments were used to detect molecular changes in the expression of core circadian genes in gingival tissues of normal and model mice at multiple time points within a 24-hour cycle.A composite model of circadian disruption and experimental periodontitis was established,and the impact of circadian disruption on periodontitis was assessed by using micro-CT,H&E staining,and Masson staining.2.Inflammatory gingival tissue derived from mice with normal or disrupted circadian rhythm was extracted,and transcriptome sequencing was conducted to identify key molecular pathways involved in this process.To verify the sequencing results,RT-q PCR and Western Blot were performed to analyze the expression of potential key genes Bmal1,Nr1d1,and Gsdmd.3.The histological distribution of pyroptosis in inflammatory periodontal tissue was identified by immunofluorescence staining.Primary m GFs were extracted by tissue digestion using enzymes and identified by immunofluorescence staining.CCK8 and Western Blot were employed to determine the optimal concentration of P.gingivalis-LPS for simulating an inflammatory environment in vitro.Bmal1 overexpression plasmid or si RNA was transfected either before or after P.gingivalis-LPS treatment;The cell morphology was observed using scanning electron microscopy,and cell rupture and death were evaluated through PI staining and LDH activity detection;The changes at molecular level related to the NLRP3 inflammasome pathway and GSDMD-mediated pyroptosis were evaluated by RT-q PCR,Western Blot,and immunofluorescence staining.To investigate whether BMAL1’s regulation of the NLRP3 inflammasome signaling pathway depends on NR1D1,m GFs were treated with SR8278,which acts as both an NR1D1 antagonist and an BMAL1 agonist,after P.gingivalis-LPS exposure;RT-q PCR,Western Blot,cell immunofluorescence,PI staining,LDH activity detection,and double luciferase reporter assays were used to clarify the regulatory mechanisms of BMAL1 on GSDMD-mediated pyroptosis.4.A pyroptosis inhibitor was injected into the periodontal tissue after constructing an experimental periodontitis model in mice;The effectiveness of the model was confirmed through Western Blot and immunofluorescence experiments;micro-CT and H&E staining were used to evaluate the impact of GSDMD-mediated pyroptosis on periodontitis progression.Based on the model of circadian disruption complicated with periodontitis,circadian recovery and SR8278 ZT4 intraperitoneal injection models were constructed;Western Blot and immunofluorescence were employed to assess its effects on BMAL1/GSDMD axis,and micro-CT and H&E staining were used to demonstrate the therapeutic role of BMAL1-upregulation in circadian disruption-aggravated periodontitis.[Results] 1.The circadian disruption model can be successfully constructed in mice by controlling the light-dark cycle that simulated the work shift schedule.Compared with the normal mice,the circadian behaviors were perturbed in the model mice as evidenced by a decrease in activity intensity and diurnal difference,as well as an activity phase shift.At the molecular level,the expressions of core circadian genes in the gingival tissue of model mice were disrupted,manifesting a loss of rhythmicity.The assessment of periodontitis progression revealed that,in comparison to the mice with normal rhythm,the mice with circadian disruption caused by shift work exhibited more pronounced alveolar bone resorption,inflammatory cell infiltration,and collagen fiber fracture and disorganized arrangement.These findings suggest that circadian disruption caused by shift work exacerbated the progression of experimental periodontitis.2.The transcriptome sequencing results of inflammatory gingival tissue with normal and disrupted rhythm showed that circadian entrainment and pyroptosis were closely related to circadian disruption-aggravated periodontitis.The verification on the chronological expressions of central circadian genes Bmal1 and Nr1d1,as well as the corresponding levels of pyroptosis effector Gsdmd was conducted,and the results showed that compared to mice with normal rhythm,mice with rhythm disruption exhibited decreased levels of BMAL1 and NR1D1,along with increased pyroptosis activity in gingival tissue.And the expressions of BMAL1 and GSDMD showed an opposite trend over time,suggesting that BMAL1 may play a role in regulating GSDMD-mediated pyroptosis.3.Immunofluorescence staining showed that the distribution of GSDMD in inflammatory periodontal tissues was closely related to gingival fibroblasts,and its expression level was higher in mice with rhythm disruption than that in mice with normal rhythm.Primary mouse gingival fibroblasts were successfully extracted,and 5 μg/m L was screened as the optimal concentration of P.gingivalis-LPS to simulate the inflammatory environment in vitro.Subsequent studies found that interfering with BMAL1 expression upregulated the NLRP3 inflammasome signaling pathway,exacerbating pyroptosis induced by P.gingivalis-LPS.Conversely,overexpression of BMAL1 inhibited the NLRP3 inflammasome signaling pathway,alleviating P.gingivalis-LPS-induced pyroptosis.Further studies revealed that SR8278 inhibited NR1D1 and activated the NLRP3 inflammasome signaling pathway,while increased the expression of BMAL1 and effectively suppressed GSDMD-induced pyroptosis,suggesting that BMAL1 regulated NLRP3 inflammasome signaling through NR1D1,and other ways may exist for BMAL1 to modulate the pyroptosis effector GSDMD.It was subsequently discovered that BMAL1 could directly bind to the promoter of Gsdmd and inhibit its transcriptional activity,as demonstrated by a double luciferase reporter assay.4.The promotion effect of GSDMD-mediated pyroptosis on periodontitis was demonstrated by the periodontitis pyroptosis inhibition model.Circadian recovery and SR8278 treatment at ZT4 improved the decreased BMAL1 level,the increased GSDMD activity,the alveolar bone resorption,the inflammatory cell infiltration,and the collagen fiber damage,caused by circadian disruption,indicating that BMAL1-upregualtion can relieve the circadian disruption-related periodontitis progression by inhibiting GSDMDmediated pyroptosis.[Conclusion] 1.Circadian rhythm disruption by simulating shift work aggravates the progression of experimental periodontitis;2.Circadian protein BMAL1 and GSDMD-mediated pyroptosis play an important role in circadian disruption-aggravated periodontitis;3.BMAL1 inhibited the fibroblast pyroptosis mediated by GSDMD in an inflammatory environment,which not only depends on the regulation of NR1D1 on the NLRP3 inflammasome pathway,but also attributes to the transcriptional modulation of BMAL1 on Gsdmd;4.Restoring the duly expression of BMAL1 alleviates the circadian disruption-related periodontitis progression through inhibiting GSDMD-mediated pyroptosis.BMAL1/GSDMD signaling acts as a coordinator of shift work-related circadian disruption and periodontitis progression,providing a potential guidance for the development of intervention strategies targeting circadian clock against periodontitis. |
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