Porphyromonas gingivalis disrupts hippocampal circadian clock via PI3K/AKT pathway, exacerbating Alzheimer‐like pathology

Abstract

INTRODUCTION

Periodontal pathogen Porphyromonas gingivalis is epidemiologically linked to Alzheimer's disease (AD), yet how oral infection disrupts the central circadian clock to drive hippocampal neurodegeneration remains unknown.

METHODS

C57BL/6 mice received oral P. gingivalis for 6 months; hippocampal clock gene oscillations, phosphorylated protein kinase B (p‐AKT), glial fibrillary acidic protein (GFAP)/Ionized calcium‐binding adapter molecule 1 (Iba1), and amyloid beta (Aβ) load were quantified. C8‐D1A astrocytes and BV2 microglia were infected with P. gingivalis ± phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT inhibitor or agonist; Bmal1 (brain and muscle ARNT‐like 1)/Clock (circadian locomotor output cycles kaput) were knocked down by lentivirus.

RESULTS

P. gingivalis‐induced periodontitis dampened hippocampal Bmal1 rhythms, lowered p‐AKT, activated glia, and elevated Aβ and interleukin 1β (IL‐1β). In glial cells, P. gingivalis flattened Bmal1 oscillation; PI3K blockade mimicked these effects, whereas AKT agonist restored rhythms and suppressed GFAP/Iba1/IL‐1β. Bmal1 knockdown alone triggered glial activation and cytokine release.

DISCUSSION

P. gingivalis oral infection suppresses PI3K/AKT signaling, destabilizing glial circadian clocks and unleashing neuroinflammation that fosters hippocampal AD‐like pathology; rescuing PI3K/AKT or clock function may mitigate the oral–brain axis in AD.

Highlights

• P. gingivalis oral infection triggers alveolar bone loss, serum/cerebral inflammation, and hippocampal Aβ₄₂ deposition and p‐tau181 (Tau protein hyperphosphorylation at threonine 181) in mice.

• Hippocampal Bmal1 amplitude collapses and peak phase shifts > 6 h, while Cry1 rhythm disappears, indicating clock disruption by P. gingivalis.

• P. gingivalis suppresses p‐AKT; PI3K/AKT inhibitors downregulate BMAL1 and upregulate GFAP/Iba1/IL‐1β, whereas AKT agonist reverses these effects.

• Lentiviral Bmal1 knockdown alone suffices to activate glia and release IL‐1β, pinpointing Bmal1 as an anti‐inflammatory node in P. gingivalis ‐driven neurodegeneration.

A graphical abstract is provided to visually summarize the key findings of this study.

1. BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment, and pathological hallmarks such as extracellular amyloid beta (Aβ) plaques, neurofibrillary tangles of hyperphosphorylated tau, and sustained neuroinflammation. ¹ Chronic periodontitis, a bacterially driven inflammatory disease in which P. gingivalis acts as the keystone pathogen, is a widespread oral disease with systemic implications. P. gingivalis has been detected in the brains of AD patients and shown to promote neuroinflammatory responses and exacerbate AD pathology. The 16S rRNA of P. gingivalis, as well as virulence factors such as lipopolysaccharides (LPSs) and gingipains, have been detected in the brains of patients with AD. ² , ³ , ⁴ P. gingivalis can damage the blood–brain barrier (BBB) and promote tau protein phosphorylation, Aβ deposition, glial cell activation, and neuroinflammation in the hippocampal tissue of mice. ⁵ , ⁶ More research is needed to determine the exact function and molecular processes of P. gingivalis infection in the pathophysiology of AD. A comprehensive understanding of this process is essential for the development of preventive measures and targeted therapeutics against AD.

In parallel, there is a bidirectional interaction relationship between the circadian clock and AD. ⁷ , ⁸ , ⁹ The circadian clock of mammals hinges on the regulation of the transcription–translation feedback loop (TTFL), constituted by a group of core clock genes, and plays an important role in various aspects such as sleep, metabolism, immunity, and diet. The main core clock genes discovered so far include Bmal1, Clock, Per (Per1, Per2, Per3), and Cry (Cry1, Cry2). ¹⁰ Patients with AD often experience sleep disruptions and circadian rhythm disorders. ¹¹ , ¹² Sleep disorders and circadian rhythm disorders may lead to neuroinflammation, low efficiency of amyloid beta (Aβ) clearance, increased concentration of reactive oxygen species (ROS), and damage to the BBB. ¹³ , ¹⁴ These research results indicate that the disorder of the circadian clock is closely related to the occurrence and development of AD.

Recent evidence suggests that the phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) signaling pathway is involved in modulating circadian components and that dysregulation of this pathway may exacerbate neuroinflammation. ¹⁵ There exists a significant bidirectional association between the PI3K/AKT signaling pathway and the core clock components. Under both physiological and pathogenic circumstances, the core clock components prompt the rhythmic activation of PI3K and AKT. Meanwhile, the PI3K/AKT signaling pathway can reciprocally modulate the expression or activity of the core clock components. ¹⁶ The activation of the PI3K/AKT signaling pathway is associated with protecting neurons against Aβ‐induced neurotoxicity. Conversely, the inhibition of PI3K/AKT can trigger neuronal apoptosis. ¹⁷ P. gingivalis, a keystone pathogen in periodontitis, has been causally implicated in the pathogenesis of AD. However, whether P. gingivalis disrupts circadian clock or modulates the PI3K/AKT signaling pathway to precipitate AD‐related neuropathology remains to be experimentally elucidated.

In this study, we hypothesized that P. gingivalis would disrupt hippocampal circadian gene expression via inhibition of the PI3K/AKT pathway, contributing to glial activation and neurodegeneration. Using a combination of in vivo and in vitro models, we investigated how P. gingivalis exposure influenced circadian regulation in the brain, evaluated the consequential influence of circadian rhythm on glial cell activation and neuroinflammation, and identified potential molecular links between oral infection and central nervous system pathology.

2. METHODS

2.1. Bacterial culture

P. gingivalis W83 was inoculated on sheep blood agar plates (Huankai Microbiology, China) in an anaerobic system (Gene Science, Cambridge, MA, USA) for 5 to 7 days. Single colonies were cultured on brain‐heart infusion broth (OXOID, UK) supplemented with hemin (0.5 mg/mL) and vitamin K (10 mg/mL) for 48 h at 37°C anaerobically. The bacterial cells were washed and resuspended in phosphate‐buffered saline (PBS). Finally, the bacterial cells were diluted with PBS containing 3% carboxymethyl cellulose (CMC) to a concentration of 10⁹ CFU/mL and stored at 4°C. The inoculum concentration and frequency were selected based on commonly used chronic oral infection protocols for P. gingivalis in mice. ¹⁸

RESEARCH IN CONTEXT

1. Systematic review: We searched PubMed for preclinical studies examining the relationship between Porphyromonas gingivalis infection and AD pathology, focusing on circadian disruption and glial activation. Evidence indicates that oral infection of P. gingivalis can reach the hippocampus and exacerbate Aβ/tau pathology, yet how P. gingivalis affects central clock gene expression – especially Bmal1‐driven glial rhythms – remains undefined. Moreover, the specific contribution of the PI3K/AKT–BMAL1 axis to P. gingivalis‐induced neuroinflammation remains unknown.

2. Interpretation: Our study mimics the periodontal–oral–brain axis in AD by chronically administering Pg to C57BL/6 mice and by infecting/activating cultured astrocytes and microglia. Pg flattened hippocampal BMAL1 rhythms, lowered p‐AKT, activated glia, raised Aβ42 and p‐tau181, and impaired memory; all effects were replicated by PI3K/AKT inhibition and reversed by an AKT agonist. Knockdown of Bmal1 alone was sufficient to trigger glial activation and IL‐1β release, indicating that Pg suppresses the PI3K/AKT–BMAL1 pathway to initiate neuroinflammation and AD‐like pathology.

3. Future directions: The proposed framework invites further investigation of (a) which Pg virulence factors (gingipains vs LPSs) selectively inhibit PI3K/AKT–BMAL1; (b) whether pharmacological restoration of BMAL1 can reverse established glial activation and Aβ burden; (c) longitudinal clinical cohorts relating periodontal intervention to CSF clock–protein changes and ARIA incidence; and (d) the potential of chronotherapeutics combined with anti‐Aβ immunotherapy to minimize micro‐hemorrhagic events.

   Our findings identify the PI3K/AKT–BMAL1 module as a druggable checkpoint in Pg‐induced neurodegeneration. Future work should determine which Pg virulence factors (gingipains vs LPSs) preferentially inhibit PI3K/AKT, test whether restoring BMAL1 (via REV‐ERB antagonists or bright‐light chronotherapy) can reverse established glial activation and Aβ burden, and evaluate periodontal or circadian interventions in AD patients who are APOE ε4 carriers and at high risk for ARIA during anti‐Aβ immunotherapy.

2.2. Mouse treatment

All animal experiments were conducted at the State Key Laboratory of Oral Diseases and approved by the Research Ethics Committee of West China Hospital of Stomatology (WCHSIRB‐D‐2021‐174). The animal research: reporting of in vivo experiments (ARRIVE) criteria were strictly followed when reporting all animal research. Fifty 8‐week‐old C57BL/6 male mice were purchased from Dassy, China. All mice were kept in a standard environment with controlled temperature and humidity under specific pathogen‐free conditions on a 12:12 light/dark cycle (LD12:12) with free access to food and water. They were randomly divided into a sham infection group (blank control) and a P. gingivalis‐infected group (experimental group) (n = 25 per group). The sample size was calculated by G*Power 3.1 software ¹⁹ and was based on the data from our previous study. ²⁰ Before the first application of P. gingivalis, all mice were given 1 mg/mL kanamycin in drinking water for 3 days of antibacterial treatment. The experimental group was given the bacterial solution obtained from the preparation above (10⁹ CFU/mL/50 µL) at a frequency of twice a week for 24 weeks, while the blank control group received an equal volume of PBS buffer. The sham group and the infection group were subjected to the same frequency, identical volume (50 µL), and identical oral handling procedures. Mechanical stimulation and stress were therefore fully controlled for between the two groups. The study design for mouse treatment is provided in Figure S1.

Samples were collected after 24 weeks, with five of each group collected at the same time of day, and the other 20 collected at indicated time points of the day (8:00, 14:00, 20:00, and 2:00). The aforementioned mice were subjected to Morris water maze (MWM) (BD, Germany) experiments. Then the mice were anesthetized, and their blood and tissues were collected. The hippocampus was dissected, the right hemisphere was fixed with 4% paraformaldehyde (PFA), and the left hemisphere was stored at −80°C for further analysis. The upper and lower jaws of the mice were taken and fixed with 4% PFA and then stored in 70% alcohol at 4°C for detection.

2.3. MWM test

The water maze consisted of a pool (120 cm in diameter) containing opaque water (25°C ± 1°C) and a platform (10 cm in diameter) submerged 1.0 cm under the water. During the training trial, mice were allowed to swim for 60 s to locate the hidden platform, and mice unable to locate the platform were guided to it and stayed for 20 s. All mice were trained with four trials per day over five consecutive days. Approximately 24 h after the last training trial, a probe trial was administered for memory retention in the absence of the platform. The experimental data were recorded by the EthoVision XT9 image acquisition and analysis system.

2.4. Measurement of alveolar bone loss

The maxilla was trimmed and fixed to a foam plate. Scanning was done with a Micro‐CT instrument (SCANCO Medical AG uCT50, Switzerland). Three‐dimensional (3D) model reconstruction of the maxillary alveolar bone was performed using SCANCO Evaluation software. The distance from the cemento‐enamel junction (CEJ) to the alveolar bone crest (ABC) was measured at the mesial, middle, and distal sites three times with ImageJ software. The alveolar bone loss (ABL) was calculated by averaging the figures from these three sites.

2.5. Hematoxylin and eosin staining and immunohistochemistry

The fixed mouse alveolar bone and brain tissues were rinsed of surface fixative, then gradient dehydrated and embedded with paraffin wax, cooled, and moved to a −20°C refrigerator for solidification. Coronal 5‐µm slices were made using a vibrating slicer (Leica, Germany). The slices were deparaffinized and hydrated, stained with hematoxylin staining solution (Solarbio, China) for 8 min followed by differentiation, and then stained with eosin staining solution (Solarbio, China) for 1 min. The slices were subsequently dehydrated, cleared twice with xylene, mounted with mounting medium, and coverslipped. Images were acquired using a VS200 scanning system (Olympus, Japan).

Sections of the hippocampal region were dewaxed and rehydrated and received antigenic repair in sodium citrate buffer for 20 min. Then all sections were treated with 3% hydrogen peroxide for 15 min to inhibit endogenous peroxidase and incubated with primary rabbit BMAL1 monoclonal antibody (1:100, Abcam, UK) and rabbit CLOCK polyclonal antibody (1:100, SAB, USA) overnight at 4°C. After washing away excess primary antibody, horseradish peroxidase (HRP)‐labeled goat anti‐rabbit immunoglobulin G (IgG) secondary antibody (SAB, USA) conjugated with HRP was added and incubated for 30 min. The DAB substrate solution was then used to induce the formation of a colored precipitate at the tissue antigen binding sites. All sections were exposed to DAB substrate at the same time to prevent intensity bias. Hematoxylin was re‐stained to improve visualization. The images were captured by the VS200 scanning system.

2.6. Cell culture and treatment

Mouse brain microglia cell line (BV2) and mouse brain astrocyte cell line (C8‐D1A) were purchased from Shanghai Enzyme Research Biological Company. BV2 and C8‐D1A were cultured in Dulbecco's modified Eagle's medium (DMEM, Zeta Life, USA), which contains 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin‐streptomycin (HyClone, USA) at 37°C in a 5% CO2 humidified incubator. The experimental group was co‐incubated with P. gingivalis (multiplicity of infection [MOI] 1:100), while the control group was co‐incubated with DMEM containing 10% FBS, and samples were collected at different time points according to the experimental design. The MOI of 1:100 was selected based on previous studies of P. gingivalis infection models as well as preliminary optimization experiments in our laboratory. This infection ratio ensured sufficient bacterial exposure while maintaining acceptable cell viability. ²¹ , ²² , ²³

2.7. Western blotting

Protein extracts from cells or hippocampi were prepared in a modified RIPA buffer supplemented with protease inhibitors (Signalway Antibody, Greenbelt, MD, USA). The BCA method was used to determine the concentration of protein. The protein extracts were boiled after being diluted in sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) protein loading buffer (5×) (Beyotime, Shanghai, China) at a ratio of 4:1. Proteins from the boiled samples were separated by SDS‐PAGE and transferred to polyvinylidene fluoride membranes (polyvinylidene fluoride). Then the membranes were blocked in tris‐buffered saline with Tween 20 buffer (20 mM Tris‐HCl, pH 7.4,137 mM NaCl, and 0.1% Tween‐20) with 5% non‐fat milk at 37°C for 1 h, and incubated with primary rabbit polyclonal IgG antibodies against p‐tau181 (1:1000), glial fibrillary acidic protein (GFAP) (1:2000), IL‐1β (1:2000), rabbit monoclonal antibody against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (1:5000) (all Signalway Antibody, USA), or rabbit polyclonal IgG antibody against Iba1 (1:5000), AKT (1:5000), rabbit monoclonal IgG antibody against p‐AKT (1:5000) (all Hua An Biological, China) overnight at 4°C. After extensive rinsing, the membranes were incubated with appropriate HRP‐conjugated secondary antibody or Cy3‐conjugated secondary antibody (all Signalway Antibody, USA), then visualized using an enhanced chemiluminescence detection kit (Yase, China). Quantification of bands by the optical density using ImageJ software (1.41v, US National Institutes of Health, Bethesda, MD, USA) after imaging the blots.

2.8. Enzyme‐linked immunosorbent assay

After rinsing, the hippocampal tissues were homogenized with the protease inhibitor‐containing PBS at a ratio of 1:9, then centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was collected for testing. The levels of Aβ42, IL‐1β, TNF‐α, and IL‐6 in hippocampal tissues were measured using mouse Aβ42, IL‐1β, TNF‐α, and IL‐6 ELISA kits (Ruixin Bio, China) according to the manufacturer's instructions. The reaction was recorded at 450 nm using an enzyme marker (Thermo Fisher Scientific, USA).

2.9. Quantitative reverse transcription polymerase chain reaction

The procedure was performed according to minimum information for publication of quantitative real‐time PCR experiments (MIQE) guidelines. Total RNA was extracted from mouse hippocampus or cells using the RNApure Total RNA Rapid Isolation Kit (Takara, Japan) and then reverse transcribed using the Takara Reverse Transcription Kit according to the manufacturer's instructions. The resultant cDNA served as a template for quantitative PCR analysis using gene‐specific primers (TSINGKE, Beijing, China). Real‐time quantitative polymerase chain reactions were performed with TB Green Premix Ex Taq II (TAKARA, Tokyo, Japan) using an Applied Biosystems QuantStudio 6 Flex Real‐Time PCR System under the following cycling conditions: initial denaturation at 95°C for 30 s, 40 cycles of 95°C for 5 s, followed by 60°C for 10 s, and 95°C for 15 s, 1 min at 60°C, then 15 s at 95°C. Fluorescence intensity was monitored at the end of each amplification step. Quantitative measurements of target gene levels were normalized to GAPDH, and the results were expressed as fold changes of the threshold cycle (Ct) value relative to control using the 2^−∆∆Ct method. Primers and amplicon size are shown in Table S1.

2.10. Immunofluorescence and imaging

Sections of the hippocampal region of the brain were deparaffinized and rehydrated and received antigenic repair in sodium citrate buffer for 20 min. Then all sections were treated with 3% hydrogen peroxide for 15 min to inhibit endogenous peroxidase and incubated with primary rabbit GFAP polyclonal antibody (1:200, SAB, USA) and rabbit Iba1 polyclonal antibody (1:100, Huaan Biologicals, China) overnight at 4°C. After washing with PBS, the sections were incubated with Cy3 fluorescently conjugated secondary antibody at room temperature for 1 h. Following washing with PBS three times, after the slides were dried, the nuclei of the cells were re‐stained with DAPI in mounting solution. The fluorescent images were viewed under a fluorescence‐inverted microscope (Leica, Germany) and captured with the VS200 scanning system.

2.11. Lentivirus‐mediated gene transductions

The construction and synthesis of negative control virus, Bmal1, and Clock knockdown lentivirus with GFP gene and puromycin resistance gene were done by Shanghai Jikai Gene. Mouse brain microglia cell line (BV2) and mouse brain astrocyte cell line (C8‐D1A) were cultured. After screening puromycin concentration and lentivirus titer, the lentiviruses were transfected with BV2 and C8‐D1A cells. Bmal1 and Clock knockdown efficiency were detected by qRT‐PCR, and glial cell activation was detected by Western blot.

2.12. Statistical analysis

Data are presented as the mean ± standard deviation (SD) and analyzed using the SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA). GraphPad Prism 8.0 was applied for statistical graphing. One‐way analysis of variance and the Student–Newman–Keuls tests are used to evaluate the differences between groups. P < 0.05 was considered a statistically significant difference. The signs of significance were denoted by ns = non‐significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. RESULTS

3.1. P. gingivalis oral infection induced alveolar absorption and AD‐like pathological changes

The 3D reconstruction and analysis of the mouse maxilla revealed significant alveolar bone resorption in mice subjected to P. gingivalis infection compared to the sham group (Figure 1A,B). The MWM experiment demonstrated that the learning and memory abilities and spatial exploration capacity of P. gingivalis‐infected mice were inferior to those of the sham group (Figure 1C,D). The serum levels of inflammatory factors in mice were elevated by the periodontal infection of P. gingivalis (Figure 1E). The hippocampi of mice showed neuronal degeneration and necrosis in the P. gingivalis‐infected group (Figure 1F). The levels of p‐tau181 and Aβ42 accumulation were significantly higher than in the sham group (Figure 1G). Additionally, the levels of associated inflammatory factors notably increased more than in the sham group (Figure 1H). Collectively, these results suggested that P. gingivalis periodontal infection could lead to alveolar bone loss, peripheral circulatory system inflammation, cognitive impairment, and AD‐like pathological alterations.

FIGURE 1.

Porphyromonas gingivalis oral infection induced alveolar absorption and AD‐like pathological changes. (A) Three‐dimensional reconstruction images of the maxilla of mice obtained by Micro‐CT. The yellow boxed area represents the alveolar bone resorption region. (B) Statistical analysis of alveolar bone resorption height in the maxillary second molar of mice. (C) Movement trajectories of mice in the Morris water maze during spatial exploration test. (D) P. gingivalis infection leads to cognitive dysfunction in mice. The bar charts respectively show the latency time of mice during the hidden platform period, the percentage of time that mice stayed in the target quadrant in the spatial exploration test, and the number of times that mice crossed the platform in the spatial exploration test. (E) Levels of inflammatory factors such as interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor‐α (TNF‐α) in the serum of mice. (F) P. gingivalis infection leads to neuronal damage in the CA1 region of the hippocampus of mice. The HE staining images of hippocampal tissue sections of mice are shown. The neuronal cell nuclei are stained blue, and nuclear pyknosis can be observed (indicated by red arrows in figure). (G) P. gingivalis infection results in AD‐like pathological changes in the hippocampal tissue of mice. Electrophoresis images of p‐tau181 protein in hippocampal tissue of mice and grayscale quantitative analysis; ELISA quantitative results of amyloid beta 42 (Aβ42) in hippocampal tissue of mice. (H) ELISA quantitative results of expression levels of IL‐1β, IL‐6, and TNF‐α in hippocampal tissue of mice. *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. P. gingivalis infection led to the activation of astrocyte and microglia

Neuroinflammation is a crucial part of AD‐like pathological changes, and activation of astrocytes and microglia is an important component of neuroinflammation. We examined the expression levels of GFAP (an astrocyte activation indicator) and Iba1 (a microglia activation indicator) in mouse hippocampi. Six months after periodontal infection with P. gingivalis, the expression levels of GFAP (Figure 2A) and Iba1 (Figure 2B) in the hippocampi became more abundant and stronger than in the sham group (Figure 2C). We also measured GFAP, Iba1, and inflammatory factor levels in the P. gingivalis‐infected C8‐D1A cells and BV2 cells in vitro. The expression levels of GFAP and IL‐1β were significantly elevated in the C8‐D1A cells (Figure 2D), and in the BV2 cells both Iba1 and IL‐1β expression levels were significantly elevated (Figure 2E). Our results confirmed that P. gingivalis infection induced the activation of astrocytes and microglia and prompted their secretion of inflammatory factors.

FIGURE 2.

P. gingivalis infection led to the activation of astrocyte and microglia. (A) Immunofluorescence staining images of GFAP in astrocytes of mouse hippocampal tissue. (B) Immunofluorescence staining images of Iba1 in microglia of mouse hippocampal tissue. (C) Electrophoresis images of GFAP and Iba1 proteins in mouse hippocampal tissue and quantitative analysis of protein grayscale. (D) Electrophoresis images of GFAP and IL‐1β proteins in C8‐D1A cells and quantitative analysis of protein grayscale. (E) Electrophoresis images of Iba1 and IL‐1β proteins in BV2 cells and quantitative analysis of protein grayscale. *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. P. gingivalis disrupted circadian clock gene expression in hippocampus

In the sham group, BMAL1 levels peaked at ZT6 and were lowest at ZT0, with rhythmic changes in expression levels, whereas in the P. gingivalis infection group, BMAL1 levels peaked at ZT12 and were lowest at ZT6, with no rhythmic changes in expression levels (Figure 3A). There were no rhythmic alterations in the expression levels of CLOCK, another essential circadian clock protein, in any group (Figure 3B). Further investigation of the expression levels of circadian clock genes in hippocampal tissues revealed that the expression of several circadian clock genes in mouse hippocampi differed from the sham group, including in showing a decrease in the amplitude of Bmal1 and Cry1 oscillations, the disappearance of rhythmicity, and a delay in the clock's peak period (Figure 3C). These results indicated that P. gingivalis infection disrupted circadian clock gene expression in the hippocampi, especially the Bmal1 gene.

FIGURE 3.

P. gingivalis disrupted the circadian clock gene expression in the hippocampi. In the present study, Zeitgeber time (ZT) 0 was aligned with 02:00 h, rendering all subsequent circadian references relative to this nocturnal reference point. (A) P. gingivalis infection leads to the disappearance of the rhythmic expression of BMAL1 in the hippocampal tissue of mice. On the left is the immunohistochemical staining image of BMAL1 in the CA3 region of the hippocampal tissue of mice. The nuclei are stained blue, and the positive immunoreaction of BMAL1 protein is brownish‐yellow. On the right is the semi‐quantitative analysis of BMAL1 protein in the CA3 region of the hippocampal tissue of mice. (B) The impact of P. gingivalis infection on the expression level of CLOCK in the hippocampal tissue of mice. On the left is the immunohistochemical staining image of CLOCK in the CA3 region of the hippocampal tissue of mice. The nuclei are stained blue, and the positive immunoreaction of CLOCK protein is brownish‐yellow. On the right is the semi‐quantitative analysis of CLOCK protein in the CA3 region of the hippocampal tissue of mice. (C) Impact of P. gingivalis infection on transcriptional levels of circadian clock genes Bmal1, Clock, Cry1, Per1, Per2, and Nr1d1 in the hippocampal tissue of mice. *p < 0.05, ***p < 0.001, ****p < 0.0001.

3.4. PI3K/AKT signaling regulates BMAL1 expression and glial activation

The PI3K/AKT signaling pathway has been shown to play an important role in the central nervous system, ¹⁵ which regulates the expression and activity of circadian clock genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes from hippocampal transcriptome data of P. gingivalis‐infected mice was performed, and the top 10 enriched pathways are presented in Figure 4A. The complete KEGG analysis is presented in Figure S2. We detected the expression levels of AKT, p‐AKT, and key proteins of the PI3K/AKT signaling pathway in the mouse hippocampi and glial cells after P. gingivalis infection by Western blot. The results demonstrate that the expression level of p‐AKT in the hippocampi was significantly lower in the P. gingivalis infection group than in the sham group (Figure 4B). Similarly, in the C8‐D1A cells and BV2 cells, the expression level of p‐AKT was considerably lower in the P. gingivalis infection group (Figure 4C,D). We subsequently treated C8‐D1A and BV2 cells with the PI3K inhibitor LY294002, the AKT inhibitor MK‐2206, and the AKT activator SC79 to examine the function of the PI3K/AKT signaling pathway in circadian clock alterations and cell activation. The results showed that AKT inhibitor treatment caused a significant decrease in BMAL1 level in C8‐D1A cells (Figure 4E). PI3K inhibitor and AKT inhibitor treatment caused a significant decrease in BMAL1 and an increase in Iba1 expression in BV2 cells (Figure 4F). Further, the addition of AKT activator reversed the effects of P. gingivalis infection, deducing increases in activation and inflammation levels (Figure 4E,F). These findings imply that P. gingivalis infection may impede the PI3K/AKT pathway, which in turn cause the circadian clock protein BMAL1 to be downregulated and the expression of glial cell activation indicators and inflammatory factors to be elevated.

FIGURE 4.

PI3K/AKT signaling regulates BMAL1 expression and glial activation. (A) Top 10 KEGG pathways enriched among differentially expressed genes in hippocampal tissue of P. gingivalis‐infected mice. (B) Electrophoresis images of p‐AKT and AKT proteins in mouse hippocampal tissue and quantitative analysis of protein grayscale (with GAPDH as the reference standard). (C) Electrophoresis images of p‐AKT and AKT proteins in C8‐D1A cells and quantitative analysis of protein grayscale. (D) Electrophoresis images of p‐AKT and AKT proteins in BV2 cells and quantitative analysis of protein grayscale. (E) Electrophoresis images of BMAL1, GFAP, and IL‐1β proteins in each group of C8‐D1A cells and quantitative analysis of protein grayscale. (F) Electrophoresis images of BMAL1, Iba1, and IL‐1β proteins in each group of BV2 cells and quantitative analysis of protein grayscale. The analysis was carried out with GAPDH as the reference standard. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.5. Bmal1 knockdown significantly enhanced neuroinflammatory responses in glial cells

To further explore whether the low expression of Bmal1 and Clock has an impact on glial cell activation and inflammatory factor secretion. We created a lentiviral vector for the knockdown of the circadian clock genes Bmal1 and Clock, which led to a decrease in the expression of the two genes in astrocytes and microglial cells. Figure 5A displays the results of fluorescence screening for appropriate lentiviral infection titers. Infecting C8‐D1A at a viral titer of MOI = 100 and BV2 at a titer of MOI = 50 resulted in an infection efficiency of roughly 80%. After lentiviral transfection, the Bmal1 and Clock transcript levels of C8‐D1A and BV2 were significantly lower than those of the negative control group (Figure 5B). When Bmal1 was knocked down, the expression levels of GFAP and IL‐1β were found to be significantly increased in C8‐D1A cells (Figure 5C), and Iba1 and IL‐1β were found to be significantly increased in BV2 cells (Figure 5D). These findings confirm that Bmal1 plays a critical anti‐inflammatory role in glial regulation and that its suppression may contribute to P. gingivalis‐induced neuroinflammation and AD‐like changes.

FIGURE 5.

Bmal1 knockdown significantly enhanced neuroinflammatory responses in glial cells. (A) The infection efficiency of different multiplicities of infection (MOI) virus titers on C8‐D1A and BV2 cells, and the images of the two types of cells under a fluorescence microscope. (B) The mRNA expression levels of Bmal1 (upper) and Clock (lower). (C) Electrophoresis images of GFAP and IL‐1β proteins in C8‐D1A cells and quantitative analysis of protein grayscale (with GAPDH as reference standard). (D) Electrophoresis images of Iba1 and IL‐1β proteins in BV2 cells and quantitative analysis of protein grayscale (with GAPDH as reference standard). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

4. DISCUSSION

P. gingivalis, as a key pathogen in periodontitis, destroys periodontal tissue by expressing various virulence factors and triggers the host's immune‐inflammatory response, releasing cytokines that participate in tissue damage. ²⁴ Pathogens and inflammatory factors caused by periodontitis enter the bloodstream, increasing the systemic inflammatory burden, and are associated with an increased risk of various systemic inflammatory diseases, including AD. ²⁵ The significantly increased level of Aβ₄₂ in the hippocampus of P. gingivalis‐infected mice indicates that peripheral inflammation may increase the brain's Aβ burden by increasing Aβ synthesis and disrupting the blood‐brain barrier, ²⁶ , ²⁷ and this is consistent with our findings. Excessive phosphorylation of tau protein destroys neuronal microtubule function and is a key factor in the pathogenesis of AD. ²⁸ P. gingivalis infection triggers an inflammatory response in the circulatory system, activating neuroinflammation in the hippocampus, leading to excessive phosphorylation of tau protein. ⁵ In addition, P. gingivalis periodontal infection increases the levels of inflammatory factors such as TNF‐α, IL‐6, and IL‐1β in the mouse hippocampus, further promoting tau protein phosphorylation and Aβ deposition. ²⁹ , ³⁰ Consistent with these findings, our in vivo experiment confirmed that long‐term oral infection with P. gingivalis results in cognitive deficits, glial activation, increased hippocampal levels of Aβ₄₂ and p‐tau, and microstructural damage – hallmarks of AD pathology.

Exploring how P. gingivalis infection causes AD‐like pathology is significant and offers new strategies for AD prevention and treatment. Changes in the circadian clock play a key role in AD pathology, with circadian rhythm disorders believed to activate microglia and astrocytes, induce neuroinflammation, and lead to neurodegeneration. ³¹ This study delved into the effects of P. gingivalis periodontal infection on circadian rhythms and neuroinflammation, as well as how these changes relate to the pathological alterations in AD. The experiment revealed that P. gingivalis infection led to altered expression of circadian genes in the hippocampus of mice, particularly a decrease in Bmal1 expression levels and the disappearance of rhythmicity. Additionally, the inhibition of the PI3K/AKT signaling pathway and the activation of astrocytes and microglia were observed. Existing evidence indicates that the AKT/GSK3 signaling axis regulates clock protein stability. Specifically, GSK3β phosphorylates BMAL1 to facilitate its ubiquitination and proteasomal degradation, and AKT signaling also governs the subcellular localization of core clock components. ³² , ³³

Circadian rhythms govern key physiological processes, including sleep, metabolism, and immune function. Bmal1 is crucial for maintaining a normal circadian rhythm, and periodontitis can lead to the disappearance of Bmal1 expression rhythms, consistent with changes in Bmal1 expression under inflammatory conditions. ³⁴ , ³⁵ Li et al. found that BMAL1 could directly bind to the TNF‐α promoter, increasing the expression of the proinflammatory cytokine TNF‐α. ³⁶ P. gingivalis periodontal infection may interfere with the expression and activity of clock genes by affecting the PI3K‐AKT signaling pathway, participating in the pathological process of AD. ¹⁵ , ¹⁷ , ³⁷ AKT phosphorylates BMAL1 and CLOCK at distinct residues, thereby curtailing their nuclear import. Concurrently, AKT‐mediated inhibition of glycogen synthase kinase‐3 (GSK3) indirectly stabilizes BMAL1 by restraining its GSK3‐dependent phosphorylation at Ser‐17 and Thr‐21, a modification that otherwise targets the protein for ubiquitination and proteasomal degradation. ³⁸ , ³⁹ In experiments, AKT inhibitor treatment led to decreased BMAL1 levels and increased inflammatory factor levels in C8‐D1A and BV2 cells, while AKT activator SC79 could reverse the effects of P. gingivalis infection, revealing the complex interactions between P. gingivalis infection, the PI3K/AKT signaling pathway, and the circadian clock. ⁴⁰ , ⁴¹ In our study, P. gingivalis exposure led to the suppression of core circadian genes in the hippocampus, particularly Bmal1 and Clock, and dampened oscillatory expression. These alterations coincided with increased glial activation and proinflammatory cytokine release, suggesting a causative role of circadian dysfunction in mediating P. gingivalis‐induced neuroinflammation. We further elucidated the role of the PI3K/AKT signaling pathway as an upstream regulator of the circadian machinery in glial cells. Inhibition of this pathway recapitulated the BMAL1 downregulation and proinflammatory phenotype induced by P. gingivalis, while AKT activation reversed these effects. This regulatory relationship highlights a molecular mechanism by which periodontal pathogens may perturb central circadian control, linking oral inflammation to neurodegenerative cascades. Experiments by knocking down Bmal1 and Clock genes showed that Bmal1 knockdown significantly enhanced the activation of C8‐D1A and BV2 cells and the expression of inflammatory factors IL‐1β, while Clock knockdown had no significant effect. Bmal1 plays an important role in the activation and inflammatory response of astrocytes; knocking out Bmal1 leads to astrocyte proliferation, increased oxidative stress, synaptic damage, and inflammation. ⁴² Astrocyte‐specific Bmal1 knockout can exacerbate astrocyte activation around Aβ plaques but does not affect Aβ deposition. ⁴³ The response of microglia to Bmal1 knockout is inconsistent, with some studies showing increased immune reactivity and number of microglia, while other studies find that Bmal1 knockout reduces the expression of inflammatory genes. ⁴⁴ , ⁴⁵ Bmal1 knockdown induces neuroinflammation through specific inflammatory pathways. Based on the current literature, the primary mechanism involves the NF‐κB signaling pathway and its downstream regulation of the NLRP3 inflammasome. ⁴⁶ , ⁴⁷ For the Clock gene, although some studies suggest it may play a role in the immune suppression of microglia, ⁴⁸ the knockdown of Clock in this experiment did not cause significant inflammation. CLOCK is functionally redundant with NPAS2 in glial cells. In contrast, CLOCK has a well‐characterized paralog NPAS2, which is abundantly expressed in brain tissue, including hippocampal, forebrain, and glial cell populations. ⁴⁹ , ⁵⁰ When Clock is knocked down, NPAS2 can transcriptionally and functionally compensate by heterodimerizing with the remaining BMAL1 protein to reconstitute a functional transcription complex and maintain core clock output. ⁵¹ This compensatory mechanism buffers against inflammatory dysregulation, explaining why Clock knockdown alone does not produce a significant inflammatory phenotype.

These results emphasize the importance of Bmal1 in the regulation of glial cell function and indicate the need for further research on the role of Clock in neuroinflammation. A critical unanswered question is the reversibility of this circadian disruption. Future longitudinal studies should investigate whether eradicating P. gingivalis infection can rescue the molecular clock and halt AD progression.

In conclusion, our findings demonstrate that P. gingivalis infection disrupts hippocampal circadian clock regulation via inhibition of the PI3K/AKT signaling pathway, resulting in glial activation and AD‐like pathology. This study provides new mechanistic insight into how peripheral infections can drive central neurodegeneration, underscoring the therapeutic potential of targeting circadian regulators to counteract inflammation‐driven neurodegenerative processes. In the future, clinical samples may be collected to further confirm the P. gingivalis/PI3K/AKT/circadian axis in AD pathology.

5. CONCLUSION

P. gingivalis periodontal infection can disrupt hippocampal circadian clock expression and generate AD‐like pathological changes in the hippocampus. The possible mechanism is that P. gingivalis infection inhibits the PI3K/AKT signaling pathway, resulting in the disturbance of the circadian clock expression of glial cells and the activation of glial cells to release inflammatory factors and participate in the occurrence of neuroinflammation, thereby promoting the occurrence and development of AD‐like lesions.

AUTHOR CONTRIBUTIONS

Chunmei Huang and Qin Cai contributed to the conception and design of the study, data acquisition, analysis, and interpretation, and drafted and critically revised the manuscript. Zeru Feng, Chenze Zhang, and Zhigiang Luo contributed to the study's conception and data interpretation and critically revised the manuscript. Xingqun Cheng and Hongkun Wu contributed to conception, design, and data interpretation and critically revised the manuscript. All authors gave their final approval and agreed to be accountable for all aspects of the work. The data are available from the corresponding author upon reasonable request.

CONFLICT OF INTEREST STATEMENT

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author disclosures are available in the Supporting Information.

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