Porphyromonas gingivalis lipopolysaccharide induces cognitive dysfunction, mediated by neuronal inflammation via activation of the TLR4 signaling pathway in C57BL/6 mice

Abstract

Background: Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) is one of the major pathogenic factors of chronic periodontitis (CP). Few reports on the correlation between P. gingivalis-LPS and cognitive function exist. Thus, the present study aimed to investigate the effects of P. gingivalis-LPS on cognitive function and the associated underlying mechanism in C57BL/6 mice.

Methods: The C57BL/6 mice were injected with P. gingivalis-LPS (5 mg kg^-1) either with or without Toll-like receptor 4 (TLR4) inhibitor (TAK-242, 5 mg kg^-1). After 7 days, behavioral alterations were assessed with the open field test (OFT), Morris water maze (MWM) test, and passive avoidance test (PAT). The activation of astrocytes and microglia in the cerebral cortex and hippocampus of mice was observed by immunohistochemistry. The expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8), TLRs (TLR2, TLR3, and TLR4), and CD14 and the activation of the NF-κB signaling pathway (IRAK1, p65, and p-p65) in the cerebral cortex of the mice were evaluated by RT-PCR, ELISA, and western blot.

Results: The OFT showed that P. gingivalis-LPS did not affect the initiative and activity of mice. Administration of P. gingivalis-LPS significantly impaired spatial learning and memory during the MWM test and attenuated the ability of passive avoidance learning during the PAT. Both astrocytes and microglia were activated in the cortex and hippocampus. The messenger RNA (mRNA) and protein expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) was upregulated by P. gingivalis-LPS in the cortex. In addition, the TLR4/NF-κB signaling pathway was activated (TLR4, CD14, IRAK1, and p-p65). These effects were effectively alleviated by TAK-242.

Conclusions: Administration of P. gingivalis-LPS can lead to learning and memory impairment in C57BL/6 mice. This impairment is mediated by activation of the TLR4 signaling pathway. Our study suggests that P. gingivalis-LPS-induced neuroinflammation plays an important role in cognitive impairment. It also reveals that endotoxins of periodontal pathogens could represent a risk factor for cognitive disorders.

Keywords: Cognition; Lipopolysaccharide; Neuroinflammation; Porphyromonas gingivalis; TLR4.

Fig. 1

Experimental design. P.gingivalis-LPS (5 mg kg⁻¹, i.p.) was administered in the P.gingivalis-LPS group and P.gingivalis-LPS plus TAK group. Seven days after P.gingivalis-LPS administration, cognitive function was assessed by the open field test (OFT), Morris water maze (MWM) test, and passive avoidance test (PAT). The underlying mechanism was further detected by RT-PCR, ELISA, western blot, and immunohistochemistry

Fig. 2

Effects of P.gingivalis-LPS on animal activity. The open field test (OFT) was used to evaluate the spontaneous activities of mice after 7 days of administration, including total distance covered (a), percentage of time spent on central grid (b), percentage of distance covered on central grid (c), number of rearings (d), number of defecations (e), and frequency of grooming (f). Overall, no significant differences were observed between treatment groups

Fig. 3

Effects of P.gingivalis-LPS on spatial learning of mice during the Morris water maze (MWM) test. Effects of treatment with P.gingivalis-LPS (5 mg kg⁻¹, i.p.) alone or in combination with TAK-242 (5 mg kg⁻¹, i.p.) on latency to find the platform during the acquisition phase of the MWM test. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001, compared to the control group; ^#p < 0.05 and ^##p < 0.01, compared to the P.gingivalis-LPS group

Fig. 4

Effects of P.gingivalis-LPS on spatial memory and trajectories in the Morris water maze (MWM) test. The platform was removed on the sixth day, and the distance traveled in the target quadrant was compared between the groups. The following parameters were assessed: percentage of the distance covered in the target quadrant (a), percentage of time spent in the target quadrant (b), and number of platform crossings in the target quadrant (c). The percentage of distance, percentage of time, and number of platform crossings in the target quadrant were significantly reduced by P.gingivalis-LPS. The typical trajectories of the P.gingivalis-LPS group approximated the arc of a circle, without any crossings over the original platform (d). Data are presented as mean ± SEM; **p < 0.01, ***p < 0.001, compared to the control group; ^##p < 0.01, compared to the P.gingivalis-LPS group

Fig. 5

Effects of P.gingivalis-LPS on response and memory of mice in the passive avoidance test (PAT). The PAT was used to evaluate passive avoidance learning in mice following the Morris water maze (MWM) test. The following parameters were determined: latency to enter the dark compartment (a) and error times to enter the dark compartment (b). Administration of P.gingivalis-LPS reduced the latency and increased error times to enter the dark compartment. Data are presented as mean ± SEM; ***p < 0.001, compared to the control group; ^##p < 0.01 and  ^###p < 0.001, compared to the P.gingivalis-LPS group

Fig. 6

Effects of P.gingivalis-LPS on microglia in the hippocampus. Histopathological analysis of brain sections was performed using immunohistochemistry. Microglia were visualized with ionized calcium-binding adaptor molecule 1 (Iba1) (arrows). Activated microglia with irregular protrusions were observed in the P.gingivalis-LPS group (bar = 50 μm)

Fig. 7

Effects of P.gingivalis-LPS on microglia in the cortex. Histopathological analysis of brain sections was performed using immunohistochemistry. Microglia were visualized with ionized calcium-binding adaptor molecule 1 (Iba1) (arrows). Activated microglia with irregular protrusions were observed in the P.gingivalis-LPS group (bar = 50 μm)

Fig. 8

Effects of P.gingivalis-LPS on astrocytes in the hippocampus and cortex. Histopathological analysis of brain sections was performed using immunohistochemistry. Astrocytes were visualized with the glial fibrillary acidic protein (GFAP) (a, arrows). Quantification of GFAP levels in the hippocampus and cortex are shown (b, c). Activated astrocytes were significantly increased following P.gingivalis-LPS administration. Activation was attenuated by TAK-242 (bar = 50 μm). Data are presented as mean ± SEM; ***p < 0.001, compared to the control group; ^###p < 0.001, compared to the P.gingivalis-LPS group

Fig. 9

Effects of P.gingivalis-LPS on mRNA and protein expression of inflammatory cytokines. RT-PCR and ELISA were performed to detect mRNA (a, c, e, g), and protein (b, d, f, h) levels of inflammatory cytokines. Administration of P.gingivalis-LPS induced high expression of inflammatory factors on genes and proteins, in comparison to the control group, whereas these changes were reversed by TAK-242. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01,  and ***p < 0.001, compared to the control group; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, compared to the P.gingivalis-LPS group

Fig. 10

Effects of P.gingivalis-LPS on mRNA expression of TLR2, TLR3, TLR4, and CD14. The RT-PCR was performed to detect mRNA levels of TLR2, TLR3, TLR4, and CD14 (a–d). The mRNA expression of TLR4 and CD14 was upregulated by P.gingivalis-LPS; however, similar effects were not observed in TLR2 and TLR3. Data are presented as mean ± SEM; ***p < 0.001, compared to the control group; ^##p < 0.01, compared with the P.gingivalis-LPS group; ⁺⁺⁺p < 0.001, P.gingivalis-LPS group compared with the E.coli-LPS group

Fig. 11

P.gingivalis-LPS-induced neuroinflammatory processes via the TLR4/NF-κB signaling pathway. Expression of CD14, TLR4, IRAK1, p65, and p-p65 was measured by western blot analysis (a). Graphs show the semiquantitative analysis of protein levels (b–e). Expression of CD14, TLR4, IRAK1, and p-p65/p65 proteins was upregulated by P.gingivalis-LPS. High expression of these proteins (TLR4, IRAK1, and p-p65/p65) was effectively inhibited by TAK-242. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, compared to the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared to the P.gingivalis-LPS group