Aerobic exercise attenuates LPS-induced cognitive dysfunction by reducing oxidative stress, glial activation, and neuroinflammation

Abstract

Physical activity has been considered an important non-medication intervention in preserving mnemonic processes during aging. However, how aerobic exercise promotes such benefits for human health remains unclear. In this study, we aimed to explore the neuroprotective and anti-inflammatory effects of aerobic exercise against lipopolysaccharide (LPS)-induced amnesic C57BL/6J mice and BV-2 microglial cell models. In the in vivo experiment, the aerobic exercise training groups were allowed to run on a motorized treadmill 5 days/week for 4 weeks at a speed of 10 rpm/min, with LPS (0.1 mg/kg) intraperitoneally injected once a week for 4 weeks. We found that aerobic exercise ameliorated memory impairment and cognitive deficits among the amnesic mice. Correspondingly, aerobic exercise significantly increased the protein expressions of FNDC5, which activates target neuroprotective markers BDNF and CREB, and antioxidant markers Nrf2/HO-1, leading to inhibiting microglial-mediated neuroinflammation and reduced the expression of BACE-1 in the hippocampus and cerebral cortex of amnesic mice. We estimated that aerobic exercise inhibited neuroinflammation in part through the action of FNDC5/irisin on microglial cells. Therefore, we explored the anti-inflammatory effects of irisin on LPS-stimulated BV-2 microglial cells. In the in vitro experiment, irisin treatment blocked NF-κB/MAPK/IRF3 signaling activation concomitantly with the significantly lowered levels of the LPS-induced iNOS and COX-2 elevations and promotes the Nrf2/HO-1 expression in the LPS-stimulated BV-2 microglial cells. Together, our findings suggest that aerobic exercise can improve the spatial learning ability and cognitive functions of LPS-treated mice by inhibiting microglia-mediated neuroinflammation through its effect on the expression of BDNF/FNDC5/irisin.

Keywords: Aerobic exercise; FNDC5/Irisin; Microglial activation; Neuroinflammation; Neuroprotection.

Graphical abstract

Fig. 1

Effects of aerobic exercise on LPS-induced spatial learning and cognitive function associated with muscle strength were assessed using the MWM, Y-maze, and muscle strength tests. (A) Shows a summary of the experimental design. (B–D) Total traveled distance and mean escape latency during the probe test. (E) Percentage of spontaneous alterations, (F) total number of arm entries, and (G) muscle strength. Data are expressed as means ± SDs (n = 6 per group). ^###p < 0.001, ^##p < 0.01, and ns—not significant: LPS-treated group vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001, and ns—not significant: LPS + Ex and Ex alone groups vs LPS alone group.

Fig. 2

(A, B) Effects of aerobic exercise on the protein expression of BDNF/FNDC5/p-CREB and Nrf2/HO-1 in the hippocampus and cerebral cortex of the LPS group. Representative Western blot bands and quantitative analysis of (C, D) FNDC5, (E, F) BDNF, (G, H) p-CREB, (I, J) Nrf2, and (K, L) HO-1 (n = 3 per group). Data are presented as means ± SDs. ^###p < 0.001, ^##p < 0.01, ^#p < 0.01 and ns—not significant: LPS-treated group vs. control group; *p < 0.05, **p < 0.01, and ***p < 0.001: LPS + Ex and Ex alone groups vs. LPS alone group. *p < 0.05, **p < 0.01, ***p < 0.001, and ns—not significant: Ex alone group vs. non-treated control group. (A, B) FNDC5; hippocampus and cerebral cortex [F(3, 8) = 151.0 and 13.91], BDNF; hippocampus and cerebral cortex F(3, 8) = 18.66 and 2.412], p-CREB; hippocampus and cerebral cortex F(3, 8) = 10.19 and 4.054], Nrf2; hippocampus and cerebral cortex [F(3, 8) = 5.579 and 8.733], HO-1; hippocampus and cerebral cortex [F(3, 8) = 12.63 and 27.92].

Fig. 3

(A, B) Effects of aerobic exercise on the mRNA expression of proinflammatory mediators in the hippocampus and cerebral cortex of the LPS group. Representative RT-PCR bands and quantitative analysis of (C, D) iNOS, (E, F) COX-2, (G, H) IL-1β, (I, J) IL-10, and (K, L) IFN-γ (n = 3 per group). Data are presented as means ± SDs. ^###p < 0.001: LPS-treated group vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001: LPS + Ex and Ex alone groups vs. LPS alone group. (A, B) iNOS; hippocampus and cerebral cortex [F(3, 8) = 52.11 and 16.33], COX-2; hippocampus and cerebral cortex [F(3, 8) = 36.26 and 275.1], IL-1β; hippocampus and cerebral cortex [F(3, 8) = 61.93 and 117.0], IL-10; hippocampus and cerebral cortex [F(3, 8) = 24.90 and 72.33], IFN-γ; hippocampus and cerebral cortex [F(3, 8) = 4.352 and 81.45].

Fig. 4

(A–J) Effects of aerobic exercise on BACE-1 and microglial activation in the hippocampus and cerebral cortex of the LPS group. Representative Western blot and RT-PCR bands and quantitative analysis of (C, D) BACE-1, (G, H) iba-1, and (K, L) GCN5 (n = 3 per group). Data are presented as means ± SDs. ^###p < 0.001 and ^##p < 0.01: LPS-treated group vs. control group; **p < 0.01 and ***p < 0.001: LPS + Ex and Ex alone groups vs. LPS alone group. (A, B) BACE-1; hippocampus and cerebral cortex [F(3, 8) = 18.44 and 61.72], (E, F) iba-1; hippocampus and cerebral cortex [F(3, 8) = 37.42 and 347.6], (I, J) GCN5; hippocampus and cerebral cortex [F(3, 8) = 19.13 and 31.16].

Fig. 5

(A, B) Effects of aerobic exercise on the expressions of proapoptotic and antiapoptotic proteins in the hippocampus and cerebral cortex of the LPS group. Representative Western blot bands and quantitative analysis of (C, D) Bcl-2, (E, F) Bax, (G, H) caspase-3, and (I, J) PARP-1 (n = 3 per group). Data are presented as means ± SDs. ^###p < 0.001, ^##p < 0.01, ^#p < 0.05, and ns—not significant: LPS-treated group vs. control group; *p < 0.05, **p < 0.01, and ***p < 0.001: LPS + Ex and Ex alone groups vs. LPS alone group. (A, B) Bcl2; hippocampus and cerebral cortex [F(3, 8) = 16.80 and 11.52], Bax; hippocampus and cerebral cortex [F(3, 8) = 5.289 and 11.61], caspase-3; hippocampus and cerebral cortex [F(3, 8) = 10.9 and 18.49], PARP-1; hippocampus and cerebral cortex [F(3, 8) = 16.66 and 24.94].

Fig. 6

Effects of irisin on the cell viability, antioxidant biomarkers, and inflammatory responses in BV-2 microglial cells induced by LPS. (A) The cells were pretreated with irisin (12.5, 25, and 50 nM) for 12 h, followed by LPS (200 ng/L) stimulation for 24 h. (B) The antioxidant protein expression of Nrf2 and HO-1 was estimated using Western blot analysis, and the bands were measured using the ImageJ software to quantify the expression of (C) Nrf2 and HO-1. (D) The inflammatory protein expression of iNOS and COX-2 was estimated using Western blot analysis, and the bands were measured using the ImageJ software to quantify the expression of (E) iNOS and (F) COX-2. (G) The protein expression of phosphorylated NF-κB, IκB-α, and IRF3 was estimated using Western blot analysis, and the bands were measured using the ImageJ software to quantify the expression of p–NF–κB, p-IκB-α, and p-IRF3 (H). (I) The immunofluorescence staining analysis of p65 nuclear translocation in BV-2 microglial cells. Nuclei were stained with DAPI. (J) The protein expression of phosphorylated p38, ERK, and JNK was estimated using Western blot analysis, and the bands were measured using the ImageJ software to quantify the expression of (K) p-p38, (L) p-ERK, and (M) p-JNK. All experiments were independently conducted at least three times. Data are presented as means ± SDs. ^###p < 0.001 and ns—not significant: LPS-treated group vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 and ns—not significant: LPS + Ex and Ex alone groups vs. LPS alone group. (B, D) Nrf2; [F(4,10) = 10.94], HO-1; [F(4,10) = 11.62], iNOS; [F(4,10) = 50.56], COX-2; [F(4,10) = 8.415], (G, J) p–NF–κB; [F(4,10) = 14.96], p-IκB-α; [F(4,10) = 46.59], p-IRF3; [F(4,10) = 18.97], p-p38; [F(4,10) = 10.36], p-Erk; [F(4,10) = 8.536], p-JNK; [F(4,10) = 48.26].