Icariin Attenuates M1 Activation of Microglia and Aβ Plaque Accumulation in the Hippocampus and Prefrontal Cortex by Up-Regulating PPARγ in Restraint/Isolation-Stressed APP/PS1 Mice

Abstract

Background: Studies have shown that psychosocial stress is involved in Alzheimer's disease (AD) pathogenesis; it induces M1 microglia polarization and production of pro-inflammatory cytokines, leading to neurotoxic outcomes and decreased β-amyloid (Aβ) clearance. Icariin has been proven to be an effective anti-inflammatory agent and to activate peroxisome proliferator-activated receptors gamma (PPARγ) which induces the M2 phenotype in the microglia. However, whether restraint/isolation stress reduces the clearance ability of microglia by priming and polarizing microglia to the M1 phenotype, and the effects of icariin in attenuating the inflammatory response and relieving the pathological changes of AD are still unclear.

Methods: APP/PS1 mice (male, aged 3 months) were randomly divided into a control group, a restraint/isolation stress group, and a restraint/isolation stress + icariin group. The restraint/isolation stress group was subjected to a paradigm to build a depressive animal model. Sucrose preference, open field, elevated plus maze, and Y maze test were used to assess the stress paradigm. The Morris water maze test was performed to evaluate spatial reference learning and memory. Enzyme-linked immunosorbent assay and immunohistochemistry were used to identify the microglia phenotype and Aβ accumulation. Western blotting was used to detect the expression of PPARγ in the hippocampus and prefrontal cortex (PFC).

Results: Restraint/isolation stress induced significant depressive-like behaviors in APP/PS1 mice at 4 months of age and memory impairment at 10 months of age, while 6 months of icariin administration relieved the memory damage. Restraint/isolation stressed mice had elevated pro-inflammatory cytokines, decreased anti-inflammatory cytokines, increased Aβ plaque accumulation and more M1 phenotype microglia in the hippocampus and PFC at 10 months of age, while 6 months of icariin administration relieved these changes. Moreover, restraint/isolation stressed mice had down-regulated PPARγ expression in the hippocampus and PFC at 10 months of age, while 6 months of icariin administration reversed the alteration, especially in the hippocampus.

Conclusion: Restraint/isolation stress induced depressive-like behaviors and spatial memory damage, over-expression of M1 microglia markers and more severe Aβ accumulation by suppressing PPARγ in APP/PS1 mice. Icariin can be considered a new treatment option as it induces the switch of the microglia phenotype by activating PPARγ.

Keywords: Alzheimer’s disease; PPARγ; cytokine; hippocampus; icariin; microglia; prefrontal cortex; stress.

Figure 1

The experimental paradigm. Thirty male APP/PS1 mice were randomly divided into three groups (10 mice per group): control group (Ctr), restraint/isolation stress group (RIS), and restraint/isolation stress + icariin group (RIS+ICA). Mice in both the RIS and RIS+ICA groups were subjected to 28 days of restraint/isolation stress procedure, from 3 to 4 months of age. Mice in the RIS+ICA group were administered icariin (60 mg/kg) daily for 6 months, from 4 to 10 months of age. The weight of the mice was recorded at baseline, every week during the stress procedure and every month during the period of drug administration. The behavioral tests performed after stress included the sucrose preference test (SPT), open field test (OFT), elevated plus maze (EPM) and Y maze test in this order. After drug administration, the Morris water maze (MWM) test was performed. Enzyme-linked immunosorbent assay and immunohistochemistry were used to assess the microglia phenotype and western blotting was used to measure the expression of PPARγ in the hippocampus and PFC.

Figure 2

Effect of RIS on behavioral changes in SPT (A), OFT (B), EPM (C), and Y-maze test (D). (A) Stress exposure significantly reduced the percentage of sucrose consumption in stressed APP/PS1 mice, in both the RIS and the RIS+ICA groups, compared with the control animals. (B) APP/PS1 mice in the two stress groups showed decreased horizontal and vertical movements compared with the control group. (C) Four weeks of RI stress exposure did not significantly change the anxiety score of mice in the two stress groups. (D) Four weeks of RI stress exposure did not significantly reduce the ratio entry. The results are expressed as the mean ± SEM, n = 10. ^∗p < 0.05, ^∗∗p < 0.01 vs. the control group.

Figure 3

Effect of icariin on learning and memory in MWM. (A) The escape latency of training days. (B) The tracks in the MWM test. (C) Target quadrant time. (D) Ratio entry in the target quadrant. Mice in RIS group spent less time in the target quadrant and showed decreased entry ratio in the target quadrant in the MWM test, in comparison with the control group (p < 0.01). Icariin administration significantly relieved the behavioral alterations of the mice in the RIS+ICA group, compared with the RIS group. The results are expressed as the mean ± SEM, n = 10. ^∗∗p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. RIS group.

Figure 4

Effect of icariin on cytokines and NF-κB in the hippocampus. (A–C) The level of IL-1β (A), IL-6 (B), and TNF-α (C) increased in the RIS group, and icariin relieved the alteration. (D–F) The level of IL-4 (D), IL-10 (E), and TGF-β1 (F) decreased in the RIS group, and icariin relieved the alteration. (G) The level of NF-κB increased in the RIS group, and icariin relieved the alteration. The results are expressed as the mean ± SEM, n = 6. ^∗∗p < 0.01 vs. control group; ^##p < 0.01 vs. RIS group.

Figure 5

Effect of icariin on cytokines and NF-κB in the PFC. (A–C) The level of IL-1β (A), IL-6 (B), and TNF-α (C) increased in the RIS group, and icariin relieved the alteration. (D) The levels of IL-4 decreased in the RIS group, but icariin failed to relieve the alteration. (E,F) RIS significantly decreased the level of IL-10 (E) and TGF-β1 (F) compared with controls, and icariin could reverse the change. (G) NF-κB levels increased in the RIS group, and icariin relieved the alteration. The results are expressed as the mean ± SEM, n = 6. ^∗p < 0.05, ^∗∗p < 0.01 vs. control group; ^##p < 0.01 vs. RIS group.

Figure 6

Effect of icariin on Aβ plaques and microglia phenotype in the hippocampus. (A–C) The immunohistochemical staining results of Aβ, Iba-1, and iNOS (400×). (D) RIS significantly increased the levels of Aβ in the RIS group compared with the controls, and administration of icariin decreased its levels in the RIS+ICA group. (E) The number of Iba-1+ microglia positive cells was not significantly different among the three groups. (F) The number of iNOS+ microglia positive cells in the hippocampus of stressed mice was higher than that of control mice, and icariin administration reversed the change in the RIS+ICA group. (G) The RIS group showed higher ratio of iNOS+ to Iba-1+ microglia, while the RIS+ICA group showed a lower ratio. The results of Aβ are expressed as the mean ± SEM, n = 6; the results of Iba-1 and iNOS are expressed as the mean ± SEM, n = 4. ^∗p < 0.05, ^∗∗p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. RIS group.

Figure 7

Effect of icariin on Aβ plaques and microglia phenotype in the PFC. (A) Results of immunohistochemical staining (400×). (B) RIS significantly increased the level of Aβ compared with controls, and icariin decreased it. (C–E) The number of Iba-1+ (C) and iNOS+ (D) microglia cells was not significantly different among the three groups. As a result, the ratio of iNOS+ to Iba-1+ microglia (E) was not significantly different either among the three groups. The results of Aβ are expressed as the mean ± SEM, n = 6; the results of Iba-1 and iNOS are expressed as the mean ± SEM, n = 4. ^∗p < 0.05 vs. control group; ^#p < 0.05 vs. RIS group.

Figure 8

Effect of icariin on PPARγ in the hippocampus and PFC. (A) PPARγ expression in the hippocampus using western blotting. The RIS procedure induced a marked decrease in PPARγ levels in APP/PS1 mice compared with controls, and icariin could reverse the alteration. (B) PPARγ expression in the PFC using western blotting. The RIS procedure induced a marked decrease in PPARγ level in APP/PS1 mice compared with controls, and icariin could partially reverse the alteration. The results of hippocampus are expressed as the mean ± SEM, n = 4; the results of PFC are expressed as the mean ± SEM, n = 5. ^∗p < 0.05 vs. control group; ^#p < 0.05 vs. RIS group.