Paeoniflorin exerts neuroprotective effects by modulating the M1/M2 subset polarization of microglia/macrophages in the hippocampal CA1 region of vascular dementia rats via cannabinoid receptor 2

Abstract

Background: Cerebral hypoperfusion is a pivotal risk factor for vascular dementia (VD), for which effective therapy remains inadequate. Persistent inflammatory responses and excessive chemotaxis of microglia/macrophages in the brain may accelerate the progression of VD. Endocannabinoids are involved in neuronal protection against inflammation-induced neuronal injury. Cannabinoids acting at cannabinoid receptor 2 (CB₂R) can decrease inflammation. Based on the identification of paeoniflorin (PF) as a CB₂R agonist, we investigated the neuroprotective and microglia/macrophages M1 to M2 polarization promoting effects of PF in a permanent four-vessel occlusion rat model.

Methods: One week after surgery, PF was intraperitoneally administered at a dose of 40 mg/kg once a day for 28 successive days. The effects of PF on memory deficit were investigated by a Morris water maze test, and the effects of PF on hippocampal neuronal damage were evaluated by light microscope and electron microscope. The mRNA and protein expression levels of key molecules related to the M1/M2 polarization of microglia/macrophages were assessed by RT-qPCR and Western blotting, respectively.

Results: Administration of PF could significantly attenuate cerebral hypoperfusion-induced impairment of learning and memory and reduce the morphological and ultrastructural changes in the hippocampal CA1 region of rats. Moreover, PF promoted an M1 to M2 phenotype transition in microglia/macrophages in the hippocampus of rats. In addition to its inhibitory property against proinflammatory M1 mediator expression, such as IL-1β, IL-6, TNF-α and NO, PF dramatically up-regulated expression of anti-inflammatory cytokines IL-10 and TGF-β1. Importantly, CB₂R antagonist AM630 abolished these beneficial effects produced by PF on learning, memory and hippocampus structure in rats, as well as the polarization of microglia/macrophages to the M2 phenotype. Additionally, PF treatment significantly inhibited cerebral hypoperfusion-induced mTOR/NF-κB proinflammatory pathway and enhanced PI3K/Akt anti-inflammatory pathway. Effects of PF on these signaling pathways were effectively attenuated when rats were co-treated with PF and AM630, indicating that the mTOR/NF-κB and PI3K/Akt signaling pathways were involved in the PF effects through CB₂R activation.

Conclusion: These findings demonstrated PF exerts its neuroprotective effect and shifts the inflammatory milieu toward resolution by modulation of microglia/macrophage polarization via CB₂R activation.

Keywords: Cannabinoid receptor 2; Neuroprotection; Paeoniflorin; Vascular dementia.

Fig. 1

Effects of paeoniflorin on learning and memory abilities, histopathology and ultrastructure of the hippocampal CA1 area of rats after cerebral ischemia. One week after four-vessel occlusion (4-VO) surgery, rats were intraperitoneally administered saline (4-VO), paeoniflorin (4-VO+PF; 40 mg/kg/day), paeoniflorin+AM630 (4-VO+PF+AM630; 40 + 3 mg/kg/day), AM630 (4-VO+AM630; 3 mg/kg/day) or HU308 (4-VO+HU308; 3 mg/kg/day) for consecutive 28 days. Sham-operated group (Sham) was performed using the same surgical procedures, except that vertebral artery and bilateral common carotid arteries were not occluded. A Mean escape latency of rats in the water maze task was recorded during the last 5 days (23–28 days) of treatment. The spatial probe trial was performed on the last day (day 28) of water maze training, and the parameters measured included (B) the number of times rats crossed the platform and (C) relative path length in the target quadrant/path length in the whole pool within 120 s. D Rats were sacrificed after 28 days consecutive drug treatment. Representative photomicrographs of hematoxylin–eosin-stained hippocampal regions of rats are shown in different groups: (a, g) sham-operated group, (b, h) 4-VO-operated group, (c, i) 4-VO+PF group, (d, j) 4-VO+PF+AM630 group, (e, k) 4-VO+AM630 group or (f, l) 4-VO+HU308 group. Boxed regions in a–f are shown in j–l, respectively. Scale bar: 50 µm. E Neuronal cell density in CA1 region was measured by hematoxylin–eosin staining and cell counting. F Representative photomicrographs with a transmission electron microscope showing ultrastructural changes in hippocampus CA1 regions of rats in each group. Scale bar: 1 µm. Each bar represents mean ± SD of three independent experiments. n = 6 or 10 rats per group. ^#P < 0.05 versus sham group, *P < 0.05 versus 4-VO model group, ^§P < 0.05 versus 4-VO+PF group

Fig. 2

Effects of paeoniflorin on the expression of CB₂R and M1/M2 polarization markers in microglia/macrophage in hippocampi of rats after cerebral ischemia. After 28 days consecutive drug treatment, a real-time quantitative PCR analysis of the mRNA level of CB₂R in the hippocampus tissues of rats from different groups: sham (sham operated), 4-VO (four-vessel occlusion operated), 4-VO+PF (four-vessel occlusion operated plus paeoniflorin 40 mg/kg/day), 4-VO+PF+AM630 (four-vessel occlusion operated plus paeoniflorin 40 mg/kg/day and AM630 3 mg/kg/day), 4-VO+AM630 (four-vessel occlusion operated plus AM630 3 mg/kg/day) or 4-VO+HU308 (four-vessel occlusion operated plus HU308 3 mg/kg/day). b Proteins were extracted from hippocampi of rats and subjected to Western blotting analysis using primary antibody specific for CB₂R. Blots were stripped and re-probed with antibody against β-actin to correct for differences in protein loading. Fold-change in the relative level of CB₂R protein is shown after normalizing with β-actin. c CB₂R protein level in hippocampi of rats was analyzed by Western blotting from different groups: sham (sham operated), 4-VO (four-vessel occlusion operated), 4-VO+PF (four-vessel occlusion operated plus paeoniflorin 10, 20, 40 mg/kg/day) or 4-VO+HU308 (four-vessel occlusion operated plus HU308 3 mg/kg/day). d Hippocampus tissue sections were co-stained for CD68 (M1 marker; green) or CD206 (M2 marker; red) and DAPI (blue). The images were observed and captured by a confocal laser scanning microscope. Scale bar: 50 μm. e, f Quantitative analysis of the number of CD68-positive and CD206-positive cells per visual field in the hippocampal CA1 region of rats. Each bar represents mean ± SD of three independent experiments. n = 6 rats per group. ^#P < 0.05 versus sham group, *P < 0.05 versus 4-VO model group, ^§P < 0.05 versus 4-VO+PF group

Fig. 3

Effects of paeoniflorin on expression products of microglia/macrophage in hippocampi of rats after cerebral ischemia. One week after four-vessel occlusion (4-VO) surgery, rats were intraperitoneally administered saline (4-VO), paeoniflorin (4-VO+PF; 40 mg/kg/day), paeoniflorin+AM630 (4-VO+PF+AM630; 40 + 3 mg/kg/day), AM630 (4-VO+AM630; 3 mg/kg/day) or HU308 (4-VO+HU308; 3 mg/kg/day) for consecutive 28 days. a After treatment, real-time quantitative PCR analysis and b ELISA were used to detect the mRNA and protein levels of the proinflammatory cytokines IL-1β, TNF-α and IL-6, respectively. c Western blotting analysis was performed to further detect iNOS protein expression level. β-Actin served as an internal control. d The protein level of iNOS was expressed as arbitrary densitometric unit and normalized by the value of β-actin and finally expressed relative to the protein level in the sham-operated group (defined as 1-fold). e The content of nitrite in the hippocampus tissues of rats were determined by the Griess reaction. f Real-time quantitative PCR analysis was used to detect the mRNA levels of the anti-inflammatory cytokines IL-10 and TGF-β1; and the M2-associated markers arginase-1 and Ym1. Each bar represents mean ± SD of three independent experiments. n = 6 rats per group. ^#P < 0.05 versus sham group, *P < 0.05 versus 4-VO model group, ^§P < 0.05 versus 4-VO+PF group

Fig. 4

Effects of paeoniflorin on mTOR/NF-κB signaling pathway in hippocampi of rats after cerebral ischemia. One week after four-vessel occlusion (4-VO) surgery, rats were intraperitoneally administered saline (4-VO), paeoniflorin (4-VO+PF; 40 mg/kg/day), paeoniflorin+AM630 (4-VO+PF+AM630; 40 + 3 mg/kg/day), AM630 (4-VO+AM630; 3 mg/kg/day) or HU308 (4-VO+HU308; 3 mg/kg/day) for consecutive 28 days. a Representative Western blotting photographs showing protein levels of p-mTOR, mTOR, p-IκBα, IκBα and β-actin in cytoplasmic fractions and NF-κB p65 and lamin B1 in nuclear fractions of hippocampus tissues of rats. The β-actin and lamin B1 protein levels served as internal controls, respectively, for cytoplasmic extracts and nuclear extracts. Levels of phosphorylated b mTOR and c IκBα in cytoplasmic extracts, and of d NF-κB p65 in nuclear extracts, were converted to arbitrary densitometric units, normalized by the value of the corresponding loading control and expressed relative to the phosphorylation ratio or to the protein level in sham-operated group (defined as 1-fold). Each bar represents mean ± SD of three independent experiments. n = 6 rats per group. ^#P < 0.05 versus sham group, *P < 0.05 versus 4-VO model group, ^§P < 0.05 versus 4-VO+PF group

Fig. 5

Effects of paeoniflorin on PI3K/Akt signaling pathway in hippocampi of rats after cerebral ischemia. One week after four-vessel occlusion (4-VO) surgery, rats were intraperitoneally administered saline (4-VO), paeoniflorin (4-VO+PF; 40 mg/kg/day), paeoniflorin+AM630 (4-VO+PF+AM630; 40 + 3 mg/kg/day), AM630 (4-VO+AM630; 3 mg/kg/day) or HU308 (4-VO+HU308; 3 mg/kg/day) for consecutive 28 days. a After treatment, whole tissue lysates of hippocampus were prepared and subjected to Western blotting analysis using antibodies specific for phosphorylated forms or all forms of PI3K and Akt. β-Actin was used as a loading control. b The protein levels of phosphorylated PI3K and Akt were converted to arbitrary densitometricunits, normalized by the value of the corresponding total protein level and expressed relative to the phosphorylation ratio in sham-operated rats (defined as 1-fold). Each bar represents mean ± SD of three independent experiments. n = 6 rats per group. ^#P < 0.05 versus sham group, *P < 0.05 versus 4-VO model group, ^§P < 0.05 versus 4-VO+PF group