Emodin Rescued Hyperhomocysteinemia-Induced Dementia and Alzheimer's Disease-Like Features in Rats

Abstract

Background: Hyperhomocysteinemia is an independent risk factor for dementia, including Alzheimer's disease. Lowering homocysteine levels with folic acid treatment with or without vitamin B12 has shown few clinical benefits on cognition.

Methods: To verify the effect of emodin, a naturally active compound from Rheum officinale, on hyperhomocysteinemia-induced dementia, rats were treated with homocysteine injection (HCY, 400 μg/kg/d, 2 weeks) via vena caudalis. Afterwards, HCY rats with cognitive deficits were administered intragastric emodin at different concentrations for 2 weeks: 0 (HCY-E0), 20 (HCY-E20), 40 (HCY-E40), and 80 mg/kg/d (HCY-E80).

Results: β-Amyloid overproduction, tau hyperphosphorylation, and losses of neuron and synaptic proteins were detected in the hippocampi of HCY-E0 rats with cognitive deficits. HCY-E40 and HCY-E80 rats had better behavioral performance. Although it did not reduce the plasma homocysteine level, emodin (especially 80 mg/kg/d) reduced the levels of β-amyloid and tau phosphorylation, decreased the levels of β-site amyloid precursor protein-cleaving enzyme 1, and improved the activity of protein phosphatase 2A. In the hippocampi of HCY-E40 and HCY-E80 rats, the neuron numbers, levels of synaptic proteins, and phosphorylation of the cAMP responsive element-binding protein at Ser133 were increased. In addition, depressed microglial activation and reduced levels of 5-lipoxygenase, interleukin-6, and tumor necrosis factor α were also observed. Lastly, hyperhomocysteinemia-induced microangiopathic alterations, oxidative stress, and elevated DNA methyltransferases 1 and 3β were rescued by emodin.

Conclusions: Emodin represents a novel potential candidate agent for hyperhomocysteinemia-induced dementia and Alzheimer's disease-like features.

Figure 1.

Cognitive deficits in homocysteine-injected rats were rescued by emodin. The experimental workflow is shown in (A). Sixty-five male rats were randomly divided into 2 groups, 52 rats were administered 2 weeks of homocysteine (Hcy) injections (400 μg/kg/d, via the vena caudalis, HCY rats, n=52), and 13 rats were administered normal saline (NS) injections (control [CON] rats, n=13) in the same volume as the Hcy treatment. The injections were performed with an insulin syringe (29 gauge, 0.33×12.7 mm) at 9:00 am every day. After the final injection, the animals completed the open field test (OFT) and novel object recognition test (NORT) to assess their activities of locomotion and cognition. The numbers of zone crossings in OFT (B) and the recognition indexes (RI) during the acquisition trial and retention trial in NORT (C) of HCY and CON rats were recorded. Then, HCY rats were randomly divided into 4 groups (HCY-E0, HCY-E20, HCY-E40, and HCY-E80, n=13/group) to be administered intragastric emodin at different concentrations (e.g., 0, 20, 40, and 80 mg/kg/d, respectively). HCY-E0 rats received a vehicle control (0.5% carboxymethylcellulose sodium). Two weeks later, the RI during the acquisition trial and retention trial in NORT (D), the escape latencies on the 5th day in the learning phase (E), and the times crossing the platform in the memory tests in Morris water maze (F) of the rats were recorded. The plasma Hcy levels of the rats were detected by ELISA (n=4) (G). Data were expressed as the means±SEM. ^□P<.05, ^□□P<.01, vs the data of same rats in acquisition trial. ^$P<.05, ^$$P<.01 vs HCY-E0 (HCY). ^△△P<.01 vs HCY-E20. ^*P<.05, ^**P<.01 vs CON. ^#P<.05 vs HCY-E0.

Figure 2.

Hippocampal neurons and synapse-related proteins. Neurons in the CA1, CA2, CA3, CA4, and dentate gyrus regions of the hippocampi were detected with Nissl staining (A, bar=50 μm) and quantified by ImageJ. Differences were only found in the numbers of neurons in the CA3 region (B, n=6). Levels of CREB, its phosphorylation at S133 (pCREB) (C–D), and synaptic proteins including synaptophysin, synapsin1, glutamate receptor 1 (GluR1), N-methyl D-aspartate receptor (NR) subtype 1 (NR1), and NR2B (E–G) were measured by western blotting and quantitatively analyzed (n=6). Data were expressed as the means±SEM. ^*P<.05, ^**P<.01, ^***P<.001 vs CON. ^#P<.05, ^##P<.01, ^###P<.001 vs HCY-E0.

Figure 3.

Emodin eliminated Aβ overproduction and tau hyperphosphorylation. Hippocampal Aβ40 (A) and Aβ42 (B) were detected by ELISA (pg/g tissue) (n=6). Levels of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) (C–D), total tau (Tau5), tau unphosphorylation at Ser198/199/202 (Tau1) and tau phosphorylated at Thr231 (pT231), Ser396 (pS396) and Ser214 (pS214) (E–F) were measured by western blotting and quantitatively analyzed (n=6). Levels of the catalytic subunit of protein phosphatase 2A (PP2Ac), its Tyr307-phosphorylation form (p-PP2Ac), and its demethylated form (DM-PP2Ac) (G–H) were measured by western blotting and quantitatively analyzed (n=6). Data were expressed as the means±SEM. ^*P<.05, ^**P<.01, ^***P<.001 vs CON. ^#P<.05, ^##P<.01, ^###P<.001 vs HCY-E0.

Figure 4.

Emodin reversed hippocampal oxidative stress and upregulation of DNA methylation methyltransferases. Hippocampal malondialdehyde (MDA) (A) and superoxide dismutase 1 (SOD1) (B) were detected by ELISA (ng/g tissue) (n=4). Levels of DNA methyltransferases (DNMTs), e.g., DNMT1, DNMT3α and DNMT3β (C–D), were measured by western blotting and quantitatively analyzed (n=6). Hippocampi with Tau1-based immunohistochemical staining were shown (E), and the densities of Tau1 positive cells were quantitatively analyzed (F) (bar=200 μm, n=6). Data were expressed as the means±SEM. ^*P<.05, ^**P<.01, ^***P<.001 vs CON. ^#P<.05, ^##P<.01, ^###P<.001 vs HCY-E0.

Figure 5.

Morphological changes of cerebral microvessels. Cerebral vessels were visualized by immunohistochemical staining of RECA-1 (an endothelial marker, Figure 5A–C), the fluorescence of fluorescein isothiocyanate (FITC) (Figure 5D–E), and HE staining (Figure 5F). The locations of the hippocampi, primary somatosensory cortex (S1Cx), and corpus callosum were shown in A (bar=1 mm). The decreased lumen diameters of microarterioles by an intermittent dilatation manner in the S1Cx (B) and hippocampi (C) were imaged by RECA-1-based immunohistochemical staining (bar=50 μm). Extravascular leakage (D) and angiemphraxis (E) of microvessels were shown by the fluorescence of FITC (bar=50 μm). The hyalinosis (1), increased perivascular space (perivascular dilatation, (2) and splitting of the walls (double barrel formation, (3) of microvessels were shown by HE is staining (F) (bar=50 μm).

Figure 6.

Microglia activation and inflammation in HCY rats was inhibited by emodin. Microglia in the hippocampi and S1Cx were shown by immunohistochemical staining with Iba1, a marker of microglia (A, bar=25 μm). The solidities of microglia in the S1Cx (B) and hippocampi (C) were calculated to assess the activation of microglia (n=6). Levels of IL-6 (D) and TNF-α (E) in the hippocampi were detected (pg/g tissue, n=4) by ELISA. Levels of NF-κB p65 and 5-lipoxygenase (5-LO) in the hippocampi were measured by western blotting (F, n=6) and quantitatively analyzed (G–H). Hippocampi and S1Cx with 5-LO-based immunohistochemical staining were shown in (I) (bar=25 μm). Data were expressed as the means±SEM. ^*P<.05, ^***P<.001 vs CON. ^#P<.05, ^##P<.01, ^###P<.001 vs HCY-E0.