Bioactive Peptide VHVV Upregulates the Long-Term Memory-Related Biomarkers in Adult Spontaneously Hypertensive Rats

Abstract

Hypertension is one of the growing risk factors for the progression of long-term memory loss. Hypertension-mediated memory loss and treatment remain not thoroughly elucidated to date. Plant-based natural compounds are an alternative solution to treating human diseases without side effects associated with commercial drugs. This study reveals that bioactive peptides extracted from soy hydrolysates mimic hypertension-mediated memory loss and neuronal degeneration and alters the memory molecular pathway in spontaneously hypertensive rats (SHR). The SHR animal model was treated with bioactive peptide VHVV (10 mg/kg/oral administration) and angiotensin-converting-enzyme (ACE) inhibitors (5 mg/kg/oral administration) for 24 weeks. We evaluated molecular level expression of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and survival markers phospho-protein kinase B (P-AKT) and phosphoinositide 3-kinase (PI3K) after 24 weeks of treatment for SHR in this study. Western blotting, hematoxylin and eosin (H&E) staining, and immunohistochemistry showed long-term memory loss and neuronal degeneration in SHR animals. Bioactive peptide VHVV-treated animals upregulated the expression of long-term memory-relate proteins and neuronal survival. Spontaneously hypertensive rats treated with oral administration of bioactive peptide VHVV had activated CREB-mediated downstream proteins which may reduce hypertension-mediated long-term memory loss and maintain neuronal survival.

Keywords: angiotensin-converting-enzyme inhibitor; bioactive peptide; blood–brain barrier; long-term memory.

Figure 1

The hematoxylin and eosin (H&E) stain of the cerebral cortex (magnification: 400×): (A) The control Wistar Kyoto (WKY) group showed a good arrangement of neurons with clear round nucleus; (B) the spontaneously hypertensive rats (SHR) group showed neuronophagia, (short arrow), neurodegeneration (large arrow), and abnormal neuronal size (arrowhead; swollen). Of the neurons, (C) SHR rats treated with VHVV showed the rearranged neurons with better histopathological arrangement; (D) SHR rats treated with Angiotensin-converting enzyme (ACE) showed a moderate level of neurodegeneration (small arrow) and neuronal swollen (arrowhead).

Figure 2

Immunohistochemistry (IHC) illustrating the expression level of brain-derived neurotrophic factor (BDNF) in the cortex. (A) The brown color indicated the expression and distribution of BDNF in tissue section. The scale bar is 20 μm. The expression of BDNF in the control WKY group. (B) SHR rat brain cortex shows visibly decreased BDNF levels compared to the WKY, VHVV, and ACE groups. (C) VHVV-treated rats with visibly enhanced BDNF compared to all other groups. (D) ACE-treated rats show more enhanced BDNF levels than the SHR group but visibly decreased levels compared to VHVV.

Figure 3

Protein expression levels of L-glutamate receptor 1 (Glu-R-1), postsynaptic density protein 95 (PSD-95), phosphor-Ca²⁺/calmodulin-dependent protein kinase II (P-CamKII), phosphor- cAMP response element binding protein (CREB)/ brain-derived neurotrophic factor (BDNF), C-Fos, and b-cell lymphoma-2 (BCL-2) and the corresponding Western blot analysis in the cortex of SHRs and WKY rats. (A) Long-term memory (LTM) progressively increased in bioactive peptide VHVV-treated animals compared with the positive control ACE inhibitor and control WKY groups. The 24-week SHR groups were significantly different from the VHVV- and ACE inhibitor-treated animals. (B) Quantification of Figure 3A. β-actin was used to monitor protein quantification as an internal control. The data are presented as the mean ± standard error. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 4

Effect of VHVV on neuronal cell survival through the PI3K-Akt-mTOR signaling pathway: Representative western blot results for tropomyosin receptor kinase B (Trk-β), phospho-protein kinase B (P-AKT), phosphoinositide 3-kinase (PI3K), and phospho-mechanistic target of rapamycin (p-mTOR). (A) Proteins are overexpressed in the VHVV group animals compared with all other groups and were significantly downregulated in the SHR groups compared with the positive control ACE and control WKY groups. (B) Western blot analysis of the relative optical density of protein expression was conducted by ImageJ software. The data are presented as the mean ± standard error. * p < 0.05; ** p < 0.01, and *** p < 0.001.

Figure 5

Photomicrographs of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the experimental groups are shown. (A) Sections were labeled with TUNEL (green) to assess apoptotic brain cells. Sections were counterstained with DAPI (blue) to detect the nuclei. The arrowhead of SHR indicates TUNEL-positive cells; however, there is no TUNEL-positive cells in WKY and VHVV and a fewer number of positive cells in ACE. Scale bar showed as 100 μM. (B) Quantification of Figure 5A by image J software. ** p < 0.01.