Neuroprotective Effects of Casein-Derived Peptide Met-Lys-Pro (MKP) in a Hypertensive Model

Abstract

We have previously reported that casein hydrolysate, CH-3, from bovine milk and casein-derived tripeptide Met-Lys-Pro (MKP) has ACE inhibitory activity and reduces blood pressure. In this study, we investigated the therapeutic effects of MKP in a hypertensive rat model (7-week-old male SHRSP/Izm rats). For long term evaluation, rats were fed either a diet containing CH-3 or normal diet. The survival rate of SHRSP rats was significantly improved by intake of CH-3 for 181 days. For short term evaluation, rats were orally administered synthetic tripeptide MKP or distilled water for 4 weeks. MRI study demonstrated that hemorrhagic lesions were observed in two of five rats in the control group, while no hemorrhagic lesions were observed in the MKP group. Volumetric analysis using MRI revealed that MKP administration inhibited atrophy of diencephalic regions. Histological examinations revealed that hemorrhage areas and astrogliosis in the hippocampus and cerebral cortex were lower in the MKP group than in the control group. Gene expression analysis indicated that MKP administration reduced expression of genes related to cerebral circulation insufficiency such as immune responses (Cd74 and Prkcd), response to hypoxia (Ddit4, Apold1, and Prkcd), reactive oxygen species metabolic process (Ddit4 and Pdk4), and apoptotic process (Ddit4, Prkcd, and Sgk1), suggesting that MKP administration prevented cerebral ischemia associated with hypertension. In addition, some genes encoding responses to hormone stimulus (Fos, Dusp1, and Sik1) were also downregulated. Serum aldosterone and corticosterone levels were also significantly decreased following MKP administration. The present study indicates that MKP shows neuroprotective effects in SHRSP rats by regulating cerebral circulation insufficiency and corticoid levels. MKP administration may therefore be a potential therapeutic strategy for hypertensive brain diseases such as cerebrovascular disease.

Keywords: ACE inhibitor; MKP; Met-Lys-Pro; brain; hypertension; peptide.

FIGURE 1

Survival rate of SHRSP/Izm rats up to 181 days. Survival curves were generated using Kaplan-Meier methods. Data were analyzed by log-rank test to compare groups. n = 10 per group.

FIGURE 2

T₂WI coronal MRI. The images of MRI slices in the rat indicate hemorrhage legions. Two individuals in the control group with lesions are shown in (A) and (B), respectively. Cerebral hemorrhage (a–d) are depicted as regions showing a clear low signal near regions with a faint high signal intensity of inflammation or edema on T₂WI. a–d show different lesion, respectively.

FIGURE 3

Expression of GFAP in the hippocampus. Immunohistochemistry images of control (A) and MKP (B) are shown. Percentage of immunoreactive area in each group is represented in (C). Values are presented as mean ± SEM. Data were analyzed by Student’s t-test. *p < 0.05 compared to control group (Control: n = 4, MKP: n = 5).

FIGURE 4

Expression of GFAP in the cerebral cortex. Immunohistochemistry images of control (A) and MKP (B) groups are shown. Percentage of immunoreactive area in each group is represented in (C). Values are presented as mean ± SEM. Data were analyzed by Student’s t-test. *p < 0.05 compared to control group (Control: n = 4, MKP: n = 5).

FIGURE 5

Coronal slices of gray matter and white matter detected using tissue probability maps (TPMs). MR images of gray matter (A) and white matter (B). Horizontal slices of tissue segmentation slices are shown in (C). The rat label atlas was transformed into the brain morphology of each individual using ANTs. The volume of each brain region was measured.

FIGURE 6

Microarray analysis in the hippocampus. (A) A heat map generated in MeV showing genes that were two-fold downregulated and significantly lower in limma analysis by administration of MKP. (B) GO term enrichment analysis of the downregulated genes. The vertical coordinates are the enriched GO terms, and the horizontal coordinates are the numbers of the downregulated genes in these GO terms. The green, purple, and orange columns represent the biological process GO terms, cellular component GO terms, and molecular function GO terms, respectively. *p-value < 0.05. The p-values were obtained from the DAVID 2.1 method of statistical function classification tool. n = 3 per group.

FIGURE 7

RT-qPCR validation of expression changes of selected genes in the hippocampus and cerebral cortex in SHRSP/Izm rats. The normalized fold expression of each gene in the hippocampus (A) and cerebral cortex (B) are represented in each bar as mean ± SEM. Data were analyzed by Student’s t-test. *p < 0.05 compared to control group (Control: n = 4, MKP: n = 5).

FIGURE 8

Serum corticoids in SHRSP/Izm rats. The concentration of aldosterone (A) and corticosterone (B) are represented in each bar as mean ± SEM. Data were analyzed by Student’s t-test. *p < 0.05 compared to control group (Control: n = 9, MKP: n = 9).