Gao-Zi-Yao Improves Learning And Memory Function in Old Spontaneous Hypertensive Rats Part 2

Experimental animals

Twelve-month-old and eight-week-old male spontaneous hypertensive rats (SHR) were purchased from the Nanjing Animal Model Center. 

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In this model, hypertension starts developing by 4 months of age with the appearance of hypertension-related changes in the brain microvasculature by 6 months of age [37]. 

Rats were housed under optimal conditions with standard hygiene, kept at a temperature of 25 °C with a 12/12 light/dark cycle, and fed with standard rat chow and water ad libitum. 

All procedures were approved by and performed according to guidelines for the care and use of animals established by Soochow University, which is consistent with our previous report [35]. 

The experiments were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals (NIH, publication number 85–23, revised 1996). The present study is reported by ARRIVE guidelines.

Eight-week-old SHR was applied for aging control (SHR-Young, n=10). Twelve-month-old rats were treated with or without Gao-Zi-Yao for distilled water (Old SHR-C, n=10), low dose (Old SHR-L, n=10), medium dose (Old SHR-M, n=10), high dose (Old SHR-H, n=10) by gastric feeding. 

Dosages of low, medium, and high Gao-Zi-Yao equal to 1/2-, 1-, threefold dosages for clinical patient application calculated according to body surface area. Gao-Zi-Yao was dissolved in 5 mL distilled water before application and was administrated one-time dairy for 4  weeks. 

Blood pressure and heart rate were monitored every week by the tail-cuff method, and body weight was also monitored every week as per previous our observation [35]. 

After 4  weeks, the Morris water maze experiment was performed, and rats were sacrificed under anesthesia by sodium pentobarbital (50 mg/kg i.p.), hippocampus tissue was collected for further analysis. All experimental protocols were approved by the ethics licensing committee of Soochow University.

Morris water maze experiment

Morris water maze experiment (BW-MWM101, Shanghai Bio-will Co., Ltd., Shanghai, China) was performed as described previously with some modifications [38]. The test was planned for 6 days, days 1 to 5 were for the training period, and day 6 was for testing results. 

Rats received training for 2  min dairy, recording time for boarding on the platform as the latency time. On day 6 the platform was removed. Rats were put into the farthest distance quadrant and the swimming trajectory was recorded for 2  min. 

Parameters for learning and memory function were calculated by the software.

Measurement of serum levels of NO and inflammatory factors by ELISA

Serum levels of NO (Catalog No: S0023), IL-1β (Catalogue number: A301BH80153), IL-2 (Catalogue number: A31038348), IL-6 (Catalogue number: A30681042), and TNF-α (Catalogue number: A38280855) were measured using commercially available ELISA kits (Biotechnology Co., Ltd. Shanghai enzyme research. Shanghai, China). All steps were performed according to the manufacturer's instructions.

Nissl staining

Rat hippocampus tissue was isolated, fixed, paraffin-embedded, then incubated in 1% toluidine blue staining solution for 5–10  min at room temperature. Ten sections were rinsed in distilled water, soaked in 95% ethanol for 5–30 min, and dehydrated in 100% ethanol. 

After dehydration, the slice was placed in xylene and cover-slipped using a resin medium. The number of neurons in the CA1, CA2, and dentate gyrus (DG) regions of the hippocampus were observed and analyzed using the ImageJ analysis program.

Western blot for learning and memory-related proteins

Western blot for learning and memory-related proteins was carried out as described in our previous report with some modifications [39]. 

Hippocampus tissues were homogenized with RIPA buffer (50  mm Tris, 150  mM NaCl, 1% Triton-X-100, pH 7.0) containing phenylmenthanesulfonyl fluoride (R&D Systems Inc., Minneapolis, US). Homogenates were centrifuged at 12,000 × g for 10 min at 4 °C. 

Cell proteins were separated by SDS-PAGE and transferred to PVDF membranes (Hybond TM-ECL; Amersham Pharmacia Biotech, Inc.). The membranes were blocked in 5% nonfat milk in PBS and 0.1% Tween-20 at room temperature. 

The blots were then incubated with primary antibody: Anti-glutamate receptor 1 (1:1000, Abcam, Inc., Catalog No: ab183797), AntiNMDAR2B antibody (1:1000, Abcam, Inc., Catalog No: ab28373), Anti-phospho-CaMKII antibody (1:1000, Abcam, Inc., Catalog No: ab171095), Antiphospho-CREB antibody (1:1000, Abcam, Inc., Catalog No: 32096) or anti-GAPDH (Santa Cruz Biotech, Inc., Catalog No: sc-47724). 

Next, membranes were incubated for 1  h with a secondary antibody (HRPconjugated anti-rabbit Ig-G, 1:2000, Abcam, Inc. Catalog No: ab205718). Membranes were then three times washed for 15  min using TBS-T to remove excess antibodies before incubation for 1  min with chemiluminescent reagents (ECL, R&D Systems Inc., Minneapolis, MN, USA). 

Further, immunoreactive bands were detected by an electrophoresis gel analysis system (GL2200 Pro, Crestream Inc. USA). The intensity of the bands was analyzed by Image J software. The quantity of target proteins was normalized by GAPDH expression [39].

Statistical analysis

The SPSS 18.0 software was used for statistical analysis. Data are presented as the mean±S.E.M. Grouped data were analyzed using a one-way analysis of variance followed by the Student–Newman–Keuls test. A P value < 0.05 was considered as statistically significant as our previous report [35].

Results

Systolic blood pressure (SBP), heart rate, and body weight between young SHR and old SHR

Yong SHR showed lower SBP in the first two weeks compared with old SHR, then increased to no difference between the two groups in the later three weeks (Fig. 2A). 

On the contrary, heart rate was no difference between the two groups at the first two weeks, then young SHR increased markedly compared with old SHR in the later three weeks (Fig.  2B). Young SHR showed lower body weight compared with old SHR during the whole observing period (Fig. 2C).

Morris water maze parameters between young SHR and old SHR

To determine hippocampal-dependent learning and memory, the Morris water maze experiment was performed. Old SHR showed longer escape latency in the first four days compared with young SHR (Fig.  3A). 

Times of crossing the target quadrant were less in old SHR than in young SHR (Fig. 3B).

The percentage of time at the target platform quadrant was shorter in old SHR compared with young SHR (Fig.  3C). Percentage of path length in the quadrant was also shorter in old SHR compared with old SHR (Fig.  3D). These results suggest old SHR has impairment in cognitive function.

Number of neurons in the hippocampus between young SHR and old SHR

The number of neurons in different areas of the hippocampus was counted by Nissl staining. Results showed that there is a smaller number of neurons in CA1, CA2, and DG regions of old SHR compared with young SHR (Fig. 4).

Learning and memory-related protein expressions at the hippocampus between young SHR and old SHR

Western blot results showed that expression of learning and memory-related proteins (GluR1, NMDAR 2B, phosphorylated-CaMK II, and phosphorylated-CREB) in old SHR hippocampus was lower than that in young SHR hippocampus (Fig. 5).

Effect of Gao‑Zi‑Yao on SBP, heart rate, and body weight in old SHR 

The effect of Gao-Zi-Yao on SBP was analyzed. Treatment with a low dosage of Gao-Zi-Yao markedly decreased SBP in old SHR from the second week to the fourth week compared with control old SHR group, medium and high dosages of Gao-Zi-Yao markedly decreased SBP in old SHR from the first week to the fourth week compared with control old SHR (Fig. 6A). Tese results indicate Gao-Zi-Yao exerts anti-hypertensive effect in old SHR.

We also compared the data on the effects of Gao-Zi-Yao on heart rate and body weight. Our data demonstrated that there was no significant difference in heart rate and body weight among all groups during the whole observation period (Fig. 6B, C).

Effect of Gao‑Zi‑Yao on serum levels of NO, IL‑1β, IL‑2, and TNF‑α in old SHR

Treatment with medium and high dosages of GAO-ZIYAO increased the serum NO levels in comparison with the levels in the SHR control group (Fig.  7A). Treatment with all tested dosages of GAO-ZI-YAO reduced the serum levels of IL-1β, IL-2 in comparison with the levels in the SHR control group (Fig. 7B, C); however, only the high dosage of GAO-ZI-YAO suppressed the serum levels of TNF-α (Fig.  7D). 

These results demonstrate that GAO-ZI-YAO could regulate oxidative stress and inflammation.

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