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

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, hypertensionstarts developing by 4 months of age with the appearanceof hypertension-related changes in the brain microvasculature by 6 months of age [37]. 

Rats were housed underoptimal conditions with standard hygiene, kept at a temperature of 25  degree with a 12/12 light/dark cycle, and fed withstandard rat chow and water ad libitum. 

All procedureswere approved by and performed according to guidelinesfor the care and use of animals established by SoochowUniversity, which is consistent with our previous report[35]. 

The experiments were performed according tothe National Institutes of Health Guidelines for the Useof 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 weretreated 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 calculatedaccording to body surface area. Gao-Zi-Yao was dissolved in 5 mL distilled water before application and wasadministrated 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 everyweek as per previous our observation [35]. 

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

Morris water maze experiment (BW-MWM101, Shanghai Bio-will Co., Ltd., Shanghai, China) was performedas 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 receivedtraining for 2  min dairy, recording time for boardingon the platform as the latency time. On day 6 the platformwas removed. Rats were put into the farthest distancequadrant and the swimming trajectory was recorded for2  min. 

Serum levels of NO (Catalog No: S0023), IL-1 (Catalogue number: A301BH80153), IL-2 (Catalogue number:A31038348), IL-6 (Catalogue number: A30681042), andTNF- (Catalogue number: A38280855) were measuredusing commercially available ELISA kits (BiotechnologyCo., Ltd. Shanghai enzyme research. Shanghai, China).All steps were performed according to the manufacturer's instructions.

Rat hippocampus tissue was isolated, fixed, paraffin-embedded, then incubated in 1% toluidine blue stainingsolution 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. 

Afterdehydration, the slice was placed in xylene and cover-slippedusing a resin medium. The number of neurons in the CA1,CA2, and dentate gyrus (DG) regions of the hippocampuswere observed and analyzed using the ImageJ analysisprogram.

Western blot for learning and memory-related proteins

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

Hippocampustissues were homogenized with RIPA buffer (50  mmTris, 150  mM NaCl, 1% Triton-X-100, pH 7.0) containing phenylmenthanesulfonyl fluoride (R&D Systems Inc., Minneapolis, US). Homogenates werecentrifuged at 12,000 × g for 10 min at 4  degree . 

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

The blots were then incubatedwith 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 wereincubated 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 excessantibodies before incubation for 1  min with chemiluminescent reagents (ECL, R&D Systems Inc., Minneapolis, MN, USA). 

Further, immunoreactive bandswere 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 byGAPDH expression [39].

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

Yong SHR showed lower SBP in the first two weekscompared 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 differencebetween the two groups at the first two weeks, then young SHR increased markedly compared with old SHR inthe later three weeks (Fig.  2B). Young SHR showedlower body weight compared with old SHR during thewhole observing period (Fig. 2C).

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

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

The percentage oftime at the target platform quadrant was shorter inold SHR compared with young SHR (Fig.  3C). Percentage of path length in the quadrant was also shorterin old SHR compared with old SHR (Fig.  3D). Theseresults suggest old SHR has impairment in cognitivefunction.

The number of neurons in different areas of the hippocampuswas counted by Nissl staining. Results showed that thereis a smaller number of neurons in CA1, CA2, and DGregions of old SHR compared with young SHR (Fig. 4).

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

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

The effect of Gao-Zi-Yao on SBP was analyzed. Treatmentwith a low dosage of Gao-Zi-Yao markedly decreasedSBP in old SHR from the second week to the fourth week compared with control old SHR group, mediumand high dosages of Gao-Zi-Yao markedly decreasedSBP in old SHR from the first week to the fourth weekcompared with control old SHR (Fig. 6A). Tese resultsindicate Gao-Zi-Yao exerts anti-hypertensive effect inold SHR.

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

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

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

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

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