Rb1 (95% purity) was obtained from Dr. Yanping Chen at the Department of Natural Medicinal Chemistry, School of Chemistry, Jilin University (Jilin, China) and dissolved in double-distilled water (ddH₂O) prior to use. Rg3 (95% purity) was obtained from Jilin Yatai Pharmaceutical Co., Ltd. (Changchun, China) and suspended in 0.5% sodium carboxymethyl cellulose solution for use. All other chemicals were analytical reagents.

A total of 24 SHR and 8 WKY rats (male, 16-17-week-old, 250-300 g) were purchased from Vital River Laboratories Co., Ltd. (Beijing, China). All rats were kept in a specific pathogen-free experimental animal workshop (25°C, 10/14-h light/dark cycle), and had free access to food and water. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Jilin University and approved by the Ethics Committee of Jilin University.

The rats were randomly divided into 4 groups (each, n=8): i) A WKY group, consisting of WKY rats orally administered ddH₂O; ii) a SHR group, consisting of SHR rats orally administered ddH₂O; iii) a Rb1 group consisting of SHR rats orally administered 20 mg/kg Rb1; and iv) a Rg3 group Rg3 consisting of SHR rats orally administered 20 mg/kg Rg3. All treatments were administered once a day over 42 consecutive days.

After treatment for 6 weeks with ddH₂O or ginsenosides, all rats were anesthetized with chloral hydrate (300 mg/kg, intraperitoneally), and blood samples were obtained from the abdominal aorta before the rats were sacrificed. Hearts were obtained and weighed after the rats were sacrificed, and myocardium tissue samples were then fixed in 4% buffered paraformaldehyde solution (25°C, 24 h) for histopathological examination or frozen in liquid nitrogen and stored at −80°C prior to reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse pressure (PP) of rats were measured using the tail-cuff method and a small animal sphygmomanometer (BP-2010A; Softron Biotechnology Ltd., Beijing, China) following a previously described protocol (22) on the first and the last days of the 6-week treatment.

On the first and last days of the 6-week treatment, transthoracic echocardiography was performed as previously described (23), using a standard setting with a 10S transducer (Vivid-i; GE Healthcare, Chicago, IL, USA). Animals were anesthetized with chloral hydrate (300 mg/kg, intraperitoneally) and two-dimensional and M-mode echocardiographic measurements were conducted. A short-axis two-dimensional image of the left ventricle was obtained at the position of the papillary muscles. Subsequently, M-mode images were acquired at a sweep speed of 100 mm/s and digitally stored. The left ventricular internal dimension at diastole (LVIDd) and left ventricular internal dimension at systole (LVIDs) were acquired from M-mode images; subsequently, left ventricular fractional shortening (FS) and left ventricular ejection fraction (EF) were calculated automatically by the equipment. The parameters were measured by an experienced echocardiographer blinded to the treatment groups.

Blood samples were collected and left at room temperature for 2 h to allow complete clotting and then centrifuged at 1,500 × g, 4°C for 15 min. The serum was removed and stored at −80°C prior to ELISA. ACE (CSB-E04490r) and Ang II (CSB-E04494r) ELISA kits were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China) and the assays were completed by this company.

Myocardial tissue samples were fixed in 4% buffered paraformaldehyde solution and then embedded in paraffin. Paraffin-embedded sections 4-μm thick were stained with hematoxylin and eosin (H&E) and Masson trichrome stain. Sections were examined using a Nikon E100 light microscope (Nikon Corporation, Tokyo, Japan) and photomicrograph images were captured.

Primary antibodies against ACE (bs-0439R), Ang II (bs-0587R), Ang II receptor type 1 (AT1, bs-0438R) and transforming growth factor β1 (TGF-β1, bs-0103R) were purchased from Bioss Antibodies (Beijing, China). Peroxidase-conjugated goat anti-rabbit IgG (ZB-2301), DAB kit (ZLI-9018) and two step rabbit IHC kit (PV-6001) were purchased from ZSGB-BIO (Beijing, China). IHC was performed following the manufacturer's protocols of the IHC kit and DAB kit. Photomicrograph images were then captured, and Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used for image analysis.

Total RNA was isolated from frozen myocardium tissue samples using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. Total RNA was reverse-transcribed and qPCR was conducted using the TransScript Green Two-Step qRT-PCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China) on the Stratagene Mx3000P (Agilent Technologies, Inc., Santa Clara, CA, USA) and following the manufacturer's protocol (94°C for 5 sec, 60°C for 15 sec and 72°C for 10 sec, 40 cycles). The relative fold changes in the mRNA levels of the target genes were determined using the 2^−ΔΔCq method (24) and β-actin was used as a housekeeping gene. Primer sequences are provided in Table I.

SPSS 15.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. All data are expressed as the mean ± standard deviation. One-way analysis of variance with Tukey's post hoc test was used to analyze differences among groups and P<0.05 was considered to indicate a statistically significant difference.

The effects of Rb1 and Rg3 on cardiac structure and function were evaluated using echocardiography. As depicted in Fig. 2, compared with the WKY group prior to the 6-week treatment, the three groups of SHR rats exhibited slight cardiac function injury; they had a significantly lower FS and EF compared with the WKY group (P<0.05; Fig. 2D and E). However, LVIDd and LVIDs were similar among all groups, indicating that the cardiac structure of SHR rats was unaffected prior to the 6-week treatment.

Following the 6-week treatment, the FS and EF of the three SHR groups were all significantly reduced compared with the WKY group (P<0.05). However, the FS and EF of the Rb1 and Rg3 groups were significantly higher than those of the SHR group (P<0.05; Fig. 2D and E). Rb1 and Rg3 also exhibited protective effects on cardiac structure. Following treatment, the LVIDd and LVIDs of the SHR group were significantly higher than those of the WKY group (P<0.05); however, those of the Rb1 and Rg3 groups were significantly lower than the SHR group and did not differ significantly between those of the WKY group (Fig. 2B and C). Notably, the cardiac protective effects of Rb1 and Rg3 were comparable.

Rb1 and Rg3 did not significantly affect the blood pressure of SHR rats. The SBP and DBP of the three groups of SHR rats were all significantly higher than the WKY group prior to and following 6-week treatment (P<0.05) and there was no significant difference in blood pressure between the SHR group and the Rb1 or Rg3 groups (Fig. 3A and B). Regarding PP, there were no significant differences between any groups prior to or following treatment (Fig. 3C).

The body weights and heart weights of all four groups are presented in Fig. 3D and E. CWI was subsequently calculated using the following formula: Heart weight/body weight. The CWI of the SHR group was significantly higher than that of the WKY group (P<0.05), while those of groups Rb1 and Rg3 were significantly lower than the SHR group (P<0.05; Fig. 3F). This result demonstrated that hypertension induces cardiac structural changes and that Rb1 and Rg3 significantly attenuate these changes.

Rb1 and Rg3 had no significant effects on RAS activity in the serum. ACE and Ang II levels of the three SHR groups were all significantly higher than those of the WKY group (P<0.05) and there were no significant differences between ACE and Ang II levels between the three SHR groups (Fig. 3G and H). This may explain why Rb1 and Rg3 did not significantly reduce blood pressure in SHR rats.

Representative H&E and Masson staining histology photomicrographs are presented in Fig. 4. According to the H&E photomicrographs, myocardium tissues from the SHR group exhibited increased myocardial cell size, myocardial structural disorder and intercellular space dilatation, which are the typical pathological changes of ventricular remodeling induced by hypertension. Inflammatory cell infiltration was also observed. The Masson photomicrographs indicated increased collagen deposition (blue area) in the SHR group compared with the WKY group. However, treatment with Rb1 and Rg3 markedly improved all these histopathological changes.

The expression of ACE, Ang II, AT1 and TGF-β1 in the myocardium was evaluated using IHC. Representative photomicrographs are presented in Fig. 5A and quantitative results are presented in Fig. 5B-E. Compared with the WKY group, levels of ACE, Ang II, AT1 and TGF-β1 were significantly increased in myocardium samples from the SHR group (all P<0.05). However, these increases were significantly attenuated following treatment with Rb1 and Rg3 (all P<0.05). Indeed, the expression of Ang II and TGF-β1 in the Rb1 and Rg3 groups did not differ significantly between that of the WKY group (Fig. 5D and E). The downregulation of local RAS activity in the myocardium reduced the expression of TGF-β1, which is also a key factor to myocardial fibrosis.

Levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and ET-1 mRNA were measured (Fig. 6). In the SHR group, the expression of TNF-α, IL-6, IL-1β and ET-1 mRNA were all significantly higher than in the WKY group (all P<0.05). However, in the Rb1 and Rg3 groups, the levels of all four mRNAs were all significantly lower than the SHR group (P<0.05). These results suggest that Rb1 and Rg3 may attenuate inflammation and endothelial injury in the myocardium.