The aim of this work is to investigate the effect of electroacupuncture (EA) at PC6 on the hypertension and myocardial hypertrophy in spontaneously hypertensive rats (SHRs). Thirty SHRs were randomized into model, SHR + EA, and SHR + Sham EA group with WKY rats as normal control. EA was applied once a day in 8 consecutive weeks. The blood pressure (BP), cardiac function, and hypertrophy as well as the underlying mechanisms were investigated. After EA treatment, the enhanced BP in SHR + EA group was significantly lower compared to both the period before EA and model group. Echocardiographic, morphological studies showed that the enhanced left ventricular anterior and posterior wall end-diastolic (LVAWd and LVPWd) thickness, diameters and cross-sectional area (CSA) of cardiac myocyte, as well as the ratio of heart weight to body weight (HW/BW), were markedly diminished in SHR + EA group, while the reduced left ventricular ejection fraction, left ventricular short axis fraction shortening, and E/A ratio were significantly ameliorated. The levels of Angiotensin-converting enzyme (ACE) and Angiotensin II Type 1 and 2 receptors (AT1R, AT2R) in SHRs were also significantly attenuated by EA. The results suggest that EA at bilateral PC6 could arrest the hypertension development and ameliorate the cardiac hypertrophy and malfunction in SHRs, which might be mediated by the regulation of ACE, AT1R, and AT2R.
Hypertension is a key risk factor for various cardiovascular disorders, which affects approximately 20–50% adult populations in developed countries [
Increasing evidence has suggested that both the overactive sympathetic nervous system and endocrine factors paly crucial roles in the pathological progression of persistent hypertension, in which the function of renin-angiotensin system (RAS) has been highly emphasized due to its impact on the circulating volume and electrolyte balance, as well as the haemodynamic stability [
Although novel antihypertensive pharmacological therapies have been developed nowadays, only 5–30% of patients with hypertension achieve adequate blood pressure control, which is partially caused by low compliance to the combination of multiple antihypertensive medicines and their side effects [
Thirty male SHRs at the age of 12 weeks and 10 male Wistar-Kyoto (WKY) rats of the same age, weighing 240–270 g, were obtained from Vital River Laboratories (Certificate number SCXK 2012-0001, Beijing, China). The rats were housed in cages at 24 ± 1°C and humidity of 50 ± 5% under a 12-hour light/dark cycle and received standard diet and water ad libitum. The experiments were conducted in accordance with the Guide for Use and Care of Medical Laboratory Animals from Ministry of Public Health of China.
The rats were randomly divided into 4 groups: WKY group (
Under conscious condition, blood pressure levels were recorded by using a CODA Mouse & Rat Tail-Cuff Blood Pressure System (Kent Scientific Co., Connecticut, USA), including the systolic blood pressure (SBP), diastolic blood pressure (DBP), and the mean arterial blood pressure (MAP), as described in previous study [
The structural and functional changes among the four groups were tested by Vevo 770 High Resolution Imaging Systems (Visual Sonics, Toronto, Canada) with a 17.5 MHz linear array transducer (model 716) once every two weeks. All rats were anesthetized with 1.5–2.0% isoflurane. Two-dimensional cine loops and guided M-mode frames were recorded from the parasternal short and long axis to assess the left ventricular anterior wall end-diastolic (LVAWd) thickness, left ventricular end-diastolic internal diameter (LVIDd), left ventricular posterior wall end-diastolic (LVPWd) thickness, left ventricular ejection fraction (LVEF), and left ventricular short axis fraction shortening (LVFS) [
Six rats in each group were killed after 8 weeks of EA treatment, and the hearts were removed and washed with 4°C saline. Both body weight (BW) and heart weight (HW) were determined, and the HW/BW ratio was calculated to evaluate the hypertrophic response to overload blood pressure. The other 3 rats in each group were anaesthetized by 10% urethane and transcardially perfused with 250 mL of 0.9% saline immediately followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffered solution (PB, pH 7.4). The left ventricular (LV) section was cut off transversely at the midventricular level for paraffin sectioning [
Concentrations of ACE in heart tissue and serum were measured by commercial radioimmunoassay kits (Beijing Sino-UK Institute of Biological Technology Company, China) following the company’s protocol.
Heart tissues were lysed in RIPA buffer containing phosphatase and protease inhibitors (Roche Complete, Roche Diagnostics, Mannheim, Germany). The protein concentration in the supernatant was determined using the BCA method with a bovine serum albumin standard. Equal amount of total protein was subjected to SDS-PAGE and blotted on NC membrane (Millipore, Billerica, MA, USA). The blots were blocked with 5% defatted milk powder in Tris-buffered saline (TBS) buffer and then incubated with the respective primary antibodies (Mouse Anti-Angiotensin II Type 1 Receptor 1 : 500, Abcam, UK; Rabbit Anti-Angiotensin II Type 2 Receptor 1 : 1000, Abcam, UK; Mouse Anti-GAPDH 1 : 20000, TDY Biotech Co., Ltd., Beijing, China) for overnight at 4°C. The membrane was washed with TBS and incubated with horse radish peroxidase-conjugated goat anti-mouse or rabbit IgG (1 : 10000; TDY Biotech Co., Ltd., Beijing, China) for 40 min at room temperature. The targeted proteins were detected by using enhanced chemiluminescence system (Millipore, Billerica, MA, USA). The quantification of band intensity was carried out using Image-Pro Plus software. Band densities were normalized to individual GAPDH internal control.
All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Turkey post hoc test or repeated measures ANOVA with Bonferroni post hoc test was used for multiple comparisons. Changes of blood pressure in the same group were compared statistically by a paired
Noninvasive blood pressure recording showed that the blood pressure of SHR significantly elevated and remained on an increasing trend as compared with the WKY controls (
The antihypertensive effects of EA on the systolic (a), diastolic (b), and mean arterial pressure (c) in WKY and SHR. Blood pressure was measured by tail-cuff method before and after 1, 2, 3, 4, 5, 6, 7, and 8 weeks of EA treatment.
By using echocardiographic analysis, the structural and functional alterations of left ventricle in SHR rats were observed, including LVAWd, LVPWd, LVIDd, LVEF, LVFS, and E/A ratio. As shown in Figure
EA restored the structural and functional impairment of left ventricle in SHR. (a) Representative echocardiograms of rat hearts before and after 2, 4, 6, and 8 weeks of EA treatment for determination of the ventricle wall thickness. (b), (c), (d), (e), and (f) showed the improvement of myocardial hypertrophy and malfunction in SHR, in terms of the LVAWd, LVPWd, LVIDd, LVEF, and LVFS, respectively.
EA improved the pulsed-wave Doppler early to late transmitral peak diastolic flow velocity (E/A) ratio in SHR. (a) Representative pulse-wave Doppler echocardiograms of mitral inflow before and after 2, 4, 6, and 8 weeks of EA treatment. (b) E/A ratio was decreased in SHR when compared with that in WKY, which was reversed with EA treatment.
The histological alteration in myocardial tissue was assessed by hematoxylin and eosin staining. As shown in Figures
Effect of EA treatment on histology of myocardial tissue. The tissue was taken after 8 weeks of EA treatment from SHR or at the same time course from WKY and stained by hematoxylin and eosin. (a) H&E stained cross-sections of rat hearts in WKY, SHR, SHR + EA, and SHR + Sham group (bar = 1000
Immunoradiometric assay showed that the levels of ACE in SHR were significantly higher than WKY, in both serum and heart tissue (Figures
The roles of ACE, AT1R, and AT2R in the cardiac protective effects of EA on SHR. The concentration of ACE in serum (a) and heart tissue (b) measured by immunoradiometric assay. Western blotting analysis of AT1R (c) and AT2R (d) protein in left ventricular myocardium after 8 weeks of EA treatment.
Since the hypertension in most of the patients primarily or genetically caused enhancement of blood pressure, we selected SHR, a typical genetic hypertension animal model, to investigate the antihypertensive and antihypertrophic effects and the underlying mechanisms in the present study. Although previous studies showed a significant inhibition of hypertension was achieved by acupuncture in SHRs, the antihypertensive effect of acupuncture is actually limited more or less, which may be caused by the fact that the acupoints being stimulated in their studies are not optimal ones.
In the present study, we investigated the antihypertensive and antihypertrophic effects of EA at PC6 on SHR and the underlying mechanisms. Our data showed that, as compared with WKY controls, the systolic, diastolic, and mean arterial pressure were significantly elevated in SHR, which results in obvious structural and functional impairments in the myocardial tissue, including increased myocyte diameter, cross-sectional area, and anterior and posterior wall thickness, as well as reduced LVFS and E/A ratio. However, the increased blood pressure and hypertrophy and malfunction of myocardium were diminished by 8-week EA treatment. More interestingly, during the period from 4th to 8th weeks of EA treatment the increased blood pressure of the rats in SHR + EA group was significantly reduced, not only as compared with model group, but also even as compared with the rats themselves in SHR + EA group before the EA treatment, which means that the course of the hypertension development was arrested. Obviously, acupuncture applied at PC6 acupoints produced stronger antihypertensive effect than the aforementioned experimental studies in which the acupoints including Taichong (LV3), Baihui (GV20), and Zusanli (ST36) were selected only referring to the theory of Traditional Chinese Medicine without consideration of the anatomic or neurological aspects.
Morphological studies revealed that in the dorsal roots or dorsal horns there are dichotomizing and/or convergent neurons which are distributed to both somatic tissues and corresponding visceral organs [
Clinical studies have suggested that pharmacological inhibition on RAS is a critical component in the treatment of hypertension, in which the ACE inhibitors and Angiotensin 2 receptor blockers are the effective and most commonly used agents [
Currently, increasing evidence demonstrated the promising effectiveness of acupuncture treatment on blood pressure control in both humans and animals [
In conclusion, the results of the present study suggests that 8-week EA at PC6 attenuates significantly the increased blood pressure and the myocardial hypertrophy in SHR, which might be mediated by downregulation of enhanced both ACE and AT1R, as well as upregulation of the diminished expression of AT2R.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The experiments were done by Juan-Juan Xin, Yuan- Yuan Wang, and Feng-Yan Lu. Yu-Xue Zhao and Xiang-Hong Jing provided advice on the statistical analyses and data interpretation. Juan-Juan Xin, Xiao-Chun Yu, and Jun-Hong Gao drafted and finalized the paper. Xiao-Chun Yu was responsible for the conception, design, and supervision of the implementation of the study.
This work was supported by National Natural Science Foundation of China (no. 81473781).