Paeonol protects against hypertension in spontaneously hypertensive rats by restoring vascular endothelium

ABSTRACT

The present study focused on the effect of paeonol, one of the main components of Guizhi Fuling Pill, on blood pressure, cerebral blood flow, and vascular endothelium injury in spontaneously hypertensive rats to provide theoretical basis for the treatment of hypertension. After treatment with paeonol, the mean arterial pressure (MAP) of LSHRT and HSHRT rats decreased gradually with the prolongation of treatment time. The systolic blood flow velocity (Vs), diastolic blood flow velocity (Vd) and mean blood flow velocity (Vm) were significantly increased after paeonol treatment (p < 0.05). Paeonol effectively improved the blood pressure and increased the cerebral blood flow velocity in spontaneously hypertensive rats. This may be related to the fact that paeonol reduced the blood viscosity and the oxidative stress and improved the antioxidant capacity. Moreover, paeonol protected vascular endothelial cells and reduced vascular endothelial injury in spontaneously hypertensive rats.

Graphical abstract

Paeonol protects against hypertension in spontaneously hypertensive rats by restoring vascular endothelium

KEYWORDS:

• Spontaneously hypertensive rats
• paeonol
• blood pressure
• cerebral blood flow velocity
• oxidative stress

Hypertension still remains a major risk factor of heart failure, renal failure, stroke, and myocardial infarction despite dramatic progress in clinical hypertension diagnosis and management [1,2]. Current anti-hypertensive medications in clinic include calcium channel blockers, phosphodiesterase inhibitors, and nitric oxide (NO), prostaglandin and endothelial receptor antagonists [3,4]. However, current therapeutic strategies are often not sufficient to prevent cardiovascular events and side-effects like affecting kidney and liver function and the occurrence of drug-resistance after long-term medication has hindered these treatments to achieve satisfactory therapeutic effects [5], so it is an urgent requirement to identify new potential effective treatments for the treatment of hypertension.

Paeonol has been found to be the main phenolic compound of a Chinese herbal medicine which is prepared from the roots of the plant Paeonia suffruticosa Andrews [6]. Paeonol is traditionally used in clinic in China to improve blood circulation, amenorrhea, dysmenorrhea, and fever as an anti-inflammatory, anti-bacterial, anti-oxidant, antipyretic and analgesic agent [7,8]. Paeonol has been previously suggested to protect against acetaminophen-induced hepatotoxicity in mice and improve Parkinson’s disease in mouse model and diabetic encephalopathy in streptozotocin-induced diabetic rats by attenuating oxidative stress [9–13]. It has been demonstrated that paeonol could reduce lipid peroxidation on mitochondrial membrane by scavenging free radicals, and inhibit permeability and transmission of mitochondrial membrane by reducing thiol oxidation [12–14]. Previous studies have shown that paeonol antagonized acute myocardial ischemia and infarction in rat [15,16]. The vascular endothelium plays an important role in the regulation of vascular tone, tissue blood flow, inflammatory responses, and maintenance of blood fluidity [17]. However, it is rare to investigate the effects of paeonol on the vascular endothelium and whether paeonol could be a potential therapeutic strategy of hypertension is still unknown.

Spontaneously hypertensive rats (SHRs) have been widely used as a primary hypertension animal model [18–20], this study examined the effects of paeonol on blood pressure and blood flow in the artery of spontaneously hypertensive rats and the underlying mechanism via vasomotion was explored. The present study further investigated the endothelial protective effects of paeonol against oxidative stress in rats. We concluded that treatment of paeonol for 12 weeks normalized blood pressure increased the cerebral blood flow through protecting vascular endothelium against oxidative stress and endothelium injury. These results may provide new insights into the role of paeonol to improve cardiovascular diseases such as hypertension, heart failure, ischemic heart diseases, and atherosclerosis.

Materials and methods

Reagent

Paeonol was purchased from the China Food and Drug Testing Institute (catalog number: 110708–201407). The corresponding concentration was prepared in 0.5% sodium carboxymethyl cellulose (CMC-Na) before use.

Animal group

Thirty male spontaneously hypertensive rats (SHR, 8-week-old, 200–300 g) and 10 Wistar-Kyoto rats (WKY, 8-week-old, 200–300 g) were purchased from Pengyue (license number: SCXK (Lu) 2014-0007, Jinan, China). Rats were maintained in a quiet room with 12 h light and dark cycle light, 24 ± 2°C, 60% humidity and free food and water. All animal work was approved by Animal Experimental Ethics Committee of the Yantai Hospital of Traditional Chinese Medicine. Four animal groups including normal blood pressure rat group (WKY), hypertensive control group (SHR), low dose paeonol group (LSHRT, 2 mg/kg) and high dose paeonol group (HSHRT, 5 mg/kg) with 10 rats per group were studied in our paper. Each group of rats was orally administered once a day for 12 consecutive weeks [21–23].

Determination of rat tail artery blood pressure

The blood pressure of the tail artery was measured before and after the experiment using the animal noninvasive blood pressure analysis system (BP-2000, Visitech, US). The mean arterial pressure (MAP) was calculated after the measurement of diastolic blood pressure (DBP), systolic blood pressure (SBP), 3 times for each rat. The measurement was done at 0, 2, 4, 6, 8, 10, and 12 weeks (at 8, 10, 12, 14, 16, 18, and 20 weeks of age) after paeonol treatment.

Transcranial doppler measurement of cerebral blood flow index

Cerebral blood flow parameters were measured after 12 weeks of administration. Transcranial Doppler system (Multi-Dop x, DWL, Germany) was used to measure the middle cerebral artery blood velocity in model rats. The systolic blood flow velocity (Vs), diastolic blood flow velocity (Vd), mean blood flow velocity (Vm), pulsatility index (PI = (Vs-Vd)/Vm), and resistance index (RI) were measured in each group.

Hemorheological examination

Abdominal aorta blood samples were harvested. A fully automated blood flow rheometer (FASCO-3010, Chongqing University, Weiduo Institute of Biological Engineering, China) was used following the manufacturer’s instruction and low-shear, high-shear whole blood viscosity, plasma viscosity, and fibrinogen determination were examined.

ELISA

1.5 mL of venous blood was drawn from the rat and serum was separated and then stored at −80°C until use. The levels of vWF (CSB-E08438r), ET-1 (CSB-E06979r), AT1 (CSB-E13746r), and SOD (CSB-EL022397RA) in rat serum were determined by ELISA following the manufacturer’s instruction. All the above kits were purchased from CUSABIO (Wuhan, China).

Colorimetric determination of NO

Serum was separated as mentioned above and NO levels in the serum were measured using the NO kit (A012-1, Jiancheng, Nanjing, China) following the manufacturer’s instruction.

Determination of MDA

Serum was separated from venous blood and MDA content in the serum was measured using MDA kit (A003-1, Jiancheng, Nanjing, China) and the operation was strictly performed according to the instructions.

HE staining

Rats were anesthetized with 2% sodium pentobarbital (50 mg/kg, New Asiatic Pharmaceutical, China). The rat’s sternum was cut and the aorta was isolated. The blood vessels were cut and rinsed in cold saline to drain the blood from the blood vessels. Then, the aorta with a length of about 1 cm was fixed in formaldehyde solution and HE staining was performed to observe the morphology of rat aortic tissue cells.

Western blot

Tissue samples from the blood vessels were ground in liquid nitrogen and the protein samples were extracted with RIPA lysis buffer (R0020, Solarbio, Beijing, China). The protein concentration was determined with a BCA protein quantification kit (23225, Thermo Fisher Scientific, Waltham, USA). Total protein of 40 μg was loaded and separated by SDS-PAGE (Mini-Protean-3, Bio-Rad, Hercules, CA, USA), transferred to PVDF membrane (Millipore, Massachusetts, USA) and blocked with 5% skimmed milk for 1 h. The primary antibody against each protein (1:1000) was diluted with 5% BSA (rabbit anti-eNOS antibody, ab76198; rabbit anti-NOX4 antibody, ab133303; rabbit anti-VCAM1 antibody, ab134047; Abcam, UK) and incubated overnight at 4°C. The membranes were then washed 3 times with 1 × TBST (TBS, 1 mL/L Tween-20) for 10 min each time, and goat anti-rabbit IgG HRP (1:2000, ab6721, Abcam) was incubated at room temperature for 2 h. The membranes were washed 3 times for 10 min each time and ECL chemiluminescence was used for detection. Quantification was performed using Image J software (NIH). Protein expression levels were normalized to β-actin.

Statistical analysis

SPSS19.0 statistical software was used to analyze the data. The results were expressed as mean ± SD. One-way analysis of variance analysis (ANOVA) followed by Tukey’s test was used for the analysis of data among groups. A value of p < 0.05 was considered to be statistically significant.

Results

Paeonol reduces blood pressure of the tail artery in rats

To study the effect of paeonol on the blood pressure of rats, blood pressure of the tail artery of the rats was measured at 0, 2, 4, 6, 8, 10, and 12 weeks (at 8, 10, 12, 14, 16, 18, and 20 weeks of age) after paeonol administration. The MAP results are shown in Table 1. Before treatment, the MAP of rats in SHR, LSHRT and HSHRT groups was significantly higher than that in WKY group (p < 0.05). The MAP in the SHR group was significantly higher than the other three groups during the entire treatment period. Interestingly, the MAP of LSHRT and HSHRT rats gradually decreased after treatment. No significant difference was observed between HSHRT and WKY groups at 14 weeks of age and between LSHRT group and WKY group at 16 weeks of age, respectively. Both SBP and DBP were significantly reduced after paeonol treatment (Figure 1(a,b)). These data suggested that paeonol reduced the blood pressure of the tail artery in rats.

Table 1. Mean arterial pressure (mmHg) in various groups after 12 weeks.

Age (week)	8	10	12	14	16	18	20
WKY	94.57 ± 4.43^a	92.60 ± 2.63^a	92.17 ± 6.36^a	90.73 ± 3.72^a	90.30 ± 4.35^a	92.91 ± 5.34^a	92.97 ± 6.54^a
SHR	128.53 ± 7.35^b	132.03 ± 8.14^b	131.53 ± 6.98^b	130.63 ± 10.76^b	127.87 ± 6.58^b	131.10 ± 7.33^b	130.97 ± 6.89^b
LSHRT	128.27 ± 8.83^b	121.83 ± 7.02^c	112.43 ± 5.74^c	104.00 ± 8.45^c	98.33 ± 7.32^a	93.67 ± 6.55^a	92.37 ± 7.37^a
HSHRT	128.83 ± 7.26^b	111.73 ± 4.88^d	103.27 ± 5.23^d	96.80 ± 7.34^a	90.70 ± 5.61^a	91.97 ± 6.32^a	89.30 ± 7.35^a

N = 10, Different alphabets indicated significant differences.

Figure 1. The blood pressure measurement of each group at different time point. (a) Systolic blood pressure. (b) Diastolic blood pressure. N = 10, Different alphabets indicated significant differences.

Paeonol increases cerebral blood flow index in rats

The cerebral blood flow was measured in rats at 12 weeks after paeonol administration (at 20 weeks of age). The results are shown in Table 2. Our results showed that the Vs, Vd, and Vm in SHR group were significantly lower than those in WKY group (p < 0.05) and significantly increased after treatment with high and low doses of paeonol (p < 0.05). PI and RI in both LSHRT and HSHRT group were significantly lower than those in SHR group (p < 0.05), but not significantly different from WKY group. These results suggested that paeonol increased cerebral blood flow velocity in hypertensive rats by increasing vascular compliance and diastolic status while reducing vascular resistance.

Table 2. Cerebral blood flow in various groups after 12 weeks.

Group	WKY	SHR	LSHRT	HSHRT
Vs（cm/s）	83.45 ± 4.35^a	60.32 ± 5.21^b	78.31 ± 6.78^a	81.32 ± 6.28^a
Vd（cm/s）	46.47 ± 3.12^a	33.45 ± 4.32^b	41.51 ± 3.24^a	44.68 ± 4.56^a
Vm（cm/s）	62.38 ± 5.32^a	38.91 ± 3.41^b	59.42 ± 6.14^a	60.48 ± 4.87^a
PI	0.59 ± 0.06^a	0.69 ± 0.05^b	0.62 ± 0.07^a	0.60 ± 0.06^a
RI	0.61 ± 0.03^a	0.83 ± 0.05^b	0.68 ± 0.04^a	0.67 ± 0.04^a

N = 10, Different alphabets indicated significant differences.

Paeonol improves hemorheological parameters in rats

The low-shear whole blood viscosity, high-shear whole blood viscosity and plasma viscosity in SHR group were significantly higher than those in WKY group and significantly decreased after a high dose or low dose of paeonol treatment (Table 3) (p < 0.05). However, no significant difference was observed between high dose treatment and low dose treatment. This result suggested that paeonol could effectively reduce the blood viscosity of hypertensive rats. Moreover, fibrinogen is a protein that is synthesized by the liver and could cause blood coagulation [24]. The blood fibrinogen levels in the hypertensive group treated with paeonol (2.88 ± 0.52 g/L in LSHRT group, 2.76 ± 0.51 g/L in HSHRT group) were significantly lower than those in SHR group (3.96 ± 0.73 g/L, p < 0.05), whereas no significant difference was observed compared with WKY rats (2.53 ± 0.35 g/L).

Table 3. Hemorheology indexes in various groups after 12 weeks.

Group	WKY	SHR	LSHRT	HSHRT
Low shear blood viscosity (mPa·s)	12.89 ± 0.5^a	19.32 ± 0.82^b	14.02 ± 0.91^a	13.84 ± 0.63^a
High shear blood viscosity (mPa·s)	5.28 ± 1.01^a	8.46 ± 0.82^b	6.15 ± 0.62^a	6.39 ± 0.82^a
Plasma viscosity (mPa·s)	2.36 ± 0.52^a	4.68 ± 0.93^b	3.09 ± 0.83^a	2.81 ± 0.75^a
Fibrinogen (g/L)	2.53 ± 0.35^a	3.96 ± 0.73^b	2.88 ± 0.52^a	2.76 ± 0.51^a

N = 10, Different alphabets indicated significant differences.

Paeonol protects against oxidative stress in rats

To study the effect of paeonol on the oxidative stress, the oxidative stress factors SOD and MDA in the serum of each group were detected. The serum SOD content in SHR group (51.61 ± 2.47 U/mL) was significantly lower than that in WKY group (60.34 ± 1.41 U/mL, p < 0.05). The SOD content was significantly increased after a high dose or low dose of paeonol treatment (Figure 2(a), p < 0.05). The MDA content in SHR group is significantly higher than that in WKY group (p < 0.05), and it is obviously decreased after the paeonol treatment (Figure 2(b)). These data suggested that paeonol effectively increased the antioxidant capacity of rats.

Figure 2. The content of serum SOD (a) and MDA (b) in each group. N = 10, Different alphabets indicated significant differences.

Paeonol restores the function and structure of vascular endothelium in rats

It is well known that vWF is involved in coagulation, reflecting the degree of endothelial dysfunction [25]; AT1 could induce vasoconstriction and result in hypertension, which can cause endothelial injury, extracellular matrix deposition and atherosclerosis [26]; ET-1 has a strong vasoconstriction activity which can cause vascular damage in hypertensive rats [27]. ELISA results showed the serum levels of vWF (Figure 3(a)), AT1 (Figure 3(b)), and ET-1 (Figure 3(d)) in SHR group were significantly higher than those in WKY group (p < 0.05). After treatment with high dose or low dose of paeonol, the levels of vWF (Figure 3(a)), AT1 (Figure 3(c)), and ET-1 (Figure 3(d)) were significantly decreased although still higher than WKY group, indicating that paeonol significantly reduced blood levels of vWF, AT1, and ET-1 in hypertensive rats.

Figure 3. Serum levels of vWF (a), NO (b), AT1 (c), and ET-1 (d) in different groups of rats. N = 10, Different alphabets indicated significant differences.

NO is an important vasodilating factor synthesized by endothelial cells, and its content indirectly reflects the functional status of endothelial cells [19]. The NO content in SHR group (6.34 ± 0.47 U/mL) was significantly lower than that of WKY group (17.38 ± 0.41 U/mL, p < 0.05, Figure 3(b)), and significantly increased after paeonol treatment (p < 0.05). These results showed that paeonol significantly reduced serum levels of vWF, AT1, and ET-1 and improved serum NO level. Moreover, paeonol protected the vascular endothelium and lower blood pressure.

Then the structure of aorta was examined. Results of HE staining showed that the aortic intima was smooth and the endothelial cells were intact in WKY group (Figure 4). However, the aortic intima was destroyed and the endothelial cells were defective in SHR group. Compared with SHR group, the rat aortic intima was significantly improved and no obvious defect was observed in the endothelial cells after paeonol treatment.

Figure 4. Structure of the vascular endothelium determined by HE staining in the group of WKY (a), SHR (b), LSHRT (c) and HSHRT (d). N = 10, scale bar = 100 μm.

Paeonol decreased eNOS, NOX4, and VCAM-1 proteins in rats

NO is synthesized by eNOS in endothelial cells [28]. The expression of eNOS in SHR group was significantly lower than that in WKY group. However, the expression of eNOS was significantly increased after paeonol treatment (Figure 5; p < 0.05). NOX4 is also involved in the physiological functions of the vasculature as well as hypertension and may function as the major catalytic component of an endothelial NADPH oxidase [29]. Several studies have indicated that NOX4 activity is increased in cerebral arteries during chronic hypertension, which is associated with greater production of superoxide and vasodilatation in spontaneously hypertensive rats [18,30,31]. In addition, ET-1 stimulates VCAM-1 expression in spontaneously hypertensive rats [32]. Compared with WKY rats, the expressions of NOX4 and VCAM-1 in the aortic tissues of SHR group were significantly increased (Figure 5; p< 0.05) and the expressions of NOX4 and VCAM-1 were significantly decreased after paeonol treatment (Figure 5; p < 0.05). These data demonstrated that paeonol protected endothelial cells by down-regulating the expression of NOX4 and VCAM-1 protein [6,33,34].

Figure 5. The protein expression of eNOS, NOX4, and VCAM-1 in different groups of rats. N = 10, Different alphabets indicated significant differences.

Discussion

Hypertension still remains a major risk factor of heart failure, renal failure, stroke, and myocardial infarction. However, current therapeutic strategies are often not sufficient to prevent cardiovascular events and side-effects [35–37]. Chinese herbal medicine paeonol is traditionally used in clinic in China to improve blood circulation [16]. Increased blood pressure and decreased cerebral blood flow were observed in spontaneously hypertensive rats with increased ROS generation and increased nitric oxide (NO) bioavailability in aortae, which caused damage to vascular endothelium. The present study demonstrated that chronic treatment with paeonol in vivo confers vascular protection by alleviating oxidative stress and restoring vascular endothelium.

Previous studies have shown that paeonol antagonized acute myocardial ischemia and infarction in rat [15,16]. In this study, long-term administration with paeonol at both low (LSHRT) and high (HSHRT) doses significantly decreased the blood pressure and improved cerebral blood flow compared with WKY and SHR group. Hypertension in human is associated with oxidative stress [38,39]. Paeonol has been previously suggested to attenuate oxidative stress in other disease models such as diabetes, hepatotoxicity [9,11]. In this study, paeonol treatment increased SOD content and decreased MDA content in both low (LSHRT) and high (HSHRT) group.

NO synthesized by eNOS in endothelial cells serves a key role in the cardiovascular system through dilating blood vessels to relieve hypertension [40]. Hypertension induced by NO inhibition promotes oxidative stress [41]. In the present study, the production of NO was remarkably decreased in SHR group compared with WKY group. Interestingly, paeonol treatment significantly improved serum NO level, which may be associated with the increased SOD content and decreased MDA content after paeonol treatment. The intact structure and function of vascular endothelium are critical in the regulation of vascular tone, tissue blood flow, inflammatory responses, and maintenance of blood fluidity [17]. Paeonal treatment reduced vWF, AT1, and ET-1 levels which play important roles in maintaining the intact structure and function. Therefore, paeonol treatment protected the vascular endothelium to lower blood pressure.

To further illuminate the mechanism of the protective effect of paeonol on endothelial cells, our results showed that the expression of eNOS which synthesizes NO was up-regulated and the activity of NOS was increased. However, the expression of NOX was increased in hypertension but decreased after paeonol treatment. The expression of VCAM-1, as the downstream effector of ET-1 signaling, was increased in hypertension, and paeonol treatment significantly decreased the expression of VCAM-1.

In summary, the present results demonstrated that long-term administration of paeonol in spontaneously hypertensive rats confers protection against endothelial dysfunction and normalized blood pressure by alleviating oxidative stress and protecting vascular endothelium. These data provided further evidence which supported the potential use of paeonol as a novel therapeutic agent or health supplement for patients with cardiovascular diseases, particularly in the treatment of hypertension.

Author contribution

Zhonghui Gai, Zhenxing Wang, and Qiao Zhu conducted program design, experimental operation, data analysis, manuscript writing, and manuscript review.

Lei Zhang and Jun Ma conducted experimental operation, data analysis, and manuscript writing.

Disclosure statement

No potential conflict of interest was reported by the authors.