ABSTRACT
Background
Endothelial dysfunction is a major pathophysiology observed in hypertension. Ghrelin, a key regulator of metabolism, has been shown to play protective roles in cardiovascular system. However, whether it has the effect of improving endothelial function and lowering blood pressure in Ang II-induced hypertensive mice remains unclear.
Methods
In this study, hypertension was induced by continuous infusion of Ang II with a subcutaneous osmotic pumps and ghrelin (30 μg/kg/day) was intraperitoneal injection for 4 weeks. Acetylcholine-induced endothelium-dependent relaxation in aortae was measured on wire myograph and superoxide production in mouse aortae was assessed by fluorescence imaging.
Results
We found that ghrelin had protective effects on Ang II-induced hypertension by inhibiting oxidative stress, increasing NO production, improving endothelial function, and lowering blood pressure. Furthermore, ghrelin activated AMPK signaling in Ang II-induced hypertension, leading to inhibition of oxidative stress. Compound C, a specific inhibitor of AMPK, reversed the protective effects of ghrelin on the reduction of oxidative stress, the improvement of endothelial function and the reduction of blood pressure.
Conclusions
our findings indicated that ghrelin protected against Ang II-induced hypertension by improving endothelial function and lowering blood pressure partly through activating AMPK signaling. Thus, ghrelin may be a valuable therapeutic strategy for hypertension.
Introduction
Hypertension and related complications are one of the leading causes of death worldwide (Citation1). Hypertension can cause stroke, chronic renal failure and chronic cardiac insufficiency, which bring extremely heavy economic burden and pressure (Citation2). A better understanding of the pathogenesis of hypertension is very important for the prevention and treatment of hypertension. Angiotensin II (Ang II), is a principle component of the renin-angiotensin-aldosterone system (RAAS), which-induced vascular remodeling is a major contributor to hypertension (Citation3). More importantly, vascular endothelium is the primary site of organ damage caused by hypertension (Citation4). As such, early improvement of vascular endothelial function has important clinical significance for inhibiting the progression of hypertension and related complications.
A large number of previous studies have shown that endothelial dysfunction exists in the early stage of hypertension, and endothelial function becomes worse with the progression of hypertension (Citation5,Citation6). The occurrence of endothelial dysfunction is closely related to the increase of oxidative stress (Citation7). Inhibition of oxidative stress can significantly improve endothelial function and lower blood pressure in spontaneously hypertensive rats (Citation8), obesity-related hypertensive rats (Citation9) and aged rats (Citation10), etc. Ghrelin is a growth hormone-releasing peptide produced mainly by P/D1 cells lining the fundus of the stomach, which is involved in regulation of energy balance, food intake and insulin resistance (Citation11). In recent years, studies have found that ghrelin has a cardiovascular protective effect (Citation12). Ghrelin inhibited pressure overload-induced cardiac hypertrophy by promoting autophagy via CaMKK/AMPK signaling pathway (Citation13) and restores nitric oxide availability in resistance circulation of essential hypertensive patients via inhibition of NAD(P)H oxidase activation (Citation14). At present, whether ghrelin can improve endothelial function and lower blood pressure in Ang II-induced hypertensive mice is unclear. Therefore, the primary aim of this study was to investigate the effects of ghrelin on improving endothelial function and lowering blood pressure in Ang II-induced hypertensive mice.
Materials and methods
Animal protocols
Male C57Bl/6 mice (12 weeks, n = 30) were purchased from Vital River Laboratory Animal Technology in Beijing, China. Animals were housed under a temperature-controlled room with a 12-h light cycle and provided with standard laboratory chow and tap water. All procedures and experimental protocols were approved by the Committee on Animal Care of the Chongqing Medical University, which were conformed to the NIH guidelines for the care and use of laboratory animals.
Hypertension was induced through Ang II treatment as described previous study. Briefly, osmotic pumps (Alzet Model 2002, CA, USA) were subcutaneously implanted into mice to deliver either saline (control, n = 6) or Ang II (400 ng/kg/min, MedChemExpress, Shanghai, China, n = 6) for 4 weeks. At the same time, the mice were received vehicle (saline, 1 ml/day, n = 6) or ghrelin (30 μg/kg/day, Belmont, CA, USA, n = 6) or Compound C (0.25 mg/kg/day, MedChemExpress, n = 6) by intra-peritoneal injection for 4 weeks.
Blood pressure measurement
Blood pressures including systolic blood pressure (SBP) and diastolic blood pressure (DBP) and heart rate were measured by the tail-cuff method (BP-98A, Softron, Japan) weekly from 12 weeks to 16 weeks. Briefly, the mice were placed on a heating table. After the mice were in a quiet state, blood pressure and heart rate were measured continuously for 3 times, and the average value was taken.
Analysis of oxidative stress and Ang II concentration
To assess the level of oxidative stress, the superoxide dismutase (SOD) and lipid peroxidation product malondialdehyde (MDA) in plasma were measured by using a commercial kit (Beyotime Institute of Biotechnology, Shanghai, China). Ang II concentrations in plasma were measured by using enzyme immunoassay kit (Cloud Clone, Wuhan, China).
Artery preparation and functional assay
Mice were euthanized by CO2 inhalation. The thoracic aortas from mice were removed and placed in ice-cold Krebs solution. Arteries were cleaned of adhering tissue and cut into ring segments of 2 mm in length and suspended in myograph (Danish Myo Technology, Aarhus, Denmark) for parallel studies. To measure the endothelium-dependent or -independent relaxations, the rings were incubated with acetylcholine chloride (Ach, 10−8.5–10−5M; Sigma-Aldrich) or sodium nitroprusside (SNP, 10−9–10−5M; Sigma-Aldrich). To determine the role of eNOS in the endothelium-dependent relaxations, rings with endothelium were exposed for 30 min to 100 mmol/L L-NAME (Sigma-Aldrich) before the incubation of Ach. In some experiments, the rings were incubated with compound C (10 mmol/L, AMPKα inhibitor, MedChemExpress) for 12 h before the incubation of Ach.
Dihydroethidium (DHE) staining
Frozen sections of thoracic aortas were loaded with 5 mmol/L DHE (Beyotime Institute of Biotechnology, Shanghai, China) at 37 C for 10 min in a light-ptotected humidified chamber. Reactive oxygen species (ROS) fluorescence was measured by a confocal scanning unit (Olympus) at excitation 515 nm and emission 585 nm.
Measurement of NO metabolites
The rings were incubated with Ach (10−6 M) for 5 min, rapidly dried and weighed. The incubation solution was measured for the stable end-products of NO using the nitrate reductase method (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Immunoblot analysis
Thoracic aortas homogenates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies against phosphorylated AMPKα at Thr172, t-AMPKα, phosphorylated eNOS and t-eNOS (1:500, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight and secondary antibody for 2 h at room temperature. Immunoreactive bands were visualized by chemiluminescence and protein expressions were analyzed by NIH image software.
Statistical analysis
The data are expressed as mean ± SEM. The relaxation was calculated as percentage reduction of the PE-induced contraction. Data were analyzed by one-way ANOVA, followed by Student – Newman–Keuls test. Differences between specific groups were analyzed using a non-parametric Mann – Whitney test comparing two groups. Value of P < .05 was considered significant.
Results
Ghrelin lowered blood pressure in Ang II-induced hypertensive mice
To determine whether ghrelin can reduce blood pressure in Ang II-induced hypertensive mice, the Ang II-induced hypertensive mice were treated with ghrelin by intra-peritoneal injection for 4 weeks. Our results found that Ang II induced significant increases in blood pressure and heart rate in mice, ghrelin treatment reversed the above abnormalities by lowering blood pressure and heart rate (). Ghrelin had no significant effect on blood pressure and heart rate of control mice. To determine whether ghrelin reduced blood pressure and heart rate by directly decreasing Ang II levels, we measured Ang II levels in mice with or without ghrelin treatment. The results showed that there was no significant difference in Ang II levels among all groups (), indicating that reduction of blood pressure by ghrelin was not related to the reduction of Ang II levels.
Figure 1. Ghrelin lowered blood pressure in Ang II-induced hypertensive mice. Ang II-induced hypertensive mice received ghrelin (30 μg/kg/day) by intra-peritoneal injection for 4 weeks. Systolic blood pressure (SBP) (a) and diastolic blood pressure (DBP) (b) and heart rate (c) were measured by the tail-cuff method. *P<.05 vs Con+Vel; #P<.05 vs Ang II+Vel. (d) Ang II levels in plasma were measured by enzyme immunoassay kit. Data were expressed as the means ± S.E.M (n = 6/group). *P<.05 vs Con+Vel or Con+Ghr, one-way ANOVA followed by Newman-keuls post hoc test.
Ghrelin improved vascular endothelial function in Ang II-induced hypertensive mice
Previous studies have reported that hypertension can cause vascular endothelial dysfunction, and improving vascular endothelial function is one of the important strategies for treatment of hypertension (Citation15). To assess the endothelium-dependent relaxations of thoracic aortas in Ang II-induced hypertensive mice, thoracic aortas were treated with Ach. The endothelium-dependent relaxations were attenuated in Ang II-induced hypertensive mice compared with control mice, which were reversed by ghrelin treatment (). Pretreatment with L-NAME inhibited the Ach-induced relaxation in both Ang II group and Ang II+ghrelin group that the Ach-induced relaxation was no longer different between them (). We further observed the endothelium-independent relaxations and found that no significant difference in SNP-induced endothelium-independent relaxations among all groups (). The impaired endothelium-dependent relaxation is associated with decreased production of NO. We found that the decreased NO production by measuring the nitrate/nitrite level and the activity of eNOS by measuring the phosphorylation of eNOS in Ang II-induced hypertensive mice. Ghrelin treatment reversed the decreased NO production and activity of eNOS in Ang II-induced hypertensive mice ().
Figure 2. Ghrelin improved vascular endothelial function in Ang II-induced hypertensive mice. Ang II-induced hypertensive mice received ghrelin (30 μg/kg/day) by intra-peritoneal injection for 4 weeks. Vascular endothelial function included Ach-induced relaxation (a and b) and SNP-induced relaxation (c) in thoracic aortas from control and Ang II-induced hypertensive mice. Data were expressed as the means ± S.E.M (n = 6/group). *P<.05 vs others, one-way ANOVA followed by Newman-keuls post hoc test.
Figure 3. Ghrelin increased the NO production and activity of eNOS in thoracic aortas from Ang II-induced hypertensive mice. Ang II-induced hypertensive mice received ghrelin (30 μg/kg/day) by intra-peritoneal injection for 4 weeks. NO production was assessed by measuring Ach-stimulated NO metabolites and the activity of eNOS was assessed by measuring the phosphorylation of eNOS in thoracic aortas from control and Ang II-induced hypertensive mice. (a) NO production in thoracic aortas. (b) Phosphorylation of eNOS in thoracic aortas. Data were expressed as the means ± S.E.M (n = 6/group). *P<.05 vs others, one-way ANOVA followed by Newman-keuls post hoc test.
Ghrelin reduced oxidative stress, which contributed to the improvement of endothelial function in Ang II-induced hypertensive mice
Increased levels of oxidative stress are associated with endothelial dysfunction in hypertensive animal models (Citation16). Firstly, we measured the oxidative stress indexes in plasma, including SOD and MDA levels, and the results showed that the antioxidant index SOD level was decreased, while the pro-oxidant index MDA level was increased in Ang II-induced hypertensive mice. Ghrelin treatment could significantly increase SOD level and decrease MDA level (). Furthermore, we measured ROS level of thoracic aortas and found that the ROS level was increased in Ang II-induced hypertensive mice. Treatment with ghrelin reduced the increased ROS level in Ang II-induced hypertensive mice ().
Figure 4. Ghrelin reduced oxidative stress in Ang II-induced hypertensive mice. Ang II-induced hypertensive mice received ghrelin (30 μg/kg/day) by intra-peritoneal injection for 4 weeks. Oxidative stress markers included SOD (a) and MDA (b) levels in plasma were measured by ELISA. (C1 and C2) Representative images and quantified DHE fluorescence in thoracic aortas. Data were expressed as the means ± S.E.M (n = 6/group). *P<.05 vs others, one-way ANOVA followed by Newman-keuls post hoc test.
Ghrelin reduced oxidative stress and improved endothelial dysfunction dependent on AMPK activation
Previous studies show that treatment of ghrelin protects cells against hypoxia/reoxygenation-induced cell death via AMPK pathway (Citation17). To determine the role of AMPK in ghrelin-induced decreased oxidative stress and improved endothelial function in Ang II-induced hypertensive mice, isolated thoracic aortas were stimulated by AMPK inhibitor (compound C). We found that AMPKα phosphorylation at Thr172 was decreased, which was reversed by ghrelin treatment (). Compound C significantly inhibited the effect of ghrelin-induced the reduction in ROS levels in thoracic aortas from Ang II-induced hypertensive mice (), indicating that decreased oxidative stress with ghrelin treatment was AMPK-dependent. Furthermore, compound C blocked the ability of ghrelin to improve endothelial function and blood pressure in Ang II-induced hypertensive mice (). These results indicated that ghrelin reduced oxidative stress and improved endothelial function dependent on AMPK activation.
Figure 5. The role of AMPK signaling in ghrelin reducing oxidative stress and improving endothelial function in Ang II-induced hypertensive mice. (a) the phosphorylation of AMPKα were analyzed by western blotting. Data were expressed as the means ± S.E.M (n = 6/group). *P<.05 vs others. ROS was measured by DHE fluorescence (b1 and b2) and Ach-induced relaxation (c) in thoracic aortas incubated with or without Compound C. Systolic blood pressure (SBP) (d) was measured by the tail-cuff method at the age of 16 weeks with or without Compound C treatment. Data were expressed as the means ± S.E.M (n = 6/group). *P<.05 vs Con+Vel or Ang II+Ghr, one-way ANOVA followed by Newman-keuls post hoc test.
Discussion
In this study, we demonstrated that ghrelin improved endothelial dysfunction and lowered blood pressure in Ang II-induced hypertensive mice, which was dependent on AMPK activation. To the best of the authors’ knowledge, the present study is the first to examine the effects of ghrelin on vascular endothelial function in Ang II-induced hypertensive mice.
A large number of previous studies have shown that vascular endothelial dysfunction, especially diastolic dysfunction, may be one of the initiating factors of hypertension (Citation18,Citation19). In both hypertensive patients and animal models, NO production was significantly reduced and vasodilation function was impaired (Citation20,Citation21). More importantly, studies have found that in the early stage of hypertension, there has been a microvascular diastolic dysfunction (Citation22). With the progression of hypertension, the diastolic function of blood vessels shows a progressive worsening trend, suggesting that diastolic dysfunction in blood vessels is both the cause and result of hypertension (Citation23). Therefore, improving vascular endothelial function has become one of the important strategies in the treatment of hypertension. Ghrelin is a physiologically active peptide that is secreted by P/D1 cells lining the fundus of stomach. Ghrelin plays an important cardiovascular protective role through the suppression of sympathetic activity (Citation24), alleviation of vascular endothelial dysfunction (Citation25) and regulation of inflammation (Citation26), apoptosis (Citation27) and autophagy (Citation28). Previous reports have showed that ghrelin evokes endothelium-dependent dilation in systemic arteries from humans (Citation29), coronary arteries from pigs (Citation30) and mesenteric artery from rats (Citation31). Our study found that ghrelin improved the impaired endothelium-dependent relaxations in Ang II-induced hypertensive mice. However, a few studies have failed to demonstrate vasodilator effect of ghrelin on the isolated thoracic aorta. The apparent discrepancy in different studies is likely related to different animal models.
Previous studies have shown that oxidative stress is one of the important factors causing endothelial dysfunction (Citation32). Oxidative stress can lead to the uncoupling of eNOS and the reduction of NO production (Citation33). In both hypertensive patients and animal models, oxidative stress levels are significantly increased, and antioxidant therapy can significantly reduce endothelial function and blood pressure (Citation34). Our study also found that oxidative stress levels were increased in Ang II-induced hypertensive mice, which were reversed by ghrelin treatment. Studies have shown that ghrelin exerts antioxidant effects by preventing the peroxidation and enhancing the activity of antioxidant enzymes in several tissues and organs (Citation35). Ghrelin attenuates secondary brain injury after intracerebral hemorrhage by inhibiting NLRP3 and increasing expressions of antioxidative genes (Citation36) and alleviates paclitaxel-induced peripheral neuropathy by reducing oxidative stress (Citation37). Consistent with the modulatory effect of ghrelin on the production of NO, we found that phosphorylation of eNOS and production of NO were reduced in thoracic aortas from Ang II-induced hypertensive mice and ghrelin treatment increased phosphorylation of eNOS and production of NO. Therefore, ghrelin may improve endothelial function by reducing oxidative stress and thereby increasing the phosphorylation of eNOS and production of NO.
AMPK is a key molecule in the regulation of biological energy metabolism (Citation38). It can be activated by various stimuli in the body, including cell stress, exercise and many hormones and substances that can affect cell metabolism (Citation39). Extensive studies have shown that AMPK can improve endothelial function in obese mice (Citation40), diabetic mice (Citation41) and hypertensive rats (Citation42) by reducing downstream endoplasmic reticulum stress, oxidative stress and inflammation signaling pathways. Ghrelin ameliorates vascular calcification (Citation43) and oxidative stress, and neuronal apoptosis in a rat model of neonatal hypoxic-ischemic encephalopathy through AMPK activation (Citation43). In this study, we found that AMPK was down-regulated in thoracic aortas from Ang II-induced hypertensive mice. This result agrees with previous studies showing that AMPKα phosphorylation is down-regulated in aortas from hypertensive animal models (Citation19). Ghrelin treatment increased AMPKα phosphorylation, reduced oxidative stress and improved endothelial function in Ang II-induced hypertensive mice, effects that were inhibited by AMPK antagonist Compound C. Thus, our studies provide solid evidence that ghrelin reduced oxidative stress and improved endothelial function dependent on AMPK activation.
In conclusion, administration of ghrelin reduced oxidative stress and improved endothelial function, as well as lowering blood pressure in Ang II-induced hypertensive mice, which could be mediated via the AMPK signaling. Due to limited current therapies, this study provides a basis for ghrelin as a promising therapeutic candidate for patients with hypertension.
Author contribution
DJ conceived and designed the experiments and wrote the manuscript; HY and ZY performed the experiments and analyzed the data; YF approved the final version of the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Data availability statement
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
References
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