Yucinda YY Khor, Siew-Keah Lee, Dharmani Devi M, Wei Chih Ling✉

1Department of Pre-clinical Sciences, M.Kandiah Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Selangor, Malaysia

2Department of Pharmacology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia

ABSTRACT

Objective: To investigate the effect of epigallocatechin-3-gallate(EGCG) on endothelial dysfunction in spontaneously hypertensive rats (SHR).

Methods: Wistar-Kyoto (WKY) rats and SHR were divided into four groups; WKY control, SHR control and SHR treated with EGCG (50 mg/kg/day) or losartan (10 mg/kg/day).The treatment was given daily for 4 weeks by oral gavage and the blood pressure was monitored by tail-cuff method every 3 days.Acetylcholineinduced endothelium-dependent relaxations were assessed in isolated phenylephrine-precontracted aortic rings at the end of treatment.The vascular levels of reactive oxygen species, nitric oxide,tetrahydrobiopterin, and cyclic guanosine monophosphate were also measured.Moreover, the expression of angiotensin Ⅱ type 1 (AT1)receptor protein was determined.

Results: The systolic blood pressure was significantly decreased in SHR treated with EGCG.The impaired endothelium-dependent relaxation was significantly improved in aortic ring isolated from the EGCG-treated SHR group.EGCG also significantly increased the levels of nitric oxide, tetrahydrobiopterin, and cyclic guanosine monophosphate, while decreasing the level of reactive oxygen species and the protein expression of AT1 receptor in SHR.

Conclusions: EGCG attenuates endothelial dysfunction in SHR by decreasing oxidative stress and increasing vascular nitric oxide bioavailability, which may be modulated partly by inhibition of vascular AT1 receptors.An increase in endothelium-dependent relaxation may contribute to a decrease in blood pressure in hypertensive animals.

KEYWORDS: Epigallocatechin-3-gallate; Vascular protection;Antihypertension; Tetrahydrobiopterin; Cyclic guanosine monophosphate; Angiotensin type Ⅰ receptor; Spontaneously hypertensive rats

1.Introduction

Cardiovascular diseases are a major public health problem that is approaching epidemic proportions globally[1].Hypertension is a well-established risk factor for cardiovascular diseases and sooner will become a public health burden if left unattended due to increasing number of death and disability among people as the prevalence of hypertension goes higher[2].One of the variables that contribute the increase in peripheral resistance are the abnormalities in the structure and function of the vasculature driving the event of endothelial dysfunction[3].

Significance

Epigallocatechin-3-gallate (EGCG) is a polyphenol that exhibits antihypertensive effects due to its vasoprotective effect.The current work demonstrated that treatment with EGCG attenuates endothelial dysfunction and the expression of vascular AT1receptors which may contribute to the vasoprotective effect of EGCG.

Endothelial dysfunction can be defined as an imbalance in production of vasodilator and vasoconstrictor molecules, hence resulting in the loss of its normal function[4].It can be characterized by reduced release and/or the depletion of the bioavailability of nitric oxide (NO), a key regulator in the vasculature, leading to a compromised NO-cyclic GMP (cGMP) signaling pathway which causes vasodilation[5].

NO acts as an endothelium-derived hyperpolarizing factor (EDHF)which leads to vascular relaxation[6].Endothelial nitric oxide synthase (eNOS) is the main source of NO and plays a crucial role in maintaining contraction and relaxation of the vascular tissues[7].Tetrahydrobiopterin (BH4) is one of the important cofactors for the proper functioning of eNOS to generate NO from its precursor L-arginine[8].The produced NO is then released into vascular smooth muscle cells and activates the soluble guanylyl cyclase(sGC) by converting guanosine 5’-triphosphate to cyclic guanosine monophosphate (cGMP).The increased production of cGMP will induce smooth muscle relaxation, resulting in vasodilation[9].

Oxidative stress or a decrease in BH4level will disrupt the coupled state of eNOS[10].The uncoupling of eNOS is linked to the enhanced oxidation of BH4by reactive oxygen species (ROS) which is derived from vasculature nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases[11].Under this condition, O2-scavenges NO to produce the toxic radical peroxynitrite (ONOO-) which further decreases the bioavailability of NO and worsens endothelial dysfunction[12].A previous study has shown endothelial dysfunction is associated with the inhibition of oxidative stress injury and the downregulation of the angiotensin Ⅱ/AT1receptor pathway in D-galactose-induced aging rats[13].Since elevation of O2-is strongly associated with a decrease in BH4level, an increase in angiotensinⅡ type 1 (AT1) receptors will in part lead to endothelial dysfunction partly due to eNOS uncoupling.Indeed, exposure to angiotensinⅡ has been demonstrated to cause endothelial dysfunction through NADPH-derived ROS production in several previous studies.An animal study also demonstrated angiotensin Ⅱ-induced endothelial dysfunction in mouse ophthalmic arteries via activation of AT1receptor and NOX2-dependent ROS formation[14].

Tea obtained from Camellia sinensis plant is always used as a substance that promotes human health.The use of green tea is of great interest to researchers due to its good health effects and various bioactive compositions[15].Epigallocatechin gallate (EGCG), the most abundant tea polyphenol found in green tea[16], has been demonstrated to improve vascular function and has antioxidant and anti-inflammatory properties which can reduce the risk of cardiovascular disease[17].An in vivo study has demonstrated that green tea administration in spontaneously hypertensive rat (SHR)controlled the development of hypertension due to the antioxidative properties of catechins[18].Further, Álvarez-Cilleros and colleagues have proven that EGCG improved endothelial dysfunction by decreasing the production of ROS and the activation of stress-related pathways in human umbilical vein endothelial cells[19].In line with that, EGCG also alleviated endothelial dysfunction by downregulating HMGB1 in hypoxic trophoblast cells[20].Besides, Mohd Sabri et al.reported that EGCG improved vascular function by reducing oxidative stress and eNOS uncoupling, thus increasing NO production in angiotensin Ⅱ-induced hypertensive mice[21].

The activation of NADPH oxidase due to stimulation of AT1receptor is one of the main mechanisms leading to oxidative stress.Although studies have shown that treatment with EGCG attenuates endothelium dysfunction via decreasing oxidative stress and eNOS uncoupling, the modulatory effect of EGCG on AT1receptors is yet to be elucidated.Therefore, this study aimed to investigate the effect of EGCG on the modulation of AT1receptors in improving endothelial dysfunction in a genetically hypertensive animal model.

2.Materials and methods

2.1.Drugs and chemicals

Phenylephrine (PE), acetylcholine (ACh), sodium nitroprusside(SNP), and Tween-20 were purchased from Sigma Chemicals (St Louis, MO, USA).EGCG was purchased from Cayman Chemicals.NaCl was purchased from Calbiochem®Merck (Darmstadt,Germany).MgSO4, KCl, KH2PO2, glucose, and CaCl2were purchased from BDH Laboratory Supplies (Poole, UK).Bovine serum albumin was purchased from Santa Cruz (Dallas, Texas,USA).All chemicals were dissolved in deionized water.

2.2.Experimental animals and treatment

Briefly, 10-week-old male Wistar-Kyoto rats (WKY) (n=8) and SHR (n=24) were used for in vivo study.The experimental rats were purchased from the Animal Experimental Unit, Universiti Malaya, Malaysia.All animals were housed at the Animal Holding Facility, Universiti Tunku Abdul Rahman.They were maintained at a temperature of (24±1) ℃, and a 12 h:12 h light/dark cycle and had free access to standard chow and water.Four weeks of treatment were given to the animals and they were randomly divided into four groups, namely WKY control, SHR control, SHR treated with EGCG (50 mg/kg/day; Cayman Chemical Company, Ann Arbor,MI, USA), and SHR treated with losartan (10 mg/kg/day; Sigma Company, St Louis, MO, USA).

2.3.Measurement of systolic blood pressure (SBP) by tailcuff method

A modified tail-cuff method utilizing the CODA monitoring system(Torrington, CT, USA) was used to measure the SBP of the rats every three days starting from day 0 of the treatment period.All animals were trained for a week before the actual measurement of blood pressure to secure that they were adapted to the procedure.This minimized the stress associated with blood pressure measurements thus ensuring a more reproducible result.The animals were restrained and pre-heated in a chamber for 30 min before the blood pressure measurements were performed.At least five to six successive measurements were recorded and the average values of all readings were calculated and reported.

2.4.Collection of samples

The animals were sacrificed by excessive inhalation of carbon dioxide.Blood samples of the animals were collected immediately via cardiac puncture.The thoracic aorta was isolated and placed immediately in ice-cold Krebs solution (in mM: NaCl 118.93,NaHCO325.00, MgSO41.18, KCl 4.69, KH2PO41.03, glucose 11.10, CaCl22.38).After that, the adhering connective tissues and adipose tissues were removed.The aorta was cut into approximately 5 mm segments for vascular function study while a portion of the isolated aortic tissues was placed in optimal cutting temperature compound for NO and ROS measurements or snapped-frozen into liquid nitrogen and stored in -80 ℃ for further processing.

2.5.Vascular function study (organ bath)

For the measurement of isometric tension, an aortic ring was suspended in an organ bath containing 5 mL of Krebs solution maintained at 37 ℃ and continuously aerated with 95% O2and 5% CO2throughout the experiment.The change in the isometric tension was recorded using a Powerlab recording system (AD Instrument, Sydney, NSW, Australia).The rings were stretched to their optimal basal tension for 1 h to maintain the equilibrium of the rings.After that, each ring was contracted with 60 mM KCl until a stable contraction was achieved to test its viability.The rings were then washed three times with Krebs solution before the endothelial function of the endothelium was investigated by cumulative addition of ACh (3 nM-10 μM) to PE (300 nM-1 μM)-contracted rings to induce endothelium-dependent relaxation.A cumulative concentration-response curve to SNP (10 nM-10 μM) was also obtained to investigate if there are any changes in the vascular smooth muscle sensitivity to NO.

2.6.Measurement of NO level

A segment of the thoracic aorta embedded in optimal cutting temperature (OCT) compound (Sakura Finetik, AJ Alphen aan den Rijn, Netherlands) was cut into cryostat sections 10 μm thick.The prepared glass slide was washed using phosphate-buffer saline (PBS) (Sigma, Aldrich, USA) three times every 5 minutes before incubation with 5 μM DAF-FM diacetate fluorescence stain (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 ℃.After incubation, the aortic segments were washed again with PBS three times every 5 minutes to remove excessive staining dye.A cover slip was placed on the slides and the sections were observed by using a fluorescence microscope (ZIESS, Oberkochen, Germany)with excitation at 495 nm and emission at 515 nm for NO intensity detection.

2.7.Measurement of reactive oxygen species (ROS) level

A segment of the thoracic aorta embedded in OCT compound(Sakura Finetik, AJ Alphen aan den Rijn, Netherlands) was cut into cryostat sections 10 μm thick.The prepared glass slide was washed using PBS (Sigma, Aldrich, USA) three times every 5 min before incubation with 5 μM dihydroethidium (DHE) fluorescence stain (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 ℃.After incubation, the aortic segments were washed again with PBS three times every 5 min to remove excessive staining dye.A cover slip was placed on the slides and the sections were observed by using a fluorescence microscope (ZIESS, Oberkochen, Germany) with excitation at 516 nm and emission at 606 nm for ROS intensity detection.

2.8.Measurement of cGMP

The frozen samples of isolated aortic tissues were homogenized in PBS and centrifuged at 15 000 ×g for 15 min at 4 ℃.The supernatant was collected for measurement of total cGMP using commercially available ELISA assay kits (Cayman Chemical Company, Ann Arbor, MI, USA).The plate was read at a wavelength of 405 nm using a plate reader (Hidex, Turku Finland, Sweden) immediately.The results are expressed as pg/mL and compared with the controls.

2.9.Measurement of BH4 level

The frozen samples of isolated aortic tissues were homogenized in PBS and centrifuged at 15 000 ×g for 15 min at 4 ℃.The supernatant was collected for measurement of total vascular BH4level using a commercially available BH4ELISA assay kit (Elabscience, USA).The plate was read at a wavelength of 450 nm (Hidex, Turku Finland, Sweden) immediately.The results are expressed as pg/mL and compared with the controls.

2.10.Western blotting

Treated aortic tissues were homogenized in ice-cold radioimmunoprecipitation (RIPA) lysis buffer (1 μg/mL leupeptin,5 μg/mL aprotinin, 100 μg/mL phenylmethanesulfonyl fluoride,1 mM sodium orthovanadate, 1 mM egtazic acid, 1 mM ethylenediamine tetraacetic acid, 1 mM NAF, 2 mg/mL glycerol phosphate).The lysates were centrifuged and the supernatant was collected for Western blot.The protein concentration was determined by a modified Lowry assay (Bio-Rad Laboratories,Hercules, CA, USA).Twenty microgram protein concentrations for each sample were loaded and separated in a 10% sodium dodecyl sulfate-polyacrylamide gel, then transferred to an immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA,USA) at 110 V for 1 h 30 min.The blots were blocked with 5% bovine serum albumin at room temperature for 1 h.Then the blots were incubated with primary antibodies against AT1receptor (1:250,Santa Cruz Biotechnology; sc515884) and β-actin (1:10 000, Santa Cruz Biotechnology; sc-47778) at 4 ℃ overnight.The next day, the blots were washed three times with tris-buffered saline with 0.1% Tween®20 detergent and incubated with respective HRP-conjugated secondary antibodies at room temperature for 2 h.The membranes were developed with an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA), and the protein expression images were captured under c600 Ultimate Western System (Azure biosystems) (Dublin, CA, USA).The protein expression was quantified using Quantity One software (Bio-Rad Laboratories,Hercules, CA, USA).The targeted protein level was normalized to β-actin and then compared with the control.

2.11.Statistical analysis

All results were presented as mean ± standard error of mean(SEM).GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA)was used to analyze the concentration-response curves by nonlinear regression fitting.One-way ANOVA followed by Bonferroni’s multiple comparison tests (for more than two groups) was performed using the same statistical software.A P value of less than 0.05 was considered a statistically significant difference.

2.12.Ethical statement

All animal studies complied with the UK Animals (Scientific Procedures) Act 1986 which was agreed upon and approved by the Ethics Committee of Universiti Tunku Abdul Rahman (U/SERC/38/2020).

3.Results

3.1.Treatment with EGCG decreases SBP of SHR

Treatment with EGCG decreased SBP of SHR in a time-dependent manner.The basal SBP of SHR from different treatment groups was similar before treatment and was significantly higher than in WKY rats (P＜0.05) (Figure 1).There was a sustained decrease in SBP of SHR treated with EGCG and losartan from day 3 onwards.The SBP of WKY and SHR control was not significantly altered before and after treatment.

Figure 1.Average systolic blood pressure (SBP) in spontaneously hypertensive rats (SHR) treated with epigallocatechin-gallate (EGCG; 50 mg/kg/day) for four weeks.Data are expressed as mean ± SEM (n=4-6) and analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison tests.#P＜0.05 compared to WKY control, *P＜0.05 compared to SHR control.WKY: Wistar Kyoto rats.

3.2.Treatment with EGCG improves vascular relaxation in SHR

There was a significant decrease in the relaxation to and the pEC50of ACh in the aortic rings from SHR control compared to that of WKY control.Treatment with EGCG improved the relaxation to and the pEC50of ACh in aortic rings of SHR compared to that of SHR control (Figure 2A-C and Table 1).The relaxation to SNP,an exogenous nitric oxide donor, was similar in the aorta of all experimental groups (Figure 2D and E).

Table 1.Sensitivity (pEC50) to acetylcholine (ACh) and sodium nitroprusside(SNP) of aortic rings of spontaneously hypertensive rats (SHR) treated with epigallocatechin-gallate (EGCG) (Log M).

Figure 2.Relaxation curve to increasing concentration of acetylcholine (ACh) and sodium nitroprusside (SNP) of aortic rings from SHR treated with EGCG.(A)Representative tracings of ACh-induced relaxation in phenylephrine (PE)-contracted aortic rings of all experimental groups.(B) Relaxation curve and (C) area under the curve for percentage of relaxation to ACh of all experimental groups.(D) Relaxation curve and (E) area under the curve to SNP of all experimental groups.Data are expressed as mean ± SEM (n=4-6) and analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison tests.#P＜0.05 compared to WKY control, *P＜0.05 compared to SHR control.

3.3.Treatment with EGCG increases the level of vascular NO in SHR

The measurement of DAF fluorescence indicates that SHR exhibited a significantly lower level of vascular NO compared to the WKY control group (P＜0.05) (Figure 3).The intensity of DAF fluorescence was significantly higher in aortic tissues of SHR treated with EGCG compared to that of SHR control (P＜0.05), implying that four weeks of treatment with EGCG significantly increased the level of vascular NO in SHR.

Figure 3.Level of 4-amino-5-methylamino-2',7'-dichlorofluorescein diacetate (DAF) fluorescence intensity in aortic rings of SHR after treatment with EGCG(50 mg/kg/day).(A) The representative images of DAF-stained aortic rings.(B) Quantified fluorescence intensity for all treatment groups.Data are expressed as mean ± SEM (n=4-6) and analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison tests.#P＜0.05 compared to WKY control, *P＜0.05 compared to SHR control.

3.4.Treatment with EGCG decreases the level of vascular ROS in SHR

The measurement of DHE fluorescence intensity showed that SHR exhibited a significantly higher level of vascular ROS compared to the WKY control group (P＜0.05) (Figure 4).Treatment with EGCG for four weeks decreased the intensity of DHE fluorescence significantly (P＜0.05), indicating a significant decrease in the level of ROS in SHR aorta.

Figure 4.Level of reactive oxygen species (ROS) in aortic rings of SHR treated with 50 mg/kg EGCG for four weeks by dihydroethidium (DHE) fluorescence.(A) The representative images of DHE-stained aortic rings.(B) Quantified fluorescence intensity.Data are expressed as mean ± SEM (n=4-6) and analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison tests.#P＜0.05 compared to WKY control, *P＜0.05 compared to SHR control.

3.5.Treatment with EGCG increases vascular BH4 levels in SHR

The SHR control group exhibited a significantly lower level of vascular BH4compared to the WKY control group (P＜0.05) (Figure 5).Treatment with EGCG for four weeks in SHR increased the level of BH4significantly compared to non-treated SHR (P＜0.05).

Figure 5.(A) Vascular tetrahydrobiopterin (BH4) and (B) cyclic guanosine monophosphate (cGMP) levels as well as (C) angiotensin Ⅱ type 1 receptor (AT1 receptor) protein level in aortic rings of SHR treated with 50 mg/kg EGCG.Data are expressed as mean ± SEM (n=4-6) and analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison tests.#P＜0.05 compared to WKY control, *P＜0.05 compared to SHR control.

3.6.Treatment with EGCG increases vascular cGMP level in SHR

A significantly lower level of cGMP was observed in aorta of the SHR control group compared to that of the WKY control group(P＜0.05) (Figure 5).Treatment with EGCG significantly increased the level of cGMP in SHR aorta (P＜0.05).

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3.7.Treatment with EGCG decreases AT1 receptor protein expression

The SHR control group had a slightly higher level of AT1protein compared to the WKY control group with no significant difference(P＞0.05) (Figure 5).Four weeks of treatment with EGCG resulted in a decreasing trend of the AT1receptor in the aortic tissues of SHR.

4.Discussion

The present findings demonstrated that treatment with EGCG for four weeks significantly reduced SBP of hypertensive animals.This is followed by an improvement in endothelium-dependent relaxation in SHR aorta.Besides that, treatment with EGCG resulted in an increase in the level of total vascular NO and a decrease in the level of vascular ROS in SHR.In addition, an increase in the level of vascular BH4and cGMP was observed in the aortic tissues of SHR treated with EGCG.In line with these results, it is also observed that there is a decreasing trend in the protein expression of AT1receptor in SHR treated with EGCG.The current findings suggest that treatment with EGCG decreases the expression of AT1receptors,leading to a decrease in ROS level thus decreasing eNOS uncoupling via increasing the level of BH4which in turn increases the level of NO.This in part improves the vascular relaxation of SHR that contributes to a decrease in the blood pressure of hypertensive animals.

EGCG is a major catechin found in green tea.It has been demonstrated to have many beneficial effects on cardioprotection,antioxidant, and vascular remodeling[20].Since EGCG is a major component in green tea, the antihypertensive effect of green tea may be attributed to the effect of EGCG in green tea.Indeed, four weeks of treatment with EGCG has lowered SBP in hypertensive animals in our study.This result is in line with a recent study that has shown that treatment with EGCG significantly reduced SBP in angiotensinⅡ-infused hypertensive mice[21] and SHR[22].Although EGCG has been shown to exert an antihypertensive effect, its vascular protective mechanism is yet to be determined.

Hypertension is strongly associated with endothelial dysfunction[3].A study done by Lau’s team showed that SBP and body weight have been normalized and improved endothelial-dependent relaxation in C57BL/6 J tunicamycin-treated mice by 3′,4′-dihydroxyflavonol[23].Another study also showed that a decrease in SBP is followed by an increase in endothelium-dependent relaxation[24].Similarly,the present study demonstrated that a decrease in SBP of EGCGtreated SHR is accompanied by an improvement in the endotheliumdependent relaxation in SHR aorta.This is in line with a study done by Potenza and colleagues whereby 3 weeks of treatment with EGCG in SHR can improve vasodilation and insulin resistance by stimulating endothelial production of NO[25].An experiment done by Mohd Sabri and colleagues has demonstrated that 14 days of treatment with EGCG significantly improved impaired vasodilation induced by ACh in angiotensin Ⅱ-infused mice[21].These studies imply that the improvement in vascular function may partly contribute to the decrease in SBP of the treated hypertensive animals.

NO is a highly reactive molecule that plays an important role in vascular homeostasis and blood pressure regulation[26].The release of NO leads to the activation of sGC to produce cGMP in vascular smooth muscle to induce vasodilation[27].The DAF fluorescence staining on the vascular tissues of the animals in the current study has indicated that there is an increase in the level of vascular NO in EGCG-treated SHR compared to untreated SHR control.In addition to this, the current study has shown that there is an elevated level of BH4in SHR treated with EGCG.BH4is a cofactor of eNOS that is essential in the production of endogenous NO to maintain normal endothelial function[28].Depletion of BH4resulted in the uncoupled state of eNOS and thus reduced NO bioavailability[29].The elevated level of NO in SHR treated with EGCG is accompanied by an increase in the level of BH4in this current study which is tallying with the work done by Zhang and colleagues.Zhang et al.have demonstrated that 10-50 μM of EGCG treatment elevated the expression of BH4and eNOS (dimer) as well as increased NO production and attenuated ROS formation in high glucose-treated human umbilical vein endothelial cells[30].Our current data imply that treatment with EGCG can increase the level of BH4in the aortic tissues of hypertensive animals, leading to increased eNOS coupling and thus increasing production of the NO level.

The NO produced in the endothelium diffuses into the underlying smooth muscle cells and binds to the heme moiety of sGC.This leads to the production of cGMP and thus activates the cGMPdependent protein kinases and causes vascular relaxation[31].In line with the increase in the level of vascular NO and BH4, an elevated level of cGMP was observed in SHR treated with EGCG.A previous study has shown oral administration of EGCG (100 mg/kg) after 2 h increased the plasma cGMP level in both male and female C57BL/6J mice[32].Hence, our current finding implies that treatment with EGCG improves endothelial dysfunction partly via stimulation of the NO-sGC-cGMP pathway.

Excessive production of ROS leads to oxidative stress and subsequently causes vascular cell damage[33].The current study has demonstrated that there is a lower DHE fluorescence intensity in aortic ring of EGCG-treated SHR.This indicates that EGCG can scavenge free radicals and reduce the formation of ROS.This result is in line with the result obtained from DAF staining whereby there is an increase in NO level following treatment of EGCG.An increase in total vascular NO level accompanied by a decrease in the production of ROS cumulatively leads to decreased oxidative stress,thus contributing to the increase in vasodilation which partly reduces the blood pressure of SHR.The current finding is in agreement with the finding of the previous study in which two weeks of treatment with EGCG in angiotensin Ⅱ-infused mice successfully reduced the DHE fluorescence intensity, indicating a decrease in ROS formation and oxidative stress[21].

AT1receptor is a major bioactive peptide in the RAAS and its activation by angiotensin Ⅱ leads to the increased activation of NOx[34].Activation of NOx through the stimulation of AT1receptor leads to the production of O2-thus elevating the level of ROS and oxidative stress in vasculature[35].Han showed the protective effect of EGCG in vascular endothelial cells via decreasing NOx expression[36].However, there have been no studies on the effect of EGCG on the upstream of NOx activation.The current study showed treatment with EGCG reduced the expression of AT1receptor, albeit statistically insignificant, in aortic tissues of SHR compared to the untreated hypertensive animals.Hence, EGCG may also partly improve the endothelial function of SHR by attenuating AT1receptor-dependent signaling pathway.

Further, angiotensin Ⅱ-AT1receptor binding activates the proinflammatory transcription factor nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB), which is mediated by NOXderived ROS production and MAPK activation.

In summary, the findings from this study showed that EGCG improved endothelial dysfunction in hypertensive animals,accompanied by a decrease in oxidative stress and BH4-eNOS uncoupling and an increase in NO bioavailability.Besides, treatment with EGCG also caused a decrease in the protein expression of vascular AT1receptors.

Despite mixed outcomes from clinical trials of green tea and EGCG, the good safety profile of EGCG and its vasoprotective effect should provide new impetus to conduct preliminary investigations of the effectiveness of the polyphenol for treatment and prevention of hypertension.Besides, longer treatment durations of EGCG should be studied to investigate its long-term effect.Finally, other potential studies with EGCG should also examine other mechanisms including potential anti-inflammatory and the central actions of the polyphenol.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

The authors thank Universiti Tunku Abdul Rahman and Universiti Malaya for financial and material support.

Funding

This study was funded by the Universiti Tunku Abdul Rahman Research fund (IPSR/RMC/UTARRF/2019-C2/L08).

Authors’ contributions

YYYK and WCL performed the research and analyzed the data.WCL, SKL, and DDM designed the research study.WCL, SKL, and DDM contributed to essential reagents and tools.All authors wrote and reviewed the manuscript.