APP下载

Interaction mechanism of egg white-derived ACE inhibitory peptide TNGIIR with ACE and its effect on the expression of ACE and AT1 receptor

2020-05-28ZhipengYuHuiGuoDvidShiunChensiXiWenzhuZhoLongDingFupingZhengJingboLiu

食品科学与人类健康(英文) 2020年1期

Zhipeng Yu,Hui Guo,Dvid Shiun,Chensi Xi,Wenzhu Zho,*,Long Ding,Fuping Zheng,Jingbo Liu,*

a College of Food Science and Engineering,Bohai University,Jinzhou 121013,PR China

b Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Technology and Business University(BTBU),Beijing 102488,PR China

c Lab of Nutrition and Functional Food,Jilin University,Changchun 130062,PR China

Keywords:

ABSTRACT

1.Introduction

Hypertension is the most common health problem and is a risk factor for cardiovascular disease.The development of hypertension involves a complex interaction of genes and improper life style.It has been proposed that abnormalities in the intrarenal renin-angiotensin system(RAS)are one of the major mechanisms of hypertension[1].Abnormalities in the renin-angiotensin system(RAS)play the key role in the development of hypertension.A plasma sodium concentration lower than normal triggers the renal juxtaglomerular cells to release renin into the circulation where this enzyme cleaves a decapeptide from angiotensinogen(a plasma protein produced by the liver with its first 12 amino acids critical for activity).The short peptide angiotensin I(Ang I,10 amino acids long)is then further cleaved by the enzyme ACE(angiotensin converting enzyme,secreted by the lungs and the epithelial cells of the kidneys)to form the octapeptide angiotensin II(Ang II),which causes arterioles to constrict and increase arterial blood pressure[2].Ang II also stimulates the secretion of aldosterone,causing the tubular epithelial cells of the kidneys to increase the reabsorption of sodium ions from the tubular fluid back into the blood.Therefore,all of these components of the RAS have become targets for developing anti-hypertensive drugs because they can effectively control high blood pressure.

Because ACE is an important component in the intrarenal RAS,synthetic ACE inhibitors have been frequently utilized to treat patients with hypertension and other related diseases.Recently,there has been growing interest in using bioactive peptides as antihypertensive functional food products to lower high blood pressure[3],and most of them target ACE.Many ACE inhibitors have been isolated from various foods[4].Most of them function in vitro,and some even exhibit antihypertensive potential in vivo[5,6].

Of these,the egg white-derived peptides have been extensively studied.Some of them such as RADHPFL,RADHPF,YAEERYPIL,IVF,RADHP,YPI,RVPSL,and IQW also exhibit significant antihypertensive activity in spontaneously hypertensive rats(SHR).Wu et al.[7]evaluated the antihypertensive effects of hen egg ovotransferrinderived tripeptide IRW,which reduced Ang II synthesis as well as endothelial cell inflammation and endothelial dysfunction.Miguel et al.[8]found that the inhibition of ACE activity in vivo plays an important part in the antihypertensive effect produced by hen egg protein hydrolyzed with the typical enzyme pepsin in SHR.However,the ACE inhibitory activity does not necessarily correlate well with the antihypertensive potential,and some peptides with significant ACE inhibitory activity showed low or no antihypertensive effects in SHR by oral administration.

We previously found that the egg white-derived hexapeptide TNGIIR(Thr-Asn-Gly-Ile-Ile-Arg)exhibited significant ACE inhibitory activity[9].Because the ACE inhibitory activity does not guarantee an antihypertensive potential,the in vivo antihypertensive effect of ACE inhibitory peptides on SHRs should be validated.

In this study,we investigated the potential antihypertensive effect of orally administered TNGIIR in SHR,the concentration of circulating Ang-I and Ang-II,and the mRNA expression of ACE and the angiotensin type 1(AT1)and type 2(AT2)receptors in rat kidney.Molecular docking experiments were used to examine the interaction of TNGIIR with ACE.

2.Materials and methods

2.1.Animals

The SHRs(aged 10 weeks and weighing 210-260 g),the most widely studied model of human essential hypertension,and Wistar rats were supplied by Beijing Vital River Laboratory Animal Technology Co.,Ltd(Beijing,China).SHRs and Wistar rats were housed in an air-conditioned room with a 12 h light-12 h dark cycle,tap water and standard diets were consumed ad libitum[10].Solutions(1 mL)were administered orally to all animals using disposable plastic syringes.

2.2.Synthesis of peptide TNGIIR identified from egg white

TNGIIR was synthesized chemically and its structure confirmed by LC-MS analysis[9].

2.3.In vitro ACE activity assay

The ACE activity of peptide TNGIIR was assayed using HPLC[11].

2.4.Potential antihypertensive effect of TNGIIR

All animal experiments were approved by the Institutional Animal Care and Use committee of Jilin University(Approval No:201702003),and all efforts were made to ameliorate the suffering of animals.Thirty SHRs and twelve normotensive Wistar rats were acclimated to measurements of systolic blood pressure(SBP)[12].SHRs with tail SBP≥180 mm Hg were randomly divided into five groups with six rats per group.The negative group of rats was treated with normal saline,and the positive group was treated with captopril(10 mg/kg body weight).The low,medium,and high dose treatment groups received 2 mg/kg of TNGIIR,10 mg/kg of TNGIIR,and 50 mg/kg of TNGIIR,respectively.

2.5.Evaluation of blood pressure

Indirect evaluation of blood pressure was performed with a blood pressure monitor using previously-established methods[12].

2.6.Effects of peptide TNGIIR on gene expression in kidney

Kidney tissues of SHRs were processed,and first strand cDNA was synthesized as previously described[12].RT-PCR was applied for ACE,AT1 and AT2 receptors,and glyceraldehyde-3-phosphate dehydrogenase(GAPDH),with Premix Ex TaqTMKits as previously described[12].The amounts of ACE,AT1,and AT2 mRNA were assayed and are expressed as a relative expression ratio versus GAPDH mRNA.

2.7.Quantitation of Ang I and Ang II

The serum Ang I and Ang II concentrations were measured with the iodine angiotensin I radioimmunoassay kit,iodine angiotensin II radioimmunoassay kit[12](Beijing North Biotechnology Research,Beijing,China)according to the guidelines of the manufacturers,respectively.

2.8.Molecular docking of hexapeptide TNGIIR and ACE

The crystal structure of ACE(PDB ID:1O86)was used as a receptor to conduct docking studies.The structure of hexapeptide TNGIIR was generated using Discovery Studio 2017 Client software(DS),and the energy was minimized using the CHARMm force field.Molecular docking was applied using the CDOCKER tool of DS.The center of the input site sphere was positionedatcoordinates41.849,36.245,48.185,with a radius of 9.0Å.The CDOCKER Energy of the top 10 conformations was calculated.Furthermore,DS was used to identify the hydrogen bonds and the coordination interactions between the residues in the ACE(PDB ID:1O86)active site.

2.9.Statistical analysis

The programs Origin 8.0 and SPSS 13.0 were used for the major data processing throughout this work.Statistical significance was set at P<0.05.

3.Results and discussion

3.1.In vivo antihypertensive effects of hexapeptide TNGIIR

The peptide TNGIIR was synthesized,and its purity was proven to be 97.4%.As shown in Fig.1,the LC-MS results for the hexapeptide TNGIIR confirmed its molecular weight and amino acid sequences.Prior to administration,the SBP value of SHRs was determined to be(180±5)mm Hg.The SBPs of the SHRs in different groups after short-term administration are shown in Fig.2A.The results indicated that the oral administration of saline in SHRs did not lower the SBP values.From the fifth day after administration,the SBP of the captopril group was significantly lower than that of the saline group.The strongest antihypertensive effect was observed on the sixth day,when the values of reduced SBP for the positive group were(18±1)mm Hg lower than those of the original.

In our long-term experiments(Fig.2B),captopril(10 mg/kg bw)exerted the strongest SBP reduction effects compared to all treatment groups.In the fourth week,the SBP reduction by captopril exceeded(55±6)mm Hg compared to the saline group.However,the reduction of SBP in the treatment groups was not significantly higher than that of the saline group from the first week through the fourth week.The results demonstrated that peptide TNGIIR did not significantly lower the SBP of SHRs during short-and long-term administration,while our previous work showed that TNGIIR exerted high activity against ACE.However,some other hexapeptides with in vitro ACE inhibitory activity exerted antihypertensive effects.Some results also suggested that the DBP in SHRs that received ADVFNPR,VVLYK,and LPILR was significantly decreased(P<0.05),However,the order of antihypertensive effect of these peptides was not in accordance with their ACE inhibitory activity in vitro[13].The phenomenon indicated that ACE inhibitors with different biological characteristics expressed different blood-pressure lowering effects.In addition,absorption of bioactive peptides involves one or more routes,such as passive transport,active transport,or transcytosis.The absorbed peptides may be either hydrolyzed in cells from peptidase activity or may resist proteolysis and be transported via a basal transporter[14].In normotensive Wistar rats,none of the treatment or positive groups exhibited any significant blood pressure effects(data not shown).

Fig.1.The mass spectrum and chemical structure of the hexa-peptide TNGIIR(Thr-Asn-Gly-Ile-Ile-Arg)isolated from egg white protein.

Fig.2.The effects of ACE inhibitors on systolic blood pressure(SBP).The SHRs were treated with the hepta-peptide TNGIIR(2,10 mg/kg,and 50 mg/kg),captopril(10 mg/kg),or saline.(A)Short-term treatment,1-6 days.(B)Long-term administration,1-4 weeks.*P<0.05 compared to saline-treated rats.

3.2.Effects of TNGIIR on the gene expression of ACE and the AT1 and AT2 receptors

Evidence has been recently emerging that the occurrence of high levels of ACE and the AT1 receptor and/or AT2 receptor implies their vital roles in the vascular RAS.To understand the actions of peptide TNGIIR on the expression of ACE and the AT1 and AT2 receptors,mRNAs were determined by real-time PCR.To establish the antihypertensive effects of ACE inhibitory peptide TNGIIR in kidney tissue,relative ACE and AT1 and AT2 receptor mRNAs were determined for long-term animal experiment.The relative ACE,AT1,and AT2 receptor mRNA levels in SHR kidneys were quantified by real-time PCR(shown in Fig.3A,B and C).A significant decrease in relative ACE and AT1 receptor mRNA levels in the treatment group with oral administration of TNGIIR(50 mg/kg bw)and in the captopril group(10 mg/kg bw)were revealed.However,as compared with those of the positive group,the relative ACE mRNA level in rats influenced by TNGIIR was significantly higher.

Currently,the standardized therapy for treating hypertensive patients includes AT1 receptor blockers with ACE inhibitors.It has been demonstrated that Ang II acts mainly at the AT1 receptor in rodents and humans and is antagonized by ACE inhibitors;thus,AT1 inhibitors are termed as angiotensin receptor blockers[15].The AT2 receptor mRNA levels under TNGIIR and captopril treatments,however,maintained contrary trends compared to those of ACE and the AT1 receptor.It has also been reported that SBP increases when the AT2 receptor is absent[16,17].However,other studies suggested that blood pressure was unaffected in the absence of AT2 receptor[18,19].Thus,an intriguing question arises regarding the role of the AT2 receptor in hypertension regulation.The peptide TNGIIR did not significantly lower blood pressure,but it affected ACE,AT1,and AT2 gene expression levels.The stability of mRNA also depends on growth conditions,environmental signals,and the efficiency of translation.Furthermore,we do not know how the peptide TNGIIR changed the concentrations of ACE,AT1,and AT2.Further study is required to investigate the effects of hexapeptide TNGIIR on the concentrations of ACE,AT1,and AT2.

Fig.3.The effects of the hepta-peptide TNGIIR(10 mg/kg)on the relative mRNA levels of(A)ACE,(B)AT1 receptor,and(C)AT2 receptor in the SHR kidney by real-time PCR,comparing with that of GAPDH(glyceraldehyde-3-phosphate dehydrogenase),using captopril(10 mg/kg)and saline as the positive and negative controls,respectively.*P<0.05 compared to saline-treated rats.#P<0.05 compared to positive controls.

Fig.4.The concentrations of Ang I and Ang II in the serum of SHRs,determined and quantified by the radioimmunoassay(RIA)counter.*P<0.05 compared to salinetreated rats.#P<0.05 compared to positive controls.

3.3.Effects of TNGIIR on the concentrations of Ang I and Ang II of the RAS

It is well known that ACE exists in the vascular endothelium of various organs and converts Ang I to Ang II,which also participates in the regulation of blood pressure[12].Thus,the serum concentrations of Ang I and Ang II were evaluated and quantified.No significant difference was observed in the Ang I concentrations between the treatment groups and control groups(shown in Fig.4).The levels of Ang II in the positive and treatment groups significantly decreased by 9.5% and 10.3%,respectively,versus that of the negative group.The tendency of change in serum Ang II concentrations was similar to that described in a previous study[12,20].Similarly,IQP(Ile-Glu-Pro),derived from spirulina(Spirulina platensis),has also shown significant regulation of the expression of Ang II of the RAS accompanying the blood pressure reduction[20].In addition,there was no significant difference between the captopril group and treatment group for Ang II concentrations in serum.The effects of egg white-derived peptide TNGIIR on major RAS components were also evaluated,and the results suggested that Ang II levels of the positive group significantly decreased[12].

3.4.Insight into the molecular docking simulation

Peptide TNGIIR was docked into the site pockets of ACE(PDB:1O86)through CDOCKER to explore the ACE-TNGIIR interaction mechanism(shown in Fig.5).The three-dimensional structure of the TNGIIR-ACE is shown in Fig.5A,where peptide TNGIIR is surrounded by an electron cloud of hydrogen bond interactions(Fig.5B).There are eight hydrogen bonds between peptide TNGIIR and the amino acid residues Gln 281,Tyr 520,Glu 162,Ala 354,Ala 356,Lys 511,and Arg 522 of ACE.Moreover,research has also revealed that six classical hydrogen bonds were linked between VHW and the residues His 353,Ala 354,Glu 384,Glu 411,and Tyr 520 of ACE[21].These results suggested that the hydrogen bonds were the main interaction forces that formed a stable TNGIIR-ACE complex.Interactions of the peptide TNGIIR and residues of ACE involved a salt bridge,carbon hydrogen bond,attractive charge,conventional hydrogen bond,and alkyl and Pi-alkyl interactions(Fig.5C).There are three main active site pockets in a molecule of ACE,including the S1 pocket(i.e.,Ala 354,Glu 384,and Tyr 523),S2 pocket(i.e.,Gln 281,His 353,Lys 511,His 513,and Tyr 520),and S1′pocket(Glu 162)[22].The molecular docking study revealed that TNGIIR established hydrogen bonds with the S1(Ala 354)pocket,S2 pocket(Gln 281,His 513,Tyr 520,and Lys 511),and S1′pocket(Glu 162)of ACE.Particularly,peptide TNGIIR can coordinate directly with Zn(A:701)atoms via metal-acceptor interaction.Similarly,lisinopril and other ACE inhibitory peptides were able to interact with the residues of three ACE pockets.Previous work has demonstrated that the ACE inhibitory peptide was able to interact with at least one residue of the three ACE pockets[23].These results indicated that ACE inhibitory peptides effectively interacted with the key residues of the ACE active site,which greatly contributed to its activity(Table 1).

Fig.5.Interaction between peptide TNGIIR and ACE(PDB:1O86)amino acids.(A)3D diagram of molecular docking of TNGIIR at the ACE active site complex.(B)The binding mode of the TNGIIR-ACE complex surrounded by H bonds.(C)3D diagram of molecular docking of TNGIIR at the ACE active site complex.

Table 1 Primers and probes used in real-time PCR.

4.Conclusion

At doses of 50 mg/kg,excellent activity against ACE in vitro was exhibited by the egg white-derived peptide TNGIIR.An excellent inhibitory effect on ACE and AT1 receptor mRNA expression in SHR was observed,and the peptide also significantly decreased the serum Ang II concentration.However,the relative mRNA levels for the AT2 receptor treated with TNGIIR exhibited contrary tendencies compared to ACE and the AT1 receptor.The results also suggested that ACE inhibitors with different biological characteristics expressed different lower blood pressure effects.Furthermore,the exact role of the AT2 receptor in hypertension regulation deserves to be further investigated.Although most ACE inhibitory peptides were characterized based on in vitro ACE inhibitory activity,a relationship between in vitro ACE inhibition and the in vivo antihypertensive effect is not apparent,indicating the involvement of other mechanisms of action.Future research is expected to focus on emerging antihypertensive mechanisms of ACE inhibitory peptides.

Declaration of Competing Interest

None.

Acknowledgement

This study was supported by the National Natural Science Funds of China(No.31901635)and Beijing Advanced Innovation Centre for Food Nutrition and Human Health(Grant No.20181036).