Identification,Inhibition Mechanism And Anti-Hypertension Molecular Mechanism Of The Angiotensin-Ⅰ Converting Enzyme Inhibitory Peptide Derived From Ganoderma Lingzhi Mycelia

As a valuable traditional Chinese medicine,Ganoderma lingzhi(G.lingzhi)is important for exploitation of innovative and natural ingredients.The existing studies are mainly related with pharmacological function and fermentation of polysaccharide and triterpene in G.lingzhi,but few researches on protein-based compound.Oligopeptide is composed of 2 to 10 amino acids.So,it has small molecular weight with advantages of good tissue permeability and weak immunogenicity,which presents greater development potential.In this study,submerged fermentation was used to prepare G.lingzhi mycelia.Natural angiotensin converting enzyme inhibitory peptide(ACEIP)in G.lingzhi mycelia was isolated and finally screened.The antihypertension effect of the obtained ACEIP from G.lingzhi mycelia was evaluated in vivo,and the regulation molecular mechanism of SYP in hypertension theraphy was further clarified.It is expected to provide a theory basis for development of G.lingzhi in innovation medicine and health food,and makes a reference for others derived from natural sources.1.Isolation and structure identification of ACEIPs from G.lingzhi myceliaThirty-two peptides were identified from G.lingzhi mycelia by liquid chromatography-mass spectrometry(LC-MS),and three peptides such as Gln-Leu-Val-Pro(QLVP),Gln-Asp-Val-Leu(QDVL)and Gln-Leu-Asp-Leu(QLDL)were obtained using a series of column chromatography.Based on ACE inhibitory activity and yield,six peptides from G.lingzhi mycelia,including Ser-Tyr-Pro(SYP),Glu-Gln-Val-Leu(EQVL),Asp-Leu-Gln-Leu-Val-Pro(DLQLVP),Pro-Leu-Glu-Leu-Val-Pro(PLELVP),Gln-Arg-V al-Cys-Glu(QRVCE)and Ala-Phe-Gly-Ser-Asn-Ala-Leu-Pro-Val(AFGSNALPV)with potent ACE inhibitory activity(≥50%)were screened for further characterization including the binding free energy and ACE inhibitory activity(IC50).It was noted that SYP showed the highest binding free energy(34.4 kcal/mol)and ACE inhibitory activity with the IC50 values of 62.50±1.81 μg/mL.Hence,SYP is expected to be an ideal antihypertension drug.2.Mechanism analysis and structure modification of the oligopeptide SYPAccording to determination of the Michaelis constant using the double reciprocal plotting method,SYP acted as a non-competitive inhibitor against ACE.Additionally,the present result of molecular docking exhibited that SYP formed a salt bond through amino group in Ser1 with Asp507 of ACE,three hydrogen bonds through hydroxyl group in Ser1 with Lys511 of ACE,phenol hydroxyl group in Tyr2 with Gln281 of ACE,carboxyhydroxyl group in Pro3 with Lys454 of ACE respectively,and generated hydrophobic interaction with Phe457,Tyr520 and Phe524 of ACE through benzene ring in Tyr2 and closed-loop in Pro3.SYP bound firmly with ACE,which of the binding site involved amino acid residues such as Gln281,Lys511 and Tyr520 in the active S2 pocket of ACE,leading to changing conformation and subsequent blockade of ACE activity.Structure modification was performed using a series of methods such as amino acetylation,carboxyl amidation and amino acid substitution to remove the formed interactions between SYP and ACE in a signal or multiple.The comparative analysis of the ACE inhibitory activity before and after modification were carried out,and circular dichroism(CD)were used to explore the changes on ACE conformation.When suppression of the formed interactions between SYP and ACE existed,ACE inhibitory activity was significantly reduced.The influence of the bonds on ACE inhibition seemed in order:salt bond>hydrogen bond>>hydrophobic interaction.Furthermore,the result of CD analysis revealed that ACE conformation was changed with a performance of the increased content of α-helix or β-turn structure and the decreased content of Rndm.coil structure,associated with ACE inhibition.Therefore,ACE inhibitory activity depended on the interactions between SYP and ACE,especially the salt bond.By binding with ACE,SYP changed the secondary structure of ACE,such as α-helix,β-turn and Rndm.coil structure,and finally deactivated ACE.3.Analysis of biostability and bioavailability of the oligopeptide SYP It was found in simulated human gastrointestinal digestion in vitro experiment that SYP remained resistant to intestinal digestion and had no obvious cytotoxicity on intestinal epithelial cells and vascular endothelial cells,although it can be degraded by pepsin,suggesting a desirable safety and potential biostability.Antihypertensive efficacy assessment of SYP in SHRs in vivo was conducted by different administration methods,including intravenous injection,intraperitoneal injection and gavage.The antihypertension effect of SYP with intravenous and intraperitoneal administration was significantly better than the gavage administration,owing to inactivation of SYP caused by gastric digestion.It was noteworthy that the decread diastolic blood pressure(37.5 mmHg)and systolic blood pressure(30.5 mmHg)was observed after intravenous injection for a short period of time(1 h).Subsequently,blood pressure presented a pick-up over time,suggesting that SYP may be degraded by protease or aminopeptidase in the body and become ineffective.It was explained in the results of blood concentration determination and serum degradation experiment.Hence,it needs to improve the bioavailability of the oligopeptide,and make it play an efficient antihypertension function in the future.4.Understanding antihypertensive molecular mechanism of the oligopeptide SYP in SHRsThe spontaneously hypertensive rat(SHR)model was used to evaluate the antihypertension effect in vivo,and the gene chip analysis was performed to explore antihypertensive molecular mechanism response to SYP.Compared with two control groups treated with saline and the deactivated peptide AC-AAA-NH2 with ACE inhibitory activity of 8.91±0.33%,SYP was injected intravenously for a long-time period of 28 days,and demonstrated an obvious decrease on systolic pressure and diastolic pressure(P<0.05).The hemodynamic and pathology analysis presented an evident with an improvement in the cardio-pulmonary function.To clarify the underlying regulation molecular mechanism,transcriptomic analysis was performed for mRNA sequence of thoracic aorta in control and SYP treatment group.27777 genes were identified in thoracic aorta of SHRs,including 115 down-regulated genes and 103 up-regulated genes.According to gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)analysis,the regulated genes were related to resin-angiotensin system(RAS)and cGMP-PKG signaling pathway.在SYP长期给药情况下,RAS通路中ACE、AT1R基因下调,而CMA1、ACE2、AP-A、PREP等基因上调,从而激活其血管舒张作用的AT2R、MAS1等受体。Due to long-term administration with SYP,the vasoconstrictor factor ACE and AT1R were inhibited,but the increased expression of the opposite genes in RAS such as CMA1,ACE2,AP-A and PREP was observed,leading to activating two receptors(AT2R and MAS 1)responsible for vasodilation.In addition,most of the related genes such as eNOS,PKG,RhoA and ROCK in cGMP-PKG signaling pathway were up-regulated,suggesting that the activated eNOS/NO/cGMP signalling pathway were responsible for activating PKG,resulting in vasodilatation by accelerating elimination of intracellular free calcium.5.Analysis of antiprevention function and molecular mechanism of the oligopeptide SYP in HEACsBased on the results of the above-mentioned gene chip,Ang Ⅰ-induced HAEC model was established for verifying potential molecular mechanism of antihypertension according to mRNA and protein expression levels of targeted genes.It was found that SYP blocked the generation of Ang Ⅱ by inhibiting ACE activity without affecting the expression level of AT1R receptor,and then activated eNOS/NO/cGMP signaling pathway to exert vasodilation function.At the same time,the Ang Ⅱ/NOX2/ROS pathway was reduced due to the decrease of Ang Ⅱ,and the NO produced by activation of eNOS/NO/cGMP pathway was not converted into ONOO-by ROS,leading to accumulation in body and exerting final vasodilatation.