| As a common cardiovascular disease,hypertension is a serious threat to human health.Long-term hypertension can cause cardiac hypertrophy and myocardial fibrosis of patients,and eventually lead to heart failure.As an independent risk factor for cardiovascular disease,the reversal of myocardial hypertrophy and myocardial fibrosis has been set as one of the main treatment goals to control the hypertension by the World Health Organization.Hypertensive myocardial hypertrophy is a progressive disease with multiple factors and multiple links.Therefore,the treatment of myocardial hypertrophy requires taking drugs for a long time.However,the side effects caused by the drug increase the risk of hypotension,hyperuricemia,and renal insufficiency.Compared with drugs,bioactive peptides have the safe and effective characteristics without side effects.According to the theory of traditional Chinese medicine,bovine bones have a variety of medicinal values and long-term use can effectively lower human blood pressure.Therefore,the effect of bovine bone collagen-derived peptides on the pathological of hypertension-cardiac hypertrophy/fibrosis and its potential mechanism is a research area which is worthy of in-depth exploration.In this project,a controllable enzymatic hydrolysis technology was used to prepare bovine bone gelatin peptides(BGP)with antihypertensive effects in vitro and in vivo.Subsequently,an animal model was used to evaluate the effect of bovine bone gelatin peptides on the development of hypertensive myocardial hypertrophy/myocardial fibrosis.Finally,the mechanism and signal regulation pathways of BGP alleviating myocardial hypertrophy/fibrosis in rats were deeply explored using proteomics,enzyme-linked immunosorbent assay(ELISA),Western Blot and other experimental methods.The specific research contents and results are as follows:1.Extraction methods,physical and chemical structure properties of bovine bone gelatinThis chapter mainly studied the effects of different pre-treatment(acid and acid-enzyme combination pre-treatment)on the extraction rate and the physical and chemical properties of bovine bone gelatin.The results of the study showed that the extraction rate of bovine bone gelatin with different acid pre-treatment was significantly different(4.76%for hydrochloric acid;4.26%for acetic acid;6.00%for citric acid).The extraction rate of bovine bone gelatin by acid-pepsin pre-treatment was higher than acid pre-treatment,and the citric acid-pepsin pretreatment with the highest extraction rate(11.75%).Scanning electron microscopy analysis showed that citric acid-pepsin pre-treatment had the greatest destructive power on the collagen fiber structure.It was worth noting that citric acid disrupted more of the collagen structure compared with acetic acid and hydrochloric acid.SDS-PAGE analysis showed that the acid-pepsin pre-treatment had a greater effect on the degradation of collagen α1-chain than acid pre-treatment.Raman spectra revealed that acid-pepsin pre-treatment induced the transformation of α-helix intoβ-sheet and random coil in collagen.Rheological analysis indicated that gelatin extracted with citric acid exhibited the highest maximum gel strength,elastic modulus,gelling and melting temperature.Overall,the bovine bone gelatin yield was affected by the type of acid.Citric acid was more suitable for the pre-treatment of bovine bone collagen fibers than hydrochloric acid and acetic acid.Acid-pepsin pre-treatment significantly increased the yield of bovine bone gelatin.The citric acid-pepsin pre-treatment had better gelatin extraction rate than acetic acid-pepsin and hydrochloric acid-pepsin pre-treatment.2.Study on the preparation,processing characteristics and anti-hypertensive effects of bovine bone gelatin polypeptideIn this chapter,bovine bone gelatin peptides(BGP)with high angiotensin converting enzyme(ACE)inhibitory activity was prepared by controlled enzymatic hydrolysis technology,and the antihypertensive effect of BGP in animals was explored.At the same time,the effects of environmental factors,in vitro gastrointestinal simulated digestion and Maillard reaction on the biological activity of BGP were further analyzed.The response surface optimization experiment results showed that the optimal enzymatic hydrolysis process parameters of the bovine bone gelatin hydrolysate were as follows:enzymatic hydrolysis time 6.74 h;substrate concentration 79.34 mg/mL;and enzyme addition 7559.92 U/g.Under this condition,the ACE inhibition rate of the bovine bone gelatin hydrolysate was 71.65%.The bovine bone gelatin hydrolysate was ultra-filtered to obtain BGP(molecular weight ≤3 kDa).Totally 162 peptide sequences were identified from BGP by Nano-LC-MS/MS,and 56.8%of which contained hydroxyproline(Hyp).In vitro,BGP had good gastrointestinal stability,acid resistance and temperature tolerance.Animal experiments showed that BGP significantly inhibited the systolic blood pressure SBP and diastolic blood pressure DBP of spontaneously hypertensive rats(SHR)within 12 h.It was worthy to notice that low concentration of NaCl(addition ≤12%)had no significant effect on the ACE inhibition ability of BGP.However,high concentration of NaCl(addition≥15%)significantly reduced the ACE inhibitory activity of BGP.Maillard reaction results showed that low addition of xylose and glucose(≤15%)significantly improved the antioxidant activity of BGP with no significant effect on the ACE inhibitory activity.However,high levels of xylose and glucose(≥25%)significantly reduced the ACE inhibition rate of BGP.Based on the above results,it can be seen that BGP has good anti-hypertensive properties in vivo,good ACE inhibitory activity and food processing stability in vitro,which proves that it has the potential to become a high-quality antihypertensive functional food.3.The effect of bovine bone gelatin polypeptide in relieving hypertension,myocardial hypertrophy and myocardial fibrosis in SHRIn this chapter,9-week-old SHR rats were used for long-term gavage experiment of BGP to evaluate the curative effect of BGP on alleviating and reversing hypertension,myocardial hypertrophy and myocardial fibrosis in SHR rats.A non-invasive tail artery blood pressure meter was used to track the SBP and DBP during gastric administration in rats.Biochemical analysis and histopathological observation were used to evaluate the efficacy of BGP in alleviating the pathological symptoms of hypertension-myocardial hypertrophy/fibrosis in SHR rats.The results of blood pressure tracking showed that BGP could stably inhibit the increase of SBP and DBP of SHR rats during the gavage period(10 weeks).After the 10-week gavage experiment,compared with normal blood pressure rats(WKY),SHR rats showed significant myocardial hypertrophy and myocardial fibrosis pathological symptoms:The heart mass index(HMI),left ventricular mass index(LVMI),myocardial hypertrophy markers(atrial natriuretic peptide ANP,brain natriuretic peptide BNP),myocardial cell diameter,and myocardial interstitial collagen area of SHR rats were significantly higher than that of WKY rats.The treatment of BGP significantly alleviated and controlled the continuous development and deterioration of the pathological process of myocardial hypertrophy/fibrosis in SHR rats,and the therapeutic effect of BGP was dose-dependent.The specific research results are as follows:high-dose BGP treatment significantly reduced HMI,LVMI,ANP and BNP levels of SHR rats(P<0.05).HE and Masson staining showed that the diameter of cardiomyocytes and the area of interstitial fibrosis of SHR rats in high-dose BGP group were significantly lower than those in SHR control group.At the same time,Hyp concentration analysis showed that the Hyp content in the myocardial tissue of SHR rats in the high-dose BGP group was significantly lower than that in the SHR control group.In addition,BGP also significantly reduced the low-density cholesterol levels in the serum of SHR rats.In summary,BGP significantly inhibited the increase of blood pressure and the continuous development and deterioration of myocardial hypertrophy and myocardial fibrosis in SHR rats.4.The mechanism of bovine bone gelatin polypeptide alleviating and controlling the progression of myocardial hypertrophy and myocardial fibrosis in SHR ratsThis chapter mainly studied the potential mechanism of BGP alleviating and controlling the development of myocardial hypertrophy and myocardial fibrosis in SHR rats with the help of Label-free proteomics,Western blot and ELISA technology.The differentially expressed proteins identified by Label-free proteomics indicated that long-term use of BGP changed the protein expression in the left ventricle of SHR rats and the therapeutic effect of high-dose BGP was better than low-dose BGP.Go analysis,KEGG pathway analysis,and protein interaction analysis showed that the 37 differentially expressed proteins in the high-dose BGP group were involved in multiple signal transduction pathways related to myocardial hypertrophy and myocardial fibrosis,which indicated that BGP protected the heart of SHR by targeting and regulating multiple signaling pathways at protein levels.Western blot and ELISA analysis results showed that high-dose BGP treatment significantly inhibited the expression levels of ACE,angiotensinⅡ(Ang Ⅱ),angiotensin Ⅱ type 1 receptor(AT1R),phosphoinositide 3-kinase(Pi3k),phosphorylated protein kinase B(p-Akt)and transforming growth factor β1(TGF-β1)in myocardial tissue of SHR rats.Based on the above results,it can be seen that BGP could alleviate and control myocardial hypertrophy and myocardial fibrosis by inhibiting the ACE/Ang Ⅱ/TGF-β1 and Pi3k/Akt signaling pathways in SHR rats.The results of this chapter provide a theoretical basis for BGP to prevent and treat hypertensive myocardial hypertrophy/fibrosis.5.Identification of bovine bone gelatin ACE inhibitory peptide and its potential antihypertensive activity mechanismIn this chapter,we investigated the antihypertensive effects and mechanism of novel ACE inhibitory peptides purified and identified from BGP.Eight ACE inhibitory peptides were identified from BGH,among which RGL-(Hyp)-GL and RGM-(Hyp)-GF exhibited high ACE inhibition in vitro with IC50 values of 1.44 μM and 10.23 μM.The results of in vivo antihypertensive experiments showed that RGL and RGM significantly reduced the SBP of SHR rats.The maximal decrements in systolic blood pressure(SBP)in SHR rats induced by one-time gavage of RGL and RGM at 30 mg/kg were 31.3 and 38.6 mmHg.In addition,RGL had higher enzyme degradation resistance than RGM in vitro incubation in rat plasma.LC-MS identification results showed that RGL and RGM were sequentially degraded into GL-Hyp-GL,L-Hyp-GL and GM-Hyp-GF,M-Hyp-GF during the incubation of rat plasma within 2 h.Molecular docking predicted that RGM and the ACE residues of Glu3 84,His513 and Lys511 formed hydrogen-bonding interactions.RGL formed hydrogen bonds with Lys511 and Tyr523 and generated hydrogen-bonding interactions with His387 and Glu411 in the zinc(Ⅱ)complexation motif of ACE.In summary,RGL and RGM have good antihypertensive effects,both of which modulate the catalytic activity of ACE by changing the active conformation of ACE through the force of hydrogen bonding. |
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