枸杞蛋白酶解液对自发性高血压大鼠的降血压机制研究
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摘要
目的:探讨枸杞蛋白酶解液对自发性高血压大鼠(SHR)肾素血管紧张素系统(RAS)的影响及其可能的降压机制。方法:分别灌胃SPF级Wistar大鼠近交系雄性SHR大鼠生理盐水、10 mg/kg卡托普利、枸杞蛋白酶解液低剂量组(40 mg/kg)、枸杞蛋白酶解液中剂量组(80 mg/kg)、枸杞蛋白酶解液高剂量组(100 mg/kg),Wistar-Kyoto(正常大鼠,WKY)作为正常对照组灌胃生理盐水,共9周。采用酶联免疫吸附法测定其血浆中血管紧张素Ⅱ(AngⅡ)的含量,采用实时荧光定量PCR(RT-qPCR)方法测量心脏和肾脏组织中的血管紧张素转换酶(ACE)、血管紧张素Ⅱ1型受体(AT1-R)、血管紧张素转换酶2(ACE2)和Mas受体(Mas-R) mRNA的表达水平。结果:与WKY组相比,SHR大鼠的进食量显著增加而体重均显著低于WKY组(p <0.05),其收缩压(SBP)与舒张压(DBP)均显著升高(p <0.05),血浆中AngⅡ含量明显升高,ACE、AT1-R mRNA表达上调,而ACE2、Mas-R mRNA表达下调;与生理盐水处理组SHR大鼠比较,卡托普利组体重显著降低(p <0.05);卡托普利组、低、中、高剂量组SHR大鼠的SBP均显著下降(p <0.05);其中枸杞蛋白酶解液低剂量组显著降低SHR大鼠血浆中AngⅡ含量(p <0.05),下调心脏及肾脏组织中ACE mRNA表达水平(p <0.05),上调ACE2 mRNA表达水平(p <0.05)。结论:枸杞蛋白酶解液可以有效降低SHR大鼠的血压,其作用机制可能是通过抑制ACE活性,下调ACE、AngⅡ和AT1-R mRNA表达水平,上调ACE2和Mas-R mRNA表达水平起到降压作用。
Objective: The paper was to explore the effects of enzymatic hydrolysate of Lycium barbarum protein on the reninangiotensin system( RAS) in spontaneously hypertensive rat( SHR) and its possible antihypertensive mechanism. Methods: SPF wistar rat inbred line male SHR were respectively given saline solution,10 mg/kg of captopril,low-dose group of enzymatic hydrolysate of Lycium barbarum protein( 40 mg/kg),middle-dose group( 80 mg/kg),and high-dose group( 100 mg/kg) by gavage.Wistar-kyoto( normal rats,WKY) served as normal control group were given saline solution by gavage for 9 consecutive weeks.The contents of plasma angiotensin Ⅱ( Ang Ⅱ) was detected by enzyme-linked immunosorbent assay,the mRNA expression levels of angiotensin-converting enzyme( ACE),AngⅡ type 1 receptor( AT1-R),angiotensin-converting enzyme 2( ACE2) and mas receptor( Mas-R) in heart and kidney tissues were analyzed by RT-qPCR( real-time fluorescent quantitative polymerase chain reaction) method.Results: Compared with WKY,the food intake of SHR increased significantly and the body weight was significantly lower than WKY group( p < 0.05),the systolic blood pressure( SBP) and diastolic blood pressure( DBP) of SHR were significantly increased( p < 0.05). The contents of Ang Ⅱ in plasma were obviously increased. The expression of ACE and AT1-R mRNA were up-regulated,while ACE2 and Mas-R mRNA were down-regulated. Compared with saline-treated SHR,the weight of captopril-treated group decreased significantly( p < 0.05),the SBP of captopril-treated group,low-dose,middle-dose,and high-dose group were significantly decreased( p < 0.05).Among them,the low-dose group of enzymatic hydrolysate of Lycium barbarum protein could significantly decrease the contents of AngⅡ in plasma of SHR( p <0.05),down-regulation of the expression of ACE mRNA( p < 0.05) in heart and kidney tissues,and up-regulation of the expression of ACE2 mRNA( p < 0.05). Conclusion: The enzymatic hydrolysate of Lycium barbarum protein could effectively decrease the blood pressure of SHR. The mechanism might be through inhibiting the activity of ACE,down-regulation of the expression of ACE,AngⅡ and AT1-R mRNA and up-regulation of expression of ACE2 and mas-R mRNA to achieve antihypertensive effect.
Objective: The paper was to explore the effects of enzymatic hydrolysate of Lycium barbarum protein on the reninangiotensin system( RAS) in spontaneously hypertensive rat( SHR) and its possible antihypertensive mechanism. Methods: SPF wistar rat inbred line male SHR were respectively given saline solution,10 mg/kg of captopril,low-dose group of enzymatic hydrolysate of Lycium barbarum protein( 40 mg/kg),middle-dose group( 80 mg/kg),and high-dose group( 100 mg/kg) by gavage.Wistar-kyoto( normal rats,WKY) served as normal control group were given saline solution by gavage for 9 consecutive weeks.The contents of plasma angiotensin Ⅱ( Ang Ⅱ) was detected by enzyme-linked immunosorbent assay,the mRNA expression levels of angiotensin-converting enzyme( ACE),AngⅡ type 1 receptor( AT1-R),angiotensin-converting enzyme 2( ACE2) and mas receptor( Mas-R) in heart and kidney tissues were analyzed by RT-qPCR( real-time fluorescent quantitative polymerase chain reaction) method.Results: Compared with WKY,the food intake of SHR increased significantly and the body weight was significantly lower than WKY group( p < 0.05),the systolic blood pressure( SBP) and diastolic blood pressure( DBP) of SHR were significantly increased( p < 0.05). The contents of Ang Ⅱ in plasma were obviously increased. The expression of ACE and AT1-R mRNA were up-regulated,while ACE2 and Mas-R mRNA were down-regulated. Compared with saline-treated SHR,the weight of captopril-treated group decreased significantly( p < 0.05),the SBP of captopril-treated group,low-dose,middle-dose,and high-dose group were significantly decreased( p < 0.05).Among them,the low-dose group of enzymatic hydrolysate of Lycium barbarum protein could significantly decrease the contents of AngⅡ in plasma of SHR( p <0.05),down-regulation of the expression of ACE mRNA( p < 0.05) in heart and kidney tissues,and up-regulation of the expression of ACE2 mRNA( p < 0.05). Conclusion: The enzymatic hydrolysate of Lycium barbarum protein could effectively decrease the blood pressure of SHR. The mechanism might be through inhibiting the activity of ACE,down-regulation of the expression of ACE,AngⅡ and AT1-R mRNA and up-regulation of expression of ACE2 and mas-R mRNA to achieve antihypertensive effect.
引文
[1]陈伟伟,高润霖,刘力生,等.中国心血管病报告(2015)概要[J].中国循环杂志,2016,31(6):521-528.
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[3]Gu Y,Wu J. LC-MS/MS coupled with QSAR modeling in characterising of angiotensinⅠ-converting enzyme inhibitory peptides from soybean proteins[J]. Food Chemistry,2013,141(3):2682-2690.
[4]Ferreira S H.A bradykinin-potentiating factor(BPF)present in the venom of bothrops jararaca[J]. British Journal of Pharmacology and Chemotherapy,1965,24:163-169.
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[6]Majumder K,Wu J. Purification and characterisation of angiotensin I converting enzyme(ACE)inhibitory peptides derived from enzymatic hydrolysate of ovotransferrin[J].Food Chemistry,2011,126(11):1614-1619.
[7]Xie J,Chen X,Wu J,et al.Antihypertensive effects,molecular docking study and isothermal titration calorimetry assay of angiotensin I-converting enzyme inhibitory peptides from Chlorella vulgaris[J].Journal of Agricultural and Food Chemistry,2018,66(6):1359-1368.
[8]李莹,曾晓雄,周剑忠,等.泥鳅ACE抑制肽的体外活性研究[J].食品工业科技,2012,33(17):127-130.
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[13]Fraga-Silva R A,Pinheiro S V,Gonalves A C,et al.The antithrombotic effect of angiotensin-(1-7)involves masmediated NO release from platelets[J]. Molecular medicine,2008,14(1-2):28-35.
[14]Lee S H,Qian Z J,Kim S K.A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats[J].Food Chemistry,2010,118(1):96-102.
[15]Chen Q,Xuan G,Fu M,et al. Effect of angiotensin I-converting enzyme inhibitory peptide from rice dregs protein on antihypertensive activity in spontaneously hypertensive rats[J].Asia Pacific Journal of Clinical Nutrition,2007,16(S1):281-258.
[16]陈琨.枸杞ACE抑制肽的提取、分离纯化、活性测定及其结构鉴定[D].宁夏:宁夏医科大学,2016:14-24.
[17]国家卫生和计划生育委员会.GB 5009.3-2016食品中水分的测定[S].北京:中国标准出版社,2016.
[18]国家卫生和计划生育委员会,GB 5009.4-2016食品中灰分的测定[S].北京:中国标准出版社,2016.
[19]国家卫生和计划生育委员会.GB 5009.5-2016食品中蛋白质的测定[S].北京:中国标准出版社,2016.
[20]Kaszuba A M,Prejbisz A,K爧dziela J,et al.Forty-two-year-old female patient with resistant hypertension,bilateral renal fibromuscular dysplasia and intracranial aneurysm[J]. Advances in Interventional Cardiology,2016,12(4):386-388.
[21]王桃梅,赵俊香,陈琨,等.枸杞蛋白酶解液对ACE活性的抑制作用及其对高血压大鼠的降压效果研究[J].宁夏医科大学学报,2018,40(1):11-16.
[22]李小多,李学刚,张波,等.抑制血管紧张素转化酶的中药及活性成分的研究[J].食品工业科技,2013,34(1):49-52.
[23]Liu X,Zhang M,Zhang C,et al. Angiotensin converting enzyme(ACE)inhibitory,antihypertensive and antihyperlipidaemic activities of protein hydrolysates from Rhopilema esculentum[J].Food Chemistry,2012,134:2134-2140
[24]Mohebbati R,Bavarsad K,Rahimi M,et al.Protective effects of long-term administration of Ziziphus jujuba fruit extract on cardiovascular responses in L-NAME hypertensive rats[J].Avicenna Journal of Phytomedicine,2018,8(2):143-151.
[25]李鹏飞,张伟,马畅,等.自发性高血压大鼠心脏中ACE,AT1R,ACE2和MasR表达变化的研究[J].中国应用生理学杂志,2011,27(2):153-154,224.
[26]蔡冬梅,林书坡,蒋丽娟,等.替米沙坦对自发性高血压大鼠心肌ACE2表达的影响[J].山东医药,2009,49(20):43-44.
[27]Mugabo P,Raji I A. Effects of aqueous leaf extract of Asystasia gangetica on the blood pressure and heart rate in male spontaneously hypertensive Wistar rats[J]. BMC Complementary and Alternative Medicine,2013,13:283-289.
[28]Chappel M C,Ferrario C M.ACE and ACE2:Their role to balance the expression of angiotensinⅡand angiotensin-(1-7)[J].Kidney International,2006,70(1):8-10.
[29]Tanno T,Tomita H,Narita I,et al. Olmesartan inhibits cardiac hypertrophy in mice overexpressing renin independently of blood pressure:Its beneficial effects on ACE2/Ang(1-7)/Mas axis and NADPH oxidase expression[J].Journal of Cardiovascular Pharmacology,2016,67(6):503-509.
[2]Cheung I W,Nakayama S,Hsu M N,et al. Angiotensin-Ⅰconverting enzyme inhibitory activity of hydrolysates from oat(Avena sativa)proteins by in silico and in vitro analyses[J].Journal of Agricultural and Food Chemistry,2009,57(19):9234-9242.
[3]Gu Y,Wu J. LC-MS/MS coupled with QSAR modeling in characterising of angiotensinⅠ-converting enzyme inhibitory peptides from soybean proteins[J]. Food Chemistry,2013,141(3):2682-2690.
[4]Ferreira S H.A bradykinin-potentiating factor(BPF)present in the venom of bothrops jararaca[J]. British Journal of Pharmacology and Chemotherapy,1965,24:163-169.
[5]Daskaya-Dikmen C,Yucetepe A,Karbancioglu-Guler F,et al.Angiotensin-I-converting enzyme(ACE)-inhibitory peptides from plants[J].Nutrients,2017,9(4):316-355.
[6]Majumder K,Wu J. Purification and characterisation of angiotensin I converting enzyme(ACE)inhibitory peptides derived from enzymatic hydrolysate of ovotransferrin[J].Food Chemistry,2011,126(11):1614-1619.
[7]Xie J,Chen X,Wu J,et al.Antihypertensive effects,molecular docking study and isothermal titration calorimetry assay of angiotensin I-converting enzyme inhibitory peptides from Chlorella vulgaris[J].Journal of Agricultural and Food Chemistry,2018,66(6):1359-1368.
[8]李莹,曾晓雄,周剑忠,等.泥鳅ACE抑制肽的体外活性研究[J].食品工业科技,2012,33(17):127-130.
[9]Bader M. Tissue renin-angiotensin-aldosterone systems:Targets for pharmacological therapy[J]. Annual Review of Pharmacology and Toxicology,2010,50:439-465.
[10]Wu J,Liao W,Udenigwe C C.Revisiting the mechanisms of ACE inhibitory peptides from food proteins[J]. Trends in Food Science&Technology.2017,69:214-219.
[11]Cristina S E S A,Flynn J T. The renin-angiotensinaldosterone system in 2011:Role in hypertension and chronic kidney disease[J]. Pediatric Nephrology,2012,27(10):1835-1845.
[12]Vickers C,Hales P,Kaushik V,et al.Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase[J]. The Journal of Biological Chemistry,2002,277(17):14838-14843.
[13]Fraga-Silva R A,Pinheiro S V,Gonalves A C,et al.The antithrombotic effect of angiotensin-(1-7)involves masmediated NO release from platelets[J]. Molecular medicine,2008,14(1-2):28-35.
[14]Lee S H,Qian Z J,Kim S K.A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats[J].Food Chemistry,2010,118(1):96-102.
[15]Chen Q,Xuan G,Fu M,et al. Effect of angiotensin I-converting enzyme inhibitory peptide from rice dregs protein on antihypertensive activity in spontaneously hypertensive rats[J].Asia Pacific Journal of Clinical Nutrition,2007,16(S1):281-258.
[16]陈琨.枸杞ACE抑制肽的提取、分离纯化、活性测定及其结构鉴定[D].宁夏:宁夏医科大学,2016:14-24.
[17]国家卫生和计划生育委员会.GB 5009.3-2016食品中水分的测定[S].北京:中国标准出版社,2016.
[18]国家卫生和计划生育委员会,GB 5009.4-2016食品中灰分的测定[S].北京:中国标准出版社,2016.
[19]国家卫生和计划生育委员会.GB 5009.5-2016食品中蛋白质的测定[S].北京:中国标准出版社,2016.
[20]Kaszuba A M,Prejbisz A,K爧dziela J,et al.Forty-two-year-old female patient with resistant hypertension,bilateral renal fibromuscular dysplasia and intracranial aneurysm[J]. Advances in Interventional Cardiology,2016,12(4):386-388.
[21]王桃梅,赵俊香,陈琨,等.枸杞蛋白酶解液对ACE活性的抑制作用及其对高血压大鼠的降压效果研究[J].宁夏医科大学学报,2018,40(1):11-16.
[22]李小多,李学刚,张波,等.抑制血管紧张素转化酶的中药及活性成分的研究[J].食品工业科技,2013,34(1):49-52.
[23]Liu X,Zhang M,Zhang C,et al. Angiotensin converting enzyme(ACE)inhibitory,antihypertensive and antihyperlipidaemic activities of protein hydrolysates from Rhopilema esculentum[J].Food Chemistry,2012,134:2134-2140
[24]Mohebbati R,Bavarsad K,Rahimi M,et al.Protective effects of long-term administration of Ziziphus jujuba fruit extract on cardiovascular responses in L-NAME hypertensive rats[J].Avicenna Journal of Phytomedicine,2018,8(2):143-151.
[25]李鹏飞,张伟,马畅,等.自发性高血压大鼠心脏中ACE,AT1R,ACE2和MasR表达变化的研究[J].中国应用生理学杂志,2011,27(2):153-154,224.
[26]蔡冬梅,林书坡,蒋丽娟,等.替米沙坦对自发性高血压大鼠心肌ACE2表达的影响[J].山东医药,2009,49(20):43-44.
[27]Mugabo P,Raji I A. Effects of aqueous leaf extract of Asystasia gangetica on the blood pressure and heart rate in male spontaneously hypertensive Wistar rats[J]. BMC Complementary and Alternative Medicine,2013,13:283-289.
[28]Chappel M C,Ferrario C M.ACE and ACE2:Their role to balance the expression of angiotensinⅡand angiotensin-(1-7)[J].Kidney International,2006,70(1):8-10.
[29]Tanno T,Tomita H,Narita I,et al. Olmesartan inhibits cardiac hypertrophy in mice overexpressing renin independently of blood pressure:Its beneficial effects on ACE2/Ang(1-7)/Mas axis and NADPH oxidase expression[J].Journal of Cardiovascular Pharmacology,2016,67(6):503-509.