【作者】 隋海霞；

【导师】 荫士安； 严卫星； 徐海滨；

【作者基本信息】 中国疾病预防控制中心 ， 营养与食品卫生学， 2009， 博士

【摘要】 背景芹菜素（apigenin）,又称芹黄素,是天然存在的一种黄酮类化合物,广泛存在于多种水果、蔬菜和豆类中,其中芹菜中含量最高。人类食用黄酮类化合物的历史悠久,然而,高剂量的摄入黄酮类化合物,反而会作为促氧化剂,形成脂质过氧化物,诱导基因突变,引起DNA损伤和断裂。芹菜素可能会对人体产生的潜在毒性研究资料尚未见报道。高血压是严重威胁人类健康的心血管疾病之一,它是许多疾病发病和致死的主要危险因素,如心、脑血管病和肾脏病等。因此,对高血压病及其并发症的防治具有重要的临床意义。SHR大鼠长期压力超负荷对心肌细胞、血管内皮细胞等的作用在许多方面与人类高血压病相似。芹菜素具有抗炎、降压、抗过敏等多种生物学作用,然而,对其研究多集中在体外研究中,关于芹菜素降血压量效关系及作用机制的研究,尚未见报道。芹菜素对SHR血压调节机制的研究,将为芹菜素的合理使用提供科学的实验依据。目的研究大量摄入芹菜素的安全性,芹菜素降血压的量效关系并探讨其降压的可能机制,为其合理食用及开发利用提供科学依据。方法1芹菜素安全性研究根据食品毒理学安全性评价程序和方法,评价摄入芹菜素的安全性（包括急性毒性试验、遗传毒性试验、亚慢性毒性试验等）。2芹菜素降血压作用及其机制的实验研究2.1芹菜素对SHR大鼠血压的作用及其机制的实验研究选用12周龄SPF级雄性自发性高血压大鼠（spontaneously hypertensive rats,SHR）大鼠,根据血压和体重随机分为空白对照组、卡托普利阳性对照组（12mL/kgbw）以及0.007、0.026、0.104、0.417g/kg bw共4个芹菜素剂量组,实验周期为4周,每周测量收缩压和心率1次,称重2次。确定芹菜素降血压的量效关系。在上述试验末期,将所有大鼠用1%戊巴比妥钠腹腔注射麻醉后,抽取约6mL动脉血,分别研究芹菜素对SHR大鼠血浆肾素-血管紧张素-醛固酮系统（renin-angiotensin-aldosterone system,RAAS）、血压可能的敏感指标,包括尿酸（uric acid,UA）、C-反应蛋白（C-reactive protein,CRP）、白介素-6（interleukin,IL-6）以及对SHR大鼠体内氧化应激的影响;研究芹菜素对SHR大鼠左室肥厚的影响;利用实时定量PCR以及免疫组织化学方法研究芹菜素对SHR大鼠肾脏RAAS的影响。2.2芹菜素舒张血管作用及其机制的实验研究腹腔注射1%戊巴比妥钠麻醉大鼠,迅速游离大鼠胸主动脉,置于4℃含95%O₂和5%CO₂混合气体预饱和的Kreb液中,剔除周围结缔组织,剪成3～4mm的血管环。据实验需要,采用棉签磨擦血管环内表面的方法,将部分血管环制备成去除内皮的模型。血管环悬挂于20mL 95%O₂和5%CO₂饱和的37℃Kreb液中,以BIO-PAC MP150系统记录血管环张力,研究芹菜素对苯肾上腺素（phenylephrine,PE）预收缩主动脉环张力的作用、各种内皮阻断剂对芹菜素舒张血管作用的影响、钾通道阻断剂对芹菜素舒张血管作用的影响以及芹菜素舒张血管与钙通道的关系。结果1芹菜素安全性研究1.急性毒性试验:大鼠的MTD大于8g/kg bw。2.遗传毒性试验:结果为阴性。3.亚慢性毒性试验:未见芹菜素对大鼠体重、摄食量、食物利用率、血常规、血生化、尿常规有生物学意义的影响。芹菜素对大鼠脏器无明显影响。雄性8g/kg bw剂量组血清及2g/kg bw剂量组肝脏的GST显著低于对照组（P＜0.05或0.01）:雌雄4g/kg bw剂量组血清GSH-Px及雄性2、4g/kg bw剂量组的肝脏SOD、GSH-Px显著低于对照组（P＜0.05）。2芹菜素降血压作用及其机制的实验研究2.1芹菜素对SHR大鼠血压的作用及其机制的实验研究1.灌胃2周后,卡托普利阳性对照组和0.417g/kg bw芹菜素剂量组血压显著低于对照组（P＜0.05）;自灌胃第3周开始,卡托普利阳性对照组、0.026、0.104、0.417g/kg bw芹菜素剂量组的血压均显著低于对照组（P＜0.05）。各剂量组大鼠心率、体重与对照组比较均无显著性差异（P＞0.05）。2.芹菜素对SHR大鼠血浆RAAS的影响12mL/kg bw卡托普利阳性对照组、0.417g/kg bw芹菜素剂量组的血浆AngⅡ、ACE明显低于对照组（P＜0.05）,12mL/kg bw卡托普利阳性对照组的Ald显著低于对照组（P＜0.05）。3.芹菜素对SHR大鼠肾脏ACE2、ACE、eNOS、ET-1的影响给与芹菜素4周后,卡托普利阳性对照组和0.417g/kg bw芹菜素剂量组ACE2mRNA、eNOS mRNA的转录水平明显升高（P＜0.05）,ACE、ET-1 mRNA的转录水平明显降低（P＜0.05）。免疫组化测定的蛋白表达情况与基因表达情况基本一致。4.芹菜素对SHR大鼠血浆部分舒缩因子的影响0.417g/kg bw芹菜素剂量组的降钙素基因相关肽（calcitonin gene relatedprotein,CGRP）、eNOS、NO、0.026、0.104、0.417g/kg bw芹菜素剂量组的cGMP及NO/ET明显高于对照组（P＜0.05）,12mL/kg bw剂量组的卡托普利及0.007、0.104、0.417g/kg bw芹菜素剂量组的NO/AngⅡ明显高于对照组（P＜0.05）。各剂量组ET-1及PGI₂与对照组比较均无显著性差异（P＞0.05）。5.芹菜素对SHR大鼠体内氧化应激的影响灌胃芹菜素4周后,0.417g/kg bw芹菜素剂量组血浆O₂^-的含量明显低于对照组,血浆SOD的活性明显高于对照组（P＜0.05）。6.芹菜素对SHR大鼠左室肥厚的影响给予芹菜素4周后,与对照组比较,各剂量组左室肥厚指数（left ventricularhypertrophy index,LVHI）、心脏系数虽然有不同程度的下降,但均无显著性差异（P＞0.05）。7.芹菜素对SHR大鼠血浆UA、CRP、IL-6的影响给予芹菜素4周后,各剂量组血浆UA、CRP、IL-6与对照组比较,均无显著性差异（P＞0.05）。2.2芹菜素舒张血管作用及其机制的实验研究1.芹菜素不影响血管环的静息张力。2.芹菜素在有或无内皮的血管上均可剂量依赖性地减小PE预收缩的血管张力,但是在内皮完整的血管上,这一作用显著大于去内皮血管。内皮完整组的E_max=76%,EC₅₀=4.55×10^-6M,而去内皮组的E_max=44%,EC₅₀=4.84×10^-5M。3.用L-NAME、MB孵育内皮完整的血管环后,可明显抑制芹菜素引发的血管舒张,与未经处理的内皮完整组相比有显著性差异（P＜0.05）。而预先给予吲哚美辛并不能抑制芹菜素引发的血管舒张作用。4.电压敏感型K⁺通道抑制剂4-AP、线粒体ATP敏感K⁺通道抑制剂5-HD、Ca²⁺激活的K⁺通道抑制剂TEA、内向整流型钾通道抑制剂BaCl₂均能显著抑制芹菜素对PE预收缩去内皮血管的舒张作用（P＜0.05）。而非特异K_ATP抑制剂格列苯脲对芹菜素舒张血管的作用则没有影响。5.在无钾环境下,芹菜素对PE引起的收缩有显著抑制作用（P＜0.05）。芹菜素对高钾引起的血管收缩具有显著的抑制作用（P＜0.05）。无钙环境下,当PE预收缩达平台后再逐渐加入钙,芹菜素可显著降低加入Ca²⁺引起的血管张力的升高（P＜0.05）。结论1.芹菜素的NOAEL为2g/kg bw,芹菜素属实际无毒物,遗传毒性结果为阴性;2.芹菜素降低SHR大鼠血压的最小有作用剂量为0.026g/kg bw;3.芹菜素可能的降压机制1)芹菜素可以通过上调SHR大鼠肾脏ACE2的mRNA转录水平以及蛋白表达水平,下调SHR大鼠肾脏ACE的mRNA转录水平以及蛋白表达水平,改善SHR大鼠的肾素-血管紧张素-醛固酮系统;2)芹菜素可以通过上调SHR大鼠肾脏eNOS的mRNA转录水平以及蛋白表达水平,下调SHR大鼠肾脏ET-1的mRNA转录水平以及蛋白表达水平;降低血浆AngⅡ含量,增加血浆NO、eNOS、CGRP、cGMP含量,升高血浆NO/ET以及NO/AngⅡ比值;增加血浆SOD的活性,降低O₂^-的水平,改善SHR大鼠体内氧化应激状态,从而改善SHR大鼠内皮功能;3)芹菜素内皮依赖性舒张血管作用依赖于NO/cGMP通路,而前列环素并不影响芹菜素的血管舒张作用。4)芹菜素通过激活Kca、Kv、K_IR、mito-K_ATP,抑制受体操纵性钙通道、电压依赖性钙通道、抑制细胞外钙内流而舒张血管;更多还原

【Abstract】 BackgroundApigenin is one of natural flavonoids that are almost ubiquitously present in many fruits,vegetables and beans,with the highest in celery.There has been a long history for people ingesting flavonoids.However,high doses of flavonoids may act as pro-oxidant,form lipid peroxidate,induce genetic mutation,cause DNA damage and breakage.The potential toxicological properties of apigenin remain largely unkown.Hypertension is one of the cardiovascular diseases which threatens the health of human being.It is an important risk factor of many diseases such as coronary heart disease,cerebrovascular and renal disease.Prevention and treatment of hypertension and its complications have an important clinical significance.The effects of long over-loading pressure on the vascular endothelium and myocardium in spontaneously hypertension rats（SHR） are similar to those in patients with essential hypertension. Apigenin has a varity of bioactivities,such as antioxidantive,anti-inflammatory, antiallergic effects.However,the mechanisms of hypotensive effect of apigenin remain largely unkown.Therefore,the studies on effects and mechanisms of apigenin on blood pressure of SHR will contribute to supply useful experimental evidence for the reasonable use of apigenin.ObjectivesTo evaluate the safety of high ingestion of apigenin,dose-response relationship as well as possible mechanisms of apigenin in lowering blood pressure,so as to provide scientifc support for the reasonable use and development of apigenin.Methods1.Safety evaluation of apigeninAccording to the procedure and methods of food toxicological safety evaluation,a series of toxicity studies were conducted on the ingestion of apigenin（including acute toxicity,genetic toxicity as well as sub-chronic toxicity experiments）. 2.Study on the effects of apigenin on lowering blood pressure and its possible mechanisms2.1 Effects of apigenin on lowering blood pressure of SHR and its possible mechanismsAccording to body weight and blood pressure,male SHR rats aged 12 weeks were randomly divided into 6 groups,10 rats of each group,with different levels of 0,0.007, 0.026,0.104,0.417g/kg bw of apigenin as well as 12mL/kg bw captopril positive control group.Systolic blood pressure（SBP） and heart rate were measured once a week,body weight was measured twice a week.At the end of the above animal study,all rats were anesthetized with 1%sodium pentobarbital（i.p.）,and 6mL arterial blood was drawn for the determination of blood plasm renin-angiotensin-aldosterone system（RAAS）,possible sensitive indexes of blood pressure,including uric acid（UA）,C-reactive protein（CRP）,interleukin-6（IL-6）, oxidative stress level,the effect of apigenin on SHR left ventricular hypertrophy index（LVHI）,as well as the effect of apigenin on SHR kidney RAAS by real-time PCR and immunohistochemistry methods.2.2 Study on possible mechanisms of apigenin relaxing rat aortic rings effectsRats were anesthetized with 1%sodium pentobarbital（i.p.） and killed by exsanguination.The thoracic aorta was immediately isolated and put in oxygenated Kreb solution at 4℃.The adherent connective tissue was cleaned and the vessel was cut into 3-4 mm rings with special care not to damage the endothelium.In some preparations,endothelium was mechanically removed by gently rubbing the lumen with a wet cotton tip.Vascular rings were suspended in an organ bath filled with 20 mL Kreb solution which was maintained at 37℃and ventilated continuously with 95%O₂ and 5%CO₂. The contraction was measured with a multichannel acquisition and analysis system （BIO-PAC MP150,America）.Related studies were conducted on the effect of apigenin on the contraction induced by PE and role of endothelium,K⁺ channel as well as Ca²⁺ channel in PE-induced relaxation. Results1.Safety evaluation of apigenin1) For the acute toxicity,the MTD was higher than 8g/kg bw.2) No apigenin-related genetic toxicity was found.3)Except for GST in 8g/kg bw dose of serum and 2g/kg bw dose of liver homogenate of male rats was significantly lower than control group（P＜0.05 or 0.01）;4g/kg bw dose of GSH-P_X in serum of both sex as well as 2,4g/kg bw dose of SOD and GSH-Px of male rats liver homogenate were significantly lower than control groups（（P＜0.05）, there were no apigenin-related adverse effects on the body weight,food intake,food consumption,hematology,serum biochemistry,urine routine as well as histopathology.2.Study on the effects of apigenin on lowering blood pressure and its possible mechanisms2.1 Effects of apigenin on lowering blood pressure of SHR and its possible mechanisms1） After apigenin administration for 2 weeks,SBP of captopril positive group and 0.417g/kg bw group was significantly lower than control group（P＜0.05）.From the 3th of experiment,SBP of all groups except 0.007g/kg bw was significantly lower than the control group（P＜0.05）.There was no significant difference between body weight and heart rate throughout the experiment（P＞0.05）.2) Effect of apigenin on RAAS of SHR blood plasmAngⅡand ACE in blood plasm of captopril positive control group and 0.417g/kg bw group was significantly lower than control group（P＜0.05）.Ald of captopril positive control group was also significantly lower than control group（P＜0.05）.3) Effect of apigenin on ACE2,ACE,eNOS and ET-1 of SHR kidneyAfter apigenin administration for 4 weeks,transcription level of ACE2 mRNA and eNOS mRNA of captopril positive control group and 0.417g/kg bw group was significantly higher than control group（P＜0.05）.Transcription level of ACE and ET-1 mRNA was significantly lower than control group（P＜0.05）.The immunohistochemistry results were consistent with the above gene expression results. 4) Effect of apigenin on vasorelaxation and vasoconstriction factors of SHRCalcitonin gene related protein（CGRP）,eNOS and NO of 0.417g/kg bw group were significantly higher than control group（P＜0.05）.cGMP and NO/ET of 0.026、0.104、0.417g/kg bw were also significantly higher than control group（P＜0.05）.NO/AngⅡof captopril positive control group and 0.007、0.104、0.417g/kg bw groups were also significantly higher than control group（P＜0.05）.There was no significant difference between ET-1 and PGI₂ in all groups（P＞0.05）.5) Effect of apigenin on oxidative stress of SHRAfter apigenin administration for 4 weeks,O₂ level was greatly lower than control group（P＜0.05）,while SOD activity was greatly higher（P＜0.05） than control group（P＜0.05）.6) Effect of apigenin on LVHI of SHRAfter apigenin administration for 4 weeks,although there were descending tendency among LVHI and heart index,yet no significant difference was found（P＞0.05）.7) Effect of apigenin on UA,CRP and IL-6 of SHR blood plasmAfter apigenin administration for 4 weeks,there was no obvious difference among UA,CRP as well as IL-6 in all groups（P＞0.05）.2.2 Study on possible mechanisms of apigenin relaxing rat aortic rings effects1) Apigenin had no effect on the basal tension in rat aortic rings.2) Apigenin can relax PE pre-contracted rings in both endothelium-intact aortic and endothelium-denuded aortic in a dose-dependent manner,with the effect of endothelium-intact aortic significantly stronger than that of endothelium-denuded aortic（P＜0.05）(E_max=76%,EC₅₀=4.55×10^-6M with endothelium-intact aortic,while for endothelium-denuded aortic,E_max=44%,EC₅₀=4.84×10^-5M).3) Pre-incubation of endothelium-intact rings with L-NAME,an inhibitor of NOS, and methylene blue,an inhibitor of soluble guanylyl cyclase,significantly reduced apigenin-induced relaxation（P＜0.05）.However,indomethacin,a cyclooxygenase blocker,did not significantly affect the apigenin-induced relaxation in endothelium-intact rings（P＞0.05）.4) Voltage-dependent K⁺ channel blocker 4-AP,mito-ATP-sensitive K⁺ channel blocker 5-HD,Ca²⁺-activated K⁺ channel blocker TEA as well as inward rectifying K⁺ channel blocker BaCl₂ significantly inhibited apigenin-induced relaxation in endothelium-denuded rings pre-contracted by PE（P＜0.05）.However,unspecific ATP-sensitive K⁺ channel blocker glibenclamide had no significant effect（P＞0.05）.5) In the K⁺-free solution,apigenin can significantly inhibit PE pre-contracted aortic rings（P＜0.05）.Apigenin can strongly inhibit 60 mM KCl induced aorta rings contraction（P＜0.05）.In the Ca²⁺-free solution plus PE,cumulative addition of CaCl₂ induced a stepwise tension increase of aortic rings.Pretreated with apigenin for 20 min significantly attenuated CaCl₂ induced contraction.Conclusions1) The NOAEL of apigenin was 2g/kg bw.Apigenin belongs to actually non-toxic substance and it had no genetic-related effects.2) The lowest effective dose of apigenin in lowering blood pressure of SHR was 0.026g/kg bw.3) Possible mechanisms of apigenin in lowering blood pressure●Up-regulation of the expression of ACE2 and down-regulation of the expression of ACE gene in kidney,thereby improving RAAS of SHR.●Up-regulation of the expression of eNOS and down-regulation of the expression of ET-1 gene in kidney,decrease of the AngⅡcontent in blodd plasm,increase the content of blood plasm NO,eNOS,CGRP,cGMP,up-regulation the ratio of NO/ET-1 and NO/AngⅡ,increase the activity of blood plasm SOD,lowering the content of blood plasm O₂,thereby,improving endothelium fuction.●NO-cGMP pathway was involved in the relaxation of apigein in endothelium-intact aorta,whereas production of PGI₂ by the endothelial cells was not attributed to the effect.●The vasorelaxant effect of apigenin was partially mediated by the opening of the K⁺ channels in vascular smooth muscle cells,including Kca,Kv,K_IR as well as mito-K_ATP,and by inhibiting of receptor-operated Ca²⁺ channel,voltage-dependent Ca²⁺ channel as well as extracellular Ca²⁺ influx.更多还原