人参三七川芎提取物对自然衰老大鼠血管外膜重构的干预机制

Intervention Mechanism of Extracts fromRadix Ginseng,Radix Notoginseng andRhizoma Chuanxiong on Adventitia of Senescent Rats

目的观察自然衰老大鼠血管外膜的重构特点,并探讨人参三七川芎提取物的干预作用。方法将85只20月龄的自然衰老Wistar大鼠按体重水平分层后随机分为衰老组、人参三七川芎醇提物低、中、高剂量组(下称中药低、中、高剂量组)及氯沙坦对照组(Losartan组),每组17只,另选14只2月龄青年Wistar大鼠作为青年组。中药高、中、低剂量组分别给予不同剂量的人参三七川芎醇提物[1493.4、746.7、373.4mg/(kg·d)]灌胃给药,Losartan组给予氯沙坦钾混悬液[10 mg/(kg·d)]灌胃,衰老组及青年组给予同体积蒸馏水灌胃,均每日1次。干预15周后,采用HE染色法观察各组大鼠胸主动脉壁形态;采用苦味酸天狼猩红染色法观测血管壁胶原种类、分布和含量;放射免疫分析法检测血浆肾素活性(plasma renin activity,PRA)、血浆血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)浓度及外膜组织AngⅡ含量;生物化学分析法检测外膜组织羟脯氨酸水平;实时荧光定量多聚酶链式反应(real-time PCR,RT-PCR)检测外膜组织血管紧张素Ⅱ1型受体(angiotensinⅡreceptor 1,AT_1R)、血管紧张素Ⅱ2型受体(angiotensinⅡreceptor 2,AT_2R)mRNA水平;Western blot法检测外膜组织AT_1R、AT_2R蛋白表达水平。结果与青年组比较,衰老组血管外膜增厚,外膜厚度/管径增高,胶原纤维堆积,Ⅰ型胶原面积增加,Ⅲ型胶原面积减少,Ⅲ型胶原/Ⅰ型胶原降低(P<0.05),血浆PRA及AngⅡ降低(P<0.01,P<0.05),外膜组织AngⅡ及羟脯氨酸含量增高,AT_1R mRNA及蛋白表达下调,AT_2R mRNA及蛋白表达上调(P<0.01,P<0.05)。与衰老组比较,中药低、中、高剂量组均可改善衰老血管形态学改变;中药中、高剂量组及Losartan组外膜厚度/管径均减小,中药高剂量组及Losartan组Ⅰ型胶原面积减少,Ⅲ型胶原面积增加,Ⅲ型胶原/Ⅰ型胶原明显升高,血管外膜羟脯氨酸含量明显降低(P<0.05,P<0.01);中药中、高剂量及Losartan组外膜组织中AngⅡ水平降低(P<0.01,P<0.05);衰老组、中药各剂量组、Losartan组各组间PRA比较,差异无统计学意义(P>0.05)。与衰老组比较,各治疗组AT_1R mRNA表达均增加(P<0.01),中药中、高剂量组AT_2R mRNA表达亦增加(P<0.05);中药高剂量组及Losartan组AT_1R蛋白表达升高(P<0.01,P<0.05);中药中、高剂量组AT_2R蛋白表达亦升高(P<0.05)。结论衰老大鼠血管出现外膜重构,表现为外膜增厚,胶原堆积,Ⅰ型、Ⅲ型胶原比例紊乱等,其机制可能与局部肾素血管紧张素系统(renin-angiotensin system,RAS)激活有关;人参三七川芎提取物可能通过对外膜局部AngⅡ、AT_1R等多靶位的干预,改善外膜重构,进而延缓血管衰老。

Objective To observe the reconstruction features of adventitia in senescent rats,and to explore the intervention mechanism of Chinese herbs（CH,extracts from Radix Ginseng,Radix Notoginseng,and Rhizoma Chuanxiong）.Methods Totally 85 20-month senescent rats were randomly divided into 5 groups according to body weight,i.e.,the aging model group,the high dose CH group,the middle dose CH group,the low dose CH group,the Losartan group,17 in each group.Another 14 2-month old Wistar rats were selected as a young group.Extracts of CH at the daily dose of 1493.4,746.7,and 373.4 mg/kg were administered to rats in the 3 CH groups respectively by gastrogavage.Losartan suspension at the daily dose of 10 mg/kg was administered to rats in the Losartan group by gastrogavage.Equal volume of distilled water was administered to rats in the aging model group and the young group.All medication was performed once daily.After 15-week intervention,morphological changes of thoracic aorta were observed by HE staining.The types,distribution,and contents of vessel wall collagens were determined using picric acid picrosirius red staining.The plasma renin activity（PRA）,the concentration of rennin angiotensin Ⅱ（Ang Ⅱ）,and the content of Ang Ⅱ in adventitia were detected by radioimmunoassay.The content of hydroxyproline（Hyp） was detected by biochemical analysis.mRNA contents and protein expressions of angiotensin Ⅱ receptor 1（AT_1 R） and angiotensin Ⅱ receptor 2（AT_2 R） were detected by real-time PCR（RT-PCR） and Western blot.Results Compared with the young group,thickened adventitia,increased adventitia thickness/caliber accumulated collagen fiber,increased area of type Ⅰ collagen,decreased area of type Ⅲ collagen,decreased type Ⅲ /Ⅰ collagen area ratio（P〈0.05）,decreased plasma PRA and Ang Ⅱ（P〈0.01,P〈0.05）,increased contents of Ang Ⅱ and Hyp in adventitia,down-regulated mRNA and protein expressions of AT_1R,and up-regulated mRNA and protein expression of AT_2R could be seen in the aging model group（P〈0.05）.Compared with the aging model group,morphological changes could be improved in the 3 CH groups.Adventitia thickness/caliber was reduced in middle and high dose CH groups,as well as the Losartan group.The area of type Ⅰ collagen was reduced and the area of type Ⅲ collagen was enlarged,type Ⅲ /Ⅰ collagen area ratio obviously increased,contents of adventitia Hyp was obviously lowered in the high dose CH groups and the Losartan group（P〈0.05,P〈0.01）.Ang Ⅱ levels in adventitia decreased in middle and high dose CH groups and the Losartan group（P〈0.05 P〈0.01）.There was no statistical difference in PAR among all groups（P〈0.05）.Compared with the aging model group,mRNA expression of AT_1R all increased in each treatment group[P〈0.01）;mRNA expression of AT_2R also increased in middle and high dose CH groups（P〈0.05）.Protein expression of AT,R increased in the high dose CH group and the Losartan group（P〈0.01,P〈0.05）;protein expression of AT_2R also increased in middle and high dose CH groups（P〈0.05）.Conclusions Adventitia remodeling occurred in aged rats,manifested as thickened adventitia and accumulated collagens,disordered ratios of collagen Ⅰ and Ⅲ.Its mechanism might be possibly associated with aactivation of renin-angiotensin system（RAS）.Extracts from Radix Ginseng,Radix Notoginseng,and Rhizoma Chuanxiong could improve adventitial remodeling possibly by interfering multi-targets,such as Ang Ⅱ and AT_1R,thereby delaying vascular aging.