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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 remodeling characteristics of natural adventitial rat adventitia and to explore the intervention of ginseng and rhizoma chuanxiong rhizome extract. Methods 85 aged Wistar rats aged 20 months were stratified according to their body weight. The rats were randomly divided into aging group, low, medium and high dose groups of Ginseng extract of Panax notoginseng ) And Losartan group (17 rats in each group). Another 14 young Wistar rats of 2 months old were selected as the youth group. Chinese medicine high, medium and low dose groups were given different doses of ginseng notoginseng alcohol extract [1493.4,746.7,373.4mg / (kg · d)] intragastric administration, Losartan group was given losartan potassium suspension [ 10 mg / (kg · d)] intragastric administration, the aging group and the young group were given the same volume of distilled water, gavage once daily. After 15 weeks of intervention, the morphology of the thoracic aorta in each group was observed by HE staining; the type, distribution and content of collagen in the vascular wall were observed by picric acid Sirius red staining; the plasma renin was detected by radioimmunoassay activity and PRA, AngiotensinⅡ (AngⅡ) and AngⅡ in epicardium. The levels of hydroxyproline in epicardial tissue were detected by biochemical methods. Real-time PCR (real-time PCR) RT-PCR) was used to detect the mRNA expression of AT1R and AT2R in epicardium and the expression of AT1R and AT2R in epicardium by Western blot Level. Results Compared with young group, the thickening of adventitia, the increase of adventitial thickness / diameter, the accumulation of collagen fibers, the increase of type I collagen, the decrease of type Ⅲ collagen and the decrease of type Ⅲ collagen / collagen Ⅰ (P <0.05) (P <0.01, P <0.05). The contents of AngⅡ and hydroxyproline in epicardium were increased, the mRNA and protein expressions of AT1R were down-regulated and the expressions of AT2R mRNA and protein were up-regulated ). Compared with the aging group, the Chinese medicine low, medium and high dose groups can improve aging vascular morphological changes; Chinese medicine, high dose group and Losartan group of outer membrane thickness / diameter were reduced, Chinese medicine high-dose Losartan group and type Ⅰ The area of collagen decreased, the area of type Ⅲ collagen increased, the type Ⅲ collagen / type Ⅰ collagen was significantly increased, and the content of hydroxyproline in vascular adventitia was significantly decreased (P <0.05, P <0.01) There was no significant difference in the levels of AngⅡ in the membrane between the aging group, the Chinese medicine groups and Losartan group (P <0.01, P <0.05). Compared with the aging group, the expression of AT1R mRNA in each treatment group increased (P <0.01), and the expression of AT2R mRNA in the medium and high dose groups also increased (P <0.05). The expression of AT1R protein in high dose group and Losartan group increased <0.01, P <0.05). The AT 2 R protein expression was also increased in medium and high dose groups (P <0.05). CONCLUSION: The adventitia remodeling occurs in the blood vessels of aging rats, which is characterized by thickening of outer membrane, accumulation of collagen and imbalance of type I and III collagen. The mechanism may be related to renin-angiotensin system (RAS) Activation; ginseng notoginseng Chuanxiong extract may be through the outer membrane of Ang Ⅱ, AT_1R and other multi-target intervention to improve the outer membrane remodeling, thereby delaying vascular aging.