低氧性肺动脉高压和肺血管结构重构以及葛根素的干预效应

Hypoxic pulmonary hypertension and remodeling of pulmonary vascular structure and the interventional effects of puerarin

目的:观察大鼠在低氧状态下出现的肺动脉高压以及肺小动脉结构的重建,以及葛根素对这两因素的干预效应。方法:实验于2003-06/2003-09在东南大学实验动物中心完成。①选用雄性SD大鼠24只。将大鼠按区组随机法分为3组,每组8只,分别为对照组、低氧组和葛根素干预组。②间断性低氧,在(10.0±0.5)%氧浓度的常压低氧舱内建立肺动脉高压大鼠模型。低氧组及葛根素干预组在常压条件下进行间歇性缺氧,每天8h,连续3周。葛根素干预组大鼠每天低氧前即刻腹腔内注射葛根素500mg/kg,对照组及低氧组大鼠每天腹腔内注射相同容积的生理盐水。③采用压力传感器和MacLab/4sADInstru-ments生物信号采集系统测定平均肺动脉压。④剪去心房组织,沿室间隔边缘分离出右心室游离壁,左心室及室间隔,用[右心室游离壁/(左心室+室间隔)]的质量比作为右心室肥厚的指标。⑤取大鼠左肺沿肺门横断取材,石蜡包埋切片,进行常规苏木精-伊红染色,观察肺小动脉形态学变化。⑥用SP-2000病理图文报告系统进行图像采集,IMAGE-PROplus4.5软件进行图像分析,测量与呼吸性细支气管及肺泡管伴行的肺小动脉外径、管壁厚度、血管总面积及管壁面积,计算出管壁厚度占血管外径的百分比和管壁面积占血管总面积的百分比,反映肺小动脉管壁增厚程度。⑦血浆及肺组织匀浆一氧化氮测定、一氧化氮合酶、原生型一氧化氮合酶和诱导型一氧化氮合酶测定按试剂盒南京建成生物医学工程研究所说明书操作。⑧组间比较采用两样本均数比较的t检验。结果:大鼠24只均进入结果分析。①大鼠平均肺动脉压:对照组和葛根素干预组均明显低于低氧组[(15.01±1.47),(20.43±2.86),(29.74±2.32)mmHg,P<0.01]。②右心室游离壁与(左心室+室间隔)的重量比:低氧组和葛根素干预组明显高于对照组[(33.33±1.43)%,(30.48±2.17)%,(23.86±0.94)%,P<0.05]。③低氧大鼠肺血管形态学改变:常压低氧3周后对照组大鼠肺小血管管壁较薄、管腔较大,低氧组大鼠肺小动脉普遍增厚,管腔狭窄,葛根素干预组的肺小动脉管壁及血管管腔狭窄程度较低氧组显著减轻。④常压低氧3周后管壁厚度占血管外径的百分比和管壁面积占血管总面积的百分比:对照组和葛根素干预组明显低于低氧组(P<0.01)。⑤常压低氧3周后大鼠血浆一氧化氮水平:对照组和葛根素干预组明显高于低氧组(P<0.01)。⑥常压低氧3周后大鼠肺组织一氧化氮含量及一氧化氮合酶、原生型一氧化氮合酶活性:对照组和葛根素干预组明显高于低氧组(P<0.05～0.01)。⑦常压低氧3周后大鼠肺组织诱导型一氧化氮合酶活性:对照组和葛根素干预组明显低于低氧组(P<0.01,0.05)。结论:①慢性缺氧可导致肺动脉壁增厚、管腔狭窄等血管再建病理改变。②葛根素能够明显升高低氧性肺动脉高压大鼠血浆一氧化氮水平,从而抑制胶原合成,阻止肺动脉外膜成纤维细胞的增生,在一定程度上抑制了低氧肺动脉高压的形成和发展以及肺血管结构的重构。

AIM: To investigate occurrence of pulmonary hypertension and the remodeling of pulmonary arteriolar structure in rats under hypoxia, and the interventional effects of puerarin on the two factors. METHODS: The experiment was completed in the experimental animal center of Southeast University between June and September 2003.① Twenty-four male SD rats were randomly allocated into 3 groups: control group (n=8), hypoxic group (n=8) and puerarin interventional group. ② Rat models of pulmonary hypertension were established under discontinuous hypoxia in the normobaric hypoxic chamber with the oxygen concentration of (10.0±0.5)%. Rats in the hypoxic group and puerarin interventional group were treated with discontinuous hypoxia under normal pressure, 8 hours every day for 2 continuous weeks. Intraperitoneal injection of puerarin (500 mg/kg) was given to the rats in the puerarin interventional group every day immediately before hypoxia, and those in the control group and hypoxic group received intraperitoneal injection of saline of the same volume. ③ Average pulmonary artery pressure was determined with pressure sensor and MacLab/4s Austrilia AD Instruments biological signal collection system. ④ Atrial tissues were cut, right ventricular free wall, left ventricle and interventricular septum were isolated along the edge of interventricular septum, and then the mass ratio of [right ventricular free wall/(left ventricle + interventricular septum)] was taken as the index for right ventricular hypertrophy. ⑤ The left lungs of rats were taken along the hilum of lung, slices were embedded with olefin and stained with hematoxylin-eosin to observe the morphological changes of pulmonary small artery.⑥ Images were collected with the SP-2000 pathological picture and writing report system, and the images were analyzed with IMAGE-PRO plus 4.5 software; The outer diameter, thickness of vascular wall, total vascular area and area of vascular wall of the pulmonary small arteries accompanied by respiratory bronchiole and alveolar duct, and then the percentage of the outer diameter to the thickness of vascular wall and the percentage of the area of vascular wall to total vascular area were calculated to reflect the thickening degree of pulmonary small arterial vascular wall.⑦The nitric oxide, nitric oxide synthase, constitutive nitric oxide synthase and inducible nitric oxide synthase in plasma and homogenate of lung tissue were detected according to the introduction of the kit produced by Nanjing Jiancheng Biomedical Engineering Institute. ⑧ The intergroup comparison was undertaken with the t test for the mean of two samples. RESULTS: All the 24 rats were involved in the analysis of results. ① Average pulmonary artery pressure: It was obviously lower in the control group and control group and puerarin interventional group than in the hypoxic group [(15.01±1.47), (20.43±2.86), (29.74±2.32) mm Hg, P < 0.01]. ② The ratio of [right ventricular free wall mass to that of (left ventricle + interventricular septum): It was obviously higher in the puerarin interventional group and hypoxic group than in the control group [(33.33±1.43)%, (30.48±2.17)%, (23.86±0.94)%, P < 0.05].③ The morphological changes of pulmonary blood vessel under hypoxia: Three weeks after hypoxia under normal pressure, the pulmonary small artery vascular wall was thinner and vascular cavity was bigger in the control group; In the hypoxic group, the pulmonary small artery was generally thickened, and the vascular cavity was constricted; In the puerarin interventional group, the constricted severities of pulmonary small artery vascular wall and vascular cavity were significantly relieved in the puerarin interventional group than in the hypoxic group. ④The percentage of the outer diameter to the thickness of vascular wall and the percentage of the area of vascular wall to total vascular area after hypoxia under normal pressure for 3 weeks: Those were obviously lower in the control group and puerarin interventional group than in the hypoxic group (P < 0.01). ⑤ The plasma level of nitric oxide after hypoxia under normal pressure for 3 weeks: It was obviously higher in the control group and puerarin interventional group than in the hypoxic group (P <0.01).⑥ The content of nitric oxide and activities of nitric oxide synthase and constitutive nitric oxide synthase in lung tissue after hypoxia under normal pressure for 3 weeks: Those were obviously higher in the control group and puerarin interventional group than in the hypoxic group (P < 0.05 to 0.01).⑦ The activity of inducible nitric oxide synthase in lung tissue after hypoxia under normal pressure for 3 weeks: It was obviously lower in the control group and puerarin interventional group than in the hypoxic group (P < 0.01, 0.05). CONCLUSION: ① Chronic hypoxia can result in the pathological changes of vascular reconstruction, such as thickening of pulmonary arterial wall and constriction of vascular cavity.② Puerarin can obviously increase the plasma level of nitric oxide in rats with hypoxic pulmonary hypertension, and then restrain the synthesis of collagen, arrest the hyperplasia of fibroblasts in pulmonary artery ectoblast; It can inhibit the formation and development of hypoxic pulmonary hypertension and the remodeling of the pulmonary vascular structure to a certain extent