【作者】 苏琛；

【导师】 杨仁旭；

【作者基本信息】 成都中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：通过建立低氧性肺动脉高压(HPH)大鼠模型,探讨HPH形成的病理机制,观察具有宣肺逐瘀化痰法的麻杏芎葶合剂对HPH大鼠肺动脉压增高和血管结构重建的干预作用。并通过观察rock-1基因及蛋白在HPH大鼠肺组织中的表达水平,探讨Rho/ROCK信号传导通路对低氧性肺动脉高压的调控作用及麻杏芎葶台剂对低氧性肺动脉高压干预的分子机制。方法：Sprague-Dawley大鼠50只,雌雄各半,随机均分为正常组、HPH模型组和法舒地尔对照组、中药中剂量组、中药高剂量组。采用常压间断低氧法：(10±0.5)%O2,每天间断低氧6-8小时,每周缺氧6天,持续3周,建立大鼠HPH模型。每日放入低氧舱前给药：法舒地尔组腹腔注射10mg/(kg·d)盐酸法舒地尔注射液；中药中剂量组给予10g/kg-d麻杏芎葶合剂药液灌胃；中药高剂量组给予20g/kg·d麻杏芎葶合剂药液灌胃；正常组、HPH模型组按每100g体重1ml的生理盐水灌胃。在第21天测定各组大鼠平均肺动脉压力(mean pulmonary artery pressure, mPAP)、平均颈动脉压力(mean cervical arterial pressure, mCAP)、右心肥厚指数(right ventricular hypertrophy index, RVHI);观察光镜下肺小动脉的形态学改变,利用图像分析计算血管壁厚度占外径的百分比(WT%)、管壁面积占血管总面积的百分比(WA%)及管腔面积与管总面积的百分比(LA%)。测定肌型动脉、部分肌型动脉和非肌型血管所占比例及肌型肺小动脉中膜细胞核密度,观察肺血管结构重建情况。在证实HPH建模成功后,应用Real time PCR、Westen bolt等技术,以分子生物学方法测定各组大鼠肺组织中Rho激酶ROCK-1基因及蛋白的表达量,并测定各组大鼠肺组织中Rho激酶底物——MBS Thr-697位的磷酸化的表达情况。结果：(1)低氧21天后,HPH模型组大鼠mPAP、RVHI、WT%、WA%。LA%、肌型动脉、部分肌型动脉和非肌型血管所占比例、肌型肺小动脉中膜细胞核密度分别为(30.27±1.47)mmHg、(41.41±3.71)%、(55.23±3.07)%、(83.12±5.90)%、(16.88±1.74)%、(57.26±0.26)%、(17.46±2.78)%、(25.28±3.07)%、(9.41±1.90),与正常组(16.14±0.38)mmHg、(24.65±1.85)%、(33.41±3.65)%、(54.23±4.61)%、(45.76±3.23)%、(7.88±0.83)%、(25.76±2.05)%、(66.36±3.65)%、(7.05±1.37)比较均有差异(P均<0.05)。(2)低氧21天后大鼠mPAP、RVHI、WT%、WA%、LA%、肌型动脉、部分肌型动脉和非肌型血管所占比例、肌型肺小动脉中膜细胞核密度,法舒地尔组大鼠分别为(18.85±0.65) mmHg、(30.24±1.82)%、(38.15±2.96)%、(68.49±4.16)%、(31.50±3.53)%、(12.49±0.65)%、(36.58±2.55)%、(50.94±2.96)%、(13.13±2.61)；中药中剂量组大鼠分别为(21.61±0.38)mmHg、(28.19±1.67)%、(43.12±3.61)%、(65.49±4.15)%、(34.51±3.38)%、(20.71±0.21)%、(30.86±2.03)%、(48.43±3.61)%、(11.28±1.08)；中药高剂量组大鼠分别为(23.25±1.19)mmHg、(27.6411.55)%、(44.5313.76)%、(69.9014.22)%、(30.10±3.37)%、(20.77±0.19)%、(29.08±2.57)%、(50.15±3.76)%、(7.26±1.68)：以上三组分别与HPH模型组(30.27±1.47)mmHg、(41.41±3.71)%、(55.2313.07)%±(83.12±5.90)%、(16.88±1.74)%、(57.26±0.26)%、(17.46±2.78)%、(25.28±3.07)%、(9.41±1.90)比较均有差异性(P均<0.05)。(3)各组间mCAP比较未见显著性差异(P均>0.05)。(4)HPH模型组大鼠肺小血管管壁明显增厚；出现明显肺小动脉重构特征：内皮细胞连续性破坏,血管内皮呈立方形或柱状突向血管腔,甚至坏死脱落,中膜平滑肌细胞肥大、增生,管腔变小。与HPH模型组比较,法舒地尔干预组、中药中、高剂量组大鼠肺小血管管壁较薄,管腔变宽,血管平滑肌细胞的肥大、增生程度以及内膜的增厚明显减轻。(5)经低氧干预三周(21天)后,HPH模型组大鼠Rho激酶ROCK-1mRNA (2.680±0.061)与正常组大鼠(1.094±0.051)比较增高,差别有统计学意义(P<0.05)。法舒地尔组、中药中、高剂量组大鼠Rho激酶ROCK-1mRNA(1.66110.0.90)、(2.03510.078)、(1.96210.066)较正常组(1.094±0.051)均增高,差别有统计学意义(P<0.05),较HPH模型组大鼠(2.680±0.061)均降低,差别亦有统计学意义(P均<0.05)。(6)应用Western印迹法检测,HPH模型组ROCK-1蛋白表达较正常组增加(P<0.05),经法舒地尔、麻杏芎葶合剂干预过的法舒地尔组、中药中、高剂量组ROCK-1蛋白表达较正常组升高(P<0.05),较HPH模型组有不同程度降低(P<0.05)。(7)免疫印迹结果显示,HPH模型组P-MYPT1蛋白表达较正常组增加(P<0.05),经法舒地尔、麻杏芎葶合剂干预过的法舒地尔组、中药中、高剂量组P-MYPT1蛋白表达较正常组升高(P<0.05),而较HPH模型组有所降低(P<0.05)。结论：(1)麻杏芎葶合剂可以减轻低氧引起的肺动脉压力的增加,改善右心室肥厚,肺血管结构重建,对低氧性肺动脉高压有明确防治、干预作用。(2) Rho/ROCK信号传导通路与HPH的发生、发展密切相关。麻杏芎葶合剂对低氧性肺动脉高压的干预作用,与其能调控Rho/ROCK信号传导通路,抑制肺血管Rho激酶的合成和表达有关。(3)按照传统中医理论,麻杏芎葶合剂的立方、配伍及药理分析,可知麻杏芎葶合剂具有宣肺、肃肺、泻肺、化痰、利水、活血之功效,全方将宣肺逐瘀化痰法融为一体,切中低氧性肺动脉高压的中医辨证病机,有确切疗效。更多还原

【Abstract】 Objective: through the establishment of hypoxic pulmonary hypertension (HPH) rat model, to explore the HPH pathological mechanism, observe the intervention effects for Ma Xing Xiong Ting He Ji (admixture) in releasing lung, removing blood stasis and dissolving phlegm pattern on pulmonary artery pressure increase and vascular structure reconstruction of intervention effects of HPH rats. And through the observation the expression level of rock-1gene and protein in HPH rats lung tissue, explore the control function of Rho/rock signaling pathways on hypoxic pulmonary hypertension and the intervention of molecular mechanism of hypoxic pulmonary hypertension by Ma Xing Xiong Ting He Ji (admixture) in releasing lung, removing blood stasis and dissolving phlegm pattern.Methods:50Sprague wrote-Dawley rats, half male and half female, randomly divided into normal group, the HPH model group and fasduil control group, medium dosage Chinese medicine group, high dosage Chinese medicine group. Atmospheric intermittent hypoxia method:(10±0.5)%for02, intermittent hypoxia for six to eight hours a day, anoxic for6days a week,4weeks, to establish HPH rats model. Put them in a low oxygen tank before medicine delivery, For fasudil group, intraperitoneal injection,10mg/kg, with hydroxyfasudil infection; For medium dosage Chinese medicine group,10g/kg of Ma Xing Xiong Ting He Ji(mixture) gavage; For high dosage Chinese medicine group,20g/kg of Ma Xing Xiong Ting He Ji(mixture) gavage; For normal group and the HPH model group,1ml saline lavage for every100g of body weigh. At21th day determine their mean pulmonary artery pressure (mPAP), average carotid artery pressure (mean cervical arterial pressure, mCAP), right heart hypertrophy index (right ventricular hypertrophy index, RVHI); Observe morphology changes of pulmonary arterioles under light microscopy, calculate the percentage of the thickness of vessel wall over the external diameter (WT%), the percentage of the wall area over the total area of blood vessel (WA%), the percentage of the lumen area over total area of the tube (LA%) by picture analysis. Determine the percentage of muscular arteries,partial muscular artery non muscular arteries,and muscular pulmonary arterioles and density of membrane nucleus,observe the reconstruction of pulmonary vascular structure.After confirming the successful modeling of HPH, apply Real time PCR,Westen bolt and other techniques,to determine the amount of the expression of Rho kinase gene and protein of lung tissue of the rats in each group by molecular biology method and determine Rho kinase substrate of lung tissue of the rats in each group the phosphorylated expression of MBS Thr-697.Results:(1)After21days of hypoxia,mPAP, RVHI,WT%,WA%,LA%and the portion of muscular arteries,partial muscular arteries,non-musclar arteries,and density of membrane nucleus in muscular pulmonary arterioles of rats in HPH model group are as follows respectively:(30.27±1.47mmHg,,(41.41±3.71)％,(55.23±3.07)％,(83.12±5.90)％,(16.88±1.74)％,(57.26±0.26)％,(17.46±2.78)％,(25.28±3.07)％,(9.41±1.90),and rormal group(16.14±0.38)mmHg,(24.65±1.85)％,(33.41±3.65)％,(54.23±4.61)％,(45.76±3.23)％,(7.88±0.83)％,(25.76±2.05)％,(66.36±3.65)％,(7.05±1.37).The comparison exists a dofference.(P<0.05).(2) After21days of hypoxia,mPAP, RVHI,WT%,WA%,LA%and the portion of muscular arteries,partial muscular arteries,non-muscular arteries,and density of membrane nucleus in muscular pulmonary arterioles of rats in Fasudi group are as follows respectively:(18.85±0.65)mmHg,(30.24±1.82)％,(38.15±2.96)％,(68.49±4.16)％,(31,50±3.53)％,(12.49±0.65)％,(36.58±2.55)％,(50.94±2.96)％,(13.13±2.61)；For medium dosage Chinese medicine group are:(21.61±0.38)mmHg,(28.19±1.67)％,(43.12±3.61)％,(65.49±4.15)％,(34.51±3.38)％,(20.71±0.21)％,(30.86±2.03)％,(48.43±3.61)％,(11.28±1.08)；For high dosage Chinese medicine group are(23.25±1.19)mmHg,(27.64±1.55)％,(44.53±3.76)％,(69.90±4.22)％,(30.10±3.37)％,(20.77±0.19)％,(29.08±2.57)％,(50.15±3.76)％,(7.26±1.68)；The three groups above all have comparison difference(P<0.05) respectively with HPH model group(30.27±1.47mmHg),(41.41±3.71)％,(55.23±3.07)％,(83.12±5.90)％,(16.88±1.74)％,(57.26±0.26)％,(17.46±2.78)％,(25.28±3.07)％,(9.41±1.90).(3)mCAP has no significant difference among groups(P>0.05).(4)It is obvious that the vessel wall of rats’ pulmonary arterioles in HPH model group is thicker; and there are significant remodeling of pulmonary arterioles: continuous damage on endothelial cells and vascular endothelial is generating cuboidally or columnarly to the lumen, even has necrosis and falls off, smooth muscle cells on nunica media has hypertrophy, hyperplasia and the lumen decreases. Compared with HPH model group, Fasudi intervention group, medium and high dosage Traditional Chinese medicine (TCM) group, rats’ pulmonary arterioles are with thinner walls and lumen with wider and the degree of hypertrophy and hyperplasia of vascular muscle cells and thickening of the tunica intima is reduced obviously.(5)After three weeks of hypoxia (21days) intervention, rats’ Rho kinase ROCK-1mRNA (2.680±0.061) and normal group (1.094±0.051) are increased in HPH model group, the difference was statistically significant (P<0.05). Method of shu in Bristol, in traditional Chinese medicine (TCM) and high dose group rats’ Rho kinase (ROCK-1mRNA (1.661±0.0.90),(2.035±0.078),(1.962±0.066)(1.094±0.051) in Fasudi group, medium and high dosage TCM group are higher than normal group, the difference was statistically significant (P<0.05), compared with the HPH model group (2.680±0.061),they are lower, and the difference has statistical significance (P<0.05).(6) By applying Western imprinting detection method, ROCK-1protein expression in HPH model group is increased than the normal group (P<0.05), the ROCK-1protein expression in Fasudi group, medium and high dosage TCM group, after the intervention of Ma Xing Xiong Ting He Ji(mixture),is increased than the normal group (P<0.05), compared with HPH model group there is a reduction in different degrees (P<0.05).(7) According to the results of western blot, P-MYPT1protein expression in HPH model group is increased than the normal group (P<0.05), P-MYPT1protein expression in Fasudi group, medium and high dosage TCM group, after the intervention of Ma Xing Xiong Ting He Ji(mixture), is increased than the normal group (P<0.05), while decreased compared the HPH model group (P<0.05).Conclusion:(1) Ma Xing Xiong Ting He Ji(mixture), with its lung-releasing-and-blood stasis-removing-phlegm-dissolving effect can reduce the increase of pulmonary artery pressure caused by hypoxia, improve the right ventricular hypertrophy, reconstruct pulmonary vascular structure, has specific prevention and intervention for hypoxic pulmonary hypertension.(2) the Rho/ROCK signaling pathways are closely associated with the occurrence and development of HPH.The intervention effect of Ma Xing Xiong Ting He Ji(mixture), with its lung-releasing-and-blood stasis-removing-phlegm-dissolving effect,for hypoxic pulmonary hypertension, is related to its ability to regulate the Rho/ROCK signaling pathways, inhibit the synthesis and expression of pulmonary vascular Rho kinase.(3) According to basic theory of TCM, the formula, compatibility and pharmacological analysis of Ma Xing Xiong Ting He Ji(mixture), we can declare that it can disperse the lung, astringe the lung, and reduce the lung, dissolve the phlegm, promote water, and activate the blood. This formula combine lung-releasing and blood stasis-removing and phlegm-dissolving all together, fcous on the TCM pathogenesis and syndrome differentiation of hypoxic pulmonary hypertension to achieve and exactly curative effect.更多还原