川芎嗪对肺动脉高压影响的实验与临床研究
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摘要
研究背景：
肺动脉高压是一种临床上比较常见的心血管疾病,本病以肺血管阻力进行性升高为特征,病理学以肺小动脉痉挛、内膜增生及重构和原位微血栓病灶形成为主要特征。其诊断标准：静息状态下心导管测定平均肺动脉压≥25mmHg或者运动时≥30mmHg,且肺毛细血管压或者左房压<15mmHg。根据指南的最新分类方法,肺动脉高压分为五大类：1.动脉型肺动脉高压；2.静脉性肺动脉高压；3.低氧血症相关性肺动脉高压；4.慢性血栓或栓塞所致肺动脉高压；5.混合性肺动脉高压。
本病致死、致残率高,严重威胁人民健康及生命,是医学研究的热点方向之一。其中,流行病学调查发现,动脉型肺动脉高压(以下称“肺动脉高压”)和慢性血栓和／或栓塞性疾病所引起的肺动脉高压是本病中危害我国人民身体健康最严重的类型,是引起患者致死的重要原因。根据美国国立卫生院注册研究表明肺动脉高压患者的中位生存期为2.8年、1年、3年和5年的生存率分别为68%、48%和34%,而在我国特发性肺动脉高压和家族性肺动脉高压患者1、2、3、5年的生存率分别为68%、56.9%、38.9%及20.8%。肺小动脉血管增生、重构导致肺血管阻力进行性加重,最终引起右心室功能衰竭、死亡,这是本病的主要致死原因。
以往,由于肺动脉高压的发病机理未明,传统治疗仅仅在于限制体力活动、吸氧、抗凝、利尿、强心,钙离子拮抗剂等一般处理,但在临床上这些治疗的效果往往不佳,本病的治疗曾一度停滞不前。随着发病机理研究的进展,新型靶向治疗药物不断面世,目前公认较为有效的靶向治疗药物包括：前列环素类(伊洛前列环素)、内皮素受体拮抗剂(波生坦)、5-磷酸二酯酶抑制剂(西地那非)。循证医学研究表明,上述靶向治疗药物能使肺动脉高压患者的心功能、生活质量有了很大的改善,甚至提高了生存率。由此,美国FDA批准上述药物用于治疗肺动脉高压,使许多原本绝望的患者看到再生的希望。然而,上述药物仅能改善患者临床症状与血流动力学状况,单纯扩血管的作用有限,不能减缓肺动脉压力进行性升高的趋势,病情仅仅是延迟加重,并没有根治。肺动脉高压患者肺动脉压力不断升高的原因,在于肺血管重构；而肺血管重构的基础是血管平滑肌细胞的过度增殖。因此,抑制血管平滑肌细胞增殖、促进其凋亡是肺动脉高压治疗领域的发展趋势之一。与此同时,上述药物完全依靠进口,价格昂贵,例如其中费用最低的西地那非,每月的费用需数千元,而我国实行基本药物医疗保障制度,上述药物均未能列入国家基本医疗保险药品目录,患者需自行承担所有费用,致使我国许多患者未能得到有效的治疗。基于上述原因,寻找能够抑制甚至逆转肺血管重构、改善右室心功能且价格低廉的药物具有十分重要的意义,也更适合我国的国情。
川芎嗪是中药川芎的有效成分,新近研究表明它有抗血小板、刺激NO生成、扩张血管、抗炎、抗肿瘤细胞增生等作用,与肺动脉高压的治疗有相符的地方。目前国内已经有临床研究应用川芎嗪干预由肺心病引起的低氧性肺动脉高压,结果提示川芎嗪能降低肺动脉压,提高血氧浓度,改善患者生活质量,研究显示监测的指标反应ET-1下降,NO升高,推测川芎嗪通过提高肺血管内皮NO释放达到治疗肺动脉高压,同时试验表明药物是安全的。为探讨川芎嗪对动脉型肺动脉高压、慢性血栓或栓塞性疾病所引起的肺动脉高压患者是否具有治疗作用而设计本研究。如能证实川芎嗪对肺动脉高压有治疗作用,可以为肺动脉高压的治疗提供更多的选择。
本研究分为动物基础研究及临床研究两部分,研究获得单位所在伦理委员会通过。根据伦理委员会的意见,临床研究部分不设空白对照组。
第一章川芎嗪对肺动脉高压大鼠肺血管重构的影响
目的：
通过使用川芎嗪对野百合碱(MCT)诱导的大鼠肺动脉高压进行干预,观察川芎嗪对大鼠肺动脉压力及肺血管重构的影响,并初步探讨其作用的可能机制。
方法：
1.造模：实验动物为SD大鼠,成年,雄性共30只,随机分为对照组、MCT诱导的肺动脉高压组(MCT组)、川芎嗪治疗组(治疗组)。MCT组(10只)：次皮下注射野百合碱(50mg/kg)饲养21天后,再以生理盐水(2ml/d)每日灌胃一次,共20天；对照组(10只)：一次皮下注射等量生理盐水饲养21天后,再以生理盐水(2ml/d)每日灌胃一次,共20天；治疗组(10只)：一次皮下注射野百合碱(50mg/kg),饲养21天后,以川芎嗪(80mg/(kg.d))每日灌胃一次,共20天。
2.主要检测指标：平均肺动脉压力(mean pulmonary arterial pressure, mPAP),右心室肥大指数(right ventricular hypertrophy index. RVHI),血管周围炎症评分,肺动脉中膜厚度百分比(WT%),肺组织PCNA、α-SMA及NF-κB的表达。
3.统计学方法：使用SPSS13.0软件。三组数据的组间比较,采用单向方差分析(one-way ANOVA)进行统计分析,方差齐：多个样本均数方差间比较采用最小显著差值法(LSD法)进行各组间数据的两两比较；方差不齐：多个样本均数方差间比较采用Tamhane检验法；指标间相关关系采用Pearson或Spearman相关分析法,以P<0.05为有统计学差异。
结果：
大鼠死亡情况：实验进行到第22、25天,MCT组死亡大鼠各一只,实验进行到第27天治疗组死亡大鼠一只,对照组大鼠全部生存。大鼠死亡前表现为呼吸困难。最终MCT组成模8只,治疗组成模9只,对照组10只。
1.川芎嗪对肺动脉高压大鼠mPAP以及RVHI的影响：MCT组大鼠的mPAP和RVHI均显著高于对照组(P<0.01),而治疗组大鼠的mPAP和RVHI均较MCT组明显降低(P<0.01),治疗组、对照组大鼠的mPAP和RVHI比较无差别(P>0.05)。
2.川芎嗪对肺血管周围炎症评分的影响：MCT组大鼠肺组织病理切片提示,血管周围炎症评分平均为3.5分,与对照组比较差异有显著性(P<0.01)；治疗组肺血管炎症程度评分明显降低,与MCT组比较差异有显著性(P<0.01)；治疗组、对照组组间比较无显著性差异(P=0.75)。
3.川芎嗪对肺动脉高压大鼠肺中动脉肺动脉中膜厚度百分比(WT%)的影响：MCT组WT%大于对照组,P<0.01；治疗组WT%小于MCT组P<0.01；治疗组WT%大于对照组P<0.01。
4.川芎嗪对肺动脉高压大鼠肺中小动脉PCNA表达的影响：MCT组PCNA表达大于对照组,P<0.01；治疗组PCNA表达小于MCT组P<0.01；治疗组PCNA表达大于对照组P<0.01。
5.川芎嗪对肺动脉高压大鼠肺小动脉a-SMA表达的影响：MCT组可见大鼠肺小动脉α-SMA表达明显增强,中膜增厚显著；对照组小动脉未见明显a-SMA表达;治疗组小动脉可见少量a-SMA表达。
6.川芎嗪对肺动脉高压大鼠肺组织NF-κB表达的影响：MCT组大鼠细支气管上皮细胞胞核阳性表达率较对照组明显增加(P<0.01)；治疗组大鼠细支气管上皮细胞胞核阳性表达率较MCT组少(P<0.01),较对照组多(P<0.01)。
7.大鼠PCNA表达与肺动脉中膜厚度指标的相关性：大鼠PCNA的表达与肺动脉中膜厚度百分比(WT%)呈正相关,r=0.96,P<0.01。
8.大鼠NF-κB表达与炎症及肺动脉中膜厚度指标的相关性：大鼠NF-κB表达与肺小动脉周围炎症呈正相关,r=0.73,P<0.01；大鼠NF-κB表达与炎症及肺动脉中膜厚度百分比呈正相关,r=0.94,P<0.01。
结论：
1.平均肺动脉压力增高时大鼠肺动脉中膜厚度百分比(WT%)、肺组织PCNA、α-SMA及NF-κB的表达均增高,大鼠PCNA、NF-κB表达与肺动脉中膜厚度呈正相关,提示大鼠肺动脉高压的肺血管重构可能与肺动脉平滑肌细胞过度增殖有关；
2.肺动脉高压时肺血管周围炎症严重,提示炎症可能参与影响肺动脉压力；
3.川芎嗪的干预可以降低大鼠平均肺动脉压、肺动脉中膜厚度百分比(WT%)、肺组织PCNA、α-SMA及NF-κB的表达,同时可以减轻肺血管周围炎症,提示川芎嗪对大鼠肺动脉高压有治疗作用；
4.川芎嗪降低大鼠平均肺动脉压力的机制可能与抑制肺动脉平滑肌细胞增殖,改善肺血管重构,以及减轻肺血管周围炎症有关。
第二章川芎嗪联合西地那非对肺动脉高压的临床研究
目的：
通过对肺动脉高压患者进行西地那非单药治疗与川芎嗪联合西地那非联合治疗进行对比,观察联合治疗组疗效是否优于西地那非单药组。
方法：
1.研究对象：符合入选标准的肺动脉高压患者：年龄在18-65岁之间的特发性肺动脉高压、慢性血栓栓塞性肺动脉高压、家族性肺动脉高压患者；基线6分钟步行距离≥100m且≤500m。共32例,男23例,女9例。WHO肺动脉高压功能分级为Ⅱ级；NYHA心功能分级为Ⅲ、 Ⅳ级；急性血管反应试验阴性,且未接受内皮素受体拮抗剂、前列环素类药物、治疗。
2.分组及治疗方法：本研究为前瞻性、开放性、非空白对照研究。按患者是否接受川芎嗪治疗分为2组：西地那非组(18例)、西地那非±川芎嗪联合用药组(联合用药组,14例)。用药方法：两组均口服西地那非,一次25mg,餐前1小时口服,一日3次；联合用药组在口服西地那非基础上加用磷酸川芎嗪片,一次100mg餐前1小时口服,一日3次。疗程：12周,每2周门诊随访。两组患者基线资料的比较无统计学差异。
3.疗效评价指标：①主要评价指标：6分钟步行距离(6-minute walk test,6MWT);②次要评价指标：WHO肺动脉高压功能分级、Borg呼吸困难评级、平均肺动脉压力(mPAP)。
4.统计学方法：使用SPSS13.0软件进行统计学分析。6分钟步行距离、血流动力学指标采用均数±标准差(x±s)表示,同组治疗前后用配对t检验进行分析,2组间均数比较用独立样本t检验进行分析。Borg呼吸困难评级、WHO肺动脉高压功能分级的比较,同组治疗前后用非参数配对秩和检验(Wilcoxon符号秩和检验)进行分析,2组间比较用两独立样本非参数检验(Mann-Whitney U)进行分析。P<0.05表示差异有统计学意义。
结果：
1.各药物治疗组指标前、后的变化：
西地那非组：治疗后6分钟步行距离比治疗前增加45米,P<0.01,差异有统计学意义；治疗后平均肺动脉压比治疗前下降约13mmHg,P<0.01,差异有统计学意义；治疗后WHO肺动脉高压功能分级下降,P=0.002,差异有统计学意义；Borg呼吸困难评级下降,P=0.007,差异有统计学意义。
联合治疗组：治疗后6分钟步行距离增加57米,P<0.01,差异有统计学意义；治疗后平均肺动脉压下降约25mmHg,P<0.01,差异有统计学意义；WHO肺动脉高压功能分级下降,P=0.001,差异有统计学意义；Borg呼吸困难评级下降,P=0.001,差异有统计学意义。
2.两组治疗后指标差异的比较：
6分钟步行距离：西地那非组小于联合治疗组,两者差异有统计学意义,P=0.046；平均肺动脉压：西地那非组高于联合治疗组治疗,差异有统计学意义,P=0.039；WHO肺动脉高压功能：联合治疗组下降更明显,差别有统计学意义,P=0.038；Borg呼吸困难评级：联合治疗组下降更低,差别有统计学意义,P=0.017。
结论：
1.西地那非干预后患者症状改善,平均肺动脉压力下降,6分钟步行距离增加,WHO肺动脉高压功能、Borg呼吸困难评级均改善,无严重不良反应,说明西地那非治疗肺动脉高压安全、有效,与以往研究结果相同,在伦理委员会不建议设置空白对照组的建议下,治疗效果有效且安全的西地那非作为非空白对照组是有意义的选择。
2.在使用西地那非的基础上,加用川芎嗪治疗后,患者平均肺动脉压力、6分钟步行距离、WHO肺动脉高压功能、Borg呼吸困难评级等指标的改善情况比单用西地那非更明显,同时加用川芎嗪治疗后,不良反应并未增加,提示川芎嗪对肺动脉高压具有治疗作用且安全性高,这为今后进一步的研究中选择川芎嗪单药治疗提供依据。
3.由于既往缺乏川芎嗪单药治疗肺动脉高压的研究,出于伦理方面的原因,本研究没有设置川芎嗪单药治疗组是一种不足。而本次临床研究结果提示川芎嗪对肺动脉高压患者的症状及观察指标均有明显的改善,同时安全性也得到验证。由此推测,川芎嗪可能成为治疗肺动脉高压的一种有效而廉价的选择,值得我们作更进一步的研究。
Background
Pulmonary hypertension is a common cardiovascular disease; the character of this disease is progressive elevated pulmonary vascular resistance. In pathology the main characteristics of the disease are pulmonary artery spasm, intimal hyperplasia and remodeling and in situ micro thrombus. Its diagnostic criteria:resting state mean pulmonary artery pressure over25mmHg or State of motion over30mmHg measure by cardiac catheterization, with left atrial pressure or pulmonary capillary pressure less than15mmHg. According to the latest guidelines, pulmonary hypertension is divided into five categories:1. arterial pulmonary hypertension;2. venous pulmonary hypertension;3. hypoxia-related pulmonary hypertension;4. due to chronic thrombotic or embolic pulmonary hypertension;5. mixed pulmonary hypertension.
The disease has a high disability or death rate and it is a serious threat to the people's health and lives. It is one of the hot spots of medical research. In all the five categories, the epidemiological survey found that arterial pulmonary hypertension (hereinafter referred to as "pulmonary hypertension") and chronic thrombotic and/or embolic disease pulmonary hypertension is the most serious type that caused patients die in our country. Registered study under the U.S. National Institutes of Health showed a median survival of patients with pulmonary hypertension was2.8years,1year,3year and5-year survival rates were68%,48%and34%, while in China idiopathic pulmonary arterial hypertension and familial pulmonary hypertension1,2,3,5-year survival rates were68%,56.9%,38.9%and20.8%. Pulmonary artery vascular proliferation and remodeling leads to the pulmonary vascular resistance progressive increased. It finally leads to right ventricular failure and death. That is the main cause of death in this disease.
In the past, because of the pathogenesis of pulmonary hypertension is unknown, the treatment of the disease is stagnant. Traditional treatment includes limited physical activity, giving oxygen, taking anticoagulants, taking diuretics, taking cardiac, having calcium antagonists, etc. But these treatments in the clinical often got poor effects. With the progress of research in the pathogenesis, novel targeted drugs continue to be available. It is widely recognized as more effective targeted drugs include:prostacyclin (iloprost), endothelin receptor antagonists (bosentan),5-phosphodiesterase inhibitors (sildenafil). Evidence-based medical research shows that these targeted drugs make cardiac function in patients with pulmonary hypertension, quality of life has been greatly improved, and even improved survival. Thus, the FDA_approved the drugs used to treat pulmonary hypertension, which make many patients who were desperate getting hope to regeneration. However, these drugs can only improve the clinical symptoms and hemodynamic status of patients. The limited role of vasodilator alone can not slow down the progressive of pressure increase in pulmonary artery. The condition is merely delaying pressure increase and there is no cure. The reason of pulmonary arterial pressure rising is pulmonary vascular remodeling and the basic of pulmonary vascular remodeling is the proliferation of vascular smooth muscle cells being excessive. Thus, inhibition of vascular smooth muscle cell proliferation and apoptosis is one of the trends in the field of pulmonary hypertension therapy. At the same time, these drugs completely dependent on imports, that makes them very expensive. For example, it will cost someone at lease several thousands of RMB a month in taking sildenafil. And sildenafil is the cheapest one among them. In our country, it runs basic medical security system and these drugs are failed to be included in the national essential medical insurance drug list. It means patients need to bear all of the costs for the drugs what make many of our patients can not using these drugs. Based on the above reasons, it has great significance to looking for some drugs that not only can inhibit or even reverse pulmonary vascular remodeling and improve cardiac function in the RV but also cheaper. That may be more suitable for national conditions of our country.
Ligustrazine is the active ingredient of Chinese medicine Chuanxiong, recent studies have shown that it has effects of anti-platelet, stimulate NO production, and dilate blood vessels, anti-inflammatory, anti-tumor cell proliferation and other effects. That can be effect on pulmonary hypertension. Currently, there are some clinical researches using ligustrazine to treat hypoxic pulmonary hypertension caused by chronic cor pulmonale. The results suggest that ligustrazine can reduce pulmonary artery pressure, improve blood oxygen levels, and improve the quality of life for the patients. The study showed reaction monitoring indicators decreased ET-land increased NO. It is presumably that ligustrazine can increasing NO releasing by pulmonary vascular endothelial, so that to reduce pulmonary hypertension and tests showed that the drug is safe. We design this study to explore the ligustrazine weather affect the pulmonary hypertension in patients with arterial pulmonary hypertension, chronic thrombotic or embolic disease. If ligustrazine have a therapeutic effect on pulmonary hypertension, it can provide more options for the treatment of pulmonary arterial hypertension.
This study is divided into two parts:basic research and clinical research. This study is approved by the research ethics committee. According to the ethics committee opinion, a clinical study there is no blank control group.
Chapter1:Effect of ligustrazine on pulmonary vascular remodeling in rats with pulmonary hypertension
Objective:
We treat the rats in pulmonary hypertension induced by Monocrotaline with ligustrazine. We want to observe the effect of ligustrazine on pulmonary artery pressure and pulmonary vascular remodeling in rats and to explore the possible mechanism of the effect.
Methods:
1. Make model:We divide30adult male SD rats randomly into3groups: control group, MCT-induced pulmonary hypertension group (MCT group), ligustrazine treatment group (treatment group). MCT group (10rats):once subcutaneous monocrotaline (50mg/kg) reared for21days, and then with saline (2ml/d) orally once daily for20days; the control group (10rats):once subcutaneous saline reared for21days, then saline (2ml/d) orally once daily for20days; treatment group (10rats):once subcutaneous monocrotaline (50mg/kg), reared for21days, then with ligustrazine (80mg/(kg.d)) orally once a day for20days.
2. Main testing indicators:mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), peri-vascular inflammation score(PVIS), percentage of pulmonary artery thickness(WT%), expression of proliferating cell nuclear antigen(PCNA)、Alpha-smooth muscle actin(a-SMA) and nuclear factor of kappa B(NF-kB).
3. Statistics:Using SPSS13.0software. Comparison among three groups of data using one-way ANOVA statistical analysis, when homogeneity of variance was used to compare multiple samples are among the least significant difference method (LSD method) each group of data pairwise comparison, when the heterogeneity of variance between multiple samples were used to compare the variance between two Tamhane test method; correlation analysis using Pearson or Spearman correlation analysis. P<0.05was considered statistically different.
Results:
Case of death of rats:There are two rats dead in MCT group at22ndand25thday during experiment, while treatment group got one rat dead at27th day. The rats in control group are all survive. Before they death, the rats showed difficulty breathing. Finally, MCT group got8rat models,9in treatment group and10in the control group.
1.Effect of ligustrazine on mPAP and RVHI in rats:mPAP and RVHI were significantly higher in MCT group than in control group (P<0.01); mPAP and RVHI treatment group were significantly lower than those in MCT group (P<0.01); mPAP and RVHI were undifferentiated between treatment group and control group (P>0.05).
2.Effect of ligustrazine on PVIS in rats:PVIS was significantly higher in MCT group than in control group (P<0.01); PVIS in treatment group was significantly lower than those in MCT group (P<0.01); PVIS was undifferentiated between treatment group and control group (P=G.75).
3.Effect of ligustrazine on WT%in rats:WT%was significantly higher in MCT group than in control group (P<0.01); WT%in treatment group was significantly lower than those in MCT group (P<0.01); WT%was significantly higher in treatment group than in control group (P<0.01).
4.Effect of ligustrazine on PCNA in rats:PCNA was significantly higher in MCT group than in control group (P<0.01); PCNA in treatment group was significantly lower than those in MCT group (P<0.01); PCNA was significantly higher in treatment group than in control group (P<0.01).
5.Effect of ligustrazine on α-SMA in rats:There is no expression of a-SMA on small arteries in control group; MCT group showed pulmonary arterioles a-SMA expression was significantly enhanced and the media thickening; there are a few small arteries treated a-SMA expression.
6.Effect of ligustrazine on NF-kB in rats:NF-κB was significantly higher in MCT group than in control group (P<0.01); NF-κB in treatment group was significantly lower than those in MCT group (P<0.01); NF-κB was significantly higher in treatment group than in control group (P<0.01).
7.The relevance between PCNA expression and WT%in rats:PCNA expression and WT%in rats were positively correlated, r=0.96, P<0.01.
8.The relevance between NF-κB and PVIS or WTκin rats:NF-κB and PVIS in rats were positively correlated, r=0.73, P<0.01; NF-κB and WT%in rats were positively correlated, r=0.94, P<0.01.
Conclusion:
1. When the mPAP in rats increased WT%, a-SMA, PCNA and NF-κB were increased. PCNA and NF-κB were positively correlated with WT%, suggesting that pulmonary vascular remodeling in rats with pulmonary hypertension may be related to pulmonary artery smooth muscle cell excessive proliferation.
2.When pulmonary hypertension peri-vascular inflammation was severe suggesting that inflammation may be impacted the pulmonary artery pressure.
3.Ligustrazine can reduce mPAP, WT%, expression of PCNA, α-SMA, NF-κB and also reduce peri-vascular inflammation in rats. It means that ligustrazine take part in the treatment of pulmonary hypertension.
4.The mechanism of ligustrazine reduce mean pulmonary arterial pressure may be related to inhibition of pulmonary artery smooth muscle cell proliferation what lead to improve pulmonary vascular remodeling. Reducing pulmonary peri-vascular inflammation maybe another way to bring down the mPAP.
Chapter2:Ligustrazine combine sildenafil treating pulmonary hypertension for clinical trial
Objective:
By sildenafil monotherapy in patients with pulmonary hypertension compare with ligustrazine combine sildenafil treating, we want to see that if combined treatment-group is superior to sildenafil monotherapy group.
Methods:
1. object of study:patients with pulmonary hypertension met the inclusion criteria:idiopathic pulmonary arterial hypertension, familial pulmonary hypertension, chronic thromboembolic pulmonary hypertension, between the ages of18-65, including32patients, male:23, female:9; NYHA functional class or PAH WHO functional class II and class acute vascular reactivity test was negative, or NYHA class of III, IV grade, and did not accept the drug prostacyclin, endothelin receptor antagonist therapy; baseline6-minute walk distance≥100m and≤500m.
2. Group and the treatment method:This study was based on a prospective, open, non-blank control group. The patients were divided into two treatment groups by weather accepted Ligustrazine:sildenafil group (18patients), sildenafil+ligustrazine combined treatment group (combination group,14patients). Treatment methods:oral sildenafil groups were once25mg, orally1hour before meals, three times a day; combination group were treated with oral sildenafil and Tetramethylpyrazine phosphate tablets a100mg1hour before orally,3times a day. Duration of treatment:12weeks, every two weeks outpatient follow-up. No significant difference between the two groups at baseline data.
3.Curative effect evaluation:①Main Outcome Measurements,6-minute walk test,6MWT;②Second Outcome Measurements:PAH WHO functional classes, Borg dyspnea rating, mean pulmonary arterial pressure (mPAP).
4. Statistics:Using SPSS13.0software. Variables data in the same group before and after treatment were analyzed by paired t test; between the two groups were compared using independent sample t-test analysis. Attributes data in the same group before and after treatment were analyzed using nonparametric paired rank sum test (Wilcoxon signed rank test) between two groups were compared using two independent samples nonparametric test (Mann-Whitney U) for analysis. P<0.05indicates significant difference.
Result:
1. The changes of indicator in each treatment group before and after treatment:
Sildenafil group:After treatment6-minute walk distance increased by45meters (P<0.01); after treatment mean pulmonary artery pressure decreased by about13mmHg,(P<0.01); after treatment pulmonary hypertension WHO functional class decreased,(P=0.002); after treatment Borg dyspnea ratings decline, P=0.007.
Combination group:After treatment6minutes walking distance increased57meters,(P<..01); After treatment mean pulmonary artery pressure decreased by about25mmHg,(P<0.01); After treatment pulmonary hypertension WHO functional class decreased(P=0.001); After treatment Borg dyspnea ratings decline,(P=0.001).2. The changes of indicator between two treatment group after treatment:
6minutes walking distance:Sildenafil group is less than combination group, the difference was statistically significant (P=0.046).
Mean pulmonary arterial pressure:Sildenafil group is more than combination group, the difference was statistically significant (P=0.039).
PAH WHO functional class:Sildenafil group is more than combination group, the difference was statistically significant (P=0.038).
Borg dyspnea rating:Sildenafil group is more than combination group, the difference was statistically significant (P=0.017).
Conclusion:
1.Patient's symptom was improved by the intervention of sildenafil. After treatment, mean pulmonary artery pressure decreased,6-minute walk distance increased, pulmonary hypertension WHO functional and Borg dyspnea ratings are improved. There in no serious adverse reactions, indicating the effectively and safety of sildenafil for pulmonary arterial hypertension. That is the same with previous findings. Because the Ethics Committee does not recommend setting recommend blank control group, so the treatment effect of sildenafil as an effective and safe non-blank control group is a meaningful choice.
2.0n the basis of using sildenafil, indicators in combination group are more obvious improved than sildenafil alone, while adverse reactions did not increase. It suggests that ligustrazine has a therapeutic effect on pulmonary hypertension and it is safeties, which provide the basis for further research in the future, selecting ligustrazine as a monotherapy.
3.Due to the lack of ligustrazine monotherapy in pulmonary hypertension research before. For ethical reasons, this study is not set ligustrazine monotherapy group, it is a kind of shortage. In this clinical trial, the findings suggest ligustrazine can improve the symptoms and the observation indexes in patients with pulmonary hypertension, and security is also verified. Then, ligustrazine could become an effective and cheap drug for the treatment of pulmonary hypertension. It worths we get a further research.
肺动脉高压是一种临床上比较常见的心血管疾病,本病以肺血管阻力进行性升高为特征,病理学以肺小动脉痉挛、内膜增生及重构和原位微血栓病灶形成为主要特征。其诊断标准：静息状态下心导管测定平均肺动脉压≥25mmHg或者运动时≥30mmHg,且肺毛细血管压或者左房压<15mmHg。根据指南的最新分类方法,肺动脉高压分为五大类：1.动脉型肺动脉高压；2.静脉性肺动脉高压；3.低氧血症相关性肺动脉高压；4.慢性血栓或栓塞所致肺动脉高压；5.混合性肺动脉高压。
本病致死、致残率高,严重威胁人民健康及生命,是医学研究的热点方向之一。其中,流行病学调查发现,动脉型肺动脉高压(以下称“肺动脉高压”)和慢性血栓和／或栓塞性疾病所引起的肺动脉高压是本病中危害我国人民身体健康最严重的类型,是引起患者致死的重要原因。根据美国国立卫生院注册研究表明肺动脉高压患者的中位生存期为2.8年、1年、3年和5年的生存率分别为68%、48%和34%,而在我国特发性肺动脉高压和家族性肺动脉高压患者1、2、3、5年的生存率分别为68%、56.9%、38.9%及20.8%。肺小动脉血管增生、重构导致肺血管阻力进行性加重,最终引起右心室功能衰竭、死亡,这是本病的主要致死原因。
以往,由于肺动脉高压的发病机理未明,传统治疗仅仅在于限制体力活动、吸氧、抗凝、利尿、强心,钙离子拮抗剂等一般处理,但在临床上这些治疗的效果往往不佳,本病的治疗曾一度停滞不前。随着发病机理研究的进展,新型靶向治疗药物不断面世,目前公认较为有效的靶向治疗药物包括：前列环素类(伊洛前列环素)、内皮素受体拮抗剂(波生坦)、5-磷酸二酯酶抑制剂(西地那非)。循证医学研究表明,上述靶向治疗药物能使肺动脉高压患者的心功能、生活质量有了很大的改善,甚至提高了生存率。由此,美国FDA批准上述药物用于治疗肺动脉高压,使许多原本绝望的患者看到再生的希望。然而,上述药物仅能改善患者临床症状与血流动力学状况,单纯扩血管的作用有限,不能减缓肺动脉压力进行性升高的趋势,病情仅仅是延迟加重,并没有根治。肺动脉高压患者肺动脉压力不断升高的原因,在于肺血管重构；而肺血管重构的基础是血管平滑肌细胞的过度增殖。因此,抑制血管平滑肌细胞增殖、促进其凋亡是肺动脉高压治疗领域的发展趋势之一。与此同时,上述药物完全依靠进口,价格昂贵,例如其中费用最低的西地那非,每月的费用需数千元,而我国实行基本药物医疗保障制度,上述药物均未能列入国家基本医疗保险药品目录,患者需自行承担所有费用,致使我国许多患者未能得到有效的治疗。基于上述原因,寻找能够抑制甚至逆转肺血管重构、改善右室心功能且价格低廉的药物具有十分重要的意义,也更适合我国的国情。
川芎嗪是中药川芎的有效成分,新近研究表明它有抗血小板、刺激NO生成、扩张血管、抗炎、抗肿瘤细胞增生等作用,与肺动脉高压的治疗有相符的地方。目前国内已经有临床研究应用川芎嗪干预由肺心病引起的低氧性肺动脉高压,结果提示川芎嗪能降低肺动脉压,提高血氧浓度,改善患者生活质量,研究显示监测的指标反应ET-1下降,NO升高,推测川芎嗪通过提高肺血管内皮NO释放达到治疗肺动脉高压,同时试验表明药物是安全的。为探讨川芎嗪对动脉型肺动脉高压、慢性血栓或栓塞性疾病所引起的肺动脉高压患者是否具有治疗作用而设计本研究。如能证实川芎嗪对肺动脉高压有治疗作用,可以为肺动脉高压的治疗提供更多的选择。
本研究分为动物基础研究及临床研究两部分,研究获得单位所在伦理委员会通过。根据伦理委员会的意见,临床研究部分不设空白对照组。
第一章川芎嗪对肺动脉高压大鼠肺血管重构的影响
目的：
通过使用川芎嗪对野百合碱(MCT)诱导的大鼠肺动脉高压进行干预,观察川芎嗪对大鼠肺动脉压力及肺血管重构的影响,并初步探讨其作用的可能机制。
方法：
1.造模：实验动物为SD大鼠,成年,雄性共30只,随机分为对照组、MCT诱导的肺动脉高压组(MCT组)、川芎嗪治疗组(治疗组)。MCT组(10只)：次皮下注射野百合碱(50mg/kg)饲养21天后,再以生理盐水(2ml/d)每日灌胃一次,共20天；对照组(10只)：一次皮下注射等量生理盐水饲养21天后,再以生理盐水(2ml/d)每日灌胃一次,共20天；治疗组(10只)：一次皮下注射野百合碱(50mg/kg),饲养21天后,以川芎嗪(80mg/(kg.d))每日灌胃一次,共20天。
2.主要检测指标：平均肺动脉压力(mean pulmonary arterial pressure, mPAP),右心室肥大指数(right ventricular hypertrophy index. RVHI),血管周围炎症评分,肺动脉中膜厚度百分比(WT%),肺组织PCNA、α-SMA及NF-κB的表达。
3.统计学方法：使用SPSS13.0软件。三组数据的组间比较,采用单向方差分析(one-way ANOVA)进行统计分析,方差齐：多个样本均数方差间比较采用最小显著差值法(LSD法)进行各组间数据的两两比较；方差不齐：多个样本均数方差间比较采用Tamhane检验法；指标间相关关系采用Pearson或Spearman相关分析法,以P<0.05为有统计学差异。
结果：
大鼠死亡情况：实验进行到第22、25天,MCT组死亡大鼠各一只,实验进行到第27天治疗组死亡大鼠一只,对照组大鼠全部生存。大鼠死亡前表现为呼吸困难。最终MCT组成模8只,治疗组成模9只,对照组10只。
1.川芎嗪对肺动脉高压大鼠mPAP以及RVHI的影响：MCT组大鼠的mPAP和RVHI均显著高于对照组(P<0.01),而治疗组大鼠的mPAP和RVHI均较MCT组明显降低(P<0.01),治疗组、对照组大鼠的mPAP和RVHI比较无差别(P>0.05)。
2.川芎嗪对肺血管周围炎症评分的影响：MCT组大鼠肺组织病理切片提示,血管周围炎症评分平均为3.5分,与对照组比较差异有显著性(P<0.01)；治疗组肺血管炎症程度评分明显降低,与MCT组比较差异有显著性(P<0.01)；治疗组、对照组组间比较无显著性差异(P=0.75)。
3.川芎嗪对肺动脉高压大鼠肺中动脉肺动脉中膜厚度百分比(WT%)的影响：MCT组WT%大于对照组,P<0.01；治疗组WT%小于MCT组P<0.01；治疗组WT%大于对照组P<0.01。
4.川芎嗪对肺动脉高压大鼠肺中小动脉PCNA表达的影响：MCT组PCNA表达大于对照组,P<0.01；治疗组PCNA表达小于MCT组P<0.01；治疗组PCNA表达大于对照组P<0.01。
5.川芎嗪对肺动脉高压大鼠肺小动脉a-SMA表达的影响：MCT组可见大鼠肺小动脉α-SMA表达明显增强,中膜增厚显著；对照组小动脉未见明显a-SMA表达;治疗组小动脉可见少量a-SMA表达。
6.川芎嗪对肺动脉高压大鼠肺组织NF-κB表达的影响：MCT组大鼠细支气管上皮细胞胞核阳性表达率较对照组明显增加(P<0.01)；治疗组大鼠细支气管上皮细胞胞核阳性表达率较MCT组少(P<0.01),较对照组多(P<0.01)。
7.大鼠PCNA表达与肺动脉中膜厚度指标的相关性：大鼠PCNA的表达与肺动脉中膜厚度百分比(WT%)呈正相关,r=0.96,P<0.01。
8.大鼠NF-κB表达与炎症及肺动脉中膜厚度指标的相关性：大鼠NF-κB表达与肺小动脉周围炎症呈正相关,r=0.73,P<0.01；大鼠NF-κB表达与炎症及肺动脉中膜厚度百分比呈正相关,r=0.94,P<0.01。
结论：
1.平均肺动脉压力增高时大鼠肺动脉中膜厚度百分比(WT%)、肺组织PCNA、α-SMA及NF-κB的表达均增高,大鼠PCNA、NF-κB表达与肺动脉中膜厚度呈正相关,提示大鼠肺动脉高压的肺血管重构可能与肺动脉平滑肌细胞过度增殖有关；
2.肺动脉高压时肺血管周围炎症严重,提示炎症可能参与影响肺动脉压力；
3.川芎嗪的干预可以降低大鼠平均肺动脉压、肺动脉中膜厚度百分比(WT%)、肺组织PCNA、α-SMA及NF-κB的表达,同时可以减轻肺血管周围炎症,提示川芎嗪对大鼠肺动脉高压有治疗作用；
4.川芎嗪降低大鼠平均肺动脉压力的机制可能与抑制肺动脉平滑肌细胞增殖,改善肺血管重构,以及减轻肺血管周围炎症有关。
第二章川芎嗪联合西地那非对肺动脉高压的临床研究
目的：
通过对肺动脉高压患者进行西地那非单药治疗与川芎嗪联合西地那非联合治疗进行对比,观察联合治疗组疗效是否优于西地那非单药组。
方法：
1.研究对象：符合入选标准的肺动脉高压患者：年龄在18-65岁之间的特发性肺动脉高压、慢性血栓栓塞性肺动脉高压、家族性肺动脉高压患者；基线6分钟步行距离≥100m且≤500m。共32例,男23例,女9例。WHO肺动脉高压功能分级为Ⅱ级；NYHA心功能分级为Ⅲ、 Ⅳ级；急性血管反应试验阴性,且未接受内皮素受体拮抗剂、前列环素类药物、治疗。
2.分组及治疗方法：本研究为前瞻性、开放性、非空白对照研究。按患者是否接受川芎嗪治疗分为2组：西地那非组(18例)、西地那非±川芎嗪联合用药组(联合用药组,14例)。用药方法：两组均口服西地那非,一次25mg,餐前1小时口服,一日3次；联合用药组在口服西地那非基础上加用磷酸川芎嗪片,一次100mg餐前1小时口服,一日3次。疗程：12周,每2周门诊随访。两组患者基线资料的比较无统计学差异。
3.疗效评价指标：①主要评价指标：6分钟步行距离(6-minute walk test,6MWT);②次要评价指标：WHO肺动脉高压功能分级、Borg呼吸困难评级、平均肺动脉压力(mPAP)。
4.统计学方法：使用SPSS13.0软件进行统计学分析。6分钟步行距离、血流动力学指标采用均数±标准差(x±s)表示,同组治疗前后用配对t检验进行分析,2组间均数比较用独立样本t检验进行分析。Borg呼吸困难评级、WHO肺动脉高压功能分级的比较,同组治疗前后用非参数配对秩和检验(Wilcoxon符号秩和检验)进行分析,2组间比较用两独立样本非参数检验(Mann-Whitney U)进行分析。P<0.05表示差异有统计学意义。
结果：
1.各药物治疗组指标前、后的变化：
西地那非组：治疗后6分钟步行距离比治疗前增加45米,P<0.01,差异有统计学意义；治疗后平均肺动脉压比治疗前下降约13mmHg,P<0.01,差异有统计学意义；治疗后WHO肺动脉高压功能分级下降,P=0.002,差异有统计学意义；Borg呼吸困难评级下降,P=0.007,差异有统计学意义。
联合治疗组：治疗后6分钟步行距离增加57米,P<0.01,差异有统计学意义；治疗后平均肺动脉压下降约25mmHg,P<0.01,差异有统计学意义；WHO肺动脉高压功能分级下降,P=0.001,差异有统计学意义；Borg呼吸困难评级下降,P=0.001,差异有统计学意义。
2.两组治疗后指标差异的比较：
6分钟步行距离：西地那非组小于联合治疗组,两者差异有统计学意义,P=0.046；平均肺动脉压：西地那非组高于联合治疗组治疗,差异有统计学意义,P=0.039；WHO肺动脉高压功能：联合治疗组下降更明显,差别有统计学意义,P=0.038；Borg呼吸困难评级：联合治疗组下降更低,差别有统计学意义,P=0.017。
结论：
1.西地那非干预后患者症状改善,平均肺动脉压力下降,6分钟步行距离增加,WHO肺动脉高压功能、Borg呼吸困难评级均改善,无严重不良反应,说明西地那非治疗肺动脉高压安全、有效,与以往研究结果相同,在伦理委员会不建议设置空白对照组的建议下,治疗效果有效且安全的西地那非作为非空白对照组是有意义的选择。
2.在使用西地那非的基础上,加用川芎嗪治疗后,患者平均肺动脉压力、6分钟步行距离、WHO肺动脉高压功能、Borg呼吸困难评级等指标的改善情况比单用西地那非更明显,同时加用川芎嗪治疗后,不良反应并未增加,提示川芎嗪对肺动脉高压具有治疗作用且安全性高,这为今后进一步的研究中选择川芎嗪单药治疗提供依据。
3.由于既往缺乏川芎嗪单药治疗肺动脉高压的研究,出于伦理方面的原因,本研究没有设置川芎嗪单药治疗组是一种不足。而本次临床研究结果提示川芎嗪对肺动脉高压患者的症状及观察指标均有明显的改善,同时安全性也得到验证。由此推测,川芎嗪可能成为治疗肺动脉高压的一种有效而廉价的选择,值得我们作更进一步的研究。
Background
Pulmonary hypertension is a common cardiovascular disease; the character of this disease is progressive elevated pulmonary vascular resistance. In pathology the main characteristics of the disease are pulmonary artery spasm, intimal hyperplasia and remodeling and in situ micro thrombus. Its diagnostic criteria:resting state mean pulmonary artery pressure over25mmHg or State of motion over30mmHg measure by cardiac catheterization, with left atrial pressure or pulmonary capillary pressure less than15mmHg. According to the latest guidelines, pulmonary hypertension is divided into five categories:1. arterial pulmonary hypertension;2. venous pulmonary hypertension;3. hypoxia-related pulmonary hypertension;4. due to chronic thrombotic or embolic pulmonary hypertension;5. mixed pulmonary hypertension.
The disease has a high disability or death rate and it is a serious threat to the people's health and lives. It is one of the hot spots of medical research. In all the five categories, the epidemiological survey found that arterial pulmonary hypertension (hereinafter referred to as "pulmonary hypertension") and chronic thrombotic and/or embolic disease pulmonary hypertension is the most serious type that caused patients die in our country. Registered study under the U.S. National Institutes of Health showed a median survival of patients with pulmonary hypertension was2.8years,1year,3year and5-year survival rates were68%,48%and34%, while in China idiopathic pulmonary arterial hypertension and familial pulmonary hypertension1,2,3,5-year survival rates were68%,56.9%,38.9%and20.8%. Pulmonary artery vascular proliferation and remodeling leads to the pulmonary vascular resistance progressive increased. It finally leads to right ventricular failure and death. That is the main cause of death in this disease.
In the past, because of the pathogenesis of pulmonary hypertension is unknown, the treatment of the disease is stagnant. Traditional treatment includes limited physical activity, giving oxygen, taking anticoagulants, taking diuretics, taking cardiac, having calcium antagonists, etc. But these treatments in the clinical often got poor effects. With the progress of research in the pathogenesis, novel targeted drugs continue to be available. It is widely recognized as more effective targeted drugs include:prostacyclin (iloprost), endothelin receptor antagonists (bosentan),5-phosphodiesterase inhibitors (sildenafil). Evidence-based medical research shows that these targeted drugs make cardiac function in patients with pulmonary hypertension, quality of life has been greatly improved, and even improved survival. Thus, the FDA_approved the drugs used to treat pulmonary hypertension, which make many patients who were desperate getting hope to regeneration. However, these drugs can only improve the clinical symptoms and hemodynamic status of patients. The limited role of vasodilator alone can not slow down the progressive of pressure increase in pulmonary artery. The condition is merely delaying pressure increase and there is no cure. The reason of pulmonary arterial pressure rising is pulmonary vascular remodeling and the basic of pulmonary vascular remodeling is the proliferation of vascular smooth muscle cells being excessive. Thus, inhibition of vascular smooth muscle cell proliferation and apoptosis is one of the trends in the field of pulmonary hypertension therapy. At the same time, these drugs completely dependent on imports, that makes them very expensive. For example, it will cost someone at lease several thousands of RMB a month in taking sildenafil. And sildenafil is the cheapest one among them. In our country, it runs basic medical security system and these drugs are failed to be included in the national essential medical insurance drug list. It means patients need to bear all of the costs for the drugs what make many of our patients can not using these drugs. Based on the above reasons, it has great significance to looking for some drugs that not only can inhibit or even reverse pulmonary vascular remodeling and improve cardiac function in the RV but also cheaper. That may be more suitable for national conditions of our country.
Ligustrazine is the active ingredient of Chinese medicine Chuanxiong, recent studies have shown that it has effects of anti-platelet, stimulate NO production, and dilate blood vessels, anti-inflammatory, anti-tumor cell proliferation and other effects. That can be effect on pulmonary hypertension. Currently, there are some clinical researches using ligustrazine to treat hypoxic pulmonary hypertension caused by chronic cor pulmonale. The results suggest that ligustrazine can reduce pulmonary artery pressure, improve blood oxygen levels, and improve the quality of life for the patients. The study showed reaction monitoring indicators decreased ET-land increased NO. It is presumably that ligustrazine can increasing NO releasing by pulmonary vascular endothelial, so that to reduce pulmonary hypertension and tests showed that the drug is safe. We design this study to explore the ligustrazine weather affect the pulmonary hypertension in patients with arterial pulmonary hypertension, chronic thrombotic or embolic disease. If ligustrazine have a therapeutic effect on pulmonary hypertension, it can provide more options for the treatment of pulmonary arterial hypertension.
This study is divided into two parts:basic research and clinical research. This study is approved by the research ethics committee. According to the ethics committee opinion, a clinical study there is no blank control group.
Chapter1:Effect of ligustrazine on pulmonary vascular remodeling in rats with pulmonary hypertension
Objective:
We treat the rats in pulmonary hypertension induced by Monocrotaline with ligustrazine. We want to observe the effect of ligustrazine on pulmonary artery pressure and pulmonary vascular remodeling in rats and to explore the possible mechanism of the effect.
Methods:
1. Make model:We divide30adult male SD rats randomly into3groups: control group, MCT-induced pulmonary hypertension group (MCT group), ligustrazine treatment group (treatment group). MCT group (10rats):once subcutaneous monocrotaline (50mg/kg) reared for21days, and then with saline (2ml/d) orally once daily for20days; the control group (10rats):once subcutaneous saline reared for21days, then saline (2ml/d) orally once daily for20days; treatment group (10rats):once subcutaneous monocrotaline (50mg/kg), reared for21days, then with ligustrazine (80mg/(kg.d)) orally once a day for20days.
2. Main testing indicators:mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), peri-vascular inflammation score(PVIS), percentage of pulmonary artery thickness(WT%), expression of proliferating cell nuclear antigen(PCNA)、Alpha-smooth muscle actin(a-SMA) and nuclear factor of kappa B(NF-kB).
3. Statistics:Using SPSS13.0software. Comparison among three groups of data using one-way ANOVA statistical analysis, when homogeneity of variance was used to compare multiple samples are among the least significant difference method (LSD method) each group of data pairwise comparison, when the heterogeneity of variance between multiple samples were used to compare the variance between two Tamhane test method; correlation analysis using Pearson or Spearman correlation analysis. P<0.05was considered statistically different.
Results:
Case of death of rats:There are two rats dead in MCT group at22ndand25thday during experiment, while treatment group got one rat dead at27th day. The rats in control group are all survive. Before they death, the rats showed difficulty breathing. Finally, MCT group got8rat models,9in treatment group and10in the control group.
1.Effect of ligustrazine on mPAP and RVHI in rats:mPAP and RVHI were significantly higher in MCT group than in control group (P<0.01); mPAP and RVHI treatment group were significantly lower than those in MCT group (P<0.01); mPAP and RVHI were undifferentiated between treatment group and control group (P>0.05).
2.Effect of ligustrazine on PVIS in rats:PVIS was significantly higher in MCT group than in control group (P<0.01); PVIS in treatment group was significantly lower than those in MCT group (P<0.01); PVIS was undifferentiated between treatment group and control group (P=G.75).
3.Effect of ligustrazine on WT%in rats:WT%was significantly higher in MCT group than in control group (P<0.01); WT%in treatment group was significantly lower than those in MCT group (P<0.01); WT%was significantly higher in treatment group than in control group (P<0.01).
4.Effect of ligustrazine on PCNA in rats:PCNA was significantly higher in MCT group than in control group (P<0.01); PCNA in treatment group was significantly lower than those in MCT group (P<0.01); PCNA was significantly higher in treatment group than in control group (P<0.01).
5.Effect of ligustrazine on α-SMA in rats:There is no expression of a-SMA on small arteries in control group; MCT group showed pulmonary arterioles a-SMA expression was significantly enhanced and the media thickening; there are a few small arteries treated a-SMA expression.
6.Effect of ligustrazine on NF-kB in rats:NF-κB was significantly higher in MCT group than in control group (P<0.01); NF-κB in treatment group was significantly lower than those in MCT group (P<0.01); NF-κB was significantly higher in treatment group than in control group (P<0.01).
7.The relevance between PCNA expression and WT%in rats:PCNA expression and WT%in rats were positively correlated, r=0.96, P<0.01.
8.The relevance between NF-κB and PVIS or WTκin rats:NF-κB and PVIS in rats were positively correlated, r=0.73, P<0.01; NF-κB and WT%in rats were positively correlated, r=0.94, P<0.01.
Conclusion:
1. When the mPAP in rats increased WT%, a-SMA, PCNA and NF-κB were increased. PCNA and NF-κB were positively correlated with WT%, suggesting that pulmonary vascular remodeling in rats with pulmonary hypertension may be related to pulmonary artery smooth muscle cell excessive proliferation.
2.When pulmonary hypertension peri-vascular inflammation was severe suggesting that inflammation may be impacted the pulmonary artery pressure.
3.Ligustrazine can reduce mPAP, WT%, expression of PCNA, α-SMA, NF-κB and also reduce peri-vascular inflammation in rats. It means that ligustrazine take part in the treatment of pulmonary hypertension.
4.The mechanism of ligustrazine reduce mean pulmonary arterial pressure may be related to inhibition of pulmonary artery smooth muscle cell proliferation what lead to improve pulmonary vascular remodeling. Reducing pulmonary peri-vascular inflammation maybe another way to bring down the mPAP.
Chapter2:Ligustrazine combine sildenafil treating pulmonary hypertension for clinical trial
Objective:
By sildenafil monotherapy in patients with pulmonary hypertension compare with ligustrazine combine sildenafil treating, we want to see that if combined treatment-group is superior to sildenafil monotherapy group.
Methods:
1. object of study:patients with pulmonary hypertension met the inclusion criteria:idiopathic pulmonary arterial hypertension, familial pulmonary hypertension, chronic thromboembolic pulmonary hypertension, between the ages of18-65, including32patients, male:23, female:9; NYHA functional class or PAH WHO functional class II and class acute vascular reactivity test was negative, or NYHA class of III, IV grade, and did not accept the drug prostacyclin, endothelin receptor antagonist therapy; baseline6-minute walk distance≥100m and≤500m.
2. Group and the treatment method:This study was based on a prospective, open, non-blank control group. The patients were divided into two treatment groups by weather accepted Ligustrazine:sildenafil group (18patients), sildenafil+ligustrazine combined treatment group (combination group,14patients). Treatment methods:oral sildenafil groups were once25mg, orally1hour before meals, three times a day; combination group were treated with oral sildenafil and Tetramethylpyrazine phosphate tablets a100mg1hour before orally,3times a day. Duration of treatment:12weeks, every two weeks outpatient follow-up. No significant difference between the two groups at baseline data.
3.Curative effect evaluation:①Main Outcome Measurements,6-minute walk test,6MWT;②Second Outcome Measurements:PAH WHO functional classes, Borg dyspnea rating, mean pulmonary arterial pressure (mPAP).
4. Statistics:Using SPSS13.0software. Variables data in the same group before and after treatment were analyzed by paired t test; between the two groups were compared using independent sample t-test analysis. Attributes data in the same group before and after treatment were analyzed using nonparametric paired rank sum test (Wilcoxon signed rank test) between two groups were compared using two independent samples nonparametric test (Mann-Whitney U) for analysis. P<0.05indicates significant difference.
Result:
1. The changes of indicator in each treatment group before and after treatment:
Sildenafil group:After treatment6-minute walk distance increased by45meters (P<0.01); after treatment mean pulmonary artery pressure decreased by about13mmHg,(P<0.01); after treatment pulmonary hypertension WHO functional class decreased,(P=0.002); after treatment Borg dyspnea ratings decline, P=0.007.
Combination group:After treatment6minutes walking distance increased57meters,(P<..01); After treatment mean pulmonary artery pressure decreased by about25mmHg,(P<0.01); After treatment pulmonary hypertension WHO functional class decreased(P=0.001); After treatment Borg dyspnea ratings decline,(P=0.001).2. The changes of indicator between two treatment group after treatment:
6minutes walking distance:Sildenafil group is less than combination group, the difference was statistically significant (P=0.046).
Mean pulmonary arterial pressure:Sildenafil group is more than combination group, the difference was statistically significant (P=0.039).
PAH WHO functional class:Sildenafil group is more than combination group, the difference was statistically significant (P=0.038).
Borg dyspnea rating:Sildenafil group is more than combination group, the difference was statistically significant (P=0.017).
Conclusion:
1.Patient's symptom was improved by the intervention of sildenafil. After treatment, mean pulmonary artery pressure decreased,6-minute walk distance increased, pulmonary hypertension WHO functional and Borg dyspnea ratings are improved. There in no serious adverse reactions, indicating the effectively and safety of sildenafil for pulmonary arterial hypertension. That is the same with previous findings. Because the Ethics Committee does not recommend setting recommend blank control group, so the treatment effect of sildenafil as an effective and safe non-blank control group is a meaningful choice.
2.0n the basis of using sildenafil, indicators in combination group are more obvious improved than sildenafil alone, while adverse reactions did not increase. It suggests that ligustrazine has a therapeutic effect on pulmonary hypertension and it is safeties, which provide the basis for further research in the future, selecting ligustrazine as a monotherapy.
3.Due to the lack of ligustrazine monotherapy in pulmonary hypertension research before. For ethical reasons, this study is not set ligustrazine monotherapy group, it is a kind of shortage. In this clinical trial, the findings suggest ligustrazine can improve the symptoms and the observation indexes in patients with pulmonary hypertension, and security is also verified. Then, ligustrazine could become an effective and cheap drug for the treatment of pulmonary hypertension. It worths we get a further research.
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65. Barth PJ, Kimpel C, Roy S, et at. An improved mathematical approach for the assessment of the medial thickness of pulmonary arteries. Pathol Res Pract,1993. 189(5):567-76.
66.程德云,关键,夏秀琼等.低氧性肺动脉高压大鼠肺组织核因子NF-kB表达的变化.四川大学学报(医学版),2006,37(4)：611-3.
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68.王利梅,张焕萍,柴景伟.川芎嗪对野百合碱诱导大鼠肺动脉高压的影响.中西医结合心脑血管病杂志.2012,10(1)：71-72.
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76.高琳琳.川芎嗪对ET-1诱导的脐动脉平滑肌细胞增殖的作用.黑龙江医学,2006；30(4)：259-261.
77.王宏伟,张露,赵华月.川芎嗪对血管平滑肌细胞增殖及表达c-myc基因的影响.中国动脉硬化杂志,2000；8(4)：319-321.
78. Wong KL, Wu KC, Wu RS, et al.Tetramethylpyrazine inhibits angiotensin II-increased NAD(P)H oxidase activity and subsequent proliferation in rat aortic smooth muscle cells.Am J Chin Med,2007; 35(6):1021-35.
79.王燕侠,梁杰.老年原发性高血压颈动脉粥样硬化患者动态动脉硬化指数的变化及意义[J].中国老年学杂志,2013,33(8)：1759-1761.
80.刘泽军,李晚珍,吴礼文等.川芎嗪联合厄贝沙坦对高血压早期肾损害的防治作用[J].中国循证心血管医学杂志,2011,03(1)：44-47.
81.刘薇,王志强.川芎嗪联合坎地沙坦酯对高血压早期肾损害合并高尿酸血症的临床观察[J].临床肾脏病杂志,2012,12(1)：41-43.
82. D'Alonzo GE, Barst RJ, Ayres SM, et al. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med,1991,115(5):343-349.
83. Jing ZC, Xu XQ, Han ZY, et al. Registry and survival study in Chinese patients with idiopathic and familial pulmonary arterial hypertension. Chest,2007,132(2): 373-379.
84. Macchia A, Marchioli R, Marfisi R, et al. A meta-analysis of trials of pulmonary hypertension:a clinical condition looking for drugs and research methodology. Am Heart J,2007,153(6):1037-1047.
85. ATS statement:guidelines for the six-minute walk test. Am J Respir Crit Care Med.2002; 166:111-117.
86. Wu CC, Chiou WF, Yen MH. A possible mechanism of action of tetramethylpyrazine on vascular smooth muscle in rat aorta [J]. Eur J Pharmacol. 1989,10:169(23):189-195.
87. Pang PK, Shan JJ, Chiu KW. Tetramethylpyrazine, a calcium antagonist. Planta Med,1996; 62(5):431-435.
88.史学军,李秋贵.川芎嗪对慢性肺源性心脏病相关指标的影响.上海中医药杂志,1999；(2)：10-11.
89. Kisseleva T, Song L, Vorontchikhina M, Feirt N, Kitajewski J, Schindler C. NF-kappaB regulation of endothelial cell function during LPS-induced toxemia and cancer [J]. J Clin Invest.2006,116(11):2955-2963.
90. Li X, Tupper JC, Bannerman DD, Winn RK, Rhodes CJ, Harlan JM. Phosphoinositide 3 kinase mediates Toll-like receptor 4-induced activation of NF-kappa B in endothelial cells [J]. Infection and Immunity.2003,71 (8):4414-4420.
91.刘艳凯,刘圣君,张玉平,等.川芎嗪、当归注射液对急性微循环障碍大鼠血小板功能和器官血流量的影响[J].中国医学物理学杂志,2005(5)： 660-662.
92.王玉良,巴彦坤.川芍嗦对心血管组织的药理和电生理作用—一种新的钙拮抗剂.中国中西医结合杂志,1985；5：291.
93. DuanW, Chan JH, Wong CH et al. Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model. J Immunol.2004,172:7053-7059.
94.王宏伟,张露,赵华月.川芎嗪对血管平滑肌细胞增殖及表达c-myc基因的影响中国动脉硬化杂志.2000,7：132-137.
95. Galie N, Ghofrani HA, Tothicki A, et al. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med,2005; 353:2148-57.
96.胡立刚.大剂量川芎嗪对慢性肺心病加重期血液流变学的影响[J].实用内科杂志,1992；12(10)：540.
97. Peetors P, Mets T. The6-minute walk as an aPProPriate exereise test in elderly Patients with chronic heart failure. J Gerontol A Biol Sci Med Sci,1996,51(4): M147-151.
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