The Effects Of Ligustrazine On Pulmonary Artery Hypertension In Experimental And Clinical Research

BackgroundPulmonary 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 hypertensionObjective: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 trialObjective: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.