【作者】 王文靖；

【导师】 潘毅；

【作者基本信息】 广州中医药大学 ， 中医基础理论， 2012， 博士

【摘要】 目的：高血压是危害人类生命的主要疾病之一,其治疗的最终目标是有效保护靶器官,降低病死率和病残率。传统的西药降压药,降压虽较快捷,但可出现诸多方面的不良反应,在靶器官保护方面的作用未尽人意。中药降压具有多途径、多环节、多靶点的特点,且价格相对较低,在治疗上有着少或无毒副作用的优势,因此如果能够开发出一种既可以降压又对靶器官具有一定保护作用的中成药,那么对于促进中药现代化和促进高血压病的临床康复将具有重要意义。本课题拟在以往研究的基础上,对根据导师的临床验方“调肝肾、祛痰瘀”组方所开发成的“血畅宁无糖颗粒”进行药效学研究,从心室重构、血管重塑、血管内皮功能等不同层面了解其在治疗高血压病时的作用机制,从而深化对中医治疗高血压病病机的认识,为临床防治高血压病提供新的治疗途径。方法：1.血畅宁无糖颗粒：熟地25g、龟版30g、丹参15g、瓜萎12g、田七5g、钩藤12g。以上生药材经过再次干燥,按处方比例(其中龟板打碎、先煎30min,钩藤后下煎煮5min)混匀,常温下加入蒸馏水浸泡过夜,回流提取,将过滤后的滤出液混合并浓缩,制成浸膏(含生药1g/m1),冷藏备用。2.实验动物及分组实验采用两种模型,即自发性高血压大鼠(SHR)模型和自制的“两肾一夹”肾性高血压大鼠(RHR)模型。(1)RHR按随机数字表法分成5组：假手术组、模型组、血畅宁高剂量组、血畅宁低剂量组、氯沙坦组,每组10只。(2)SHR按随机数字表法分成4组：模型组、血畅宁高剂量组、血畅宁低剂量组、氯沙坦组,每组10只；另外,加设对照组：由普通SD大鼠组成,10只。3.给药及喂养方式(1)RHR造模第4周末起,各组大鼠灌胃给药,1次/天。药物灌服的剂量根据临床等效剂量,血畅宁高剂量组为20g生药/kg/d,血畅宁低剂量组为l0g生药/kg/d,氯沙坦组为30mmg/kg/d,给药体积均为1ml/100g。假手术组和模型组均给予1ml/100g的蒸馏水灌胃,每日上午给药一次,持续5周。在实验期间,各组大鼠均以标准饲料喂养,自由饮水。(2)SHR组于SHR第10周时开始灌胃给药,给药及喂养方式同RHR相应组别的方式,其中对照组同假手术组。4.研究内容包括五个部分：①肾性高血压大鼠模型的改良与评价；②血畅宁对两种模型大鼠的血压以及血清NO和AngⅡ含量的影响；③血畅宁对两种模型大鼠心、肾、主动脉等靶器官形态学方面的影响：光镜下观察心、主动脉和肾脏的结构,电镜下观察主动脉和肾脏的超微结构,并测量左室重量和动脉管壁中膜厚度；④免疫组织化学法及RT-PCR法检测血畅宁对两种模型大鼠动脉组织中bFGF.OPN含量及bFGFmRNA、OPNmRNA表达的影响；⑤免疫组织化学法及RT-PCR法检测血畅宁对两种模型大鼠心肌组织中ACE2的含量及AT1RmRNA、 AT2RmRNA、 ACE2mRNA表达的影响。结果：1.对SD大鼠造模后,RHR模型组与假手术组比较,模型组在造模后3天收缩压开始升高,7天后血压急剧上升,14天高血压已初步形成,具有显著性差异(P<0.05)。2.在RHR模型中,治疗5周末,血畅宁高剂量组与氯沙坦组降压效果相当,均优于低剂量组,而在其他各个时间点氯沙坦组血压均比血畅宁高剂量组低。另外,在降低血清AngⅡ含量方面,血畅宁高剂量组优于低剂量组,而氯沙坦组能增高血清AngⅡ含量。在增高血清NO含量方面,血畅宁高剂量组和氯沙坦组相当,均优于低剂量组；在SHR模型中,治疗5周末,氯沙坦组降压效果最好,血畅宁高剂量组降压效果优于低剂量组,在其他各个时间点氯沙坦组血压均比血畅宁高剂量组低。在降低血清AngⅡ含量方面,血畅宁高剂量组优于低剂量组和氯沙坦组。在增高血清NO含量方面,血畅宁高剂量组作用显著,优于低剂量组和氯沙坦组。3.两种模型中,在降低LVMI、 MT和VFC方面以及对心和主动脉的形态学保护方面的结果一致,血畅宁高剂量组和氯沙坦组效果相当,均优于低剂量组；而在肾脏保护方面,血畅宁高剂量组和低剂量组均有一定的保护效应,而氯沙坦组的肾脏保护效应较差。4.两种模型中,在降低动脉组织中bFGF、 OPN含量以及bFGFmRNA和OPNmRNA表达方面结果一致,血畅宁高剂量组均优于血畅宁低剂量组和氯沙坦组,而氯沙坦组作用不稳定,有时与血畅宁高剂量组效果相当,有时又与低剂量组效果相当。5.在增加ACE2含量和AT2R mRNA方面,两种模型的血畅宁高剂量组和氯沙坦组的作用相当,均优于低剂量组,而且治疗效果接近正常对照组；而在降低AT1R mRNA方面氯沙坦组优于血畅宁高剂量组和低剂量组；同时,在ACE2mRNA的表达方面两种模型的结果不一致,在RHR模型中,血畅宁高剂量组和氯沙坦组均可以增加心肌组织中ACE2mRNA的表达,但氯沙坦组的效果更好；在SHR模型中,血畅宁高剂量组可以明显增加心肌组织中ACE2mRNA的表达,而氯沙坦组的效果与血畅宁低剂量组的效果相当,与血畅宁高剂量组比较效果较差。结论：1.血畅宁具有明显的降低RHR和SHR血压的作用,但降压较为缓慢,在治疗过程中,动物血压呈现出持续缓慢下降的趋势,虽然降压效果不及氯沙坦迅速,但却显示出了中药降压缓慢平稳,持续降压的优势；2.血畅宁能够降低RHR和SHR血清AngⅡ含量,同时提高RHR和SHR的血清NO含量,从而改善血管内皮功能；3.血畅宁可以降低RHR和SHR心肌的LVMI、增加心肌组织中ACE2的含量和ACE2mRNA、 AT2R mRNA的表达,同时还可以降低AT1R mRNA的表达。形态学观察显示血畅宁能显著改善两种模型的主要靶器官心的形态学表现,说明血畅宁有比较明显的改善心室重构的作用,从而抑制了心肌这一靶器官的损伤；4.血畅宁能够明显降低RHR和SHR主动脉管壁的MT、VFC,降低主动脉组织中bFGF、 OPN的含量以及bFGF mRNA、 OPN mRNA的表达,同时还能够显著改善两种模型的主要靶器官主动脉的形态学表现,说明血畅宁有比较明显的改善动脉血管重塑的作用,从而抑制了主动脉这一靶器官的损伤；5.血畅宁能够显著改善两种模型的主要靶器官肾脏的形态学表现,从而抑制了大鼠肾脏这一靶器官的损伤。6.从整体来讲,高剂量血畅宁优于低剂量血畅宁的效果,而且在靶器官保护方面血畅宁效果优于氯沙坦。更多还原

【Abstract】 ObjectiveHypertension is one of the major diseases that endanger human life. The ultimate goal of the treatment is protecting the target organs effectively and reducing the mortality rate and disability rate. Although the efficiency of antihypertensive by Western medicine is fast, there may be many adverse reactions. Moreover, the effect of protecting the target organs by Western medicine is not good. Traditional Chinese medicine (TCM) has characteristics of multi-channel, multi-link and multi-target in treating hypertension and its price is relatively low. In addition, it has little or no adverse reaction. Therefore, the development of proprietary Chinese medicines is of great significance in developing TCM resources and promoting modernization of TCM, especially for the pharmacological research on the active ingredients and mechanism of action.The primary purpose of this paper is to do a pharmacological research on the Xuechangning sugar free granule based on previous studies. Xuechangning sugar free granule is a new proprietary Chinese medicine that has been developed according to my instructor’s clinical prescription named "regulating the function of liver and kidney while removing phlegm and blood stasis". We discuss its Anti-hypertension mechanism from different aspects such as left ventricular remodeling, vascular remodeling and vascular endothelial function, to provide some new ideas in the prevention and treatment of hypertension in clinical practices.Methods1. Xuechangning sugar free granule Radix rehmanniae preparata25g、carapax et plastrum testudins3g、 radix salviae miltiorrhizae15g、 fructus trichosanthis12g、 radix notoginseng5g、 ramulus uncariae cum uncis12g. Raw-material medicine mixed according to recipe portion after a second dry(Break carapax et plastrum testudins into pieces、 fore-decoct30min, and ramulus uncariae cum uncis post-decoct for5min). Add distilled water to soak overnight at air temperature, reflux extraction. Then mixed and concentrated all filtrate, to prepare the extract (containing raw medicine1g per ml), refrigerated ready to use.2. The animals and groupsThe animals used in this experiment are two kinds of rat models:the spontaneously hypertensive rats (SHR) model and "two kidney-one clip" renal hypertensive rats (RHR) model.(1) RHR are divided into five groups according to the random number table:Sham operation group, Model group, High-dosage of Xuechangnin group, Low-dosage of Xuechangnin group, Losartan group (n=10).(2) SHR are divided into four groups according to the random number table:Model group, High-dosage of Xuechangnin group, Low-dosage of Xuechangnin group, Losartan group (n=10). In addition, there is a control group of10normal SD rats.3. Administration method(1) The model rats started administration from the first five weeks after model establishment. The dose of medicine was clinical equivalent dose. High-dosage of Xuechangnin group was supplied with20g raw medicine/kg/d; Low-dosage of Xuechangnin group was supplied with lOg raw medicine/kg/d; losartan group was supplied with30mg/kg/d, administration volumes were all1ml/100g. The sham operation group and model group were both supplied with1ml/100g distilled water. Administered once AM per day for five continuous weeks. During the experiment, the rats in each group were supplied with standard feed and with free access to water.(2) SHR was administrated at the tenth week. The way of delivery and feeding was the same as the corresponding groups of RHR, and the control group was same with the sham group.4. Research contentsThe research includes five parts:①The improvement and evaluation in method of renal hypertensive model rats;②The effects of Xuechangning on blood pressure and the contents of NO and AngⅡ in serum of two model rats;③The effects of Xuechangning on the morphology in target organs of two model rats, such as heart, kidney and aorta. We observed morphological changes of the heart, aorta and kidney by light microscope and observed the ultrastructure of the aorta and kidney by electron microscopy. Meanwhile, we measured the left ventricular mass and artery intima-media wall thickness;④The protein production of bFGF and OPN in the aortic tissue of two model rats were detected by immunohistochemical, while the mRNA expression of bFGF and OPN were detected by RT-PCR;⑤The protein production of ACE2in the myocardial tissue of two model rats were detected by immunohistochemical, while the mRNA expression of AT1R AT2R and ACE2were detected by RT-PCR.Results1. Compared with sham operation group, systolic blood pressure of model group began to increase on the3rd day after building the models, and then significantly increased on the7th day. And hypertension has been initially formed on the14th day; there were significant differences compared with sham operation group (P<0.05).2. In the RHR model, the antihypertensive effect on high-dosage of Xuechangnin group was equal to the effect of the losartan group after fifth week’s treatment; they are both better than the low-dosage of Xuechangnin group. Meanwhile, the blood pressure of losartan group is lower than high-dosage of Xuechangnin group in each of the other time points. In addition high-dosage of Xuechangnin group is better than the low-dosage of Xuechangnin group in reducing serum AngⅡ content. Meanwhile, losartan group increased serum AngⅡ content. High-dosage of Xuechangnin group was equal to losartan group in increasing serum NO content; they are both better than the low-dosage of Xuechangnin group; In the SHR model, the antihypertensive effect on high-dosage of Xuechangnin group is equal to losartan group after fifth weeks treatment, are both better than the low-dosage of Xuechangnin group. Meanwhile, the blood pressure of losartan group is lower than high-dosage of Xuechangnin group in each of the other time points. High-dosage of Xuechangnin group is best in reducing serum AngⅡcontent. In addition, high-dosage of Xuechangnin group has a significant effect on increasing serum NO, bettei than low-dosage of Xuechangnin group and losartan group.3. We obtained consistent results in the two models:the role of reducing LVMI, MT, and VFC and inhibiting morphological change of the heart and aorta are considerable in high-dosage of Xuechangnin group and losartan group, and both better than the low-dosage of Xuechangnin group; In addition, high-dosage of Xuechangnin group and low-dosage of Xuechangnin group had a certain protective effect on the kidney, While losartan group had a relatively poor protective effect on the kidney.4. The result suggests that RHR groups are consistent with that of SHR groups in reducing the protein production of bFGF and OPN and their mRNA expression in the aortic tissue. We found that high-dosage of Xuechangnin group is superior to low-dosage of Xuechangnin group and losartan group. While the role of the losartan group is unstable, sometimes equal to the high-dosage of Xuechangnin group, sometimes equal to the low-dosage of Xuechangnin group.5. The result shows that RHR groups are equal to that of SHR groups in increasing protein production of ACE2and AT2RmRNA expression in the myocardial tissue. We found that the effects are considerable in high-dosage of Xuechangnin group and losartan group, they are both better than low-dosage of Xuechangnin group and close to the normal control group; while the role of reducing ATlRmRNA expression in Losartan group is better than high-dosage of Xuechangnin group and low-dosage of Xuechangnin group; Besides, the results show that RHR groups are equal to that of SHR groups in ACE2mRNA expression. In the RHR model, we found that high-dosage of Xuechangnin group and losartan group both can increase ACE2mRNA expression of the myocardial tissue, but the losartan group is better; in the SHR model, we found that high-dosage of Xuechangnin group can increase ACE2mRNA expression of the myocardial tissue significantly, and is better than the losartan group and the low-dosage of Xuechangnin group, while the latter two effects are relatively poor.Conclusion1. Xuechangning has a significant effect on reducing blood pressure of the two models. The blood pressure of rats showed a slow but continued decreasing tendency in the whole period of experiment. Although the effect of antihypertensive is not as good as losartan, it shows advantages of TCM in reducing the blood pressure slowly, steadily and continuously;2. Xuechangning can reduce the contents of Angll in serum of two model rats, while increase the contents of NO in serum of two model rats. So it can improve the endothelial function; 3. Xuechangning can reduce LVMI, increase protein production of ACE2and expression of ACE2mRNA and AT2RmRNA in the myocardial tissue, while reducing the expression of ATlRmRNA. Morphological observation of myocardial tissue showed that Xuechangning can significantly improve morphological features of the heart of the two models. It is illustrated that Xuechangning can improved ventricular remodeling significantly, thus inhibiting the damage of this target organ;4. Xuechangning can significantly reduce Mt and VFC in thoracic aorta wall of RHR and SHR, reduce the protein production of bFGF and OPN and their mRNA expression in the aortic tissue, and significantly improve morphological manifestations of the aorta. It is illustrated that Xuechangning can improved arterial vascular remodeling obviously, thus inhibiting the damage of this target organ;5. Xuechangning can significantly improve the kidney morphologic features of the two models, thus inhibiting the damage of this target organ;6. Generally speaking, we obtained consistent results in the two models: the high-dosage of Xuechangnin group excels the low one. The effect on protecting the target organ of Xuechangning is superior to that of losartan.更多还原