芪参健脾方对自发性高血压大鼠降压及血管保护作用的机制研究
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摘要
目的：
本实验通过观察芪参健脾方对自发性高血压大鼠(SHR)尾动脉收缩压及血浆一氧化氮、前列环素、内皮源性超极化因子、血管紧张素Ⅱ和内皮素1含量的影响,评价其降压效果及探讨血管舒缩因子释放对血压的影响,考察芪参健脾方对血管内皮功能的修复,探求高血压进程中血管内皮功能障碍的改善机制；同时研究芪参健脾方对SHR ACE2-Ang(1-7)-Mas-AKT通路中各信号分子含量及肾激肽释放酶表达的调节,探索二者协同作用调节内皮舒缩因子释放的分子机制；进一步研究芪参健脾方对于SHR胸主动脉Ⅰ型、Ⅲ型胶原及转化生长因子β1。表达的影响,探求高血压进程中血管重构的改善机制；为临床辨证治疗施以芪参健脾方提供科学的实验数据与理论基础,部分阐释其降压及血管保护作用的分子机制。
材料与方法：
采用随机对照方法将60只24周龄SHR分为模型组(给予蒸馏水,SHR组)、培哚普利组[培哚普利.0.4mg/kg/d]、培哚普利联合中药组[培哚普利0.4mg/kg/d+中药中剂量,中加西组]、中药高、中、低剂量组(用药量按照每100g大鼠体质量分别折算为4g,2g,1g),每组10只,以同龄同种系正常血压的京都种大鼠(WKY)10只作为正常组(WKY组),大鼠日一次灌胃。智能无创血压计测量大鼠初次给药前以及给药后4h、2周、4周、6周大鼠尾动脉收缩压变化情况,每次测量时间为给药后4h。连续治疗6周。
大鼠禁食、自由饮水24小时后,各组大鼠称重,10%水合氯醛腹腔麻醉(3ml/kg体重)。大鼠麻醉后固定于鼠台上,用0.5%碘伏消毒大鼠腹部皮肤,剪开皮肤及腹膜,显现腹主动脉,注射器缓慢抽血,滴加抗凝剂,4℃静置2h后3000rpm离心10min,抽取上层淡黄色透明血浆,-20℃保存备用ELISA法检测血浆舒缩因子及Ang(1-7)含量。沿腹腔向上打开胸腔,显现胸主动脉,用手术线结扎胸主动脉上下两端lcm长度,保证血管内含有部分血液,沿结扎处将血管剪下浸入4%多聚甲醛中固定,进行血管病理形态学观察及免疫组化方法检测SHR胸主动脉壁Ⅰ、Ⅲ型胶原、转化生长因子β3,的表达；另取血管组织约50mg放入EP管中,于-70℃冰箱保存,western方法检测血管转化生长因子β1的表达。横向剪开腹腔,充分暴露肾脏,取左肾脏组织约50mg放入EP管中并加入1mL Trizol试剂,于-70℃冰箱保存,PCR方法检测肾脏ACE2、Mas、激肽释放酶基因的表达；另取左肾脏组织约50mg放入EP管中,于-70℃冰箱保存,western方法检测AKT蛋白表达。
数据采用SPSS16.0软件进行单因素方差分析,组间两两比较采用LSD法分析,结果以均数±标准差(x±s)表示,P<0.05被认为有显著性差异。
结果：
1.芪参健脾方对SHR尾动脉收缩压的影响
统计结果显示：用药前,各组大鼠收缩压均高于WKY组(P 2.芪参健脾方对SHR血浆中血管内皮舒缩因子含量的影响
统计结果显示：SHR组大鼠血浆一氧化氮(NO)、前列环素(PGI2)、内皮源性超极化因子(EDHF)含量显著低于WKY组(P＜O.01)；培哚普利组、中加西组、中药高中剂量组大鼠血浆NO含量明显高于SHR组(P<0.05)；培哚普利组、中加西组、中药高剂量组大鼠血浆PGI2含量明显高于SHR组(P<0.05)；培哚普利组、中加西组、中药高中剂量组大鼠血浆EDHF含量明显高于SHR组(P<0.05)；中加西组与培哚普利组相比舒张因子的含量无统计学意义。SHR组大鼠血浆血管紧张素Ⅱ(Ang Ⅱ)和内皮素1(ET-1)含量显著高于WKY组(P<0.05)；培哚普利组、中加西组、中药高中低各剂量组大鼠血浆Ang Ⅱ.ET-1含量明显低于SHR组(P<0.01);WKY组、中加西组、中药高剂量组血浆AngⅡ含量与培哚普利组相比无统计学意义；WKY组、中加西组、中药高中低各剂量组血浆ET-1含量与培哚普利组相比无统计学意义。
3.芪参健脾方对SHR ACE2-Ang(1-7)-Mas-AKT通路中各信号分子含量的影响
SHR组大鼠Ang(1-7)含量显著低于WKY组(P<0.01)；培哚普利组、中加西组、中药高中低各剂量组大鼠含量明显高于SHR组(P<0.05)；中药高中低各剂量组Ang(1-7)含量低于培哚普利组(P<0.05)。与WKY组比较,SHR组大鼠肾脏ACE2、Mas、激肽释放酶mRNA的表达量明显减少(P<0.01)；中加西组、培哚普利组ACE2、Mas、激肽释放酶mRNA的表达量明显高于SHR组(P<0.01,P<0.05)：中药高、中剂量组Mas mRNA(?)的表达量明显高于SHR组(P<0.01),中药高剂量组ACE2、激肽释放酶mRNA的表达量与SHR组相比P<0.05。与WKY组比较,SHR组大鼠肾脏AKT蛋白的表达量明显减少(P<0.01)；中加西组、中药高剂量组、培哚普利组AKT蛋白的表达量明显高于SHR组(P<0.0l,P<0.05),中药中、低剂量组对AKT蛋白表达与SHR组相比无统计意义。
4.芪参健脾方对SHR胸主动脉管壁形态学的影响
WKY组胸主动脉血管内膜无内皮细胞脱落,无血细胞粘附,细胞排列致密有序,无细胞增生,血管壁无增厚。SHR组胸主动脉内皮细胞部分脱落,内膜不光滑,有血细胞粘附聚集,中膜弹性纤维变直、减少、断裂,平滑肌细胞肥大增生明显,外膜细胞增生活跃,细胞核密度增加,血管管壁增厚。中加西组大鼠胸主动脉内皮细胞未脱落,无血细胞粘附,平滑肌细胞增生不明显,弹性纤维较SHR组排列有序；中药高剂量组血管病理形态观察与中加西组相似。培哚普利组、中药中低剂量组胸主动脉内皮细胞偶有脱落,较SHR组相比中膜弹性纤维排列基本有序,平滑肌细胞肥大增生减少。
5.芪参健脾方对SHR胸主动脉壁Ⅰ、Ⅲ型胶原、转化生长因子β,表达的影响
免疫组化观察：SHR组与WKY组相比Ⅰ、Ⅲ型胶原、血管转化生长因子β,表达明显增多,差异有统计学意义(P<0.01)；中加西组、培哚普利组、中药高中低剂量组表达较SHR组均减少(P<0.01)。中加西组和WKY组Ⅰ、Ⅲ型胶原、血管转化生长因子β1表达量比较差异无统计学意义。与WKY组比较,SHR组大鼠血管转化生长因子β1的蛋白表达量明显增多(P<0.01)；中加西组、培哚普利组、中药高中低各剂量组蛋白表达量明显低于SHR组(P<0.0l,P<0.05)。
结论：
1.芪参健脾方中剂量联合培哚普利治疗对SHR尾动脉收缩压表现出较好的降压效果,治疗4周达到稳定期；与培哚普利治疗组降压效果相似。
2.芪参健脾方通过调节血管内皮舒缩因子NO、PGI2、EDHF、AngⅡ和ET-1的释放平衡,改善高血压进程中血管内皮功能障碍。
3.芪参健脾方联合培哚普利及单方高剂量的应用,明显促进了ACE2-Ang(1-7)-Mas-AKT通路中各信号分子的表达,同时也增强激肽释放酶基因的表达量,从而调节血管内皮舒缩因子的分泌与释放,发挥降压及保护血管内皮功能。
4.芪参健脾方联合培哚普利及单方高剂量的应用能有效改善SHR血管病理形态结构,抑制血管Ⅰ、Ⅲ型胶原的表达,下调TGF-β1,的蛋白表达,从而逆转血管的重构。
Purposes:
This study is meant to observe the influence of qishenjianpifang on the SHR about the caudal artery's systolic pressure and the content of NO, PGI2, EDHF, AngⅡ, ET-1by which we can value its antihypertensive effect and also investigate the vessals endodermis vasomotor releasing factors'anti-influence on the prescription. Meanwhile, we can even inspect this prescription's repairing situation towards the vessal endothelium and pursue the amelioration system of the endothelial dysfunction during the process of hypertension.
At the same time, we will also discuss how.the qishenjianpifang regulates the expression of kidney Kallikrein and influences the content of signaling molecule which are in the SHR ACE2-Ang(1-7)-Mas-AKT thoroughfare, and the mutual action on the adjustment of vessals endodermis vasomotor releasing process will also be included. Besides, we will further do research about how the qishenjianpifang influences SHR thoracic aorta collagen Ⅰ, Ⅲ, and the expression of TGF-β1, meanwhile, we will also explore the reconstruction system of the vessals during the process of hypertension. All these experiments have offered the scientific basis and theories for the use of qishenjianpifang of the clinical treatment and also partially explain its molecule process of protecting the vessals.
Materials and methods:
Using the randomized controlled method to divide the6024-week-old SHR into the model group (giving water, SHR group), perindopril group [perindopril,0.4mg/(kg·d)], perindopril associated with herb group [perindopril0.4mg/(kg·d)+medium dose of herb], and another3groups individually high level, middle level and low level dose of herb (the dose is decided according to the100g weight which are4g,2g, and1g) and the normal group is10normal arterial pressure WKY at the same age and the same kind (giving pure water, WKY). The rats are fed once a day. We will detect the systolic pressure's change of their caudal artery with BP-98A intelligent sphygmomanometer before and4hours,2weeks, and6weeks after giving them the medicine, and the measuring time is
4hours after the medicine giving. It will last6weeks.
The rats will be fed only water and after24hours, these groups of rats will be weighed after given10%chloral hydrate's anesthetic. We will fix the rats on the desk after anesthetic and sterilize their belly skin with0.5%Iodophor, then we will cut off their belly skin to make the abdominal aorta seeable, we draw off the blood and drop gelation inhibitor into it, and let the blood stand2hours with the temperature of4℃, and then off-centre it with3000rpm. After all these steps, we will draw off the upper class jasmine plasma and keep it under the experiment of-20℃temperature. We will detect the dose of plasma endodermis vasomotor and releasing factors and Ang(1-7). We will widen the thoracic cavity along the abdominal cavity to make the aorta thoracica seeable, and then ligate it about1cm at the both ends of the aorta theoracica to make sure that there is blood in it. Next we will cut off the vessals at the ligature and put it into4%paraformal-dehyde to carry on the observation of pathology images and at the same time detect the expression of SHR thoracic aorta collagen Ⅰ, Ⅲ and TGF-β1,
We will take other vascular tissue about50mg to put it in the EP canal in the refrigerator under the temperature of-70℃, and the Western method will be used to detect the expression of the vessal TGF-β1, To cut off the abdominal cavity to make the kidney seeable, and take about50mg tissue of the left kidney and also put it into the EP canal combined with1mL Trizol in the refrigerator under the temperature of-70℃gene of the kidney; We will also take another50mg tissue which will be kept and dealt with in the same way to detect the expression of AKT with the Western way. The data will be checked with one factor analysis of variance using the SPSS16.0software, and LSD method will be used to make comparison in the way of (x±s), if P<0.05, it will have significant difference.
Results:
1. The influence of qishenjianpifang on the SHR about the caudal artery's systolic pressure
The statistical results explanation:before giving the medicine, the systolic pressure of these groups of rats are all higher than the group WKY (P<0.01);4hours after giving them the medicine, there is no obvious change about the systolic pressure; After2weeks, the pressure is obvious getting down for the perindopril group and the perindopril associated with herb group, and comparing with the model group, it has the statistical meaning (P<0.01); after4weeks and6weeks, the pressire is continuously getting down and it has the statistical meaning comparing with the model group (P<0.01). While for the3level dose of herb groups, the pressure is also getting down at some extent after2weeks of using the medicine, however, after4and6weeks, the pressure is not getting down clearly, and comparing with the model group, it has no statistical meaning.
2. The influence of qishenjianpifang on the dose of SHR Vessals Endodermis Vasomotor and Releasing Factors
The statistical results explanation:The content of NO in SHR group、PGI2、 EDHF is prominently lower than the WKY group; the dose of NO of the perindopril group, the perindopril associated with herb group and the high and middle level of the herb groups is clearly higher than the SHR group (P<0.05); For the PGI2the perindopril group, the perindopril associated with herb group and the high level of the herb groups are higher than the SHR group (P<0.05); For the EDHF, the perindopril associated with herb group and the high and middle level of the herb groups is clearly higher than the SHR group (P<0.05); while for the dose of releasing factors, the the perindopril associated with herb group and the perindopril group has no statistical meaning. The dose of AngⅡand ET-1are prominently higher than the WKY group (P<0.05); The dose of AngⅡand ET-1of the perindopril group, the perindopril associated with herb group and the high, middle and low level of the herb groups are also much lower than the SHR group (P<0.01); For the AngⅡ, the dose of WHK, the perindopril associated with herb group and the high level of the herb groups have no satatistical meaning comparing with perindopril group, and for the dose of ET-1, the dose of WHK, the perindopril associated with herb group and the three levels of the herb groups have no statistical meaning comparing with perindopril group.
3. The influence of the qishenjianpifang on the dose of signaling molecule which are in the SHR ACE2-Ang(1-7)-Mas-AKT thoroughfare.
The content of Ang(1-7) of SHR group is clearly lower than that of WKY group(P<0.01); The content of the perindopril group, the perindopril associated with herb group and the high, middle and low levels of the herb groups is clearly higher than the SHR group (F<0.05). The content of Ang (1-7) of the3levels groups of the herb is lower than the perindopril group (P<0.05). Comparing with the WKY group, the kidney ACE2, Mas and Kallikrein mRNA's expression quantity of the SHR group is obviously getting down; the kidney ACE2, Mas and Kallikrein mRNA's expression quantity of the perindopril associated with herb group and the perindopril group is much higher than those of the SHR group (P<0.01, P<0.05); the Mas mRNA's expression quantity of the high level and middle level groups of the herb is much higher than those of the SHR group (P<0.01); the ACE2and Kallikrein mRNA's expression quantity of the high level of the herb is much higher than those of the SHR group (P<0.05); comparing with the SHR group, the kidney AKT protein expression is decreasing at a great extent(P<0.01); while for the perindopril associated with herb group, the high level of herb group and the perindopril group, the expression of the AKT protein is prominently higher than that of the SHR group (P<0.01, P<0.05), while for the middle and low level of the herb groups, the expression has no statistical meaning comparing with the SHR group.
4. The influence of qishenjianpi fang on the SHR thoracic aortic wall morphology
For the aorta thoracica vessal endangium of the WKY group, the inner cells didn't drop off, and the blood corpuscle also didn't adhere to the endangium. The cells rank compactly and there is no cells hyperplasia, and the vascular wall isn't getting thicker. For the SHR group, the endothelial cells of the aorta thoracica are partially dropping off, and the intima isn't smooth enough, and there is also blood corpuscle compacting. The elastic fiber of the middle membrane becomes straight, declined, and ruptured and the smooth muscle cells are also becoming hypertrophied; the ectoblast cells hypertrophies actively, and the karyon reaches higher density, and the vessal inner wall is getting thicker. While for the Chinese associated western medicine group, the rats aorta thoracica the inner cells didn't drop off and the blood corpuscle also didn't adhere to the endangium and the smooth muscle cells aren't becoming hypertrophied either, and the elastic fiber is much in the order than that of the SHR group. After observing the vessal pathology forms, we get rather similar results between the high level of herb group and the perindopril associated with herb group. For the perindopril group, the middle and low level of herb group, the aorta thoracica endothelial cells are dropping off sometimes, while, comparing with the SHR group, the elastic fiber of the middle membrane can be called in a good order, and the smooth muscle cells'hypertrophy is controlled
5. The influence of qishenjianpifang on the SHR aorta thoracica collagen Ⅰ, Ⅲ and the expression of TGF-β,
The expression of collagen Ⅰ, Ⅲ and TGF-β1of the SHR group is much more than that of the WKY group (P<0.01); while for the perindopril group, the perindopril associated with herb group and the high, middle and low levels of the herb groups, it becomes less than that of the SHR group (P<0.01). The difference of the collagen Ⅰ, Ⅲ and TGF-0, expression between the perindopril associated with herb group and the WKY group has no statistical meaning. For the SHR group, the expression of the TGF-β1, is much more than that of the WKY group (P<0.01); while for the perindopril group, the perindopril associated with herb group and the high, middle and low levels of the herb groups, it is much less than that of the SHR group (P<0.01)
Conclusions:
1. The middle level dose of the qishenjianpifang combined with perindopril shows the better lowing pressure effect on the caudal artery systolic pressure, after4weeks'treatment, it will reach the stable level; it has the similar treating effect with the perindopril group.
2. By regulating the releasing balance of NO、PGI2、EDHF、AngⅡand ET-1of the vessals endodermis vasomotor and relaxing factors, the qishenjianpifang can modify the vessals'function obstacle which is caused by the high blood pressure.
3. The qishenjianpi fang combining with perindopril and high level dose of folk prescription can stimulate the expression of the signal molecule which are in the ACE2-Ang(1-7)-Mas-AKT thoroughfare. At the same time, it also increases the expression quantity of the kallikrein gene so as to regulate the releasing and secreting of the vessals endodermis vasomotor and relaxing factors to exert its function of lowing blood pressure and protecting the vessal endodermis.
4. The qishenjianpi fang combining with perindopril and high level dose of folk prescription can improve SHR vessal pathology images conformation. By inhibiting the expression of collagen Ⅰ, Ⅲ and TGF-β1, so as to reverse the reconstruction of the vessal.
本实验通过观察芪参健脾方对自发性高血压大鼠(SHR)尾动脉收缩压及血浆一氧化氮、前列环素、内皮源性超极化因子、血管紧张素Ⅱ和内皮素1含量的影响,评价其降压效果及探讨血管舒缩因子释放对血压的影响,考察芪参健脾方对血管内皮功能的修复,探求高血压进程中血管内皮功能障碍的改善机制；同时研究芪参健脾方对SHR ACE2-Ang(1-7)-Mas-AKT通路中各信号分子含量及肾激肽释放酶表达的调节,探索二者协同作用调节内皮舒缩因子释放的分子机制；进一步研究芪参健脾方对于SHR胸主动脉Ⅰ型、Ⅲ型胶原及转化生长因子β1。表达的影响,探求高血压进程中血管重构的改善机制；为临床辨证治疗施以芪参健脾方提供科学的实验数据与理论基础,部分阐释其降压及血管保护作用的分子机制。
材料与方法：
采用随机对照方法将60只24周龄SHR分为模型组(给予蒸馏水,SHR组)、培哚普利组[培哚普利.0.4mg/kg/d]、培哚普利联合中药组[培哚普利0.4mg/kg/d+中药中剂量,中加西组]、中药高、中、低剂量组(用药量按照每100g大鼠体质量分别折算为4g,2g,1g),每组10只,以同龄同种系正常血压的京都种大鼠(WKY)10只作为正常组(WKY组),大鼠日一次灌胃。智能无创血压计测量大鼠初次给药前以及给药后4h、2周、4周、6周大鼠尾动脉收缩压变化情况,每次测量时间为给药后4h。连续治疗6周。
大鼠禁食、自由饮水24小时后,各组大鼠称重,10%水合氯醛腹腔麻醉(3ml/kg体重)。大鼠麻醉后固定于鼠台上,用0.5%碘伏消毒大鼠腹部皮肤,剪开皮肤及腹膜,显现腹主动脉,注射器缓慢抽血,滴加抗凝剂,4℃静置2h后3000rpm离心10min,抽取上层淡黄色透明血浆,-20℃保存备用ELISA法检测血浆舒缩因子及Ang(1-7)含量。沿腹腔向上打开胸腔,显现胸主动脉,用手术线结扎胸主动脉上下两端lcm长度,保证血管内含有部分血液,沿结扎处将血管剪下浸入4%多聚甲醛中固定,进行血管病理形态学观察及免疫组化方法检测SHR胸主动脉壁Ⅰ、Ⅲ型胶原、转化生长因子β3,的表达；另取血管组织约50mg放入EP管中,于-70℃冰箱保存,western方法检测血管转化生长因子β1的表达。横向剪开腹腔,充分暴露肾脏,取左肾脏组织约50mg放入EP管中并加入1mL Trizol试剂,于-70℃冰箱保存,PCR方法检测肾脏ACE2、Mas、激肽释放酶基因的表达；另取左肾脏组织约50mg放入EP管中,于-70℃冰箱保存,western方法检测AKT蛋白表达。
数据采用SPSS16.0软件进行单因素方差分析,组间两两比较采用LSD法分析,结果以均数±标准差(x±s)表示,P<0.05被认为有显著性差异。
结果：
1.芪参健脾方对SHR尾动脉收缩压的影响
统计结果显示：用药前,各组大鼠收缩压均高于WKY组(P
统计结果显示：SHR组大鼠血浆一氧化氮(NO)、前列环素(PGI2)、内皮源性超极化因子(EDHF)含量显著低于WKY组(P＜O.01)；培哚普利组、中加西组、中药高中剂量组大鼠血浆NO含量明显高于SHR组(P<0.05)；培哚普利组、中加西组、中药高剂量组大鼠血浆PGI2含量明显高于SHR组(P<0.05)；培哚普利组、中加西组、中药高中剂量组大鼠血浆EDHF含量明显高于SHR组(P<0.05)；中加西组与培哚普利组相比舒张因子的含量无统计学意义。SHR组大鼠血浆血管紧张素Ⅱ(Ang Ⅱ)和内皮素1(ET-1)含量显著高于WKY组(P<0.05)；培哚普利组、中加西组、中药高中低各剂量组大鼠血浆Ang Ⅱ.ET-1含量明显低于SHR组(P<0.01);WKY组、中加西组、中药高剂量组血浆AngⅡ含量与培哚普利组相比无统计学意义；WKY组、中加西组、中药高中低各剂量组血浆ET-1含量与培哚普利组相比无统计学意义。
3.芪参健脾方对SHR ACE2-Ang(1-7)-Mas-AKT通路中各信号分子含量的影响
SHR组大鼠Ang(1-7)含量显著低于WKY组(P<0.01)；培哚普利组、中加西组、中药高中低各剂量组大鼠含量明显高于SHR组(P<0.05)；中药高中低各剂量组Ang(1-7)含量低于培哚普利组(P<0.05)。与WKY组比较,SHR组大鼠肾脏ACE2、Mas、激肽释放酶mRNA的表达量明显减少(P<0.01)；中加西组、培哚普利组ACE2、Mas、激肽释放酶mRNA的表达量明显高于SHR组(P<0.01,P<0.05)：中药高、中剂量组Mas mRNA(?)的表达量明显高于SHR组(P<0.01),中药高剂量组ACE2、激肽释放酶mRNA的表达量与SHR组相比P<0.05。与WKY组比较,SHR组大鼠肾脏AKT蛋白的表达量明显减少(P<0.01)；中加西组、中药高剂量组、培哚普利组AKT蛋白的表达量明显高于SHR组(P<0.0l,P<0.05),中药中、低剂量组对AKT蛋白表达与SHR组相比无统计意义。
4.芪参健脾方对SHR胸主动脉管壁形态学的影响
WKY组胸主动脉血管内膜无内皮细胞脱落,无血细胞粘附,细胞排列致密有序,无细胞增生,血管壁无增厚。SHR组胸主动脉内皮细胞部分脱落,内膜不光滑,有血细胞粘附聚集,中膜弹性纤维变直、减少、断裂,平滑肌细胞肥大增生明显,外膜细胞增生活跃,细胞核密度增加,血管管壁增厚。中加西组大鼠胸主动脉内皮细胞未脱落,无血细胞粘附,平滑肌细胞增生不明显,弹性纤维较SHR组排列有序；中药高剂量组血管病理形态观察与中加西组相似。培哚普利组、中药中低剂量组胸主动脉内皮细胞偶有脱落,较SHR组相比中膜弹性纤维排列基本有序,平滑肌细胞肥大增生减少。
5.芪参健脾方对SHR胸主动脉壁Ⅰ、Ⅲ型胶原、转化生长因子β,表达的影响
免疫组化观察：SHR组与WKY组相比Ⅰ、Ⅲ型胶原、血管转化生长因子β,表达明显增多,差异有统计学意义(P<0.01)；中加西组、培哚普利组、中药高中低剂量组表达较SHR组均减少(P<0.01)。中加西组和WKY组Ⅰ、Ⅲ型胶原、血管转化生长因子β1表达量比较差异无统计学意义。与WKY组比较,SHR组大鼠血管转化生长因子β1的蛋白表达量明显增多(P<0.01)；中加西组、培哚普利组、中药高中低各剂量组蛋白表达量明显低于SHR组(P<0.0l,P<0.05)。
结论：
1.芪参健脾方中剂量联合培哚普利治疗对SHR尾动脉收缩压表现出较好的降压效果,治疗4周达到稳定期；与培哚普利治疗组降压效果相似。
2.芪参健脾方通过调节血管内皮舒缩因子NO、PGI2、EDHF、AngⅡ和ET-1的释放平衡,改善高血压进程中血管内皮功能障碍。
3.芪参健脾方联合培哚普利及单方高剂量的应用,明显促进了ACE2-Ang(1-7)-Mas-AKT通路中各信号分子的表达,同时也增强激肽释放酶基因的表达量,从而调节血管内皮舒缩因子的分泌与释放,发挥降压及保护血管内皮功能。
4.芪参健脾方联合培哚普利及单方高剂量的应用能有效改善SHR血管病理形态结构,抑制血管Ⅰ、Ⅲ型胶原的表达,下调TGF-β1,的蛋白表达,从而逆转血管的重构。
Purposes:
This study is meant to observe the influence of qishenjianpifang on the SHR about the caudal artery's systolic pressure and the content of NO, PGI2, EDHF, AngⅡ, ET-1by which we can value its antihypertensive effect and also investigate the vessals endodermis vasomotor releasing factors'anti-influence on the prescription. Meanwhile, we can even inspect this prescription's repairing situation towards the vessal endothelium and pursue the amelioration system of the endothelial dysfunction during the process of hypertension.
At the same time, we will also discuss how.the qishenjianpifang regulates the expression of kidney Kallikrein and influences the content of signaling molecule which are in the SHR ACE2-Ang(1-7)-Mas-AKT thoroughfare, and the mutual action on the adjustment of vessals endodermis vasomotor releasing process will also be included. Besides, we will further do research about how the qishenjianpifang influences SHR thoracic aorta collagen Ⅰ, Ⅲ, and the expression of TGF-β1, meanwhile, we will also explore the reconstruction system of the vessals during the process of hypertension. All these experiments have offered the scientific basis and theories for the use of qishenjianpifang of the clinical treatment and also partially explain its molecule process of protecting the vessals.
Materials and methods:
Using the randomized controlled method to divide the6024-week-old SHR into the model group (giving water, SHR group), perindopril group [perindopril,0.4mg/(kg·d)], perindopril associated with herb group [perindopril0.4mg/(kg·d)+medium dose of herb], and another3groups individually high level, middle level and low level dose of herb (the dose is decided according to the100g weight which are4g,2g, and1g) and the normal group is10normal arterial pressure WKY at the same age and the same kind (giving pure water, WKY). The rats are fed once a day. We will detect the systolic pressure's change of their caudal artery with BP-98A intelligent sphygmomanometer before and4hours,2weeks, and6weeks after giving them the medicine, and the measuring time is
4hours after the medicine giving. It will last6weeks.
The rats will be fed only water and after24hours, these groups of rats will be weighed after given10%chloral hydrate's anesthetic. We will fix the rats on the desk after anesthetic and sterilize their belly skin with0.5%Iodophor, then we will cut off their belly skin to make the abdominal aorta seeable, we draw off the blood and drop gelation inhibitor into it, and let the blood stand2hours with the temperature of4℃, and then off-centre it with3000rpm. After all these steps, we will draw off the upper class jasmine plasma and keep it under the experiment of-20℃temperature. We will detect the dose of plasma endodermis vasomotor and releasing factors and Ang(1-7). We will widen the thoracic cavity along the abdominal cavity to make the aorta thoracica seeable, and then ligate it about1cm at the both ends of the aorta theoracica to make sure that there is blood in it. Next we will cut off the vessals at the ligature and put it into4%paraformal-dehyde to carry on the observation of pathology images and at the same time detect the expression of SHR thoracic aorta collagen Ⅰ, Ⅲ and TGF-β1,
We will take other vascular tissue about50mg to put it in the EP canal in the refrigerator under the temperature of-70℃, and the Western method will be used to detect the expression of the vessal TGF-β1, To cut off the abdominal cavity to make the kidney seeable, and take about50mg tissue of the left kidney and also put it into the EP canal combined with1mL Trizol in the refrigerator under the temperature of-70℃gene of the kidney; We will also take another50mg tissue which will be kept and dealt with in the same way to detect the expression of AKT with the Western way. The data will be checked with one factor analysis of variance using the SPSS16.0software, and LSD method will be used to make comparison in the way of (x±s), if P<0.05, it will have significant difference.
Results:
1. The influence of qishenjianpifang on the SHR about the caudal artery's systolic pressure
The statistical results explanation:before giving the medicine, the systolic pressure of these groups of rats are all higher than the group WKY (P<0.01);4hours after giving them the medicine, there is no obvious change about the systolic pressure; After2weeks, the pressure is obvious getting down for the perindopril group and the perindopril associated with herb group, and comparing with the model group, it has the statistical meaning (P<0.01); after4weeks and6weeks, the pressire is continuously getting down and it has the statistical meaning comparing with the model group (P<0.01). While for the3level dose of herb groups, the pressure is also getting down at some extent after2weeks of using the medicine, however, after4and6weeks, the pressure is not getting down clearly, and comparing with the model group, it has no statistical meaning.
2. The influence of qishenjianpifang on the dose of SHR Vessals Endodermis Vasomotor and Releasing Factors
The statistical results explanation:The content of NO in SHR group、PGI2、 EDHF is prominently lower than the WKY group; the dose of NO of the perindopril group, the perindopril associated with herb group and the high and middle level of the herb groups is clearly higher than the SHR group (P<0.05); For the PGI2the perindopril group, the perindopril associated with herb group and the high level of the herb groups are higher than the SHR group (P<0.05); For the EDHF, the perindopril associated with herb group and the high and middle level of the herb groups is clearly higher than the SHR group (P<0.05); while for the dose of releasing factors, the the perindopril associated with herb group and the perindopril group has no statistical meaning. The dose of AngⅡand ET-1are prominently higher than the WKY group (P<0.05); The dose of AngⅡand ET-1of the perindopril group, the perindopril associated with herb group and the high, middle and low level of the herb groups are also much lower than the SHR group (P<0.01); For the AngⅡ, the dose of WHK, the perindopril associated with herb group and the high level of the herb groups have no satatistical meaning comparing with perindopril group, and for the dose of ET-1, the dose of WHK, the perindopril associated with herb group and the three levels of the herb groups have no statistical meaning comparing with perindopril group.
3. The influence of the qishenjianpifang on the dose of signaling molecule which are in the SHR ACE2-Ang(1-7)-Mas-AKT thoroughfare.
The content of Ang(1-7) of SHR group is clearly lower than that of WKY group(P<0.01); The content of the perindopril group, the perindopril associated with herb group and the high, middle and low levels of the herb groups is clearly higher than the SHR group (F<0.05). The content of Ang (1-7) of the3levels groups of the herb is lower than the perindopril group (P<0.05). Comparing with the WKY group, the kidney ACE2, Mas and Kallikrein mRNA's expression quantity of the SHR group is obviously getting down; the kidney ACE2, Mas and Kallikrein mRNA's expression quantity of the perindopril associated with herb group and the perindopril group is much higher than those of the SHR group (P<0.01, P<0.05); the Mas mRNA's expression quantity of the high level and middle level groups of the herb is much higher than those of the SHR group (P<0.01); the ACE2and Kallikrein mRNA's expression quantity of the high level of the herb is much higher than those of the SHR group (P<0.05); comparing with the SHR group, the kidney AKT protein expression is decreasing at a great extent(P<0.01); while for the perindopril associated with herb group, the high level of herb group and the perindopril group, the expression of the AKT protein is prominently higher than that of the SHR group (P<0.01, P<0.05), while for the middle and low level of the herb groups, the expression has no statistical meaning comparing with the SHR group.
4. The influence of qishenjianpi fang on the SHR thoracic aortic wall morphology
For the aorta thoracica vessal endangium of the WKY group, the inner cells didn't drop off, and the blood corpuscle also didn't adhere to the endangium. The cells rank compactly and there is no cells hyperplasia, and the vascular wall isn't getting thicker. For the SHR group, the endothelial cells of the aorta thoracica are partially dropping off, and the intima isn't smooth enough, and there is also blood corpuscle compacting. The elastic fiber of the middle membrane becomes straight, declined, and ruptured and the smooth muscle cells are also becoming hypertrophied; the ectoblast cells hypertrophies actively, and the karyon reaches higher density, and the vessal inner wall is getting thicker. While for the Chinese associated western medicine group, the rats aorta thoracica the inner cells didn't drop off and the blood corpuscle also didn't adhere to the endangium and the smooth muscle cells aren't becoming hypertrophied either, and the elastic fiber is much in the order than that of the SHR group. After observing the vessal pathology forms, we get rather similar results between the high level of herb group and the perindopril associated with herb group. For the perindopril group, the middle and low level of herb group, the aorta thoracica endothelial cells are dropping off sometimes, while, comparing with the SHR group, the elastic fiber of the middle membrane can be called in a good order, and the smooth muscle cells'hypertrophy is controlled
5. The influence of qishenjianpifang on the SHR aorta thoracica collagen Ⅰ, Ⅲ and the expression of TGF-β,
The expression of collagen Ⅰ, Ⅲ and TGF-β1of the SHR group is much more than that of the WKY group (P<0.01); while for the perindopril group, the perindopril associated with herb group and the high, middle and low levels of the herb groups, it becomes less than that of the SHR group (P<0.01). The difference of the collagen Ⅰ, Ⅲ and TGF-0, expression between the perindopril associated with herb group and the WKY group has no statistical meaning. For the SHR group, the expression of the TGF-β1, is much more than that of the WKY group (P<0.01); while for the perindopril group, the perindopril associated with herb group and the high, middle and low levels of the herb groups, it is much less than that of the SHR group (P<0.01)
Conclusions:
1. The middle level dose of the qishenjianpifang combined with perindopril shows the better lowing pressure effect on the caudal artery systolic pressure, after4weeks'treatment, it will reach the stable level; it has the similar treating effect with the perindopril group.
2. By regulating the releasing balance of NO、PGI2、EDHF、AngⅡand ET-1of the vessals endodermis vasomotor and relaxing factors, the qishenjianpifang can modify the vessals'function obstacle which is caused by the high blood pressure.
3. The qishenjianpi fang combining with perindopril and high level dose of folk prescription can stimulate the expression of the signal molecule which are in the ACE2-Ang(1-7)-Mas-AKT thoroughfare. At the same time, it also increases the expression quantity of the kallikrein gene so as to regulate the releasing and secreting of the vessals endodermis vasomotor and relaxing factors to exert its function of lowing blood pressure and protecting the vessal endodermis.
4. The qishenjianpi fang combining with perindopril and high level dose of folk prescription can improve SHR vessal pathology images conformation. By inhibiting the expression of collagen Ⅰ, Ⅲ and TGF-β1, so as to reverse the reconstruction of the vessal.
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