氨氯地平及左旋氨氯地平对高血压内皮功能影响的实验研究
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摘要
血管内皮功能的改变在动脉粥样硬化形态学改变之前，就参与了损伤的进展及随后的临床并发症，与高血压等心血管疾病关系密切。内皮功能受损时，血管舒张因子分泌减少，血管收缩因子分泌增加，这促进了血管平滑肌细胞的增殖，导致血管壁增厚和血管结构重塑，动脉弹性下降，外周阻力升高，促进高血压及其并发症的发生发展。另外，高血压引起的血流切应力和内分泌环境的变化也可引起内皮功能受损。因此逆转高血压内皮功能不全不仅是降压之外的新靶点，同时也是阻止动脉粥样硬化发生发展的重要环节，内皮功能不全检测就更强调无创、易操作及快捷。指尖脉搏容积测定（Pulse amplitude volume，PAV）是随着指尖检测内皮功能的迅猛发展而新近出现的检测手段，但尚无相关的临床应用的报道。
氨氯地平（Amlodlpine）作为第三代钙通道阻滞剂，是临床应用最为广泛的降压药物之一。氨氯地平是由左旋氨氯地平（S(-)-amlodipine）和右旋氨氯地平（R(-)-amlodipine）以1：1的比例构成的外消旋混合物，其降压作用主要来自左旋体，其效用是右旋体的1000倍。但除了降压本身的血管功能保护作用外，因为右旋体的存在是否使氨氯地平与左旋氨氯地平相比具有额外的内皮功能保护作用尚不清楚。
本研究通过检测这两种药物治疗前后高血压患者血管内皮功能的变化，探讨两者在取得相同降压效果的情况下是否具有相同的内皮保护作用，同时探讨PAV与FMD检测高血压内皮功能的相关性及一致性；并进一步通过两种药物干预体外培养的人脐静脉内皮细胞，探讨两者影响NO的水平及其机制。方法：
1临床试验
采取随机、交叉设计，选择原发性高血压病病人24例，随机分为A组及B组，A组服用苯磺酸氨氯地平（辉瑞制药）5mg1/日，B组服用苯磺酸左旋氨氯地平（施慧达药业）2.5mg1/日，均为晨起服药，治疗6周后经过2周清洗期，待患者血压及心率恢复到第一次清洗期水平后再将两药交叉继续用药6周。在基线期和每个治疗期结束时，受试者会接受采血以及血管功能检测，检测前8小时禁食并禁止吸烟、饮酒及咖啡。
检测项目包括：全自动动脉硬化检测仪（欧姆龙科林VP1000）检测脉搏传导速度(Pulse wave velocity,PWV)及踝臂指数(Ankle-branchial index,ABI)；内皮功能诊断仪（SY2000，吉林圣晔尔科技有限公司）检测指尖脉搏容积（PAV）；彩色超声诊断仪（PHLIP IE33型，美国）检测肱动脉血流介导的血管扩张功能(Flow mediated dilation,FMD)；化学法检测一氧化氮（Nitric oxide,NO）；酶联免疫吸附剂测定（Enzyme linked immunosorbent assay,Elisa）法检测血清内皮型一氧化氮合酶（Endothelial nitric oxide synthase,eNOS）、内皮素（Endothelin,ET）及血管性血友病因子（von Willebrand Factor, vWF）。
2、体外细胞培养实验
体外培养人脐静脉内皮细胞(Human Umbilical Vein EndothelialCells,HUVEC)，进行以下检测：
2.1化学法检测在eNOS抑制剂Nw-nitro-L-arginine (L-NA)存在或缺失时，氨氯地平及左旋氨氯地平作用25分钟对内皮细胞NO水平的影响。
2.2化学法检测在0、5、15及25分钟四个时间点，经氨氯地平、左旋氨氯地平或蛋白激酶C（Protein kinase C,PKC）抑制剂Ro31-8220处理后内皮细胞产生NO的水平。
2.3蛋白质印迹法(Wsetern blot,WB)检测在0、5、15及25分钟四个时间点，经氨氯地平或左旋氨氯地平处理后内皮细胞eNOS在Ser1177、Thr495及PKC磷酸化的程度。
2.4Wsetern blot法检测在0、5、15及25分钟四个时间点，经PKC抑制剂Ro31-8220处理后内皮细胞eNOS在Ser1177及Thr495磷酸化的程度。结果：
1临床试验结果：
1.1经氨氯地平及左旋氨氯地平治疗后，与基线期相比患者收缩压、舒张压及心率均有明显下降，且均有显著的统计学差异（P<0.01）；但两种药物相比，收缩压、舒张压及心率数值接近，且无统计学差异（P>0.05）
1.2与基线期相比，两种药物治疗后PAV、FMD及ABI均有明显增加，均达到统计学差异（P<0.05）；PWV均有明显下降，均达到统计学差异（P<0.01）；NMD数值变化不大，亦无统计学差异（P>0.05）；氨氯地平治疗后与左旋氨氯地平治疗后相比，PAV、FMD、PWV及ABI测值均稍高，但均未达到统计学差异（P>0.05）；其中在FMD上两者相比有接近统计学差异的趋势（P=0.053）。
1.3与基线期相比，两种药物治疗后NO及eNOS均有明显增加，均达到统计学差异（P<0.01）；ET-1及vWF均有明显下降，均达到统计学差异（P<0.01）；氨氯地平治疗后与左旋氨氯地平治疗后相比，NO及eNOS水平均稍高，ET-1及vWF水平均稍低，但均未达到统计学差异（P>0.05），其中在NO上两者相比有接近统计学差异的趋势（P=0.064）。
1.4在基线期、氨氯地平治疗后及左旋氨氯地平治疗后这三个节点，PAV与FMD测值均有显著的相关性（P<0.01），氨氯地平治疗后与基线期PAV测值的差值ΔPAV（0.24±0.16、0.18±0.15）及左旋氨氯地平后与基线期FMD测值的差值ΔFMD（2.3±1.7%、1.6±1.2%）亦具有显著的相关性（P<0.01）。对PAV及FMD诊断高血压内皮功能不全进行一致性检验，结果示Kappa=0.647，P<0.001，有统计学意义。
2体外细胞培养实验结果：
2.1在eNOS抑制剂Nw-nitro-L-arginine (L-NA,300mmol/l)存在时，氨氯地平及左旋氨氯地平处理25分钟后NO测值与L-NA组相比数值接近，且均无统计学差异（P>0.05）；两种药物之间测值相近，亦无统计学差异（P>0.05）。在L-NA缺失时，氨氯地平及左旋氨氯地平处理25分钟后NO测值与对照组相比均有明显升高，有统计学差异（P<0.01），氨氯地平组NO测值高于左旋氨氯地平组，有统计学差异（P<0.05）。
2.2经氨氯地平（1μmol/l）处理5、15及25分钟后NO水平逐渐增加，在25分钟达到最高值，15及25分钟时NO水平与对照组相比均有明显升高，且均达到统计学差异（P<0.01）；经左旋氨氯地平（1μmol/l）处理5、15及25分钟后NO测值均增加，在15分钟达到最高值，5、15及25分钟时NO水平与对照组相比均达到的统计学差异（P<0.01）；氨氯地平组与左旋氨氯地平组相比，除5分钟外，15及25分钟时NO水平均是前者较高，其中25分钟时两者达到统计学差异（P<0.05）。内皮细胞经PKC途径抑制剂Ro31-8220（30nmol/l）处理5、15及25分钟后NO测值也逐渐增加，在25分钟达到最高值，15及25分钟时NO测值与对照组相比均有显著的统计学差异（P<0.01）。
2.3氨氯地平对内皮细胞的刺激引起eNOS在Ser1177磷酸化和Thr495去磷酸化的增长，并且作用在5、15及25分钟逐渐加强，在25分钟达到峰值；左旋氨氯地平对eNOS在Ser1177磷酸化和Thr495去磷酸化作用不大，在5、15及25分钟这三个时间点未表现出明显的趋势。
2.4氨氯地平可以使PKC本身磷酸化减弱，并且作用在5、15及25分钟逐渐加强，在25分钟达到峰值；左旋氨氯地平对PKC去磷酸化作用不大，在5、15及25分钟这三个时间点未表现出明显的趋势
2.5PKC抑制剂Ro31-8220对内皮细胞的刺激引起eNOS在Ser1177磷酸化和Thr495去磷酸化的增长，并且作用在5、15及25分钟逐渐加强，在25分钟达到峰值。
结果小结：
1、在检测治疗前后高血压患者内皮功能改善上，PAV与FMD具有明显的相关性；在诊断高血压患者内皮功能不全上，PAV与FMD具有较好的一致性。
2、在取得相似的降压效果时，氨氯地平及左旋氨氯地平均具有良好的内皮保护作用，虽然两药治疗后各项指标相比均无统计学差异，但前者似乎有更好内皮保护作用的趋势。
3、在体外培养细胞中，氨氯地平及左旋氨氯地平均可以时间依赖性的提高NO水平，左旋氨氯地平组达到峰值较早，但氨氯地平组峰值水平较高。
4、氨氯地平可通过PKC途径对eNOS在Ser1177及Thr495磷酸化产生影响有从而使eNOS活化，而左旋氨氯地平对PKC影响不大，也不能对eNOS在Ser1177及Thr495的磷酸化程度产生影响。
结论：
1、氨氯地平及左旋氨氯地平都通过增加NO水平发挥良好的内皮保护作用，但程度和途径不同。
2、PAV与FMD具有较好的相关性及一致性，可替代FMD进行内皮功能检测。
Before the development of atherosclerosis morphological changes, changes invascular endothelial function participates the progress of the injury and subsequentclinical complications, and has close relationship with hypertension and othercardiovascular diseases. Dysfunction of vascular endothelial reduces secretion ofvasodilator agent, but increase the vasoconstriction cytokine secretion. That promotesthe proliferation of vascular smooth muscle cells, leading to vessel wall thickeningand vascular remodeling, decreased arterial elasticity, increased peripheral resistance,and promote the development of both hypertension and its complications. In addition,the high blood pressure caused by fluid shear stress and endocrine changes in theenvironment can cause Endothelial dysfunction, thus reversing the hypertensionendothelial dysfunction another treatment outside the buck has become a new target.The detection of hypertension endothelial function requires to be non-invasive, easyoperation and quickly. PAV is a new non-invasive way to test the endothelial functionwhen the fingertip detection of endothelial function develop rapidly, but there are norelevant clinical applications reported.
As a third-generation calcium channel blocker, Amlodipine is one of the mostwidely used antihypertensive drugs in clinical application. Amlodipine is a racemicmixture of S(-)-amlodipine and the R(-)-amlodipine constituted the outer to the ratioof1:1, which antihypertensive effect mainly come from L-body and effect is1000-time stronger than that of R(-)-amlodipine. It is not clear that amlodipine has anadditional protective effect of endothelial functionit compared with S(-)-amlodipinebecause of the presence of R(-)-amlodipine.
A clinical study was carried out to examine whether amlodipine andS(-)-amlodipine have similar protective effects on vascular endothelial function inaddition to the similar antihypertensive effects, and we explored the association andconsistency of PAV and FMD on testing of endothelial function in patitents withhypertension. Further, we explore both its protective effect of endothelial function byaffecting the level of NO and the mechanisms in cultured human umbilical vein endothelial cells.
Method
1、Clinical trials
We took a randomized and crossover design, chosen24cases of patients withessential hypertension and randomly divided into Group A and Group B,the formertaking amlodipine besylate5mg/d (Pfizer),the latter taking the benzenesulfonicS(-)-amlodipine of2.5mg/d(Shi Huida). The two groups were both early morningmedication and went through wash-out period about2weeks after6weekstreatment,then continued the treatment for6weeks crossing the two drugs after heartrate and blood pressure of patients returned to the level of the first wash-out period.Subjects, who were fasting and Prohibited smoking, drinking and coffee the nightbefore detection, will accept the detection of blood collection,and vascularfunction in the time of baseline and the end of each treatment.
Test items include：Pulse wave velocity (PWV) and Ankle-branchial index(ABI)were tested by Omron Colin VP1000. Pulse amplitude volume（PAV）was testedby SY2000（Jilin saintyear company）. Flow mediated dilation(FMD) of brachialartery was tested by Philips IE33(America). Nitric oxide(NO)was tested by Griessmethod. Endothelial nitric oxide synthas（eeNOS）,Endothelin（ET）andvon WillebrandFactor（vWF）were tested by Elisa.
2、Cell culture experiment
We cultured human umbilical vein endothelial cells in vitro for the followingchecks:
2.1Griess method had been used to detect the endothelial cell NO levels at25minutes that affected by amlodipine or S(-)-amlodipine on presence or absence of theeNOS inhibitor Nw-nitro-L-arginine (L-NA).
2.2Griess method had been used to detect respectively the endothelial cell NOlevels at0,5,15,25minutes that treated by amlodipine, S(-)-amlodipine or proteinkinase C（PKC）inhibitor Ro31-8220.
2.3Wsetern blot(WB) had been used to detect respectively degree ofphosphorylation of eNOS Ser1177、eNOS Thr495and PKC in endothelial cells that thattreated by amlodipine or S(-)-amlodipine at0,5,15,25minutes.
2.4Wsetern blot(WB) had been used to detect respectively degree of phosphorylation of eNOS Ser1177and Thr495in endothelial cells that treated by proteinkinase C（PKC）inhibitor Ro31-8220at0,5,15,25minutes.
Result：
1Results of clinical trials
1.1After amlodipine and S(-)-amlodipine treatments, the patients’ SBP, DBPand HR significantly decreased compared with baseline data (P<0.01). However,there were no significant differences between the data of amlodipine andS(-)-amlodipine groups (P>0.05).
1.2Compared with baseline,after treatment of the two drugs the PAV,FMD andABI were significantly increased,and there was a significant difference (P<0.05)；PWV were significantly reduced, there were statistically significant (P<0.01);NMDlittle change in value, no significant difference (P>0.05)。In the amlodipine group andL-amlodipine group,PAV, FMD, PWV and ABI measured values are slightlyhigher,but it did not reach a statistically significant difference (P>0.05),Howevercomparing the two in the FMD approached statistical differences (P=0.053).
1.3Compared with baseline,NO and eNOS have increased significantly aftertreatment of the two drugs，ang it was a significant difference（P<0.01）;ET-1andvEGF were significantly reduced reach statistical significance (P<0.01). In theamlodipine group and S(-)-amlodipine group,NO and NOS were slightly higher, ET-1and vWF levels were slightly lower,but it did not reach a statistically significantdifference (P>0.05),However comparing the two in the NO approached statisticaldifferences (P=0.064).
1.4At baseline,the time after the treatment of amlodipine orS(-)-amlodipine,PAV was strongly associated with FMD（P<0.01）,and the differencebetween the baseline and the treatment ΔPAV（0.24±0.16、0.18±0.15）was stronglyassociated with ΔFMD（2.3±1.7%、1.6±1.2%）. The consistency test of diagnostic testswas done between PAV and FMD, the result is Kappa=0.647，P<0.001.
2Results of cell culture experiment：
2.1In the presence of the eNOS inhibitor L-NA (300mmol/l), the NO level ofHUVECs treated with amlodipine and S(-)-amlodipine for25minutes was similar to the L-NA group, showing no statistically significant difference (P>0.05). The NOlevel of HUVECs was also similar between amlodipine and S(-)-amlodipine groupswith no statistically significant difference (P>0.05). In the absence of L-NA, the NOlevel of HUVECs treated with amlodipine and S(-)-amlodipine for25minutes wassignificantly higher than the control group (P<0.01). In addition, the NO level ofHUVECs was obviously higher in the amlodipine group than in the S(-)-amlodipinegroup, showing statistically significant differences (P<0.05).
2.2After amlodipine treatment (1μmol/l) for5,15, and25minutes, the NO levelof HUVECs gradually increased and peaked at25minutes. As compared with that ofcontrol group, the NO level of HUVECs in the amlodipine group was higher at15min and25minutes, showing statistically significant differences (P<0.01). AfterS(-)-amlodipine treatment (1μmol/l) for5,15, and25minutes, the NO level ofHUVECs increased and peaked at15minutes. As compared with that of control group,the NO level of HUVECs in the S(-)-amlodipine group was significantly higher at5,15, and25minutes (P<0.01). Data comparison between the amlodipine andS(-)-amlodipine groups showed that the NO level of HUVECs was higher in theformer at15and25minutes but not5minutes, with statistically significant differenceobserved at25minutes only (P<0.05). After Ro31-8220treatment (30nmol/l) for5,15and25minutes, the NO level of HUVECs gradually increased and peaked at25minutes. As compared with control group, the NO level of HUVECs treated with Ro31-8220was significantly different at15and25minutes (P<0.01).
2.3The stimulation of amlodipine in endothelial cells induced the increase ofSer1177phosphorylation and Thr495dephosphorylation at eNOS, the effect increasedwith the increase in culture time from5,15to25minutes, and it reached the highestvalue at25minutes; S(-)-amlodipine did not affect the phosphorylation of eNOSSer1177and Thr495at5,15or25minutes.
2.4The stimulation of amlodipine induced the decrease of the phosphorylation ofPKC, the effect increased with the increase in culture time from5,15to25minutes,and it reached the highest value at25minutes; S(-)-amlodipine did not affect thephosphorylation of PKC at5,15or25minutes.
2.5The stimulation of PKC inhibitor Ro31-8220in endothelial cells induced theincrease of Ser1177phosphorylation and Thr495dephosphorylation at eNOS，the effectincreased with the increase in culture time from5,15to25minutes, and it reached the highest value at25minutes
Summary of results:
1、There is significant correlation between PAV and FMD in detecting improvedendothelial function before and after the treatment of the hypertension; PAV has goodconsistency with FMD in the diagnosis of hypertension patients with endothelialdysfunction.
2、With similar antihypertensive effect, both amlodipine and S(-)-amlodipinehave a good protective effects of endothelial cells. Though there is no significantdifference between the two drugs at these tests, amlodipine appears to have the trendof having a better endothelial protective effect.
2、 In culturedendothelial cells, amlodipine and S(-)-amlodipine both timedependently increase the level of NO. The level of NO in S(-)-amlodipine groupreached the highest value earlier but which was higher in amlodipine group.
4、 Amlodipine can activate eNOS though affecting the phosphorylation ofeNOS Ser1177and Thr495by PKC,but S(-)-amlodipine has little effect on thephosphorylation of PKC and the phosphorylation of eNOS Ser1177and Thr495.
Conclusion
1、Amlodipine and S(-)-amlodipine both have good effect on the protection ofendothelial cells through increasing the level of NO, but the degree and the pathwayare different.
2、There are good correlation and consistency between PAV and FMD,PAV cantest the endothelial function instead of FMD.
氨氯地平（Amlodlpine）作为第三代钙通道阻滞剂，是临床应用最为广泛的降压药物之一。氨氯地平是由左旋氨氯地平（S(-)-amlodipine）和右旋氨氯地平（R(-)-amlodipine）以1：1的比例构成的外消旋混合物，其降压作用主要来自左旋体，其效用是右旋体的1000倍。但除了降压本身的血管功能保护作用外，因为右旋体的存在是否使氨氯地平与左旋氨氯地平相比具有额外的内皮功能保护作用尚不清楚。
本研究通过检测这两种药物治疗前后高血压患者血管内皮功能的变化，探讨两者在取得相同降压效果的情况下是否具有相同的内皮保护作用，同时探讨PAV与FMD检测高血压内皮功能的相关性及一致性；并进一步通过两种药物干预体外培养的人脐静脉内皮细胞，探讨两者影响NO的水平及其机制。方法：
1临床试验
采取随机、交叉设计，选择原发性高血压病病人24例，随机分为A组及B组，A组服用苯磺酸氨氯地平（辉瑞制药）5mg1/日，B组服用苯磺酸左旋氨氯地平（施慧达药业）2.5mg1/日，均为晨起服药，治疗6周后经过2周清洗期，待患者血压及心率恢复到第一次清洗期水平后再将两药交叉继续用药6周。在基线期和每个治疗期结束时，受试者会接受采血以及血管功能检测，检测前8小时禁食并禁止吸烟、饮酒及咖啡。
检测项目包括：全自动动脉硬化检测仪（欧姆龙科林VP1000）检测脉搏传导速度(Pulse wave velocity,PWV)及踝臂指数(Ankle-branchial index,ABI)；内皮功能诊断仪（SY2000，吉林圣晔尔科技有限公司）检测指尖脉搏容积（PAV）；彩色超声诊断仪（PHLIP IE33型，美国）检测肱动脉血流介导的血管扩张功能(Flow mediated dilation,FMD)；化学法检测一氧化氮（Nitric oxide,NO）；酶联免疫吸附剂测定（Enzyme linked immunosorbent assay,Elisa）法检测血清内皮型一氧化氮合酶（Endothelial nitric oxide synthase,eNOS）、内皮素（Endothelin,ET）及血管性血友病因子（von Willebrand Factor, vWF）。
2、体外细胞培养实验
体外培养人脐静脉内皮细胞(Human Umbilical Vein EndothelialCells,HUVEC)，进行以下检测：
2.1化学法检测在eNOS抑制剂Nw-nitro-L-arginine (L-NA)存在或缺失时，氨氯地平及左旋氨氯地平作用25分钟对内皮细胞NO水平的影响。
2.2化学法检测在0、5、15及25分钟四个时间点，经氨氯地平、左旋氨氯地平或蛋白激酶C（Protein kinase C,PKC）抑制剂Ro31-8220处理后内皮细胞产生NO的水平。
2.3蛋白质印迹法(Wsetern blot,WB)检测在0、5、15及25分钟四个时间点，经氨氯地平或左旋氨氯地平处理后内皮细胞eNOS在Ser1177、Thr495及PKC磷酸化的程度。
2.4Wsetern blot法检测在0、5、15及25分钟四个时间点，经PKC抑制剂Ro31-8220处理后内皮细胞eNOS在Ser1177及Thr495磷酸化的程度。结果：
1临床试验结果：
1.1经氨氯地平及左旋氨氯地平治疗后，与基线期相比患者收缩压、舒张压及心率均有明显下降，且均有显著的统计学差异（P<0.01）；但两种药物相比，收缩压、舒张压及心率数值接近，且无统计学差异（P>0.05）
1.2与基线期相比，两种药物治疗后PAV、FMD及ABI均有明显增加，均达到统计学差异（P<0.05）；PWV均有明显下降，均达到统计学差异（P<0.01）；NMD数值变化不大，亦无统计学差异（P>0.05）；氨氯地平治疗后与左旋氨氯地平治疗后相比，PAV、FMD、PWV及ABI测值均稍高，但均未达到统计学差异（P>0.05）；其中在FMD上两者相比有接近统计学差异的趋势（P=0.053）。
1.3与基线期相比，两种药物治疗后NO及eNOS均有明显增加，均达到统计学差异（P<0.01）；ET-1及vWF均有明显下降，均达到统计学差异（P<0.01）；氨氯地平治疗后与左旋氨氯地平治疗后相比，NO及eNOS水平均稍高，ET-1及vWF水平均稍低，但均未达到统计学差异（P>0.05），其中在NO上两者相比有接近统计学差异的趋势（P=0.064）。
1.4在基线期、氨氯地平治疗后及左旋氨氯地平治疗后这三个节点，PAV与FMD测值均有显著的相关性（P<0.01），氨氯地平治疗后与基线期PAV测值的差值ΔPAV（0.24±0.16、0.18±0.15）及左旋氨氯地平后与基线期FMD测值的差值ΔFMD（2.3±1.7%、1.6±1.2%）亦具有显著的相关性（P<0.01）。对PAV及FMD诊断高血压内皮功能不全进行一致性检验，结果示Kappa=0.647，P<0.001，有统计学意义。
2体外细胞培养实验结果：
2.1在eNOS抑制剂Nw-nitro-L-arginine (L-NA,300mmol/l)存在时，氨氯地平及左旋氨氯地平处理25分钟后NO测值与L-NA组相比数值接近，且均无统计学差异（P>0.05）；两种药物之间测值相近，亦无统计学差异（P>0.05）。在L-NA缺失时，氨氯地平及左旋氨氯地平处理25分钟后NO测值与对照组相比均有明显升高，有统计学差异（P<0.01），氨氯地平组NO测值高于左旋氨氯地平组，有统计学差异（P<0.05）。
2.2经氨氯地平（1μmol/l）处理5、15及25分钟后NO水平逐渐增加，在25分钟达到最高值，15及25分钟时NO水平与对照组相比均有明显升高，且均达到统计学差异（P<0.01）；经左旋氨氯地平（1μmol/l）处理5、15及25分钟后NO测值均增加，在15分钟达到最高值，5、15及25分钟时NO水平与对照组相比均达到的统计学差异（P<0.01）；氨氯地平组与左旋氨氯地平组相比，除5分钟外，15及25分钟时NO水平均是前者较高，其中25分钟时两者达到统计学差异（P<0.05）。内皮细胞经PKC途径抑制剂Ro31-8220（30nmol/l）处理5、15及25分钟后NO测值也逐渐增加，在25分钟达到最高值，15及25分钟时NO测值与对照组相比均有显著的统计学差异（P<0.01）。
2.3氨氯地平对内皮细胞的刺激引起eNOS在Ser1177磷酸化和Thr495去磷酸化的增长，并且作用在5、15及25分钟逐渐加强，在25分钟达到峰值；左旋氨氯地平对eNOS在Ser1177磷酸化和Thr495去磷酸化作用不大，在5、15及25分钟这三个时间点未表现出明显的趋势。
2.4氨氯地平可以使PKC本身磷酸化减弱，并且作用在5、15及25分钟逐渐加强，在25分钟达到峰值；左旋氨氯地平对PKC去磷酸化作用不大，在5、15及25分钟这三个时间点未表现出明显的趋势
2.5PKC抑制剂Ro31-8220对内皮细胞的刺激引起eNOS在Ser1177磷酸化和Thr495去磷酸化的增长，并且作用在5、15及25分钟逐渐加强，在25分钟达到峰值。
结果小结：
1、在检测治疗前后高血压患者内皮功能改善上，PAV与FMD具有明显的相关性；在诊断高血压患者内皮功能不全上，PAV与FMD具有较好的一致性。
2、在取得相似的降压效果时，氨氯地平及左旋氨氯地平均具有良好的内皮保护作用，虽然两药治疗后各项指标相比均无统计学差异，但前者似乎有更好内皮保护作用的趋势。
3、在体外培养细胞中，氨氯地平及左旋氨氯地平均可以时间依赖性的提高NO水平，左旋氨氯地平组达到峰值较早，但氨氯地平组峰值水平较高。
4、氨氯地平可通过PKC途径对eNOS在Ser1177及Thr495磷酸化产生影响有从而使eNOS活化，而左旋氨氯地平对PKC影响不大，也不能对eNOS在Ser1177及Thr495的磷酸化程度产生影响。
结论：
1、氨氯地平及左旋氨氯地平都通过增加NO水平发挥良好的内皮保护作用，但程度和途径不同。
2、PAV与FMD具有较好的相关性及一致性，可替代FMD进行内皮功能检测。
Before the development of atherosclerosis morphological changes, changes invascular endothelial function participates the progress of the injury and subsequentclinical complications, and has close relationship with hypertension and othercardiovascular diseases. Dysfunction of vascular endothelial reduces secretion ofvasodilator agent, but increase the vasoconstriction cytokine secretion. That promotesthe proliferation of vascular smooth muscle cells, leading to vessel wall thickeningand vascular remodeling, decreased arterial elasticity, increased peripheral resistance,and promote the development of both hypertension and its complications. In addition,the high blood pressure caused by fluid shear stress and endocrine changes in theenvironment can cause Endothelial dysfunction, thus reversing the hypertensionendothelial dysfunction another treatment outside the buck has become a new target.The detection of hypertension endothelial function requires to be non-invasive, easyoperation and quickly. PAV is a new non-invasive way to test the endothelial functionwhen the fingertip detection of endothelial function develop rapidly, but there are norelevant clinical applications reported.
As a third-generation calcium channel blocker, Amlodipine is one of the mostwidely used antihypertensive drugs in clinical application. Amlodipine is a racemicmixture of S(-)-amlodipine and the R(-)-amlodipine constituted the outer to the ratioof1:1, which antihypertensive effect mainly come from L-body and effect is1000-time stronger than that of R(-)-amlodipine. It is not clear that amlodipine has anadditional protective effect of endothelial functionit compared with S(-)-amlodipinebecause of the presence of R(-)-amlodipine.
A clinical study was carried out to examine whether amlodipine andS(-)-amlodipine have similar protective effects on vascular endothelial function inaddition to the similar antihypertensive effects, and we explored the association andconsistency of PAV and FMD on testing of endothelial function in patitents withhypertension. Further, we explore both its protective effect of endothelial function byaffecting the level of NO and the mechanisms in cultured human umbilical vein endothelial cells.
Method
1、Clinical trials
We took a randomized and crossover design, chosen24cases of patients withessential hypertension and randomly divided into Group A and Group B,the formertaking amlodipine besylate5mg/d (Pfizer),the latter taking the benzenesulfonicS(-)-amlodipine of2.5mg/d(Shi Huida). The two groups were both early morningmedication and went through wash-out period about2weeks after6weekstreatment,then continued the treatment for6weeks crossing the two drugs after heartrate and blood pressure of patients returned to the level of the first wash-out period.Subjects, who were fasting and Prohibited smoking, drinking and coffee the nightbefore detection, will accept the detection of blood collection,and vascularfunction in the time of baseline and the end of each treatment.
Test items include：Pulse wave velocity (PWV) and Ankle-branchial index(ABI)were tested by Omron Colin VP1000. Pulse amplitude volume（PAV）was testedby SY2000（Jilin saintyear company）. Flow mediated dilation(FMD) of brachialartery was tested by Philips IE33(America). Nitric oxide(NO)was tested by Griessmethod. Endothelial nitric oxide synthas（eeNOS）,Endothelin（ET）andvon WillebrandFactor（vWF）were tested by Elisa.
2、Cell culture experiment
We cultured human umbilical vein endothelial cells in vitro for the followingchecks:
2.1Griess method had been used to detect the endothelial cell NO levels at25minutes that affected by amlodipine or S(-)-amlodipine on presence or absence of theeNOS inhibitor Nw-nitro-L-arginine (L-NA).
2.2Griess method had been used to detect respectively the endothelial cell NOlevels at0,5,15,25minutes that treated by amlodipine, S(-)-amlodipine or proteinkinase C（PKC）inhibitor Ro31-8220.
2.3Wsetern blot(WB) had been used to detect respectively degree ofphosphorylation of eNOS Ser1177、eNOS Thr495and PKC in endothelial cells that thattreated by amlodipine or S(-)-amlodipine at0,5,15,25minutes.
2.4Wsetern blot(WB) had been used to detect respectively degree of phosphorylation of eNOS Ser1177and Thr495in endothelial cells that treated by proteinkinase C（PKC）inhibitor Ro31-8220at0,5,15,25minutes.
Result：
1Results of clinical trials
1.1After amlodipine and S(-)-amlodipine treatments, the patients’ SBP, DBPand HR significantly decreased compared with baseline data (P<0.01). However,there were no significant differences between the data of amlodipine andS(-)-amlodipine groups (P>0.05).
1.2Compared with baseline,after treatment of the two drugs the PAV,FMD andABI were significantly increased,and there was a significant difference (P<0.05)；PWV were significantly reduced, there were statistically significant (P<0.01);NMDlittle change in value, no significant difference (P>0.05)。In the amlodipine group andL-amlodipine group,PAV, FMD, PWV and ABI measured values are slightlyhigher,but it did not reach a statistically significant difference (P>0.05),Howevercomparing the two in the FMD approached statistical differences (P=0.053).
1.3Compared with baseline,NO and eNOS have increased significantly aftertreatment of the two drugs，ang it was a significant difference（P<0.01）;ET-1andvEGF were significantly reduced reach statistical significance (P<0.01). In theamlodipine group and S(-)-amlodipine group,NO and NOS were slightly higher, ET-1and vWF levels were slightly lower,but it did not reach a statistically significantdifference (P>0.05),However comparing the two in the NO approached statisticaldifferences (P=0.064).
1.4At baseline,the time after the treatment of amlodipine orS(-)-amlodipine,PAV was strongly associated with FMD（P<0.01）,and the differencebetween the baseline and the treatment ΔPAV（0.24±0.16、0.18±0.15）was stronglyassociated with ΔFMD（2.3±1.7%、1.6±1.2%）. The consistency test of diagnostic testswas done between PAV and FMD, the result is Kappa=0.647，P<0.001.
2Results of cell culture experiment：
2.1In the presence of the eNOS inhibitor L-NA (300mmol/l), the NO level ofHUVECs treated with amlodipine and S(-)-amlodipine for25minutes was similar to the L-NA group, showing no statistically significant difference (P>0.05). The NOlevel of HUVECs was also similar between amlodipine and S(-)-amlodipine groupswith no statistically significant difference (P>0.05). In the absence of L-NA, the NOlevel of HUVECs treated with amlodipine and S(-)-amlodipine for25minutes wassignificantly higher than the control group (P<0.01). In addition, the NO level ofHUVECs was obviously higher in the amlodipine group than in the S(-)-amlodipinegroup, showing statistically significant differences (P<0.05).
2.2After amlodipine treatment (1μmol/l) for5,15, and25minutes, the NO levelof HUVECs gradually increased and peaked at25minutes. As compared with that ofcontrol group, the NO level of HUVECs in the amlodipine group was higher at15min and25minutes, showing statistically significant differences (P<0.01). AfterS(-)-amlodipine treatment (1μmol/l) for5,15, and25minutes, the NO level ofHUVECs increased and peaked at15minutes. As compared with that of control group,the NO level of HUVECs in the S(-)-amlodipine group was significantly higher at5,15, and25minutes (P<0.01). Data comparison between the amlodipine andS(-)-amlodipine groups showed that the NO level of HUVECs was higher in theformer at15and25minutes but not5minutes, with statistically significant differenceobserved at25minutes only (P<0.05). After Ro31-8220treatment (30nmol/l) for5,15and25minutes, the NO level of HUVECs gradually increased and peaked at25minutes. As compared with control group, the NO level of HUVECs treated with Ro31-8220was significantly different at15and25minutes (P<0.01).
2.3The stimulation of amlodipine in endothelial cells induced the increase ofSer1177phosphorylation and Thr495dephosphorylation at eNOS, the effect increasedwith the increase in culture time from5,15to25minutes, and it reached the highestvalue at25minutes; S(-)-amlodipine did not affect the phosphorylation of eNOSSer1177and Thr495at5,15or25minutes.
2.4The stimulation of amlodipine induced the decrease of the phosphorylation ofPKC, the effect increased with the increase in culture time from5,15to25minutes,and it reached the highest value at25minutes; S(-)-amlodipine did not affect thephosphorylation of PKC at5,15or25minutes.
2.5The stimulation of PKC inhibitor Ro31-8220in endothelial cells induced theincrease of Ser1177phosphorylation and Thr495dephosphorylation at eNOS，the effectincreased with the increase in culture time from5,15to25minutes, and it reached the highest value at25minutes
Summary of results:
1、There is significant correlation between PAV and FMD in detecting improvedendothelial function before and after the treatment of the hypertension; PAV has goodconsistency with FMD in the diagnosis of hypertension patients with endothelialdysfunction.
2、With similar antihypertensive effect, both amlodipine and S(-)-amlodipinehave a good protective effects of endothelial cells. Though there is no significantdifference between the two drugs at these tests, amlodipine appears to have the trendof having a better endothelial protective effect.
2、 In culturedendothelial cells, amlodipine and S(-)-amlodipine both timedependently increase the level of NO. The level of NO in S(-)-amlodipine groupreached the highest value earlier but which was higher in amlodipine group.
4、 Amlodipine can activate eNOS though affecting the phosphorylation ofeNOS Ser1177and Thr495by PKC,but S(-)-amlodipine has little effect on thephosphorylation of PKC and the phosphorylation of eNOS Ser1177and Thr495.
Conclusion
1、Amlodipine and S(-)-amlodipine both have good effect on the protection ofendothelial cells through increasing the level of NO, but the degree and the pathwayare different.
2、There are good correlation and consistency between PAV and FMD,PAV cantest the endothelial function instead of FMD.
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