孤束核中血管紧张素1-7通过Mas受体/PI3K/Akt通路上调一氧化氮释放
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摘要
【研究目的】
高血压是人类多发病。《2012年世界卫生统计》(World Health Statistics2012by World Health Organization)报告指出全球三分之一成年人患有高血压，这种病症的死亡人数约达中风和心脏病所导致的总死亡人数的一半。关于原发性高血压发病机制的研究一直是医学研究的重点内容。大量研究数据显示，肾素-血管紧张素系统(Renin-angiotensin system, RAS)的异常与高血压病的发生、发展与预后有着十分密切的关系。血管紧张素1-7[Angiotensin-(1-7),Ang-(1-7)]是RAS中的新成员。Ang-(1-7)可由血管紧张素1(Angiotensin I, Ang I)和血管紧张素2(Angiotensin II,Ang II)在血管紧张素转化酶(Angiotensin-convertion enzyme,ACE)和血管紧张素转化酶2(Angiotensin-convertion enzyme2, ACE2)的催化作用下生成。Ang-(1-7)是由七个氨基酸组成的多肽，通过与细胞膜表面特异性受体Mas受体(Mas recptor, MasR)结合，产生降低血压、减缓心律、降低交感神经活性、抗心肌纤维化与重构等心血管保护效应，与Ang II的作用相拮抗，从而维持心血管系统的稳态。高血压病状态下，Ang-(1-7)和Ang II平衡失调，Ang-(1-7)活性下降。因此，纠正Ang-(1-7)和Ang II的失衡状态，增强Ang-(1-7)的功能是高血压病防治的有效策略。
中枢神经系统心血管调节中枢对血压、心率等心血管功能具有重要调节作用。心血管调节中枢主要位于脑干，其中较为重要的核团有孤束核(Nucleus ofsolitary tract, NTS)、头端延髓腹外侧区(Rostral ventrolateral medulla, RVLM)和尾端延髓腹外侧区（Caudal ventrolateral medulla, CVLM）等。NTS是心血管调节的第一站，具有重要的心血管调节效应。NTS接收来自外周动脉压力感受器的神经传入，并对外周信号进行初步整合，发出神经纤维投射到CVLM，通过CVLM中γV氨基丁酸(Gamma-aminobutyric acid, GABA)能神经元的抑制性中转，最终调节RVLM神经元活性，RVLM是控制交感神经活动的中枢区域。同时，NTS神经元通过兴奋性纤维投射到迷走神经中枢（如疑核等），调节迷走神经活动。NTS在维持基础血压、交感神经紧张性输出，介导压力反射等心血管功能调节中具有关键作用，NTS功能异常与高血压病的发生发展具有密切的关系。中枢神经系统中存在完整的RAS，即中枢神经系统内含有生成RAS所有成分所需的底物和酶。外周组织器官生成的Ang-(1-7)不能透过血脑屏障，因而中枢神经系统内局部生成的Ang-(1-7)发挥了重要的心血管调节作用。
无论外周还是中枢神经系统，一氧化氮NO都是一个重要的信使分子，参与调节心血管、免疫等系统的功能。外周循环系统的研究已证实，Ang-(1-7)通过一氧化氮(Nitric oxide, NO)介导发挥作用。另有研究发现，NTS中NO通过调节特定神经元的活性发挥重要心血管调节效应。但中枢神经系统Ang-(1-7)的作用及机制尚不明了。NTS中Ang-(1-7)发挥心血管调节效应是否通过NO介导？其具体信号通路又是什么？本课题的研究目的在于深入探讨Ang-(1-7)在NTS发挥心血管调节功能的信号通路。
【研究方法】
实验使用300-350g雄性Sprague-Dawley (SD)大鼠，整体动物水平观察NTS局部给予Ang-(1-7)对大鼠血压、心率、肾交感神经活动及动脉压力感受性反射等指标的影响；预处理一氧化氮合酶(Nitricoxide synthase, NOS)抑制剂L-NAME后，观察Ang-(1-7)的效应是否被阻断，以判断Ang-(1-7)发挥作用是否依赖于NOS；同时预处理Ang-(1-7)，观察其对L-NAME心血管效应的影响；使用相应的分子生物学方法检测大鼠NTS急性给予Ang-(1-7)后的NO含量及NOS活性，并使用工具药和小RNA干扰技术(siRNA interferring technology)等方法进一步研究其作用机制；调研文献后选出MasR/PI3K/Akt通路作为研究对象，使用Western Blot检测大鼠NTS急性给予Ang-(1-7)后Akt、nNOS和eNOS的磷酸化水平，给予MasR阻断剂（A779）、PI3K抑制剂（LY294002）和Akt抑制剂（TCN）后Ang-(1-7)的上述磷酸化效应是否被消除；同时在整体动物水平，研究给予上述通路靶点蛋白阻断剂或抑制剂后， Ang-(1-7)的心血管效应是否被消除；使用小RNA干扰技术特异性干扰大鼠NTS区域神经细胞PI3Kp110δ，亚基的表达，15天后Western Blot检测大鼠NTS急性给予Ang-(1-7)后NO含量及Akt、nNOS和eNOS的磷酸化水平，进一步验证PI3K在信号通路中的位置；使用免疫荧光共定位方法观察NTS中是否存在MasR与nNOS的共表达，从组织分布上确定两者存在相互作用的可能性。
【结果】
一、 NTS局部注射Ang-(1-7)对心血管活动的影响
NTS单侧微量注射Ang-(1-7)(25pmol/50nl)可降低血压（-12.8±1.8mmHg）、减缓心率(-22.0±3.1beats/min)和降低肾交感神经活动(-15.8±2.0%)；NTS双侧微量注射Ang-(1-7)(25pmol/50nl)可显著增强动脉压力感受性反射(ABR)(从1.0强动脉压升高至1.8动脉压力感受性反射ats/mi。
二、 NTS预处理L-NAME可改变局部注射Ang-(1-7)对心血管活动的影响
溶剂对照组大鼠NTS局部注射Ang-(1-7)可降低血压、减慢心率和下降肾交感神经活动；预处理NOS阻断剂L-NAME后，与溶剂对照组相比，Ang-(1-7)的降压幅度减小，减慢心率的作用被逆转，降低的肾交感神经活动效应也被逆转(P<0.05)。
三、 NTS局部注射Ang-(1-7)能够增强L-NAME升血压、加快心率和升肾交感神经活动的效应，Ang-(1-7)的这一效应可被MasR阻断剂和PI3K抑制剂消除
NTS预处理溶剂(人工脑脊液, aCSF)后给予L-NAME可产生升血压、加快心率和升肾交感神经兴奋性的作用；与预处理溶剂组相比，预处理Ang-(1-7)可使L-NAME升血压、加快心率和增强肾交感神经活动的作用加强(P<0.05)；预处理MasR阻断剂A779可减弱Ang-(1-7)对L-NAME的易化作用(P<0.05)；预处理PI3K抑制剂LY294002可完全消除Ang-(1-7)对L-NAME的易化作用(P<0.05)。与溶剂对照组相比，各组大鼠NTS局部注射L-NAME前血压、心率等指标均无显著性差异（P>0.05)。
四、 Ang-(1-7)能够上调NO含量和NOS活性，这一效应通过MasR/PI3K/Akt通路介导
与溶剂对照组相比，Ang-(1-7)能够增加NO的生成（平均升高34%，P<0.05），并提高总NOS的活性（平均升高38%，P<0.05）。使用A779、LY294002和TCN能够阻断Ang-(1-7)的上述效应(P<0.05)。与溶剂对照组相比，单独给予A779、LY294002或TCN不影响NO含量（P>0.05)。
五、 Ang-(1-7)可刺激Akt和NOS磷酸化，这一效应通过MasR/PI3K/Akt通路介导
Western blot结果显示，与溶剂对照组相比，Ang-(1-7)能够增加Akt Ser473、nNOS Ser1416和eNOS Ser1177相应位点的磷酸化水平(P<0.05)。与Ang-(1-7)组相比，使用A779、LY294002能够消除Ang-(1-7)对Akt的磷酸化效应(P>0.05)，使用A779、LY294002或TCN能够消除Ang-(1-7)对nNOS/eNOS的磷酸化效应(P>0.05)。
六、慢病毒转染干扰PI3K表达后，Ang-(1-7)增加Akt、nNOS和eNOS磷酸化的效应被抑制，进而导致Ang-(1-7)增加NO生成的效应被抑制
PI3K siRNA干扰慢病毒(LenVi-PI3K KD)或空载慢病毒(LenVi-GFP)局部感染NTS，15天之后NTS急性给予Ang-(1-7)进行后续检测。Western blot结果显示，NTS局部感染LenVi-GFP后，与溶剂对照组相比，Ang-(1-7)能够增加AktSer473、nNOS Ser1416和eNOS Ser1177位点的磷酸化水平(P<0.05)，进而增加NO含量(P<0.05)；NTS局部感染LenVi-PI3K KD后，与溶剂对照组相比，Ang-(1-7)不能增加Akt Ser473、nNOS Ser1416和eNOS Ser1177相应位点的磷酸化水平(P>0.05)，也不能增加NO含量(P>0.05)。
七、 MasR和nNOS免疫荧光共定位
免疫荧光共定位结果显示，MasR(绿色荧光)和nNOS(红色荧光)可在同一NTS神经细胞内共表达，提示nNOS与Ang-(1-7)/MasR之间可能存在密切联系。
【结论】
Ang-(1-7)上调NTS中NO生成，进而产生降低血压、减缓心率、降低交感神经活性等心血管效应，这一效应是通过MasR/PI3K/Akt/NOS通路介导的。
Backgrounds and Objectives
Hypertension is a common disease. It is reported by World Health Statistics2012(World Health Statistics2012by World Health Organization) that1/3adults sufferedfrom hypertension around the globe, half of the total number of deaths from strokeand heart disease was caused by hypertension. Study on the pathogenesis of essentialhypertension has been the focus of medical research for long. A lot of research datashowed, hypertension can be caused by abnormal renin-angiotensin system (RAS).Angiotensin1-7[Ang-(1-7)] is a new member of the RAS.
Ang-(1-7) is generated from angiotensin1(Ang I) and angiotensin2(Ang II) underthe catalysis of angiotensin converting enzyme (ACE) and angiotensin convertingenzyme2(ACE2). Ang-(1-7) is a polypeptide of seven amino acids, which cancombined with the specific membrane receptors, the Mas receptors (MasR), inducingthe effects of lowering blood pressure, slowing down the heart rate, decreasing renalsympathetic nerve activity (RSNA), decreasing myocardial fibrosis and remodeling.These effects antagonize with Ang II, thus maintaining the homeostasis ofcardiovascular system. Hypertension can be caused by the unbalance betweenAng-(1-7) and Ang II. Enhancing the effect of Ang-(1-7) to rebalance Ang-(1-7) andAng II is an effective strategy for treatment of hypertension.
The cardiovascular regulation center in central nervous system has an important rolein the regulation of blood pressure. Cardiovascular center is mainly located in thebrainstem, some of the important regions are the nucleus of the solitary tract (NTS), the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla(CVLM). The NTS is the first region in central control of cardiovascular activity.NTS neurons project nerve fibers to the other centers to regulate the sympathetic andvagal nerve activity. NTS plays a key role in the regulation of cardiovascular functionby maintaining blood pressure, sympathetic tone and mediating baroreflex sensitivity.NTS was closely related with the onset and development of hypertension. Centralnervous system has a complete RAS for its own, and the local production of Ang-(1-7)in the central nervous system plays an important role in cardiovascular regulation.Nitric oxide (NO) is an important messenger both systemically and in the CNS. Studyof peripheral circulation system has proved that the effects of Ang-(1-7) can bemediated by nitric oxide. NO plays an important role in cardiovascular regulation bymodulating the activity of specific neurons in NTS. The mechanism remains unclear.Is the effect of Ang-(1-7) in NTS mediated by NO? What is the specific signalingpathway? Based on the above questions, the aim of the present study was to determinethe role of phosphatidylinositol3-kinase (PI3K) signaling pathways in mediating theeffect of Ang-(1-7) on NO generation in the NTS.
Methods
300-350g male Sprague-Dawley (SD) rats were used in this study. The effects ofAng-(1-7) on blood pressure, heart rate, renal sympathetic nerve activity(RSNA) andarterial baroreflex(ABR) were observed. We observed whether the effects of Ang-(1-7)were blocked by pretreatment of the nitric oxide synthase (NOS) inhibitor L-NAME.NO production and NOS activity were detected after treatment of Ang-(1-7) in NTS. Western blot analysis was used for detecting the effects of Ang-(1-7) on Akt, nNOSand eNOS phosphorylation in NTS. MasR blocker (A779), PI3K inhibitor (LY294002)and Akt inhibitors (TCN) were used to observe the pathway of Ang-(1-7) action.Local infection of lenti-virus in NTS was used to interfer the expression of PI3K.Fifteen days after the infection, effects of Ang-(1-7) on Akt, nNOS and eNOSphosphorylation were determined by Western blot. Immunofluorescence was used todetermine the co-localization of MasR and nNOS is NTS.
Results
1. Ang-(1-7) enhances NO generation and NOS activity via Mas/PI3K/Aktpathway in the NTS
To detect the effect of Ang-(1-7) on NO production and NOS activity in the NTS, ratswere received microinjections of vehicle (aCSF), Ang-(1-7), A779plus Ang-(1-7),LY294002plus Ang-(1-7) or TCN plus Ang-(1-7) in NTS. We found that NOproduction and NOS activity in the NTS were significantly increased by an average of34%and38%, respectively (P<0.05), in rats which were acutely injected withAng-(1-7)(25pmol) compared with the vehicle injection (aCSF,50nl). However, theAng-(1-7)-induced increase in NO generation and NOS activity were completelyprevented in the presence of the Ang-(1-7) Mas receptor antagonist A779(500pmol),LY294002(100pmol) or TCN (1mmol). In addition, we confirmed that injection ofalone A779, LY294002or TCN into the NTS had little effect on baseline level of NOproduction.
2. Ang-(1-7) stimulates NOS phosphorylation via Mas/PI3K/Akt pathway in
the NTS
To detect the signaling involved in the effect of Ang-(1-7) on NO release in the NTS,NOS phosphorylation was measured by western blot. We found that, in the NTS, thephosphorylation of nNOS at Ser1416and eNOS at Ser1177was significantlyincreased after acute injection of Ang-(1-7)(25pmol)(P<0.05), which was abolishedin the presence of A779(500pmol) or LY294002(100pmol). In addition, we alsofound that the phosphorylation of Akt, a downstream of PI3K, was significantlyincreased after injection of Ang-(1-7) into the NTS(P<0.05).
3. Ang-(1-7) enhances the cardiovascular effects evoked by NOS inhibitor inthe NTS
To determine the effect of Ang-(1-7) on release of NO involved in cardiovascularresponse, several groups were used for monitoring changes in BP, HR, and RSNAevoked by the NOS inhibitor L-NAME following microinjection of Ang-(1-7) into theNTS. We first confirmed that25pmol of Ang-(1-7) injected into the NTSsignificantly decreased basal BP, HR, and RSNA(P<0.05), whereas it enhanced thebaroreflex sensitivity. Increases in BP, HR, and RSNA evoked by L-NAME (10nmol)injected into the NTS were enhanced5min after injection of Ang-(1-7)(25pmol)(P<0.05). Furthermore, co-injection of Ang-(1-7)(25pmol) and A779(500pmol) hadlittle effect on increases in BP, HR, and RSNA normally induced by L-NAMEinjected into the NTS. Moreover, it was found that the L-NAME-induced actions werecompletely abolished in the presence of LY294002.
4. Blockade of NOS prevents the cardiovascular effects of Ang-(1-7) in the NTS Ten rats (two groups: no difference of baseline BP and HR) were used for determiningthe NO mechanism responsible for cardiovascular effects evoked by Ang-(1-7) in theNTS. Pretreatment with the NOS inhibitor L-NAME (10nmol)10min prior to NTSinjection with Ang-(1-7) prevented the reduction in BP, HR, and RSNA normallyevoked by Ang-(1-7).
5. After lenti virus infected the NTS for15days, Ang-(1-7) was given in NTSfor subsequent molecular detections.PI3K Knock Down siRNA interference lenti virus (LenVi-PI3K KD) or scramblelenti virus (LenVi-GFP) were used for locally infecting the NTS. After lenti viralinfection in the NTS for15days, bilateral injection of Ang-(1-7)(150pmol) wasgiven in the NTS for detection of Western Blot or NO production. Western blot resultsshowed the Ang-(1-7) induced increase in eNOS, nNOS and Akt phosphorylation andNO production was inhibited by LenVi-PI3K KD(P<0.05). On the contrary,LenVi-GFP had no effect on Ang-(1-7).6. Immunofluorescence for detecting the co-localization of MasR and nNOSImmunofluorescence pictures indicated MasR and nNOS were expressed in the sameNTS neural cells, suggesting that nNOS may have close links to Ang-(1-7)/MasR.Conclusion: Our findings suggest that Ang-(1-7) is capable of stimulating therelease of nitric oxide in the NTS through the MasR-PI3K-Akt-NOS signalingpathway.
高血压是人类多发病。《2012年世界卫生统计》(World Health Statistics2012by World Health Organization)报告指出全球三分之一成年人患有高血压，这种病症的死亡人数约达中风和心脏病所导致的总死亡人数的一半。关于原发性高血压发病机制的研究一直是医学研究的重点内容。大量研究数据显示，肾素-血管紧张素系统(Renin-angiotensin system, RAS)的异常与高血压病的发生、发展与预后有着十分密切的关系。血管紧张素1-7[Angiotensin-(1-7),Ang-(1-7)]是RAS中的新成员。Ang-(1-7)可由血管紧张素1(Angiotensin I, Ang I)和血管紧张素2(Angiotensin II,Ang II)在血管紧张素转化酶(Angiotensin-convertion enzyme,ACE)和血管紧张素转化酶2(Angiotensin-convertion enzyme2, ACE2)的催化作用下生成。Ang-(1-7)是由七个氨基酸组成的多肽，通过与细胞膜表面特异性受体Mas受体(Mas recptor, MasR)结合，产生降低血压、减缓心律、降低交感神经活性、抗心肌纤维化与重构等心血管保护效应，与Ang II的作用相拮抗，从而维持心血管系统的稳态。高血压病状态下，Ang-(1-7)和Ang II平衡失调，Ang-(1-7)活性下降。因此，纠正Ang-(1-7)和Ang II的失衡状态，增强Ang-(1-7)的功能是高血压病防治的有效策略。
中枢神经系统心血管调节中枢对血压、心率等心血管功能具有重要调节作用。心血管调节中枢主要位于脑干，其中较为重要的核团有孤束核(Nucleus ofsolitary tract, NTS)、头端延髓腹外侧区(Rostral ventrolateral medulla, RVLM)和尾端延髓腹外侧区（Caudal ventrolateral medulla, CVLM）等。NTS是心血管调节的第一站，具有重要的心血管调节效应。NTS接收来自外周动脉压力感受器的神经传入，并对外周信号进行初步整合，发出神经纤维投射到CVLM，通过CVLM中γV氨基丁酸(Gamma-aminobutyric acid, GABA)能神经元的抑制性中转，最终调节RVLM神经元活性，RVLM是控制交感神经活动的中枢区域。同时，NTS神经元通过兴奋性纤维投射到迷走神经中枢（如疑核等），调节迷走神经活动。NTS在维持基础血压、交感神经紧张性输出，介导压力反射等心血管功能调节中具有关键作用，NTS功能异常与高血压病的发生发展具有密切的关系。中枢神经系统中存在完整的RAS，即中枢神经系统内含有生成RAS所有成分所需的底物和酶。外周组织器官生成的Ang-(1-7)不能透过血脑屏障，因而中枢神经系统内局部生成的Ang-(1-7)发挥了重要的心血管调节作用。
无论外周还是中枢神经系统，一氧化氮NO都是一个重要的信使分子，参与调节心血管、免疫等系统的功能。外周循环系统的研究已证实，Ang-(1-7)通过一氧化氮(Nitric oxide, NO)介导发挥作用。另有研究发现，NTS中NO通过调节特定神经元的活性发挥重要心血管调节效应。但中枢神经系统Ang-(1-7)的作用及机制尚不明了。NTS中Ang-(1-7)发挥心血管调节效应是否通过NO介导？其具体信号通路又是什么？本课题的研究目的在于深入探讨Ang-(1-7)在NTS发挥心血管调节功能的信号通路。
【研究方法】
实验使用300-350g雄性Sprague-Dawley (SD)大鼠，整体动物水平观察NTS局部给予Ang-(1-7)对大鼠血压、心率、肾交感神经活动及动脉压力感受性反射等指标的影响；预处理一氧化氮合酶(Nitricoxide synthase, NOS)抑制剂L-NAME后，观察Ang-(1-7)的效应是否被阻断，以判断Ang-(1-7)发挥作用是否依赖于NOS；同时预处理Ang-(1-7)，观察其对L-NAME心血管效应的影响；使用相应的分子生物学方法检测大鼠NTS急性给予Ang-(1-7)后的NO含量及NOS活性，并使用工具药和小RNA干扰技术(siRNA interferring technology)等方法进一步研究其作用机制；调研文献后选出MasR/PI3K/Akt通路作为研究对象，使用Western Blot检测大鼠NTS急性给予Ang-(1-7)后Akt、nNOS和eNOS的磷酸化水平，给予MasR阻断剂（A779）、PI3K抑制剂（LY294002）和Akt抑制剂（TCN）后Ang-(1-7)的上述磷酸化效应是否被消除；同时在整体动物水平，研究给予上述通路靶点蛋白阻断剂或抑制剂后， Ang-(1-7)的心血管效应是否被消除；使用小RNA干扰技术特异性干扰大鼠NTS区域神经细胞PI3Kp110δ，亚基的表达，15天后Western Blot检测大鼠NTS急性给予Ang-(1-7)后NO含量及Akt、nNOS和eNOS的磷酸化水平，进一步验证PI3K在信号通路中的位置；使用免疫荧光共定位方法观察NTS中是否存在MasR与nNOS的共表达，从组织分布上确定两者存在相互作用的可能性。
【结果】
一、 NTS局部注射Ang-(1-7)对心血管活动的影响
NTS单侧微量注射Ang-(1-7)(25pmol/50nl)可降低血压（-12.8±1.8mmHg）、减缓心率(-22.0±3.1beats/min)和降低肾交感神经活动(-15.8±2.0%)；NTS双侧微量注射Ang-(1-7)(25pmol/50nl)可显著增强动脉压力感受性反射(ABR)(从1.0强动脉压升高至1.8动脉压力感受性反射ats/mi。
二、 NTS预处理L-NAME可改变局部注射Ang-(1-7)对心血管活动的影响
溶剂对照组大鼠NTS局部注射Ang-(1-7)可降低血压、减慢心率和下降肾交感神经活动；预处理NOS阻断剂L-NAME后，与溶剂对照组相比，Ang-(1-7)的降压幅度减小，减慢心率的作用被逆转，降低的肾交感神经活动效应也被逆转(P<0.05)。
三、 NTS局部注射Ang-(1-7)能够增强L-NAME升血压、加快心率和升肾交感神经活动的效应，Ang-(1-7)的这一效应可被MasR阻断剂和PI3K抑制剂消除
NTS预处理溶剂(人工脑脊液, aCSF)后给予L-NAME可产生升血压、加快心率和升肾交感神经兴奋性的作用；与预处理溶剂组相比，预处理Ang-(1-7)可使L-NAME升血压、加快心率和增强肾交感神经活动的作用加强(P<0.05)；预处理MasR阻断剂A779可减弱Ang-(1-7)对L-NAME的易化作用(P<0.05)；预处理PI3K抑制剂LY294002可完全消除Ang-(1-7)对L-NAME的易化作用(P<0.05)。与溶剂对照组相比，各组大鼠NTS局部注射L-NAME前血压、心率等指标均无显著性差异（P>0.05)。
四、 Ang-(1-7)能够上调NO含量和NOS活性，这一效应通过MasR/PI3K/Akt通路介导
与溶剂对照组相比，Ang-(1-7)能够增加NO的生成（平均升高34%，P<0.05），并提高总NOS的活性（平均升高38%，P<0.05）。使用A779、LY294002和TCN能够阻断Ang-(1-7)的上述效应(P<0.05)。与溶剂对照组相比，单独给予A779、LY294002或TCN不影响NO含量（P>0.05)。
五、 Ang-(1-7)可刺激Akt和NOS磷酸化，这一效应通过MasR/PI3K/Akt通路介导
Western blot结果显示，与溶剂对照组相比，Ang-(1-7)能够增加Akt Ser473、nNOS Ser1416和eNOS Ser1177相应位点的磷酸化水平(P<0.05)。与Ang-(1-7)组相比，使用A779、LY294002能够消除Ang-(1-7)对Akt的磷酸化效应(P>0.05)，使用A779、LY294002或TCN能够消除Ang-(1-7)对nNOS/eNOS的磷酸化效应(P>0.05)。
六、慢病毒转染干扰PI3K表达后，Ang-(1-7)增加Akt、nNOS和eNOS磷酸化的效应被抑制，进而导致Ang-(1-7)增加NO生成的效应被抑制
PI3K siRNA干扰慢病毒(LenVi-PI3K KD)或空载慢病毒(LenVi-GFP)局部感染NTS，15天之后NTS急性给予Ang-(1-7)进行后续检测。Western blot结果显示，NTS局部感染LenVi-GFP后，与溶剂对照组相比，Ang-(1-7)能够增加AktSer473、nNOS Ser1416和eNOS Ser1177位点的磷酸化水平(P<0.05)，进而增加NO含量(P<0.05)；NTS局部感染LenVi-PI3K KD后，与溶剂对照组相比，Ang-(1-7)不能增加Akt Ser473、nNOS Ser1416和eNOS Ser1177相应位点的磷酸化水平(P>0.05)，也不能增加NO含量(P>0.05)。
七、 MasR和nNOS免疫荧光共定位
免疫荧光共定位结果显示，MasR(绿色荧光)和nNOS(红色荧光)可在同一NTS神经细胞内共表达，提示nNOS与Ang-(1-7)/MasR之间可能存在密切联系。
【结论】
Ang-(1-7)上调NTS中NO生成，进而产生降低血压、减缓心率、降低交感神经活性等心血管效应，这一效应是通过MasR/PI3K/Akt/NOS通路介导的。
Backgrounds and Objectives
Hypertension is a common disease. It is reported by World Health Statistics2012(World Health Statistics2012by World Health Organization) that1/3adults sufferedfrom hypertension around the globe, half of the total number of deaths from strokeand heart disease was caused by hypertension. Study on the pathogenesis of essentialhypertension has been the focus of medical research for long. A lot of research datashowed, hypertension can be caused by abnormal renin-angiotensin system (RAS).Angiotensin1-7[Ang-(1-7)] is a new member of the RAS.
Ang-(1-7) is generated from angiotensin1(Ang I) and angiotensin2(Ang II) underthe catalysis of angiotensin converting enzyme (ACE) and angiotensin convertingenzyme2(ACE2). Ang-(1-7) is a polypeptide of seven amino acids, which cancombined with the specific membrane receptors, the Mas receptors (MasR), inducingthe effects of lowering blood pressure, slowing down the heart rate, decreasing renalsympathetic nerve activity (RSNA), decreasing myocardial fibrosis and remodeling.These effects antagonize with Ang II, thus maintaining the homeostasis ofcardiovascular system. Hypertension can be caused by the unbalance betweenAng-(1-7) and Ang II. Enhancing the effect of Ang-(1-7) to rebalance Ang-(1-7) andAng II is an effective strategy for treatment of hypertension.
The cardiovascular regulation center in central nervous system has an important rolein the regulation of blood pressure. Cardiovascular center is mainly located in thebrainstem, some of the important regions are the nucleus of the solitary tract (NTS), the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla(CVLM). The NTS is the first region in central control of cardiovascular activity.NTS neurons project nerve fibers to the other centers to regulate the sympathetic andvagal nerve activity. NTS plays a key role in the regulation of cardiovascular functionby maintaining blood pressure, sympathetic tone and mediating baroreflex sensitivity.NTS was closely related with the onset and development of hypertension. Centralnervous system has a complete RAS for its own, and the local production of Ang-(1-7)in the central nervous system plays an important role in cardiovascular regulation.Nitric oxide (NO) is an important messenger both systemically and in the CNS. Studyof peripheral circulation system has proved that the effects of Ang-(1-7) can bemediated by nitric oxide. NO plays an important role in cardiovascular regulation bymodulating the activity of specific neurons in NTS. The mechanism remains unclear.Is the effect of Ang-(1-7) in NTS mediated by NO? What is the specific signalingpathway? Based on the above questions, the aim of the present study was to determinethe role of phosphatidylinositol3-kinase (PI3K) signaling pathways in mediating theeffect of Ang-(1-7) on NO generation in the NTS.
Methods
300-350g male Sprague-Dawley (SD) rats were used in this study. The effects ofAng-(1-7) on blood pressure, heart rate, renal sympathetic nerve activity(RSNA) andarterial baroreflex(ABR) were observed. We observed whether the effects of Ang-(1-7)were blocked by pretreatment of the nitric oxide synthase (NOS) inhibitor L-NAME.NO production and NOS activity were detected after treatment of Ang-(1-7) in NTS. Western blot analysis was used for detecting the effects of Ang-(1-7) on Akt, nNOSand eNOS phosphorylation in NTS. MasR blocker (A779), PI3K inhibitor (LY294002)and Akt inhibitors (TCN) were used to observe the pathway of Ang-(1-7) action.Local infection of lenti-virus in NTS was used to interfer the expression of PI3K.Fifteen days after the infection, effects of Ang-(1-7) on Akt, nNOS and eNOSphosphorylation were determined by Western blot. Immunofluorescence was used todetermine the co-localization of MasR and nNOS is NTS.
Results
1. Ang-(1-7) enhances NO generation and NOS activity via Mas/PI3K/Aktpathway in the NTS
To detect the effect of Ang-(1-7) on NO production and NOS activity in the NTS, ratswere received microinjections of vehicle (aCSF), Ang-(1-7), A779plus Ang-(1-7),LY294002plus Ang-(1-7) or TCN plus Ang-(1-7) in NTS. We found that NOproduction and NOS activity in the NTS were significantly increased by an average of34%and38%, respectively (P<0.05), in rats which were acutely injected withAng-(1-7)(25pmol) compared with the vehicle injection (aCSF,50nl). However, theAng-(1-7)-induced increase in NO generation and NOS activity were completelyprevented in the presence of the Ang-(1-7) Mas receptor antagonist A779(500pmol),LY294002(100pmol) or TCN (1mmol). In addition, we confirmed that injection ofalone A779, LY294002or TCN into the NTS had little effect on baseline level of NOproduction.
2. Ang-(1-7) stimulates NOS phosphorylation via Mas/PI3K/Akt pathway in
the NTS
To detect the signaling involved in the effect of Ang-(1-7) on NO release in the NTS,NOS phosphorylation was measured by western blot. We found that, in the NTS, thephosphorylation of nNOS at Ser1416and eNOS at Ser1177was significantlyincreased after acute injection of Ang-(1-7)(25pmol)(P<0.05), which was abolishedin the presence of A779(500pmol) or LY294002(100pmol). In addition, we alsofound that the phosphorylation of Akt, a downstream of PI3K, was significantlyincreased after injection of Ang-(1-7) into the NTS(P<0.05).
3. Ang-(1-7) enhances the cardiovascular effects evoked by NOS inhibitor inthe NTS
To determine the effect of Ang-(1-7) on release of NO involved in cardiovascularresponse, several groups were used for monitoring changes in BP, HR, and RSNAevoked by the NOS inhibitor L-NAME following microinjection of Ang-(1-7) into theNTS. We first confirmed that25pmol of Ang-(1-7) injected into the NTSsignificantly decreased basal BP, HR, and RSNA(P<0.05), whereas it enhanced thebaroreflex sensitivity. Increases in BP, HR, and RSNA evoked by L-NAME (10nmol)injected into the NTS were enhanced5min after injection of Ang-(1-7)(25pmol)(P<0.05). Furthermore, co-injection of Ang-(1-7)(25pmol) and A779(500pmol) hadlittle effect on increases in BP, HR, and RSNA normally induced by L-NAMEinjected into the NTS. Moreover, it was found that the L-NAME-induced actions werecompletely abolished in the presence of LY294002.
4. Blockade of NOS prevents the cardiovascular effects of Ang-(1-7) in the NTS Ten rats (two groups: no difference of baseline BP and HR) were used for determiningthe NO mechanism responsible for cardiovascular effects evoked by Ang-(1-7) in theNTS. Pretreatment with the NOS inhibitor L-NAME (10nmol)10min prior to NTSinjection with Ang-(1-7) prevented the reduction in BP, HR, and RSNA normallyevoked by Ang-(1-7).
5. After lenti virus infected the NTS for15days, Ang-(1-7) was given in NTSfor subsequent molecular detections.PI3K Knock Down siRNA interference lenti virus (LenVi-PI3K KD) or scramblelenti virus (LenVi-GFP) were used for locally infecting the NTS. After lenti viralinfection in the NTS for15days, bilateral injection of Ang-(1-7)(150pmol) wasgiven in the NTS for detection of Western Blot or NO production. Western blot resultsshowed the Ang-(1-7) induced increase in eNOS, nNOS and Akt phosphorylation andNO production was inhibited by LenVi-PI3K KD(P<0.05). On the contrary,LenVi-GFP had no effect on Ang-(1-7).6. Immunofluorescence for detecting the co-localization of MasR and nNOSImmunofluorescence pictures indicated MasR and nNOS were expressed in the sameNTS neural cells, suggesting that nNOS may have close links to Ang-(1-7)/MasR.Conclusion: Our findings suggest that Ang-(1-7) is capable of stimulating therelease of nitric oxide in the NTS through the MasR-PI3K-Akt-NOS signalingpathway.
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