Statins為HMG-CoA reductase 的抑制劑，普遍用來治療降低膽固醇之藥物，statins除了有降膽固醇的效果，還具有多效作用性質，包括血管保護作用、改善內皮功能、提升一氧化氮的生成、抗氧化特性。其中statins對於內皮功能與氧化壓力的改善可能對於治療高血壓有著密切關係，然而statins對於血壓調控的機制仍尚未釐清。在本實驗室先前的研究也證實了胰島素在孤立束核的心臟血管調節作用是透過活化磷酰肌醇-3 激酶(PI3K)－蛋白質激酶B（protein kinase B）－一氧化氮合成酶（nitric oxide synthase），產生一氧化氮來調控血壓、心跳。而孤立束核在中樞神經系統中扮演著重要中樞心臟血管調控的角色，不僅接收來自周邊感壓接受器傳來的訊息以進行整合，而孤立束核本身也會受到其他物質的調控。Statins經由調控膽固醇合成中重要的中間產物isoprenoid生成，其中Ras和Rac1 GTPase蛋白質為轉譯後修飾isoprenylation的重要受質，其中statins可抑制Rac1的isoprenylation可降低NADPH氧化酶活性進而減低內皮細胞中的氧化壓力和過氧化物的生成。因此本實驗探討simvastatin在中樞藉由哪些訊息傳遞路徑影響重要訊息傳遞蛋白的表現而調控一氧化氮合成酶的活性進而調控血壓。我們將自發性高血壓大鼠分成二組，分別為控制組(側腦室注射人工腦脊髓液)、實驗組(側腦室注射simvastatin)均持續注射三天，並利用尾動脈壓量測法持續觀察期間的血壓變化，發現在側腦室注射simvastatin持續三天後，造成實驗組有顯著性的血壓下降作用，同時在孤立束核中的一氧化氮產量也有顯著性地增加，此外，我們也觀察到側腦室注射simvastatin能有效降低孤立束核中的氧化壓力並促進Ras GTPase的活化，最後取出腦幹中的孤立束核組織進行西方墨點分析法及免疫組織染色等技術來探討訊息傳遞分子之間的關係，我們進一步觀察到simvastatin在孤立束核中可能透過Ras GTPase活化下游ERK1/2、Akt訊息傳遞蛋白及一氧化氮合成酶之活化位磷酸化反應增加，進而增加一氧化氮生成而造成降低血壓反應。綜合以上結果simvastatin於孤立束核中能透過活化一氧化氮合成酶訊息傳遞路徑達到降低血壓的作用。

關鍵詞: statins, 孤立束核, 一氧化氮, 氧化壓力, 中樞心臟血管調控, isoprenylation

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are unequivocally useful for lowering cholesterol levels in patients with dyslipidemias. In addition to cholesterol lowering properties, statins exert a number of pleiotropic, vascular-protective effects include improvement of endothelial function, increased nitric oxide (NO) bioavailability, antioxidant properties. Since endothelial dysfunction and reactive oxygen species (ROS) are important pathophysiological determinants of essential hypertension, these actions of statins raise the possibility that statin therapy may be useful for simultaneously clinical hypertension management. However, the signaling mechanisms of statins that improve hypertension remain unclear. Our previous study showed, in the NTS, insulin may decrease blood pressure and heart rates through PI3K-Akt-eNOS pathway, and NTS integrates convergent information from peripheral baroreceptors and central cardiovascular regulatory center. Statins also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway, members of the Ras and Rac1 GTPase family are major substrates for posttranslational modification by isoprenylation and may be important targets for inhibition by statins. Statins could inhibit Rac1 isoprenylation and Rac1-mediated nicotinamide adenine dinucleotide phosphate oxidase activity attenuates reactive oxygen species production. The aim of this study was to investigate the possible signaling pathways involved in simvastatin-mediated blood pressure regulation in the nucleus tractus solitarii (NTS). Male 20-week-old spontaneously hypertensive rats (SHR) were divided into two groups: control group and intracerebroventricular injection with simvastatin group for three days. We found that systolic blood pressure measured with tail-cuff method of the simvastatin-treated rats decreased significantly, and the NO level in the NTS was significantly increased. In addition, we observed that simvastatin could lower the ROS level and increase Ras GTPase activity in the NTS. Immunoblotting and immunohistochemistry analysis further showed that simvastatin increased the phosphorylation ratio of ERK1/2, Akt, and endothelial nitric oxide synthase (eNOS) in the NTS. Taken together, these results suggest that eNOS signaling in the NTS may play an important role in simvastatin-induced blood pressure lowering effects.

Keywords: statins, nucleus tractus solitarii, nitric oxide, oxidative stress, central cardiovascular regulatory, isoprenylation

Chinese Abstract……………………………………………………………I
English Abstract…………………………………………………………..III
Abbreviation………………………………………………………………V
Contents…………………………………………………………………VII
1. Introduction…………………………..…………………….…………1
1.1 The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase inhibitors……………………………………………...1
1.2 Inhibition of HMG-CoA reductase……………………………...1
1.3 Statins lower blood pressure…………………………………….3
1.4 Effects of statins in central nerve system……………………….4
1.5 Regulation of small GTPase…………………………………….5
1.6 Cardiovascular regulation by brainstem nuclei…………………6
1.7 Nitric oxide signaling in central cardiovascular regulation……..8
1.8 Endotheliual NOS (eNOS) and cardiovascular regulation……...9
1.9 Statins and eNOS Expression…………………………………..10
1.10 Neuronal NOS (nNOS) and cardiovascular regulation………...10
1.11 Reactive oxygen species (ROS)………………………………..11
2. Specific Aims………………………………………………………...14
3. Materials and Methods……………………………………………….15
3.1 Experimental chemicals………………………………………….15
3.2 Animals…………………………………………………………..15
3.3 Intracerebroventricular injection procedures…………………….16
3.4 Blood pressure measurement…………………………………….17
3.5 Determination of NO in NTS……………………………….……17
3.6 ROS production in the NTS……………………………………...18
3.7 Western blot analysis……………………………………………..18
3.8 Immunohistochemistry analysis………………………………….20
3.9 Ras activation assay………………………………………………22
3.10 Statistical analysis……………………………………………….22
4. Results……………………………………….………………………..24
4.1 Cardiovascular effects of simvastatin by oral administration on SHR…………………………………………………….………..24
4.2 Effects of simvastatin by oral administration on the NO production in the NTS of SHR and SHR + simvastatin group rats…….……..24
4.3 Cardiovascular effects of simvastatin by ICV injection on SHR....25
4.4 Effects of simvastatin by ICV injection on the NO production in the NTS of SHR and SHR + simvastatin group rats…………….……26
4.5 Effects of simvastatin on the eNOSS1177 phosphorylation in the NTS of studied rats…………………………………………….….26
4.6 Effects of simvastatin on the nNOSS1416 phosphorylation in the NTS of studied rats……………………………………….……….27
4.7 Effects of simvastatin on the iNOS protein expression in the NTS of studied rats………………………………………………….……..27
4.8 In situ effects of simvastatin on the eNOS phosphorylation in the NTS of studied rats…………………………………………….….28
4.9 Simvastatin lower blood pressure through Ras activation…….......28
4.10 Effects of simvastatin on the Akt phosphorylation in the NTS of studied rats………………………………………………….……29
4.11 Effects of simvastatin on the ERK1/2 phosphorylation in the NTS of studied rats…………………………………….………………29
4.12 Effects of ICV simvastatin on ROS production in the NTS of SHR and SHR + simvastatin group rats…………………………….…30
4.13 Simvastatin decreased ROS production through Rac1 inhibition.30
5. Discussion……………………………………………………………..32
6. Conclusion………………………………………………………….…37
7. Future Perspectives…………………………………………………....38
8. References………………………………………………………….….39
9. Figures and Figure Legends……………………………………….…..47

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