法尼基焦磷酸合酶的干扰对自发性高血压大鼠氧化应激的影响
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摘要
第一部分自发性高血压大鼠甲羟戊酸途径关键酶的表达改变与心血管组织重构的关系
研究背景：
甲羟戊酸途径是细胞内重要的代谢途径,其合成了固醇类异戊二烯(如胆固醇)和非固醇类异戊二烯中间产物,包括法尼基焦磷酸(farnesylpyrophosphate, FPP)和牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate, GGPP)。而非固醇类异戊二烯中间产物是小G蛋白翻译后修饰过程不可或缺的。异戊烯转移酶催化小G蛋白的羧基末端加上法尼基或牻牛儿牻牛儿基使之活化。最新研究证明,小G蛋白Ras, Rho家族参与了自发性高血压大鼠(SHR)的心血管组织的重构。
目的：
本研究探索在SHR大鼠的不同血压水平阶段,心脏、血管和肝脏中甲羟戊酸途径关键酶的改变以及其与心血管重构的关联性。
方法：
3、16和25周龄的雄性SHR大鼠和Wistar-Kyoto (WKY)大鼠用尾套法检测收缩压,用常规酶法测定血清胆固醇水平,组织学分析法检测左心室和胸主动脉肥厚指标。用Western blot法检测组织(心脏、主动脉和肝脏)中甲羟戊酸途径关键酶的蛋白表达：HMG-CoA还原酶(HMGR)、(?)去尼基焦磷酸合酶(FDPS).角鲨烯合成酶(SQS)、法尼基转移酶α亚基(FNTA).法尼基转移酶β亚基(FNTB).牻牛儿牻牛儿基转移酶(GGTase-Ⅰ).
结果：
3周龄SHR大鼠的血压与WKY大鼠相似,16周和25周的SHR大鼠血压随年龄而增高,且明显高于WKY大鼠。3周龄的SHR大鼠的低密度脂蛋白胆固醇(LDL-C)水平明显低于WKY大鼠,而高密度脂蛋白胆固醇(HDL-C)和总胆固醇(STC)水平与WKY大鼠相似。16周和25周SHR大鼠的STC、LDL-C和HDL-C水平都显著低于WKY大鼠。3周的SHR大鼠已经出现一定程度的心室肥厚和血管重构,而在16周和25周时SHR大鼠与WKY大鼠相比,则形成了更加显著的差异。SHR大鼠在各周龄的心脏组织中HMGR、FDPS、GGTase-Ⅰ蛋白表达明显上调,而SQS表达下调。在SHR大鼠的胸主动脉中,除了与心脏组织中HMGR、FDPS、GGTase-Ⅰ和SQS相似的蛋白表达改变外,FNTA在16和25周龄时上调,FNTB在25周龄时上调。SHR大鼠的肝脏组织中,在各个周龄都表现了HMGR的上调和SQS的下调。
结论：
SHR大鼠的心血管重构出现早于血压的形成和发展,而其低水平的血清胆固醇浓度可能由于肝脏胆固醇合成分支的关键酶SQS表达的下调。而在心脏和血管中甲羟戊酸途径的非固醇类异戊二烯分支合成途径中的相关酶的表达上调,SQS表达的下调可能诱导了小G蛋白的过度活化,进而导致SHR大鼠的心血管肥厚和重构。
第二部分在体法尼基焦磷酸合酶的抑制对自发性高血压大鼠内皮功能的影响及可能机制---抗氧化应激
研究背景：
法尼基焦磷酸合酶(farnesyl pyrophosphate synthase, FDPS)是甲羟戊酸途径的关键酶之一,既往研究发现：自发性高血压大鼠(SHR)中甲羟戊酸途径的一些关键酶表达显著高于正常的Wistar-Kyoto (WKY)大鼠,其中包括FDPS。并且用二膦酸盐阿仑膦酸钠抑制FDPS从而改善了SHR大鼠的内皮功能障碍,可能机制包括通过抑制FDPS,从而减少类异戊二烯中间产物的产生包括法尼基焦磷酸(farnesylpyrophosphate, FPP)和牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate, GGPP).进而抑制了小G蛋白的类异戊烯化修饰过程,包括了法尼基化和牻牛儿牻牛基儿化,而GGPP对于小G蛋白RhoA的牻牛儿牻牛儿基化和活化非常重要。抑制了小G蛋白RhoA的活化,进而上调内皮一氧化氮合酶(eNOS)的表达。
除此,另有大量实验证明SHR中活性氧族(ROS)终产物显著增多,SHR的内皮功能损伤与过度活跃的氧化应激有关,同样,Rac1的活化也依赖于牻牛儿牻牛儿基化,活化的Rac1可正向调节烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的表达和活性,从而调控ROS终产物。
目的：
本研究主要探索FDPS抑制剂二膦酸盐伊班膦酸钠(Ibandronate)能否通过抑制过度氧化应激从而改善SHR大鼠的内皮功能,并探索可能途径。
方法：
10周龄雄性SHR大鼠和WKY大鼠用Ibandronate (1 mg/kg/day)灌胃处理30天。用离体血管环灌流技术检测内皮依赖性和非内皮依赖性血管舒张作用,观察SHR和WKY大鼠的血管环功能状况1. Ibandronate、GGTI-286、C3 exoenzyme、Y-27632、Rac1 inhibitor或apocynin预孵30 mins,检测血管环功能；2. Ibandronate与GGOH、FOH或MEV共孵2h。以上分别操作后都用AngⅡ进行预收缩,观察Ach浓度梯度(10-9to 10-5M)曲线舒张情况。用荧光分析法检测胸主动脉中活性氧02-的相对含量和NADPH氧化酶的活性,pull-down法测定胸主动脉Rac1的活性,用Western blot法检测胸主动脉NADPH氧化酶亚基p47phox的表达水平
结果：
Ibandronate能部分改善SHR大鼠受伤的内皮依赖性血管舒张作用,其干预可降低胸主动脉中Rac1的活性,下调NADPH氧化酶亚基p47phox的表达和活性氧ROS的含量。
结论：
FDPS的抑制剂Ibandronate可以改善SHR大鼠的内皮功能,其机制可能是通过抑制了Rac1的活化,下调NADPH氧化酶的表达、活性进而抑制了过度的氧化应激反应。
第三部分法尼基焦磷酸合酶在血管紧张素Ⅱ诱导的氧化应激中的作用
研究背景：
既往研究证明在血管细胞中,活性氧族(ROS)的产生可被许多体液因子如血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导,自发性高血压大鼠(SHR)中过度活跃的氧化应激与增高的AngⅡ水平有关。多种血管细胞包括平滑肌细胞参与了AngⅡ诱导的过度氧化应激,而且有多种细胞通路参与了AngⅡ诱导的氧化应激,其中包括Rac1/烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶通路。法尼基焦磷酸合酶(FDPS)能催化合成类异戊二烯中间产物牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate,GGPP),后者在小G蛋白异戊二烯化过程中是不可或缺的,小G蛋白Rac1的异戊二烯化和活化也是必需的。而活化的Rac1可正向调节NADPH氧化酶的表达和活性,从而调控ROS终产物。
目的：
本研究主要明确用二膦酸盐伊班膦酸钠(Ibandronate)抑制FDPS可否影响SHR大鼠血管平滑肌细胞(VSMCs)在AngⅡ诱导下的氧化应激反应,同时研究该作用是否与Rac1/NADPH氧化酶途径相关。
方法：
来源于SHR大鼠和WKY大鼠的原代VSMCs用AngⅡ刺激3h,或者在实验中,VSMCs在AngⅡ刺激前分别先孵育24 h的Ibandronate(10-8 to 10-5 M)、Ibandronate(10-5M)加牻牛儿牻牛儿醇(GGOH,Geranylgeraniol,3×10-5 mol/L)、Ibandronate加法尼醇(FOH,Farnesol,3×10-5 mol/L)、Ibandronate加甲羟戊酸(MEV,Mevalonate, 10-4 mol/L)、GGTI-286(牻牛儿牻牛儿基转移酶抑制剂,10-5 mol/L)或Rac1inhibitor(10-4 mol/L).然后用荧光测定法检测细胞中的ROS,用试剂盒检测细胞NADPH氧化酶活性,用pull-down(?)去检测细胞中Rac1的活性,用Western blot检测NADPH氧化酶关键亚基p47phox的蛋白表达情况和Rac1总蛋白表达情况。
结果：
(1)AngⅡ可浓度依赖性诱导氧化应激产物ROS的产生,而SHR大鼠的VSMCs比WKY大鼠在AngⅡ诱导下出现更大的氧化应激反应。FDPS抑制剂Ibandronate可浓度依赖性抑制.AngⅡ诱导的ROS的产生。也抑制了AngⅡ对Rac1, NADPH oxidase的激活和p47phox的蛋白上调。
(2) GGOH部分逆转了Ibandronate的抗氧化应激反应,而FOH或MEV没有逆转作用。
(3)抑制剂GGTI-286、Racl inhibitor可模拟Ibandronate对AngⅡ诱导的过度氧化应激反应的抑制作用。
结论：
本研究结果表明Ibandronate通过抑制FDPS可以有效逆转SHR大鼠VSMCs中AngⅡ诱导的过度氧化应激,这种作用与抑制Rac1的牻牛儿牻牛儿基化过程从而降低Rac1的活性相关。
Part One Alteration of enzymes expression in mevalonate pathway: possible role for cardiovascular remodeling in spontaneously hypertensive rats
Background:
Mevalonate pathway is an important metabolic pathway which plays a key role in multiple cellular processes by synthesizing sterol isoprenoids, like cholesterol, and non-sterol isoprenoids, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These non-sterol isoprenoid intermediates of the mevalonate biosynthetic pathway play important roles in the post-translational modification of small GTP-binding proteins (GTPases). Protein prenyltransferases catalyze the addition of the isoprenoids, farnesyl or geranylgeranyl to the C-terminal of GTPases, facilitating their activation. Recent studies have suggested that small GTP-binding proteins Ras and Rho are involved substantially in functional and structural alterations of hypertensive blood vessels and hearts in SHR.
Aims:
To determine whether the key enzymes expressions of mevalonate pathway in heart, aorta and liver during the process of hypertension in SHR are altered and whether these changes are associated with the cardiovascular remodelling.
Methods:
At the ages of 3,16 and 25 weeks, systolic blood pressure of SHR and WKY was measured by the tail-cuff method. Serum cholesterol concentrations were determined by +enzymatic methods. Hearts and thoracic aortas were removed for the study of cardiovascular remodelling. The protein expressions of enzymes in hearts, aortas and livers were analysed by western blot, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR)、farnesyl diphosphate synthase (FDPS)、squalene synthetase (SQS)、farnesyltransferase alpha (FNTA)、farnesyltransferase beta (FNTB)、geranylgeranyltransferase type I (GGTase-Ⅰ).
Results:
The systolic blood pressure developed gradually over weeks in SHR after 3-week-old, but only slightly in WKY. At 3 weeks of age, blood pressure was not significantly different between SHR and WKY, whereas in both 16-week-old and 25-week-old age groups, blood pressure was significantly higher in SHR than in WKY. SHR had a lower LDL-C level but similar values of STC and HDL-C compared with WKY at 3 weeks of age. At adult stage of 16-week-old and 25-week-old age, STC, LDL-C and HDL-C were lower in SHR than in WKY. The histological measurements showed the mass and myocyte sizes of heart, the media thickness (MT) and media cross-sectional area (MCSA) of thoracic aorta were all increased in SHR since 3 weeks. In heart, there were overexpressions of some enzymes, including HMGR, FDPS. GGTase-Ⅰ, and a downregulation of SQS in SHR since 3-week-old age. In aorta, besides similar expressions of HMGR, SQS, FDPS and GGTase-Ⅰwith heart, there were upregulations of FNTA at 16-and 25-week-old age and FNTB at 25-week-old SHR. Western blot demonstrated an overexpression of HMGR and a downregulation of SQS in SHR liver at all ages tested.
Conclusions:
The cardiovascular remodeling of SHR preceded development of hypertension. The serum cholesterol level was significantly decreased in SHR, and these findings may be associated with lower expression of key enzyme SQS in sterol pathways in liver from SHR at all ages. The up regulation expression of several key enzymes in non-sterol mevalonate pathways, and down regulation expression of SQS in heart and aorta may play a potential pathophysiological role for cardiovascular remodeling in SHR via excessive activation of small GTP-binding proteins.
Part Two Effect of farnesyl diphosphate synthase inhibition on endothelial function in spontaneously hypertensive rats vivo—antioxidative stress
Background:
Farnesyl diphosphate synthase (FDPS) is a key enzyme in the mevalonate pathway. In previous studies, we found that the expression of key enzymes in the mevalonate pathway, including FDPS, are significantly upregulated in the spontaneously hypertensive rat (SHR). Bisphosphonates inhibit farnesyl pyrophosphate (FPP) synthase, through inhibition of isoprenylation including farnesylation and geranylgeranylation by preventing the synthesis of various isoprenoids such as FPP and geranylgeranyl pyrophosphate (GGPP). Bisphosphonate alendronate was able to improve the endothelial function in SHR and the upregulation of eNOS expression by suppression of small GTPase RhoA activation, which needs geranylgeranylation by GGPP. Excess of ROS generation synthesized by NADPH oxidase is critically involved in the breakdown of endothelial function in SHR. The activated Racl by geranylgeranylation is a prerequisite of NADPH oxidase activation.
Aims:
To determine whether FDPS inhibitor ibandronate improves the endothelial function in SHR, at least in part, through inhibition of excessive oxidative response via the Rac1/NADPH oxidase pathway. Methods:
After 4-week administration of ibandronate (1mg/kg/day), endothelium-dependent and independent vasorelaxation were measured in isolated aortic rings. The level of ROS in aorta was then quantitatively measured fluorometrically. and the enzymatic activities of NADPH oxidase was evaluated spectrophotometrically with commercial assay kits. Activation of Rac1 in aorta was determined by pull-down assay. Aortic expression of p47phox in both cytoplasmic and membrane fractions and Rac1 was determined by western blot.
Conclusions:
FPP synthase inhibitor ibandronate exerted antioxidant effects in the vasculature of SHR mediated by Racl/NADPH oxidase pathway. These effects of ibandronate may contribute to the vasoprotective effects for impaired endothelium in SHR.
Part Three Role of farnesylpyrophosphate synthase in angiotensinⅡ-mediated oxidative stress
Background:
In vascular cells, ROS production can be induced by humoral factors such as angiotensinⅡ(AngⅡ). An abnormal elevation of circulating AngⅡis critically involved in the excess oxidative stress in spontaneously hypertensive rat (SHR). Many kinds of pathways are involved in AngⅡ-induced excess oxidative stress response of vascular smooth muscle cells including the Rac1/NADPH oxidase signaling.
Farnesyl pyrophosphate synthase (FDPS) synthesize of various isoprenoids such as FPP and geranylgeranyl pyrophosphate (GGPP). The latter is needed for protein isoprenylation and activation of small GTP-binding protein, including Racl. Activation and translocation of Rac1 from the cytosolic compartment to the cell membrane induce activation and upregulation of NADPH oxidase expression, which contributes to the excessive generation of ROS.
Aims:
To explore whether inhibition of FDPS synthase by ibandronate (IBAN) could interfere with the oxidative stress responses induced by AngⅡin cultured vascular smooth muscle cells (VSMCs). and whether it involves Racl/NADPH oxidase pathway.
Methods:
VSMCs from SHR and Wistar-Kyoto rats (WKY) were stimulated with AngⅡfor 3 h. In some experiments, cells were pre-exposed for 24 h to IBAN (10-5 mol/L), IBAN plus Geranylgeraniol (GGOH,3×10-5 mol/L), IBAN plus Farnesol (FOH,3×10-5 mol/L), IBAN plus Mevalonate (MEV，10"4 mol/L)，GGTI-286 (selective inhibitor of GGTase I, 10-5 mol/L) or Racl Inhibitor (10-4 mol/L). The generation of ROS for the VSMCs was evaluated by a fluorometry assay using intracellular oxidation of DCFH-DA, and the enzymatic activities of NADPH oxidase was evaluated spectrophotometrically with commercial assay kits. Activation of Rac1 was determined by pull-down assay. The expression of p47phox in both cytoplasmic and membrane fractions and Racl in VSMCs were determined by western blot.
Results:
AngiotensinⅡ(AngⅡ) concentration dependently increased ROS production in cultured VSMCs from WKY and SHR. The AngⅡ-induced responses were greater in SHR VSMCs, but significantly reduced by ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, expression of NADPH oxidase subunit p47phox, NADPH oxidase and Racl GTP-binding activity in VSMCs. Addition of GGOH, but not FOH or MEV reversed the inhibitory effects of ibandronate. Moreover, inhibitor of geranylgeranyl-transferase GGTI-286 and Racl mimicked the effect of ibandronate on this excessive oxidative response.
Conclusions:
FDPS inhibitor ibandronate exerted cellular antioxidant effects induced on AngⅡstimulation in cultured SHR VSMCs, and this effect may be mediated at least partly by reduction the activated form of Rac1 via blocking of geranylgeranylation.
研究背景：
甲羟戊酸途径是细胞内重要的代谢途径,其合成了固醇类异戊二烯(如胆固醇)和非固醇类异戊二烯中间产物,包括法尼基焦磷酸(farnesylpyrophosphate, FPP)和牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate, GGPP)。而非固醇类异戊二烯中间产物是小G蛋白翻译后修饰过程不可或缺的。异戊烯转移酶催化小G蛋白的羧基末端加上法尼基或牻牛儿牻牛儿基使之活化。最新研究证明,小G蛋白Ras, Rho家族参与了自发性高血压大鼠(SHR)的心血管组织的重构。
目的：
本研究探索在SHR大鼠的不同血压水平阶段,心脏、血管和肝脏中甲羟戊酸途径关键酶的改变以及其与心血管重构的关联性。
方法：
3、16和25周龄的雄性SHR大鼠和Wistar-Kyoto (WKY)大鼠用尾套法检测收缩压,用常规酶法测定血清胆固醇水平,组织学分析法检测左心室和胸主动脉肥厚指标。用Western blot法检测组织(心脏、主动脉和肝脏)中甲羟戊酸途径关键酶的蛋白表达：HMG-CoA还原酶(HMGR)、(?)去尼基焦磷酸合酶(FDPS).角鲨烯合成酶(SQS)、法尼基转移酶α亚基(FNTA).法尼基转移酶β亚基(FNTB).牻牛儿牻牛儿基转移酶(GGTase-Ⅰ).
结果：
3周龄SHR大鼠的血压与WKY大鼠相似,16周和25周的SHR大鼠血压随年龄而增高,且明显高于WKY大鼠。3周龄的SHR大鼠的低密度脂蛋白胆固醇(LDL-C)水平明显低于WKY大鼠,而高密度脂蛋白胆固醇(HDL-C)和总胆固醇(STC)水平与WKY大鼠相似。16周和25周SHR大鼠的STC、LDL-C和HDL-C水平都显著低于WKY大鼠。3周的SHR大鼠已经出现一定程度的心室肥厚和血管重构,而在16周和25周时SHR大鼠与WKY大鼠相比,则形成了更加显著的差异。SHR大鼠在各周龄的心脏组织中HMGR、FDPS、GGTase-Ⅰ蛋白表达明显上调,而SQS表达下调。在SHR大鼠的胸主动脉中,除了与心脏组织中HMGR、FDPS、GGTase-Ⅰ和SQS相似的蛋白表达改变外,FNTA在16和25周龄时上调,FNTB在25周龄时上调。SHR大鼠的肝脏组织中,在各个周龄都表现了HMGR的上调和SQS的下调。
结论：
SHR大鼠的心血管重构出现早于血压的形成和发展,而其低水平的血清胆固醇浓度可能由于肝脏胆固醇合成分支的关键酶SQS表达的下调。而在心脏和血管中甲羟戊酸途径的非固醇类异戊二烯分支合成途径中的相关酶的表达上调,SQS表达的下调可能诱导了小G蛋白的过度活化,进而导致SHR大鼠的心血管肥厚和重构。
第二部分在体法尼基焦磷酸合酶的抑制对自发性高血压大鼠内皮功能的影响及可能机制---抗氧化应激
研究背景：
法尼基焦磷酸合酶(farnesyl pyrophosphate synthase, FDPS)是甲羟戊酸途径的关键酶之一,既往研究发现：自发性高血压大鼠(SHR)中甲羟戊酸途径的一些关键酶表达显著高于正常的Wistar-Kyoto (WKY)大鼠,其中包括FDPS。并且用二膦酸盐阿仑膦酸钠抑制FDPS从而改善了SHR大鼠的内皮功能障碍,可能机制包括通过抑制FDPS,从而减少类异戊二烯中间产物的产生包括法尼基焦磷酸(farnesylpyrophosphate, FPP)和牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate, GGPP).进而抑制了小G蛋白的类异戊烯化修饰过程,包括了法尼基化和牻牛儿牻牛基儿化,而GGPP对于小G蛋白RhoA的牻牛儿牻牛儿基化和活化非常重要。抑制了小G蛋白RhoA的活化,进而上调内皮一氧化氮合酶(eNOS)的表达。
除此,另有大量实验证明SHR中活性氧族(ROS)终产物显著增多,SHR的内皮功能损伤与过度活跃的氧化应激有关,同样,Rac1的活化也依赖于牻牛儿牻牛儿基化,活化的Rac1可正向调节烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的表达和活性,从而调控ROS终产物。
目的：
本研究主要探索FDPS抑制剂二膦酸盐伊班膦酸钠(Ibandronate)能否通过抑制过度氧化应激从而改善SHR大鼠的内皮功能,并探索可能途径。
方法：
10周龄雄性SHR大鼠和WKY大鼠用Ibandronate (1 mg/kg/day)灌胃处理30天。用离体血管环灌流技术检测内皮依赖性和非内皮依赖性血管舒张作用,观察SHR和WKY大鼠的血管环功能状况1. Ibandronate、GGTI-286、C3 exoenzyme、Y-27632、Rac1 inhibitor或apocynin预孵30 mins,检测血管环功能；2. Ibandronate与GGOH、FOH或MEV共孵2h。以上分别操作后都用AngⅡ进行预收缩,观察Ach浓度梯度(10-9to 10-5M)曲线舒张情况。用荧光分析法检测胸主动脉中活性氧02-的相对含量和NADPH氧化酶的活性,pull-down法测定胸主动脉Rac1的活性,用Western blot法检测胸主动脉NADPH氧化酶亚基p47phox的表达水平
结果：
Ibandronate能部分改善SHR大鼠受伤的内皮依赖性血管舒张作用,其干预可降低胸主动脉中Rac1的活性,下调NADPH氧化酶亚基p47phox的表达和活性氧ROS的含量。
结论：
FDPS的抑制剂Ibandronate可以改善SHR大鼠的内皮功能,其机制可能是通过抑制了Rac1的活化,下调NADPH氧化酶的表达、活性进而抑制了过度的氧化应激反应。
第三部分法尼基焦磷酸合酶在血管紧张素Ⅱ诱导的氧化应激中的作用
研究背景：
既往研究证明在血管细胞中,活性氧族(ROS)的产生可被许多体液因子如血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导,自发性高血压大鼠(SHR)中过度活跃的氧化应激与增高的AngⅡ水平有关。多种血管细胞包括平滑肌细胞参与了AngⅡ诱导的过度氧化应激,而且有多种细胞通路参与了AngⅡ诱导的氧化应激,其中包括Rac1/烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶通路。法尼基焦磷酸合酶(FDPS)能催化合成类异戊二烯中间产物牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate,GGPP),后者在小G蛋白异戊二烯化过程中是不可或缺的,小G蛋白Rac1的异戊二烯化和活化也是必需的。而活化的Rac1可正向调节NADPH氧化酶的表达和活性,从而调控ROS终产物。
目的：
本研究主要明确用二膦酸盐伊班膦酸钠(Ibandronate)抑制FDPS可否影响SHR大鼠血管平滑肌细胞(VSMCs)在AngⅡ诱导下的氧化应激反应,同时研究该作用是否与Rac1/NADPH氧化酶途径相关。
方法：
来源于SHR大鼠和WKY大鼠的原代VSMCs用AngⅡ刺激3h,或者在实验中,VSMCs在AngⅡ刺激前分别先孵育24 h的Ibandronate(10-8 to 10-5 M)、Ibandronate(10-5M)加牻牛儿牻牛儿醇(GGOH,Geranylgeraniol,3×10-5 mol/L)、Ibandronate加法尼醇(FOH,Farnesol,3×10-5 mol/L)、Ibandronate加甲羟戊酸(MEV,Mevalonate, 10-4 mol/L)、GGTI-286(牻牛儿牻牛儿基转移酶抑制剂,10-5 mol/L)或Rac1inhibitor(10-4 mol/L).然后用荧光测定法检测细胞中的ROS,用试剂盒检测细胞NADPH氧化酶活性,用pull-down(?)去检测细胞中Rac1的活性,用Western blot检测NADPH氧化酶关键亚基p47phox的蛋白表达情况和Rac1总蛋白表达情况。
结果：
(1)AngⅡ可浓度依赖性诱导氧化应激产物ROS的产生,而SHR大鼠的VSMCs比WKY大鼠在AngⅡ诱导下出现更大的氧化应激反应。FDPS抑制剂Ibandronate可浓度依赖性抑制.AngⅡ诱导的ROS的产生。也抑制了AngⅡ对Rac1, NADPH oxidase的激活和p47phox的蛋白上调。
(2) GGOH部分逆转了Ibandronate的抗氧化应激反应,而FOH或MEV没有逆转作用。
(3)抑制剂GGTI-286、Racl inhibitor可模拟Ibandronate对AngⅡ诱导的过度氧化应激反应的抑制作用。
结论：
本研究结果表明Ibandronate通过抑制FDPS可以有效逆转SHR大鼠VSMCs中AngⅡ诱导的过度氧化应激,这种作用与抑制Rac1的牻牛儿牻牛儿基化过程从而降低Rac1的活性相关。
Part One Alteration of enzymes expression in mevalonate pathway: possible role for cardiovascular remodeling in spontaneously hypertensive rats
Background:
Mevalonate pathway is an important metabolic pathway which plays a key role in multiple cellular processes by synthesizing sterol isoprenoids, like cholesterol, and non-sterol isoprenoids, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These non-sterol isoprenoid intermediates of the mevalonate biosynthetic pathway play important roles in the post-translational modification of small GTP-binding proteins (GTPases). Protein prenyltransferases catalyze the addition of the isoprenoids, farnesyl or geranylgeranyl to the C-terminal of GTPases, facilitating their activation. Recent studies have suggested that small GTP-binding proteins Ras and Rho are involved substantially in functional and structural alterations of hypertensive blood vessels and hearts in SHR.
Aims:
To determine whether the key enzymes expressions of mevalonate pathway in heart, aorta and liver during the process of hypertension in SHR are altered and whether these changes are associated with the cardiovascular remodelling.
Methods:
At the ages of 3,16 and 25 weeks, systolic blood pressure of SHR and WKY was measured by the tail-cuff method. Serum cholesterol concentrations were determined by +enzymatic methods. Hearts and thoracic aortas were removed for the study of cardiovascular remodelling. The protein expressions of enzymes in hearts, aortas and livers were analysed by western blot, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR)、farnesyl diphosphate synthase (FDPS)、squalene synthetase (SQS)、farnesyltransferase alpha (FNTA)、farnesyltransferase beta (FNTB)、geranylgeranyltransferase type I (GGTase-Ⅰ).
Results:
The systolic blood pressure developed gradually over weeks in SHR after 3-week-old, but only slightly in WKY. At 3 weeks of age, blood pressure was not significantly different between SHR and WKY, whereas in both 16-week-old and 25-week-old age groups, blood pressure was significantly higher in SHR than in WKY. SHR had a lower LDL-C level but similar values of STC and HDL-C compared with WKY at 3 weeks of age. At adult stage of 16-week-old and 25-week-old age, STC, LDL-C and HDL-C were lower in SHR than in WKY. The histological measurements showed the mass and myocyte sizes of heart, the media thickness (MT) and media cross-sectional area (MCSA) of thoracic aorta were all increased in SHR since 3 weeks. In heart, there were overexpressions of some enzymes, including HMGR, FDPS. GGTase-Ⅰ, and a downregulation of SQS in SHR since 3-week-old age. In aorta, besides similar expressions of HMGR, SQS, FDPS and GGTase-Ⅰwith heart, there were upregulations of FNTA at 16-and 25-week-old age and FNTB at 25-week-old SHR. Western blot demonstrated an overexpression of HMGR and a downregulation of SQS in SHR liver at all ages tested.
Conclusions:
The cardiovascular remodeling of SHR preceded development of hypertension. The serum cholesterol level was significantly decreased in SHR, and these findings may be associated with lower expression of key enzyme SQS in sterol pathways in liver from SHR at all ages. The up regulation expression of several key enzymes in non-sterol mevalonate pathways, and down regulation expression of SQS in heart and aorta may play a potential pathophysiological role for cardiovascular remodeling in SHR via excessive activation of small GTP-binding proteins.
Part Two Effect of farnesyl diphosphate synthase inhibition on endothelial function in spontaneously hypertensive rats vivo—antioxidative stress
Background:
Farnesyl diphosphate synthase (FDPS) is a key enzyme in the mevalonate pathway. In previous studies, we found that the expression of key enzymes in the mevalonate pathway, including FDPS, are significantly upregulated in the spontaneously hypertensive rat (SHR). Bisphosphonates inhibit farnesyl pyrophosphate (FPP) synthase, through inhibition of isoprenylation including farnesylation and geranylgeranylation by preventing the synthesis of various isoprenoids such as FPP and geranylgeranyl pyrophosphate (GGPP). Bisphosphonate alendronate was able to improve the endothelial function in SHR and the upregulation of eNOS expression by suppression of small GTPase RhoA activation, which needs geranylgeranylation by GGPP. Excess of ROS generation synthesized by NADPH oxidase is critically involved in the breakdown of endothelial function in SHR. The activated Racl by geranylgeranylation is a prerequisite of NADPH oxidase activation.
Aims:
To determine whether FDPS inhibitor ibandronate improves the endothelial function in SHR, at least in part, through inhibition of excessive oxidative response via the Rac1/NADPH oxidase pathway. Methods:
After 4-week administration of ibandronate (1mg/kg/day), endothelium-dependent and independent vasorelaxation were measured in isolated aortic rings. The level of ROS in aorta was then quantitatively measured fluorometrically. and the enzymatic activities of NADPH oxidase was evaluated spectrophotometrically with commercial assay kits. Activation of Rac1 in aorta was determined by pull-down assay. Aortic expression of p47phox in both cytoplasmic and membrane fractions and Rac1 was determined by western blot.
Conclusions:
FPP synthase inhibitor ibandronate exerted antioxidant effects in the vasculature of SHR mediated by Racl/NADPH oxidase pathway. These effects of ibandronate may contribute to the vasoprotective effects for impaired endothelium in SHR.
Part Three Role of farnesylpyrophosphate synthase in angiotensinⅡ-mediated oxidative stress
Background:
In vascular cells, ROS production can be induced by humoral factors such as angiotensinⅡ(AngⅡ). An abnormal elevation of circulating AngⅡis critically involved in the excess oxidative stress in spontaneously hypertensive rat (SHR). Many kinds of pathways are involved in AngⅡ-induced excess oxidative stress response of vascular smooth muscle cells including the Rac1/NADPH oxidase signaling.
Farnesyl pyrophosphate synthase (FDPS) synthesize of various isoprenoids such as FPP and geranylgeranyl pyrophosphate (GGPP). The latter is needed for protein isoprenylation and activation of small GTP-binding protein, including Racl. Activation and translocation of Rac1 from the cytosolic compartment to the cell membrane induce activation and upregulation of NADPH oxidase expression, which contributes to the excessive generation of ROS.
Aims:
To explore whether inhibition of FDPS synthase by ibandronate (IBAN) could interfere with the oxidative stress responses induced by AngⅡin cultured vascular smooth muscle cells (VSMCs). and whether it involves Racl/NADPH oxidase pathway.
Methods:
VSMCs from SHR and Wistar-Kyoto rats (WKY) were stimulated with AngⅡfor 3 h. In some experiments, cells were pre-exposed for 24 h to IBAN (10-5 mol/L), IBAN plus Geranylgeraniol (GGOH,3×10-5 mol/L), IBAN plus Farnesol (FOH,3×10-5 mol/L), IBAN plus Mevalonate (MEV，10"4 mol/L)，GGTI-286 (selective inhibitor of GGTase I, 10-5 mol/L) or Racl Inhibitor (10-4 mol/L). The generation of ROS for the VSMCs was evaluated by a fluorometry assay using intracellular oxidation of DCFH-DA, and the enzymatic activities of NADPH oxidase was evaluated spectrophotometrically with commercial assay kits. Activation of Rac1 was determined by pull-down assay. The expression of p47phox in both cytoplasmic and membrane fractions and Racl in VSMCs were determined by western blot.
Results:
AngiotensinⅡ(AngⅡ) concentration dependently increased ROS production in cultured VSMCs from WKY and SHR. The AngⅡ-induced responses were greater in SHR VSMCs, but significantly reduced by ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, expression of NADPH oxidase subunit p47phox, NADPH oxidase and Racl GTP-binding activity in VSMCs. Addition of GGOH, but not FOH or MEV reversed the inhibitory effects of ibandronate. Moreover, inhibitor of geranylgeranyl-transferase GGTI-286 and Racl mimicked the effect of ibandronate on this excessive oxidative response.
Conclusions:
FDPS inhibitor ibandronate exerted cellular antioxidant effects induced on AngⅡstimulation in cultured SHR VSMCs, and this effect may be mediated at least partly by reduction the activated form of Rac1 via blocking of geranylgeranylation.
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