甲羟戊酸途径的干预对自发性高血压大鼠内皮功能的影响

In previous studies, we found that the expression of key enzymes in the mevalonate pathway was significantly unregulated in spontaneously hypertensive rat (SHR). The purpose of this study is to determine the role of small G protein, such as RhoA and Rac1, in endothelial dysfunction and whether mevalonate pathway inhibition improves the endothelial function in SHR, through reversing the imbalance of NO-ROS pathway. To investigate the underlying mechanisms, we carry out the following studies. SHR rats or endothelial cells, smooth muscle cells in vitro were treated with chemical inhibitors or shRNA of over-expressioned key enzymes including FDPS，FNTA, FNTB，GGTase-I. Then the production of NO and ROS, activity of NOS, NADPH oxidase and antioxidant enzyme were determined by fluorescence. The mRNA level, protein expression and phosphorylation level of NOS, NADPH oxidase and antioxidant enzyme were determined by Real-time PCR and western blot, respectively. The protein expression and activity of RhoA / Rac1 in downstream of mevalonate pathway were determined by western blot and fluorescence. The vascular function was detected by isolated perfused assay. Our study may open the field for novel therapeutic indications and give many possible therapeutic targets beyond arterial hypertension in the future.

本项目拟在前期发现自发性高血压大鼠（SHR）心血管组织中甲羟戊酸途径的非固醇类分子途径关键酶的表达上调的基础上，进一步观察甲羟戊酸途径的干扰是否可以逆转失衡的NO-ROS途径从而改善SHR受损的内皮功能。为明确其机制，拟开展下述研究：（1）在体SHR动物实验和体外内皮细胞、平滑肌细胞实验中采用化学抑制剂或shRNA抑制上调的FDPS，FNTA, FNTB，GGTase-I等酶, 进一步观察血清/细胞NO、ROS水平；血管/细胞NOS、NADPH氧化酶、抗氧化酶的活性及各亚基mRNA与蛋白表达、磷酸化水平；（2）观察血管/细胞甲羟戊酸途径下游活性分子小G蛋白RhoA、Rac1等相关蛋白表达和活性；（3）离体灌流实验检测血管功能。以进一步明确小G蛋白在高血压内皮功能损伤过程中的作用及甲羟戊酸途径影响内皮功能的上、下游分子信号转导机制。

既往研究我们发现在高血压大鼠中甲羟戊酸途径中关键酶法尼基焦磷酸合酶（FDPS），法尼基转移酶 (FT)、牻牛儿牻牛儿基转移酶(GGTase-I)表达明显上调。这三个酶参与了异戊二烯化合物法尼基焦磷酸（FPP）和牻牛儿牻牛儿焦磷酸(GGPP）的合成及转移过程，而这些产物是小G蛋白Ras、RhoA 、Rac1活化过程所不可或缺的。FDPS抑制剂可通过抑制小G蛋白RhoA的活化，上调eNOS的表达，从而改善SHR大鼠受损的内皮功能。研究表明烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶过度地合成氧化应激产物活性氧（ROS）也参与了SHR大鼠内皮功能的失调。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）氧化酶的活性。.本研究中我们探索了甲羟戊酸途径关键酶FDPS, FT 或 GGTase-I抑制后对SHR大鼠内皮功能的影响，并明确其是否抑制了过度的氧化应激反应及相关机制。SHR大鼠在用FDPS或FT抑制剂处理后观察内皮依赖和非内皮依赖的血管舒张反应。并在体和培养的血管平滑肌细胞中检测ROS产物、NADPH氧化酶的活性。我们发现，FDPS酶抑制剂增加了内皮依赖的血管舒张反应，而FT却没有这种效应。在血管平滑肌细胞中血管紧张素Ⅱ（AngⅡ）浓度依赖性诱导ROS的产生。SHR大鼠中ROS产物明显多于对照组WKY大鼠，而FDPS酶处理后减少了血管与平滑肌细胞中ROS含量、NADPH氧化酶亚基p47phox转位、NADPH氧化酶和Rac1的活性。加入GGOH可逆转了上述FDPS抑制剂的抗氧化应激效应，而FOH或甲羟戊酸MEV没有逆转作用。GGTase-I干预后可模拟上述FDPS酶抑制剂对过度氧化应激的抑制作用。.结论：甲羟戊酸途径关键酶FDPS 和 GGTase-I可通过抑制Rac1/NADPH氧化酶，降低SHR过度的氧化应激状态从而改善受损的内皮功能。