抑制法尼基焦磷酸合成酶改善心脏重塑作用的研究
发布时间：2018-08-02 11:27
【摘要】：第一部分:法尼基焦磷酸合成酶抑制剂改善心肌肥厚并影响连接蛋白重塑研究背景:心肌肥厚是心脏疾病中的一种病理改变,其可能与机体过度激活的神经内分泌体系和内环境失稳密切相关。心肌持续而过度的肥厚反应将最终导致心脏功能减退及慢性充血性心力衰竭。在心衰形成的整个过程中,心脏重构是一个动态发展的过程,其间涉及到细胞凋亡、纤维化、脂肪堆积、线粒体异常、离子通道改变及信号转导通路的变化等。以往的研究表明,法尼基焦磷酸合成酶(famesyl pyrophosphate synthase,FPPS)是甲羟戊酸途径中的一种关键酶,其催化合成下游产物法尼基焦磷酸(Farnesyl pyrophosphate,FPP)和r{牛儿基r{牛儿基焦磷酸(Geranylgeranylpyrophosphat,GGPP)所参与的信号转导通路直接参与心肌肥厚及心脏重构的发生。心肌细胞的纤维化是心脏重构中及其重要的特征,但其他如肌钙蛋白、缝隙连接蛋白等的变化也间接地反应了心肌重构的程度与趋势。目的:探讨法尼基焦磷酸合成酶抑制剂在腹主动脉缩窄术所致心肌肥厚的动物模型中心肌重构的影响与作用。方法:1.腹主动脉缩窄术建立心肌肥厚大鼠模型并观察手术效果;2.将腹主动脉缩窄术后雄性SD大鼠随机分为3组:腹主动脉缩窄术组、腹主动脉缩窄术加药物组和假手术对照组。分别给予生理盐水10mg/kg/day或阿伦磷酸钠10mg/kg/day 灌胃维持 3、5、8 周。3.运用超声、血流动力学等手段检测大鼠组织形态学变化;4.胫骨尺测量大鼠左侧胫骨长度;5.高效液相色谱法(HPLC)技术对大鼠心肌组织中FPP和GGPP含量进行检测;6.用BNP检测试剂盒检测大鼠血液样本中的BNP含量;7.对大鼠心肌组织切片染色并对I型纤维胶原蛋白(Col-I)、T型肌钙蛋白(t-TnT)及缝隙连接蛋白(cx43)等进行免疫组化分析。结果:1.腹主动脉缩窄术导致大鼠心肌肥厚并增加心肌组织中FPP及GGPP的含量;2.FPPS抑制剂可部分改善心肌肥厚表现,同时心肌组织中FPP及GGPP含量下降;3.心肌肥厚中大鼠胫骨长度及血BNP水平无明显变化;4.心肌重构中心肌纤维化、肌钙蛋白及缝隙连接蛋白等发生改变,抑制FPPS可以部分逆转这种重构表现。结论:FPPS参与了心肌肥厚及重塑的发生、发展;抑制FPPS的活性可以部分改善心肌肥厚。第二部分:法尼基焦磷酸合成酶抑制剂阿伦磷酸钠对心肌肥厚大鼠中RhoA通路影响的研究研究背景:心脏重塑是心肌肥厚发生与发展中重要的病理过程,并以心力衰竭为最终结局。很多研究表明被激活的RhoA/ROCK通路及其相关蛋白会介导心肌肥厚及细胞凋亡的发生。Rho激酶属于小G蛋白(蛋白激酶A,G,C)家族的丝氨酸/苏氨酸激酶,可以通过其下游靶效应分子,进一步调节ROCK,这些蛋白的调节可以对细胞收缩、黏附、迁移和增殖等多种生物学行为产生影响。而已知的事实是,RhoA/Rock信号通路在心室重塑过程中起着上调炎性细胞因子,抑制心肌收缩,促进心肌肥大与凋亡等作用。研究表明,阿伦磷酸钠是经典的FPPS抑制剂,其通过抑制FPP、GGPP的生成进而影响RhoA/Rock信号通路的转导达到改善心肌肥厚及纤维化等的病理过程。法尼基焦磷酸合成酶(FPPS)可能是治疗心肌肥厚和改善心脏重构的新思路。目的:探讨FPPS抑制剂在改善心肌肥厚及重塑时可能的RhoA通路机制。方法:1.腹主动脉缩窄术建立心肌肥厚大鼠模型;2.将雄性SD大鼠分为3组:腹主动脉缩窄术组、腹主动脉缩窄术加药物组和假手术对照组。分别给予生理盐水10mg/kg/day或阿伦酸纳10mg/kg/day灌胃维持3、5、8 周。3.运用超声、血流动力学等手段检测大鼠的组织形态学变化;4.高效液相色谱法(HPLC)技术对大鼠心肌组织中FPP和GGPP含量进行检测;5.RhoA试剂盒测定心肌组织中RhoA在490nm处的OD值;6.免疫蛋白印记检测心肌组织中FDPS、RhoA及p-ERKl/2的蛋白含量。结果:1.抑制FPPS可改善心肌肥厚及心脏重塑;2.抑制FPPS可降低心肌组织中FPP、GGPP含量;3.抑制FPPS可降低心肌组织中RhoA含量;4.抑制FPPS可通过抑制RhoA相关通路的蛋白活性抑制心肌肥厚与心脏重塑。结论:抑制FPPS可通过抑制RhoA通路上蛋白的表达从而改善心肌肥厚及心脏重塑;FPPS抑制剂可能是治疗心肌肥厚的新思路。
[Abstract]:The first part: the farnesyl pyrophosphate synthetase inhibitor improves the cardiac hypertrophy and affects the study of the remodeling of connexin: myocardial hypertrophy is a pathological change in the heart disease, which may be closely related to the excessive activation of the neuroendocrine system and the instability of the internal environment. The persistent and excessive hypertrophy of the heart muscle will eventually lead to the heart In the whole process of the formation of heart failure, cardiac remodeling is a process of dynamic development, which involves apoptosis, fibrosis, fat accumulation, mitochondrial abnormalities, changes in ion channels and signal transduction pathways. Previous studies showed that farnesyl pyrophosphate synthetase (famesyl Pyrophosphate synthase, FPPS) is a key enzyme in the metholate pathway, which catalyzes the synthesis of downstream product farnyl pyrophosphate (Farnesyl pyrophosphate, FPP) and r{bovine based r{bovine basic pyrophosphate (Geranylgeranylpyrophosphat, GGPP) involved in the direct involvement of the cardiac hypertrophy and cardiac remodeling. Cell fibrosis is an important feature of cardiac remodeling, but other changes such as troponin and gap connexin also indirectly reflect the degree and trend of cardiac remodeling. Objective: To investigate the effect of farnesyl pyrophosphate synthetase inhibitor on cardiac muscle remodeling in the animal model of cardiac hypertrophy induced by abdominal aortic coarctation. Methods: 1. abdominal aorta coarctation was used to establish the rat model of myocardial hypertrophy and to observe the effect of operation. 2. the male SD rats were randomly divided into 3 groups after the coarctation of the abdominal aorta: abdominal aorta coarctation group, abdominal aorta coarctation plus drug group and sham operation control group. The rats were given saline 10mg/kg/day or Allen phosphate sodium 10mg/kg/day irrigation respectively. Gastric maintenance 3,5,8 week.3. using ultrasound, hemodynamics and other means to detect the histomorphological changes in rats; 4. tibia length measurement of the length of the left tibia of rats; 5. high performance liquid chromatography (HPLC) technique to detect the content of FPP and GGPP in rat myocardial tissue; 6. BNP detection kit to detect the content of BNP in rat blood samples; 7. rats Myocardial tissue section staining and immunohistochemical analysis of I type fiber collagen (Col-I), T type troponin (t-TnT) and gap connexin (Cx43). Results: 1. abdominal aorta coarctation caused myocardial hypertrophy in rats and increased FPP and GGPP in myocardial tissue; 2.FPPS inhibitor could partly improve the performance of myocardial hypertrophy and myocardium. The content of FPP and GGPP in the tissue decreased, and there was no significant change in the length of tibia and blood BNP in 3. cardiac hypertrophy rats; 4. cardiac fibrosis, troponin and gap connexin were changed, and the inhibition of FPPS could partly reverse this reconstruction. Conclusion: FPPS is involved in the occurrence and development of myocardial hypertrophy and remodeling, and the inhibition of FPPS. Activity can partly improve myocardial hypertrophy. Second: the second part: the research background of the effect of farnesyl pyrophosphate synthase inhibitor Allen phosphate on the cardiac hypertrophy rats: cardiac remodeling is an important pathological process in the development and development of cardiac hypertrophy, and heart failure is the final outcome. Many studies have shown that activated RhoA/ The ROCK pathway and its related proteins mediate the occurrence of myocardial hypertrophy and apoptosis..Rho kinase belongs to the serine / threonine kinase in the family of small G protein (protein kinase A, G, C), which can further regulate ROCK through its downstream target effect molecules, which can be regulated by many biological behaviors such as cell contraction, adhesion, migration and proliferation. The fact is that the RhoA/Rock signaling pathway plays the role of up regulating inflammatory cytokines, inhibiting myocardial contraction, promoting myocardial hypertrophy and apoptosis during ventricular remodeling. The study shows that Allen phosphate is a classic FPPS inhibitor, and it affects the transduction of RhoA/Rock signaling pathway by inhibiting the formation of FPP and GGPP. To improve the pathological process of myocardial hypertrophy and fibrosis, farnesyl pyrophosphate synthetase (FPPS) may be a new idea for the treatment of myocardial hypertrophy and improvement of cardiac remodeling. Objective: To explore the possible RhoA pathway mechanism of FPPS inhibitors in improving myocardial hypertrophy and remodeling. Methods: 1. abdominal aorta coarctation was used to establish a rat model of myocardial hypertrophy; 2. Male SD rats were divided into 3 groups: abdominal aorta coarctation group, abdominal aorta coarctation and drug group and sham control group. The histomorphological changes of rats were measured by 10mg/kg/day or Allen acid, 10mg/kg/day and 3,5,8 weeks, respectively. The histomorphology of rats was detected by hemodynamics, and 4. high performance liquid chromatography (HPLC) technique. The content of FPP and GGPP in myocardium of rats was detected, 5.RhoA kit was used to determine the OD value of RhoA at 490nm in myocardial tissue, and the protein content of FDPS, RhoA and p-ERKl/2 in myocardial tissue was detected by 6. immunoglobulin. Results: 1. inhibition of FPPS could improve myocardial hypertrophy and cardiac remodeling; 2. inhibition of FPPS could reduce FPP, GGPP content and 3. of myocardium tissue. Inhibition of FPPS can reduce the content of RhoA in myocardial tissue; 4. inhibition of FPPS can inhibit cardiac hypertrophy and cardiac remodeling by inhibiting the protein activity of the RhoA related pathway. Conclusion: inhibition of FPPS can improve cardiac hypertrophy and cardiac remodeling by inhibiting the expression of protein on the RhoA pathway, and FPPS suppressor may be a new idea for the treatment of myocardial hypertrophy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54
本文编号：2159285
[Abstract]:The first part: the farnesyl pyrophosphate synthetase inhibitor improves the cardiac hypertrophy and affects the study of the remodeling of connexin: myocardial hypertrophy is a pathological change in the heart disease, which may be closely related to the excessive activation of the neuroendocrine system and the instability of the internal environment. The persistent and excessive hypertrophy of the heart muscle will eventually lead to the heart In the whole process of the formation of heart failure, cardiac remodeling is a process of dynamic development, which involves apoptosis, fibrosis, fat accumulation, mitochondrial abnormalities, changes in ion channels and signal transduction pathways. Previous studies showed that farnesyl pyrophosphate synthetase (famesyl Pyrophosphate synthase, FPPS) is a key enzyme in the metholate pathway, which catalyzes the synthesis of downstream product farnyl pyrophosphate (Farnesyl pyrophosphate, FPP) and r{bovine based r{bovine basic pyrophosphate (Geranylgeranylpyrophosphat, GGPP) involved in the direct involvement of the cardiac hypertrophy and cardiac remodeling. Cell fibrosis is an important feature of cardiac remodeling, but other changes such as troponin and gap connexin also indirectly reflect the degree and trend of cardiac remodeling. Objective: To investigate the effect of farnesyl pyrophosphate synthetase inhibitor on cardiac muscle remodeling in the animal model of cardiac hypertrophy induced by abdominal aortic coarctation. Methods: 1. abdominal aorta coarctation was used to establish the rat model of myocardial hypertrophy and to observe the effect of operation. 2. the male SD rats were randomly divided into 3 groups after the coarctation of the abdominal aorta: abdominal aorta coarctation group, abdominal aorta coarctation plus drug group and sham operation control group. The rats were given saline 10mg/kg/day or Allen phosphate sodium 10mg/kg/day irrigation respectively. Gastric maintenance 3,5,8 week.3. using ultrasound, hemodynamics and other means to detect the histomorphological changes in rats; 4. tibia length measurement of the length of the left tibia of rats; 5. high performance liquid chromatography (HPLC) technique to detect the content of FPP and GGPP in rat myocardial tissue; 6. BNP detection kit to detect the content of BNP in rat blood samples; 7. rats Myocardial tissue section staining and immunohistochemical analysis of I type fiber collagen (Col-I), T type troponin (t-TnT) and gap connexin (Cx43). Results: 1. abdominal aorta coarctation caused myocardial hypertrophy in rats and increased FPP and GGPP in myocardial tissue; 2.FPPS inhibitor could partly improve the performance of myocardial hypertrophy and myocardium. The content of FPP and GGPP in the tissue decreased, and there was no significant change in the length of tibia and blood BNP in 3. cardiac hypertrophy rats; 4. cardiac fibrosis, troponin and gap connexin were changed, and the inhibition of FPPS could partly reverse this reconstruction. Conclusion: FPPS is involved in the occurrence and development of myocardial hypertrophy and remodeling, and the inhibition of FPPS. Activity can partly improve myocardial hypertrophy. Second: the second part: the research background of the effect of farnesyl pyrophosphate synthase inhibitor Allen phosphate on the cardiac hypertrophy rats: cardiac remodeling is an important pathological process in the development and development of cardiac hypertrophy, and heart failure is the final outcome. Many studies have shown that activated RhoA/ The ROCK pathway and its related proteins mediate the occurrence of myocardial hypertrophy and apoptosis..Rho kinase belongs to the serine / threonine kinase in the family of small G protein (protein kinase A, G, C), which can further regulate ROCK through its downstream target effect molecules, which can be regulated by many biological behaviors such as cell contraction, adhesion, migration and proliferation. The fact is that the RhoA/Rock signaling pathway plays the role of up regulating inflammatory cytokines, inhibiting myocardial contraction, promoting myocardial hypertrophy and apoptosis during ventricular remodeling. The study shows that Allen phosphate is a classic FPPS inhibitor, and it affects the transduction of RhoA/Rock signaling pathway by inhibiting the formation of FPP and GGPP. To improve the pathological process of myocardial hypertrophy and fibrosis, farnesyl pyrophosphate synthetase (FPPS) may be a new idea for the treatment of myocardial hypertrophy and improvement of cardiac remodeling. Objective: To explore the possible RhoA pathway mechanism of FPPS inhibitors in improving myocardial hypertrophy and remodeling. Methods: 1. abdominal aorta coarctation was used to establish a rat model of myocardial hypertrophy; 2. Male SD rats were divided into 3 groups: abdominal aorta coarctation group, abdominal aorta coarctation and drug group and sham control group. The histomorphological changes of rats were measured by 10mg/kg/day or Allen acid, 10mg/kg/day and 3,5,8 weeks, respectively. The histomorphology of rats was detected by hemodynamics, and 4. high performance liquid chromatography (HPLC) technique. The content of FPP and GGPP in myocardium of rats was detected, 5.RhoA kit was used to determine the OD value of RhoA at 490nm in myocardial tissue, and the protein content of FDPS, RhoA and p-ERKl/2 in myocardial tissue was detected by 6. immunoglobulin. Results: 1. inhibition of FPPS could improve myocardial hypertrophy and cardiac remodeling; 2. inhibition of FPPS could reduce FPP, GGPP content and 3. of myocardium tissue. Inhibition of FPPS can reduce the content of RhoA in myocardial tissue; 4. inhibition of FPPS can inhibit cardiac hypertrophy and cardiac remodeling by inhibiting the protein activity of the RhoA related pathway. Conclusion: inhibition of FPPS can improve cardiac hypertrophy and cardiac remodeling by inhibiting the expression of protein on the RhoA pathway, and FPPS suppressor may be a new idea for the treatment of myocardial hypertrophy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54
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