模拟失重对心血管系统的影响及机制研究
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摘要
失重/模拟失重可导致心血管系统功能失调,心脏、动脉血管平滑肌结构重塑和功能变化是心血管系统功能失调的重要原因。然而,目前国内尚无有关长期模拟失重导致心血管系统功能失调的研究报道;此外,失重/模拟失重下心肌、血管结构重塑和功能变化的机制并不清楚。因此,本课题的目的是:
(1)通过60天头低位卧床模拟失重实验获取中国人群的心血管系统实验数据,同时对阻抗振动和中药太空养心方两组对抗措施的防护效果进行评价;
(2)阐明模拟失重下心肌萎缩的分子调控机制;
(3)阐明模拟失重下后身动脉血管收缩功能变化的调控机制;
(4)阐明失重和模拟失重对血管平滑肌细胞增殖的影响及β-catenin信号调控机制。
为达到以上研究目的,本课题采用以下实验方法进行研究:
(1)以60天头低位卧床模拟失重,采用彩色多普勒超声技术检测心脏和血管的结构和功能;以静脉阻断体积描记术检测下肢静脉顺应性及静脉血流阻力,同时评价对抗措施的防护效果;
(2)以尾吊鼠为模拟失重模型,以太空养心方为防护措施,采用Langendorff离体心脏灌流技术检测离体心脏功能;以FITC-Lectin对心肌细胞进行免疫荧光染色,分析心肌细胞横截面积的大小;采用Western blot技术检测心肌组织TnI、Bcl-2、Hsp20蛋白表达及Akt、GSK3-β、FAK、P38 MAPK和Hsp27蛋白磷酸化水平的变化;
(3)采用离体血管灌流技术,以Powerlab生理记录仪检测模拟失重后股动脉和腹主动脉对NE或PE的收缩反应;通过给予ERK/CaD、MLCK、PI3K、P38 MAPK、L-型钙通道特异性抑制剂PD98059、ML-7、LY-294002、SB203580、Nifedipine及肌动蛋白细胞骨架破坏剂Cytochalasin D,评价这些通路在尾吊模拟失重后血管收缩反应中的作用;
(4)采用48小时回转模拟失重和航天飞行真实失重环境培养血管平滑肌细胞,观察失重和模拟失重对血管平滑肌细胞增殖的影响;采用Western blot和免疫荧光技术检测模拟失重后β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表达及Akt、GSK-3β蛋白磷酸化水平的变化;采用免疫荧光技术检测航天飞行后血管平滑肌细胞核的变化及β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表达变化。
本课题获取的实验结果如下:
一、头低位卧床实验
(1)58天卧床导致心肌萎缩、LDVD、SV等心功能指标下降。阻抗振动和太空养心方对卧床导致的心肌质量和心功能下降均有部分防护效果,并且太空养心方的防护效果优于阻抗振动;
(2)58天卧床对颈动脉和股动脉血管直径无明显影响,但却可降低三组的门静脉横截面积,并且下降幅度在对照组达到了统计学显著性差异;
(3)58天卧床影响了血管的收缩功能:肺动脉、颈动脉和股动脉收缩速度在三组均发生下降,其中除颈动脉外,肺动脉收缩速度下降在阻抗振动组和中药组达到统计学显著性差异;三组的股动脉收缩速度在卧床后均显著低于卧床前的水平;股动脉RI在对照组和阻抗振动组有下降的趋势,而在中药组却有轻微升高趋势;胫前动脉RI在对照组和中药组有升高趋势,而在阻抗振动组则有下降趋势;
(4)卧床38天导致下肢静脉顺应性显著下降,但与对照组和阻抗振动组相比,中药组的下降幅度明显更小,太空养心方对38天卧床导致的下肢静脉顺应性下降有明显的防护效果;52天卧床后,对照组和中药组下肢静脉顺应性进一步下降,而阻抗振动组下降幅度减缓,略低于卧床38天的水平,说明阻抗振动对52天卧床导致的静脉顺应性下降有更好的防护效果;此外,中药组下肢静脉顺应性虽然发生了进行性下降,但与对照组相比,下降幅度显著降低,说明太空养心方对52天卧床导致的下肢静脉顺应性下降仍具有较好的防护效果;
(5)卧床后下肢静脉血流阻力呈进行性升高,在38天,对照组的升高幅度与阻抗振动组持平,均高于中药组,说明太空养心方对38天卧床导致的下肢静脉血流阻力升高的防护效果高于阻抗振动;但在52天后,对照组和中药组的血流阻力呈进一步升高趋势,而阻抗振动组血流阻力的增幅却低于卧床38天,说明阻抗振动对52天卧床导致的下肢静脉血流阻力升高具有比中药太空养心方更好的防护效果。
二、模拟失重对心脏结构功能的影响及防护措施研究
(1)尾吊7天模拟失重导致大鼠心脏收缩和舒张功能下降,太空养心方对心功能的下降具有较好的防护效果;
(2)尾吊7天模拟失重导致大鼠心肌萎缩,TnI蛋白表达下调,Akt/GSK-3β信号通路显著受抑制,而太空养心方能够对抗尾吊模拟失重导致的心肌细胞萎缩,TnI蛋白表达下调及Akt/GSK-3β信号通路受抑;
(3)尾吊7天、14天和21天可导致心肌组织TnI蛋白表达进行性下降;7天、21天可显著抑制Akt/GSK-3β信号通路;而尾吊14天则能增强Akt/GSK-3β信号通路活性;
(4)尾吊14天模拟失重可显著抑制心肌组织中P38 MAPK/Hsp27信号通路。三、模拟失重对血管功能的影响及机制研究
(1)尾吊模拟失重降低了大鼠股动脉的收缩反应并抑制了ERK/CaD信号通路;
(2)尾吊7天模拟失重对股动脉MLCK/MLC20信号通路影响不大,但尾吊14天却可显著抑制MLCK/MLC20信号通路;
(3)尾吊7天、14天可显著抑制股动脉L-型钙通道的功能;
(4)尾吊14天可降低腹主动脉收缩反应;同时抑制P38 MAPK/Hsp27信号通路;但对股动脉中的P38 MAPK/Hsp27信号通路无影响;
(5)尾吊7天、14天可抑制股动脉PI3K信号通路;
(6)尾吊模拟失重对股动脉血管中的细胞骨架聚合功能没有影响。
四、失重和模拟失重对血管平滑肌细胞增殖的影响及β-catenin信号通路研究
(1)失重和模拟失重可导致VSMC增殖能力下降,β-catenin信号调节通路在其中起着重要作用;
(2)失重和模拟失重导致了β-catenin信号调节通路上游信号分子N-Cadherin表达、Akt和GSK-3β活性的下降;
(3)失重和模拟失重抑制β-catenin信号,导致VSMC增殖下降的直接原因是Cyclin D1表达下调,细胞周期停滞于G1期;以及P21Cip1表达过高或过低,首次发现模拟失重和失重对P21 Cip1表达存在不同方式的调控。
本课题得到以下结论:
(1)首次在中国志愿者身上发现60天头低位卧床可导致心肌萎缩、功能下降;动脉血管收缩功能下降,下肢静脉顺应性下降和血流阻力升高;太空养心方和阻抗振动锻炼对60天卧床导致的心血管功能失调具有部分防护效果;
(2)尾吊模拟失重可通过抑制调控心肌细胞肥大的Akt/GSK-3β信号通路导致心肌萎缩,太空养心方能够增强Akt/GSK-3β信号通路的活性,抑制尾吊导致的心肌萎缩;同时还能提高心肌组织Hsp20蛋白表达水平,增强心肌的保护作用。此外,尾吊模拟失重还能通过抑制P38 MAPK/Hsp27信号通路导致心肌萎缩;
(3)尾吊模拟失重降低了大鼠股动脉和腹主动脉的收缩反应,抑制了股动脉收缩反应中ERK/CaD信号通路、MLCK/MLC20信号通路、PI3K信号通路及L-型钙通道的功能;抑制了腹主动脉收缩反应中P38 MAPK/Hsp27信号通路功能;
4)首次发现失重可导致VSMC增殖能力下降,失重和模拟失重可抑制β-catenin及其上游信号分子N-Cadherin的表达;模拟失重还可导致Akt/GSK-3β活性下降;导致VSMC增殖下降的直接原因是Cyclin D1表达下调,细胞周期停滞于G1期以及P21 Cip1表达过高或过低。首次发现模拟失重和失重对P21 Cip1蛋白的表达存在不同的调控方式。
Microgravity/simulated microgravity can induce cardiovascular deconditioning, the structural remodeling and functional changes of heart and arterial vascular smooth muscle are the important mechanisms responsible for the cardiovascular dysfunction. However, until now, there is not any research report about cardiovascular dysfunction after long-time simulated microgravity on human in China. Moreover, the mechanisms of structural remodeling and functional changes in heart and arterial vascular smooth muscle induced by microgravity/simulated microgravity aren’t fully elucidated. Thus, the objective of this study is following:
(1) Acquire the cardiovascular experimental data from Chinese volunteers by 60d HDT and evaluate the countermeasure effects of Vibration and Chinese medicine“TaiKong Yangxin Prescription”.
(2) Illustrate the molecular mechanisms of cardiac atrophy induced by simulated microgravity.
(3) Illustrate the regulating mechanisms of changes in contractile response of hindlimb arterial vascualrs after simulated microgravity.
(4) Study the effects of microgravity and simulated microgravity on VSMC proliferation and theβ-catenin signal pathway.
The following methods will be used in our experiments.
(1) 60d head down bedrest was used to simulate the physiological effects of microgravity. The echography was used to measure the structure and functions of heart and vascular. The strain gauge venous occlusion plethysmography was used to assess limb venous vascular characteristics. Moreover, the protectable effects of two kinds of countermeasures were evaluated.
(2) Tai-suspension rats were used animal model of simulated microgravity, TaiKong Yangxin Prescription was used to countermeasure the cardiac dysfunction. The isolated rat heart functions were measured by Langendorff method. The cardiac muscle cells were stained by FITC-Lectin and the cross section area were calculated. Western blot was used to detect the protein expression of TnI, Bcl-2, Hsp20 and the phosphorylation level of Akt, GSK3-β, FAK, P38 MAPK and Hsp27.
(3) The contractile responses of femoral and abdomen arteries to NE or PE were measured by powerlab recording system. By the special inhibitors, PD98059, ML-7, LY-294002, SB203580, Nifedipine and Cytochalasin D, respectively for ERK/CaD, MLCK, PI3K, P38 MAPK, L type calcium channel and actin polymerization, the contributing role of these special pathways to changes of contractile responses after simulated microgravity were evaluated.
(4) In clinostat and“Shenzhou 7”spacecraft, the VSMCs were cultured for 48h, the proliferation was measured. Western blot and immunofluorescence were used to detect the protein expression ofβ-catenin、N-Cadherin、Cyclin D1、P21Cip1 and the phosphorylation level of Akt and GSK-3βafter simulated microgravity by clinostat. Immunofluorescence was used to detect changes of nucleus in VSMCs and the expression ofβ-catenin、N-Cadherin、Cyclin D1、P21Cip1. From the experiments, the following results were acquired.
1. HDT experiment
(1) 58d HDT induced the cardiac atrophy and decreased the LDVD and SV. The decrease of cardiac mass and functions can be protected partly by countermeasures of vibration and TaiKong Yangxin Prescription. Moreover, TaiKong Yangxin Prescription had better countermeasure effects than vibration did.
(2) 58d HDT had no effect on the diameters of carotid and femoral arteries, but can decrease the cross section area of portal vein in three groups. Especially, the decreased range in control group reached the statistical difference.
(3) 58d HDT affected the contractile functions of vascular. The contractile speeds in three groups all decreased. Except carotid artery, the contractile speed of pulmonary artery significantly decreased in vibration and TaiKong Yangxin Prescription group. The contractile speed of femoral artery in three groups decreased significantly compared with those of before HDT. Moreover, there was a decreased trend of femoral arteries RI in control and vibration groups, but in TaiKong Yangxin Prescription group, femoral arteries RI were increased slightly. there was a increased trend of Tibial arteries RI in control and TaiKong Yangxin Prescription group, but in vibration group, there was a decreased trend compared with that of before HDT.
(4) 38d HDT induced the significant decrease of leg vein compliance, but compared with control and vibration groups, the decrease range in TaiKong Yangxin Prescription group was obviously smaller, which showed that TaiKong Yangxin Prescription has better countermeasure effect on the decreased leg vein compliance induced by 38d HDT. After 52d HDT, the leg vein compliance in TaiKong Yangxin Prescription group and control group had a ongoing decrease, but the decreased range in vibration group reduced to the level of below 38d HDT, which showed that vibration countermeasure had a better effects on the decreased leg vein compliance after 52d HDT.
(5) HDT increased the leg venous resistance. After 38d HDT, the increased range of leg venous resistance in control and vibration groups were higher than that of TaiKong Yangxin Prescription group, which showed that TaiKong Yangxin Prescription had a better countermeasure effect compared with vibration. However, after 52d HDT, the leg venous resistance of control and TaiKong Yangxin Prescription group had a increased trend, but the increased range in vibration reduced to the level below that of 38d HDT, which showed that vibration had better countermeasure on the increased leg venous resistance than that of TaiKong Yangxin Prescription after 52d HDT.
2. Effects of simulated microgravity on cardiac structure and function and countermeasure study
(1) Simulated microgravity induced by 7d tail-suspension decreased the rat cardiac functions which can be countermeasured by TaiKong Yangxin Prescription.
(2) Simulated microgravity after 7d tail-suspension induced the rat cardiac atrophy, decreased the protein expression of TnI and inhibited the Akt/GSK-3βsignal pathway, which can be counter- measured by TaiKong Yangxin Prescription.
(3) The protein expression of TnI was decreased ongoing after 7d, 14d and 21d tail-suspension. The Akt/GSK-3βsignal pathway significantly inhibited by 7d and 21d tail-suspension, but increased by 14d tail-suspension.
(4) Simulated microgravity induced by 14d tail-suspension decreased significantly the signal of P38 MAPK/Hsp27 pathway in cardiac muscle.
3. Effects of simulated microgravity on vascular function and mechanisms study
(1) Simulated microgravity decreased the contractile response of femoral arteries and inhibited the signal of ERK/CaD pathway.
(2) 7d tail-suspension had little effects on signal of MLCK/MLC20 pathway in femoral arteries, however, 14d tail-suspension inhibited significantly the signal of MLCK/MLC20 pathway.
(3) The function of L type calcium channel decreased significantly after 7d and 14d tail-suspension.
(4) 14d tail-suspension decreased the contractile response of abdomen arteries and inhibited the signal of P38 MAPK/Hsp27 pathway, but had little effects on the P38 MAPK/Hsp27 pathway in femoral arteries.
(5) The signal of PI3K pathway decreased significantly after 7d and 14d tail-suspension.
(6) Simulated microgravity induced by tail-suspension had no effect on the actin polymerization in femoral arteries.
4. Effects of microgravity and simulated microgravity on VSMC proliferation andβ-catenin signal pathway
(1) Microgravity and simulated microgravity decreased the VSMC proliferation, in whichβ-catenin signal pathway had important role.
(2) Microgravity and simulated microgravity inhibited the expression ofβ-catenin and N-Cadherin. Simulated microgravity decreased the activity of Akt and GSK-3β.
(3) Microgravity and simulated microgravity inhibited theβ-catenin signal pathway,but the direct reason which decreased the VSMC proliferation was the reduced expression of Cyclin D1, cell cycle retarded at G1 and different expression of P21Cip1. For the first time, we found that microgravity and simulated microgravity had different regulating mode on protein expression of P21 Cip1.
This study got following conclusions.
(1) For the first time, we found that 60d HDT can induce cardiac atrophy, functional inhibition, decreased arterial contractility, decreased leg vein compliance and increased leg venous flow resistance in Chinese volunteers. The countermeasures of TaiKong Yangxin Prescription and vibration has partly protectable effects on cardiac dysfunction induced by 60d HDT.
(2) Simulated microgravity after tail-suspension can induce the cardiac atrophy by Akt/GSK-3βsignal pathway which has important role in regulating cardiac hypertrophy. TaiKong Yangxin Prescription can inhibit cardiac atrophy by activating the signal of Akt/GSK-3βpathway. It can also increase the protein expression of Hsp20 and promote its cardioprotection effects. Moreover, simulated microgravity can also induced cardiac atrophy by inhibiting the signal pathway of P38 MAPK/Hsp27.
(3) Simulated microgravity after tail-suspension decreased the contractile responses of femoral and abdomen arteries. In femoral arteries, the functions of ERK/CaD, MLCK/MLC20, PI3K signal pathways and L type calcium channel decreased by tail-suspension. In abdomen arteries, tail-suspension inhibited the signal of P38 MAPK/Hsp27 pathway.
(4) For the first time, this experiment found microgravity can decrease the VSMC proliferation. Microgravity and simulated microgravity inhibited the protein expression ofβ-catenin and N-Cadherin. Simulated microgravity can decreased the activity of Akt/GSK-3βsignal pathway. The direct reason which decreased the VSMC proliferation was the reduced expression of Cyclin D1, cell cycle retarded at G1 and different expression of P21Cip1. For the first time, we found that microgravity and simulated microgravity had different regulating mode on protein expression of P21 Cip1.
(1)通过60天头低位卧床模拟失重实验获取中国人群的心血管系统实验数据,同时对阻抗振动和中药太空养心方两组对抗措施的防护效果进行评价;
(2)阐明模拟失重下心肌萎缩的分子调控机制;
(3)阐明模拟失重下后身动脉血管收缩功能变化的调控机制;
(4)阐明失重和模拟失重对血管平滑肌细胞增殖的影响及β-catenin信号调控机制。
为达到以上研究目的,本课题采用以下实验方法进行研究:
(1)以60天头低位卧床模拟失重,采用彩色多普勒超声技术检测心脏和血管的结构和功能;以静脉阻断体积描记术检测下肢静脉顺应性及静脉血流阻力,同时评价对抗措施的防护效果;
(2)以尾吊鼠为模拟失重模型,以太空养心方为防护措施,采用Langendorff离体心脏灌流技术检测离体心脏功能;以FITC-Lectin对心肌细胞进行免疫荧光染色,分析心肌细胞横截面积的大小;采用Western blot技术检测心肌组织TnI、Bcl-2、Hsp20蛋白表达及Akt、GSK3-β、FAK、P38 MAPK和Hsp27蛋白磷酸化水平的变化;
(3)采用离体血管灌流技术,以Powerlab生理记录仪检测模拟失重后股动脉和腹主动脉对NE或PE的收缩反应;通过给予ERK/CaD、MLCK、PI3K、P38 MAPK、L-型钙通道特异性抑制剂PD98059、ML-7、LY-294002、SB203580、Nifedipine及肌动蛋白细胞骨架破坏剂Cytochalasin D,评价这些通路在尾吊模拟失重后血管收缩反应中的作用;
(4)采用48小时回转模拟失重和航天飞行真实失重环境培养血管平滑肌细胞,观察失重和模拟失重对血管平滑肌细胞增殖的影响;采用Western blot和免疫荧光技术检测模拟失重后β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表达及Akt、GSK-3β蛋白磷酸化水平的变化;采用免疫荧光技术检测航天飞行后血管平滑肌细胞核的变化及β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表达变化。
本课题获取的实验结果如下:
一、头低位卧床实验
(1)58天卧床导致心肌萎缩、LDVD、SV等心功能指标下降。阻抗振动和太空养心方对卧床导致的心肌质量和心功能下降均有部分防护效果,并且太空养心方的防护效果优于阻抗振动;
(2)58天卧床对颈动脉和股动脉血管直径无明显影响,但却可降低三组的门静脉横截面积,并且下降幅度在对照组达到了统计学显著性差异;
(3)58天卧床影响了血管的收缩功能:肺动脉、颈动脉和股动脉收缩速度在三组均发生下降,其中除颈动脉外,肺动脉收缩速度下降在阻抗振动组和中药组达到统计学显著性差异;三组的股动脉收缩速度在卧床后均显著低于卧床前的水平;股动脉RI在对照组和阻抗振动组有下降的趋势,而在中药组却有轻微升高趋势;胫前动脉RI在对照组和中药组有升高趋势,而在阻抗振动组则有下降趋势;
(4)卧床38天导致下肢静脉顺应性显著下降,但与对照组和阻抗振动组相比,中药组的下降幅度明显更小,太空养心方对38天卧床导致的下肢静脉顺应性下降有明显的防护效果;52天卧床后,对照组和中药组下肢静脉顺应性进一步下降,而阻抗振动组下降幅度减缓,略低于卧床38天的水平,说明阻抗振动对52天卧床导致的静脉顺应性下降有更好的防护效果;此外,中药组下肢静脉顺应性虽然发生了进行性下降,但与对照组相比,下降幅度显著降低,说明太空养心方对52天卧床导致的下肢静脉顺应性下降仍具有较好的防护效果;
(5)卧床后下肢静脉血流阻力呈进行性升高,在38天,对照组的升高幅度与阻抗振动组持平,均高于中药组,说明太空养心方对38天卧床导致的下肢静脉血流阻力升高的防护效果高于阻抗振动;但在52天后,对照组和中药组的血流阻力呈进一步升高趋势,而阻抗振动组血流阻力的增幅却低于卧床38天,说明阻抗振动对52天卧床导致的下肢静脉血流阻力升高具有比中药太空养心方更好的防护效果。
二、模拟失重对心脏结构功能的影响及防护措施研究
(1)尾吊7天模拟失重导致大鼠心脏收缩和舒张功能下降,太空养心方对心功能的下降具有较好的防护效果;
(2)尾吊7天模拟失重导致大鼠心肌萎缩,TnI蛋白表达下调,Akt/GSK-3β信号通路显著受抑制,而太空养心方能够对抗尾吊模拟失重导致的心肌细胞萎缩,TnI蛋白表达下调及Akt/GSK-3β信号通路受抑;
(3)尾吊7天、14天和21天可导致心肌组织TnI蛋白表达进行性下降;7天、21天可显著抑制Akt/GSK-3β信号通路;而尾吊14天则能增强Akt/GSK-3β信号通路活性;
(4)尾吊14天模拟失重可显著抑制心肌组织中P38 MAPK/Hsp27信号通路。三、模拟失重对血管功能的影响及机制研究
(1)尾吊模拟失重降低了大鼠股动脉的收缩反应并抑制了ERK/CaD信号通路;
(2)尾吊7天模拟失重对股动脉MLCK/MLC20信号通路影响不大,但尾吊14天却可显著抑制MLCK/MLC20信号通路;
(3)尾吊7天、14天可显著抑制股动脉L-型钙通道的功能;
(4)尾吊14天可降低腹主动脉收缩反应;同时抑制P38 MAPK/Hsp27信号通路;但对股动脉中的P38 MAPK/Hsp27信号通路无影响;
(5)尾吊7天、14天可抑制股动脉PI3K信号通路;
(6)尾吊模拟失重对股动脉血管中的细胞骨架聚合功能没有影响。
四、失重和模拟失重对血管平滑肌细胞增殖的影响及β-catenin信号通路研究
(1)失重和模拟失重可导致VSMC增殖能力下降,β-catenin信号调节通路在其中起着重要作用;
(2)失重和模拟失重导致了β-catenin信号调节通路上游信号分子N-Cadherin表达、Akt和GSK-3β活性的下降;
(3)失重和模拟失重抑制β-catenin信号,导致VSMC增殖下降的直接原因是Cyclin D1表达下调,细胞周期停滞于G1期;以及P21Cip1表达过高或过低,首次发现模拟失重和失重对P21 Cip1表达存在不同方式的调控。
本课题得到以下结论:
(1)首次在中国志愿者身上发现60天头低位卧床可导致心肌萎缩、功能下降;动脉血管收缩功能下降,下肢静脉顺应性下降和血流阻力升高;太空养心方和阻抗振动锻炼对60天卧床导致的心血管功能失调具有部分防护效果;
(2)尾吊模拟失重可通过抑制调控心肌细胞肥大的Akt/GSK-3β信号通路导致心肌萎缩,太空养心方能够增强Akt/GSK-3β信号通路的活性,抑制尾吊导致的心肌萎缩;同时还能提高心肌组织Hsp20蛋白表达水平,增强心肌的保护作用。此外,尾吊模拟失重还能通过抑制P38 MAPK/Hsp27信号通路导致心肌萎缩;
(3)尾吊模拟失重降低了大鼠股动脉和腹主动脉的收缩反应,抑制了股动脉收缩反应中ERK/CaD信号通路、MLCK/MLC20信号通路、PI3K信号通路及L-型钙通道的功能;抑制了腹主动脉收缩反应中P38 MAPK/Hsp27信号通路功能;
4)首次发现失重可导致VSMC增殖能力下降,失重和模拟失重可抑制β-catenin及其上游信号分子N-Cadherin的表达;模拟失重还可导致Akt/GSK-3β活性下降;导致VSMC增殖下降的直接原因是Cyclin D1表达下调,细胞周期停滞于G1期以及P21 Cip1表达过高或过低。首次发现模拟失重和失重对P21 Cip1蛋白的表达存在不同的调控方式。
Microgravity/simulated microgravity can induce cardiovascular deconditioning, the structural remodeling and functional changes of heart and arterial vascular smooth muscle are the important mechanisms responsible for the cardiovascular dysfunction. However, until now, there is not any research report about cardiovascular dysfunction after long-time simulated microgravity on human in China. Moreover, the mechanisms of structural remodeling and functional changes in heart and arterial vascular smooth muscle induced by microgravity/simulated microgravity aren’t fully elucidated. Thus, the objective of this study is following:
(1) Acquire the cardiovascular experimental data from Chinese volunteers by 60d HDT and evaluate the countermeasure effects of Vibration and Chinese medicine“TaiKong Yangxin Prescription”.
(2) Illustrate the molecular mechanisms of cardiac atrophy induced by simulated microgravity.
(3) Illustrate the regulating mechanisms of changes in contractile response of hindlimb arterial vascualrs after simulated microgravity.
(4) Study the effects of microgravity and simulated microgravity on VSMC proliferation and theβ-catenin signal pathway.
The following methods will be used in our experiments.
(1) 60d head down bedrest was used to simulate the physiological effects of microgravity. The echography was used to measure the structure and functions of heart and vascular. The strain gauge venous occlusion plethysmography was used to assess limb venous vascular characteristics. Moreover, the protectable effects of two kinds of countermeasures were evaluated.
(2) Tai-suspension rats were used animal model of simulated microgravity, TaiKong Yangxin Prescription was used to countermeasure the cardiac dysfunction. The isolated rat heart functions were measured by Langendorff method. The cardiac muscle cells were stained by FITC-Lectin and the cross section area were calculated. Western blot was used to detect the protein expression of TnI, Bcl-2, Hsp20 and the phosphorylation level of Akt, GSK3-β, FAK, P38 MAPK and Hsp27.
(3) The contractile responses of femoral and abdomen arteries to NE or PE were measured by powerlab recording system. By the special inhibitors, PD98059, ML-7, LY-294002, SB203580, Nifedipine and Cytochalasin D, respectively for ERK/CaD, MLCK, PI3K, P38 MAPK, L type calcium channel and actin polymerization, the contributing role of these special pathways to changes of contractile responses after simulated microgravity were evaluated.
(4) In clinostat and“Shenzhou 7”spacecraft, the VSMCs were cultured for 48h, the proliferation was measured. Western blot and immunofluorescence were used to detect the protein expression ofβ-catenin、N-Cadherin、Cyclin D1、P21Cip1 and the phosphorylation level of Akt and GSK-3βafter simulated microgravity by clinostat. Immunofluorescence was used to detect changes of nucleus in VSMCs and the expression ofβ-catenin、N-Cadherin、Cyclin D1、P21Cip1. From the experiments, the following results were acquired.
1. HDT experiment
(1) 58d HDT induced the cardiac atrophy and decreased the LDVD and SV. The decrease of cardiac mass and functions can be protected partly by countermeasures of vibration and TaiKong Yangxin Prescription. Moreover, TaiKong Yangxin Prescription had better countermeasure effects than vibration did.
(2) 58d HDT had no effect on the diameters of carotid and femoral arteries, but can decrease the cross section area of portal vein in three groups. Especially, the decreased range in control group reached the statistical difference.
(3) 58d HDT affected the contractile functions of vascular. The contractile speeds in three groups all decreased. Except carotid artery, the contractile speed of pulmonary artery significantly decreased in vibration and TaiKong Yangxin Prescription group. The contractile speed of femoral artery in three groups decreased significantly compared with those of before HDT. Moreover, there was a decreased trend of femoral arteries RI in control and vibration groups, but in TaiKong Yangxin Prescription group, femoral arteries RI were increased slightly. there was a increased trend of Tibial arteries RI in control and TaiKong Yangxin Prescription group, but in vibration group, there was a decreased trend compared with that of before HDT.
(4) 38d HDT induced the significant decrease of leg vein compliance, but compared with control and vibration groups, the decrease range in TaiKong Yangxin Prescription group was obviously smaller, which showed that TaiKong Yangxin Prescription has better countermeasure effect on the decreased leg vein compliance induced by 38d HDT. After 52d HDT, the leg vein compliance in TaiKong Yangxin Prescription group and control group had a ongoing decrease, but the decreased range in vibration group reduced to the level of below 38d HDT, which showed that vibration countermeasure had a better effects on the decreased leg vein compliance after 52d HDT.
(5) HDT increased the leg venous resistance. After 38d HDT, the increased range of leg venous resistance in control and vibration groups were higher than that of TaiKong Yangxin Prescription group, which showed that TaiKong Yangxin Prescription had a better countermeasure effect compared with vibration. However, after 52d HDT, the leg venous resistance of control and TaiKong Yangxin Prescription group had a increased trend, but the increased range in vibration reduced to the level below that of 38d HDT, which showed that vibration had better countermeasure on the increased leg venous resistance than that of TaiKong Yangxin Prescription after 52d HDT.
2. Effects of simulated microgravity on cardiac structure and function and countermeasure study
(1) Simulated microgravity induced by 7d tail-suspension decreased the rat cardiac functions which can be countermeasured by TaiKong Yangxin Prescription.
(2) Simulated microgravity after 7d tail-suspension induced the rat cardiac atrophy, decreased the protein expression of TnI and inhibited the Akt/GSK-3βsignal pathway, which can be counter- measured by TaiKong Yangxin Prescription.
(3) The protein expression of TnI was decreased ongoing after 7d, 14d and 21d tail-suspension. The Akt/GSK-3βsignal pathway significantly inhibited by 7d and 21d tail-suspension, but increased by 14d tail-suspension.
(4) Simulated microgravity induced by 14d tail-suspension decreased significantly the signal of P38 MAPK/Hsp27 pathway in cardiac muscle.
3. Effects of simulated microgravity on vascular function and mechanisms study
(1) Simulated microgravity decreased the contractile response of femoral arteries and inhibited the signal of ERK/CaD pathway.
(2) 7d tail-suspension had little effects on signal of MLCK/MLC20 pathway in femoral arteries, however, 14d tail-suspension inhibited significantly the signal of MLCK/MLC20 pathway.
(3) The function of L type calcium channel decreased significantly after 7d and 14d tail-suspension.
(4) 14d tail-suspension decreased the contractile response of abdomen arteries and inhibited the signal of P38 MAPK/Hsp27 pathway, but had little effects on the P38 MAPK/Hsp27 pathway in femoral arteries.
(5) The signal of PI3K pathway decreased significantly after 7d and 14d tail-suspension.
(6) Simulated microgravity induced by tail-suspension had no effect on the actin polymerization in femoral arteries.
4. Effects of microgravity and simulated microgravity on VSMC proliferation andβ-catenin signal pathway
(1) Microgravity and simulated microgravity decreased the VSMC proliferation, in whichβ-catenin signal pathway had important role.
(2) Microgravity and simulated microgravity inhibited the expression ofβ-catenin and N-Cadherin. Simulated microgravity decreased the activity of Akt and GSK-3β.
(3) Microgravity and simulated microgravity inhibited theβ-catenin signal pathway,but the direct reason which decreased the VSMC proliferation was the reduced expression of Cyclin D1, cell cycle retarded at G1 and different expression of P21Cip1. For the first time, we found that microgravity and simulated microgravity had different regulating mode on protein expression of P21 Cip1.
This study got following conclusions.
(1) For the first time, we found that 60d HDT can induce cardiac atrophy, functional inhibition, decreased arterial contractility, decreased leg vein compliance and increased leg venous flow resistance in Chinese volunteers. The countermeasures of TaiKong Yangxin Prescription and vibration has partly protectable effects on cardiac dysfunction induced by 60d HDT.
(2) Simulated microgravity after tail-suspension can induce the cardiac atrophy by Akt/GSK-3βsignal pathway which has important role in regulating cardiac hypertrophy. TaiKong Yangxin Prescription can inhibit cardiac atrophy by activating the signal of Akt/GSK-3βpathway. It can also increase the protein expression of Hsp20 and promote its cardioprotection effects. Moreover, simulated microgravity can also induced cardiac atrophy by inhibiting the signal pathway of P38 MAPK/Hsp27.
(3) Simulated microgravity after tail-suspension decreased the contractile responses of femoral and abdomen arteries. In femoral arteries, the functions of ERK/CaD, MLCK/MLC20, PI3K signal pathways and L type calcium channel decreased by tail-suspension. In abdomen arteries, tail-suspension inhibited the signal of P38 MAPK/Hsp27 pathway.
(4) For the first time, this experiment found microgravity can decrease the VSMC proliferation. Microgravity and simulated microgravity inhibited the protein expression ofβ-catenin and N-Cadherin. Simulated microgravity can decreased the activity of Akt/GSK-3βsignal pathway. The direct reason which decreased the VSMC proliferation was the reduced expression of Cyclin D1, cell cycle retarded at G1 and different expression of P21Cip1. For the first time, we found that microgravity and simulated microgravity had different regulating mode on protein expression of P21 Cip1.
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76. Geary GG, Krause DN, Purdy RE, et al. Simulated microgravity increases myogenic tone in rat cerebral arteries. J Appl Physiol,1998,85(5): 1615–1621
77. Hwang S, Shelkovnikov SA, Purdy RE. Simulated microgravity effects on the rat carotid and femoral arteries: role of contractile protein expression and mechanicalproperties of the vessel wall. J Appl Physiol,2007,102: 1595–1603
78. Dillon, P F, Aksoy, M O, Driska, S P, et al. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science, 1981, 211:495–497.
79. Murphy,RA, Rembold, CM. The latch-bridge hypothesis of smooth muscle contraction. Can J Physiol Pharmacol, 2005, 83(10):857-864
80. D’Angelo G and Adam LP. Inhibition of ERK attenuates force development by lowering myosin light chain phosphorylation. Am J Physiol Heart Circ Physiol,2002,282: H602–H610
81. Morgan, Kathleen G., Samudra S.Gangopadhyay.Invited Review: Cross-bridge regulation by thin filament-associated proteins. J Appl Physiol,2001,91: 953–962
82.Yamboliev, Ilia A., Jason C.Hedges, et al. Evidence for modulation of smooth muscle force by the p38 MAP Kinase/HSP27 pathway. Am J Physiol Heart Circ Physiol, 2000, 278: H1899-H1907
83. Somara, Sita., Khalil N.Bitar. Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle. Am J Physiol Cell Physiol, 2004, 286: C1290-C1301
84.Komalavilas, Padmini, Shyamal Mehta, et al. PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. J Appl Physiol, 2001,91:1819-1827
85.Vasken Ohanian, Kelly Gatfield and Jacqueline Ohanian. Role of the Actin Cytoskeleton in G-Protein–Coupled Receptor Activation of PYK2 and Paxillin in Vascular Smooth Muscle. Hypertension, 2005, 46:93-99
86. Saito SY, Hori M, Ozaki H, et al. Cytochalasin D inhibits smooth muscle contraction by directly inhibiting contractile apparatus. J Smooth Muscle Res, 1996,32(2):51-60
87.张乐宁,毛秦雯,张立藩模拟失重大鼠基底动脉血管壁超微结构的变化中N-cadherin shedding and therebyβ-catenin signaling and vascular smooth muscle cell proliferation. Cardiovascular Research,2009,81(1):178-186
98. Wong, Gail A., Vincent Tang, Faten El-Sabeawy, et al. BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21Cip1/Waf1. Am J Physiol Endocrinol Metab, 2003, 284: E972–E979
99. Mark Bond, Graciela B.Sala-Newby, Yih-Jer Wu,et al. Biphasic effect of P21Cip1 on smooth cell proliferation:role of PI3K and Skp2-mediated degradation. Cardiovascular Research, 2006, 69:198-206
100. Fernando S.Santiago, Hideto Ishii, Shahida Shafi, et al. Yin Yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF1/Cip1 transcription and p21WAF1./Cip1-cdk4-cyclin D1 assembly. Circ Res, 2007,101:146-155
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