从“心主血脉”、“气血相关”探讨温脉通治疗早期肢体动脉硬化闭塞症的作用机制
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摘要
本研究在传统中医理论的基础上,结合现代医学研究成果对“心主血脉”、“气血相关”进行了从宏观到微观新的诠释。认为“心主血脉”体现了心血管系统中的心脏泵血的机械作用和血管调节功能以及心血管系统对机体的整体调节作用,“气血相关”体现了神经内分泌对心血管系统的综合调控作用。因此指出“血脉”病变是心血管系统自身和神经内分泌网络调控失常所致,应从“心”和“气”着手进行综合调治。笔者以肢体动脉硬化闭塞症(ASO)为例,运用上述理论分析其病因病机以确立治则治法。通过“温脉通”治疗 ASO 的实验研究以方示理,进一步证实“血脉”病变与“心”和“气”的相关性,以及从“心”和“气”治疗 ASO 的合理性。
温脉通是陈淑长教授治疗早期 ASO 的有效方剂,由桂枝、黄芪、当归、川芎等组成,具有温阳益气、活血通脉作用。本实验研究应用多种检测手段和方法从整体器官水平和细胞分子水平探讨了该方防治 ASO、抑制血管平滑肌细胞(VSMC)增殖和迁移作用及其机制。
整体动物实验分为正常对照组、ASO 模型组、温脉通治疗组。主要观察温脉通对实验性 ASO 家兔的治疗作用,并初步探讨其作用机理。
体外细胞实验则以体外培养的兔主动脉 VSMC 为研究对象,应用血清药理学研究方法,以血管紧张素Ⅱ(AngⅡ)为诱导因素,随机将生长良好的第 4～5 代 VSMC 分为正常对照组、AngⅡ组、温脉通高剂量组、温脉通低剂量组、温阳益气拆方组、活血通脉拆方组。从对细胞数量、细胞周期、细胞代谢产物、细胞因子蛋白表达等方面进行检测,观察温脉通对 VSMC 增殖、迁移及相关因素的影响,以阐明该方防治 ASO 的细胞生物学基础。并通过温脉通全方与温阳益气拆方和活血通脉拆方的比较,证明其立法组方的科学性、合理性。
实验结果如下:
1.温脉通具有抗家兔实验性 ASO 的作用,此作用与抑制 VSMC 增殖、诱导 VSMC 凋亡有关。
形态学检查显示,ASO 家兔动脉管腔狭窄,内膜增厚,中膜 VSMC 明显增殖,排列紊乱,部分迁移至内膜;温脉通组血管内膜表面光滑,中膜无明显增厚,VSMC 排列整齐,血管壁较模型组明显变薄。治疗组细胞增殖核抗原(PCNA)阳性细胞数明显低于正常组和模型组,而凋亡细胞数高于两组。表明该方通过调整细胞增殖速率与凋亡速率之间的动态平衡,减少内膜过度增生,减缓 AS 进程,从而发挥防治 ASO 作用。
2.温脉通具有抑制 AngⅡ诱导 VSMC 增殖和迁移的作用
MTT 法观察证实,治疗组呈剂量依赖性地抑制 AngⅡ诱导的 VSMC 增殖,而且全方较拆方明显。通过显微镜下测微尺测量观察到温脉通具有对抗 AngⅡ促 VSMC 迁移作用,以 48 h 作用明显。
3.温脉通抑制 VSMC 增殖作用与调控细胞周期及表型转化有关
应用流式细胞仪及免疫组化染色技术检测发现AngⅡ可增加细胞周期中S期、G2/M期
This study is based on the theory of Traditional Chinese Medicine (TCM), combinedwith the results of Modern Medicine on “heart governs the blood” and “qi correlates blood”which gives a new explanation from microcosmic to macroscopic. The conception of “heartgoverns the blood” shows the mechanic effects of the heart pump in cardiovascular system.On the other hand, “qi correlates blood” shows the modulation of neruoendocrine incardiovascular system. The abnormal controlling of neuroendocrine system andcardiovascular system leads to the pathological changes of meridian, so that the treatment isbased on regulation of “qi and xue”. According to the theory, we analyzed the cause andpathogenesis of ASO so as to gave the principle of the treatment and the therapeutic method.This experimental study of “WENMAITONG” which is treating on ASO proved thecorrelation between the meridian disease and “heart and qi”, as well as the rationality of thetreatment by “heart and qi”.
“WENMAITONG” is an effective formula to treat early ASO which is summed up byProf. Chen Shouchang. It includes Cassia twig, Membranous milkvetch root, Chineseangelica, Szechwan lovage rhizome. The role of it is war ming Yang and boosting qi,activating the blood circulation and invigorating the meridian. This study applied many kindsof methods to evaluate the mechanism of “WENMAITONG” in preventing ASO andinhibiting the proliferation and migration of VSMC on the levels of entirety and cell.
These rabbits were randomly divided into normal control group, ASO model group and“WENMAITONG” treatment group. The therapeutical effects of “WENMAITONG” wereobserved and the mechanisms were explored in this study.
The objective of this study is the VSMC of rabbit’s aorta by incubation in vitro. Theserum-pharmacological method was applied. The Ang II was to be the induced factor. The eraIV-V VSMC were randomly divided into normal control group, Ang II group,“WENMAITONG” high dosage group, “WENMAITONG” low dosage group, war mingYang and boosting qi group, quicken the blood and free the meridian group. The followingfactors were detected: cell number, cell circle, cell metabolite, protein expression of cytokine.This study clarified the cell biological basement of “WENMAITONG” in prevention andcure ASO from observing its influence on the proliferation and immigration of VSMC.Meanwhile, the science and rationality of this formula were proved by comparing the“WENMAITONG” whole formula with war ming Yang and boosting qi part formula, andquicken the blood and free the meridian part formula.
Results:
1. “WENMAITONG” has the effects on experimental anti ASO which related to inhibit
proliferation and induct apoptosis of VSMC.
The morphological experiments showed that the arterial canal changed narrow,membrane were proliferated and arrangements were disorder, some were moved under theendothelium in ASO rabbits. After using “WENMAITONG”, the inner membrane of theblood vessel were smoothly, middle membrane were no obvious thick, the arrangement ofVSMC was in good order, vessel wall was thinner than model group. The positive cellnumber of PCNA in the treatment group was lower than that of normal control group andmodel group, but the cell number of the apoptosis was double than that of the other twogroups. The effects of this formula to prevention and cure ASO were regulating the balancebetween the rates of speed for cell proliferation and speed for apoptosis, reducing theproliferation of the inner membrane as well as decreasing the procedure of atherosclerosis.
2. “WENMAITONG” has the effects on inhibiting proliferation and immigration ofVSMC which induced by Ang II
The method of MTT shows that the treatment group could inhibit the proliferation ofVSMC which induced by Ang II according to the dosage. The effects of the whole formulawere better than the part of this formula. The effects of “WENMAITONG” were opposingAng II and promoting immigration of VSMC and the effects was obvious in 48 hours.
3. “WENMAITONG” restrain the proliferat
温脉通是陈淑长教授治疗早期 ASO 的有效方剂,由桂枝、黄芪、当归、川芎等组成,具有温阳益气、活血通脉作用。本实验研究应用多种检测手段和方法从整体器官水平和细胞分子水平探讨了该方防治 ASO、抑制血管平滑肌细胞(VSMC)增殖和迁移作用及其机制。
整体动物实验分为正常对照组、ASO 模型组、温脉通治疗组。主要观察温脉通对实验性 ASO 家兔的治疗作用,并初步探讨其作用机理。
体外细胞实验则以体外培养的兔主动脉 VSMC 为研究对象,应用血清药理学研究方法,以血管紧张素Ⅱ(AngⅡ)为诱导因素,随机将生长良好的第 4～5 代 VSMC 分为正常对照组、AngⅡ组、温脉通高剂量组、温脉通低剂量组、温阳益气拆方组、活血通脉拆方组。从对细胞数量、细胞周期、细胞代谢产物、细胞因子蛋白表达等方面进行检测,观察温脉通对 VSMC 增殖、迁移及相关因素的影响,以阐明该方防治 ASO 的细胞生物学基础。并通过温脉通全方与温阳益气拆方和活血通脉拆方的比较,证明其立法组方的科学性、合理性。
实验结果如下:
1.温脉通具有抗家兔实验性 ASO 的作用,此作用与抑制 VSMC 增殖、诱导 VSMC 凋亡有关。
形态学检查显示,ASO 家兔动脉管腔狭窄,内膜增厚,中膜 VSMC 明显增殖,排列紊乱,部分迁移至内膜;温脉通组血管内膜表面光滑,中膜无明显增厚,VSMC 排列整齐,血管壁较模型组明显变薄。治疗组细胞增殖核抗原(PCNA)阳性细胞数明显低于正常组和模型组,而凋亡细胞数高于两组。表明该方通过调整细胞增殖速率与凋亡速率之间的动态平衡,减少内膜过度增生,减缓 AS 进程,从而发挥防治 ASO 作用。
2.温脉通具有抑制 AngⅡ诱导 VSMC 增殖和迁移的作用
MTT 法观察证实,治疗组呈剂量依赖性地抑制 AngⅡ诱导的 VSMC 增殖,而且全方较拆方明显。通过显微镜下测微尺测量观察到温脉通具有对抗 AngⅡ促 VSMC 迁移作用,以 48 h 作用明显。
3.温脉通抑制 VSMC 增殖作用与调控细胞周期及表型转化有关
应用流式细胞仪及免疫组化染色技术检测发现AngⅡ可增加细胞周期中S期、G2/M期
This study is based on the theory of Traditional Chinese Medicine (TCM), combinedwith the results of Modern Medicine on “heart governs the blood” and “qi correlates blood”which gives a new explanation from microcosmic to macroscopic. The conception of “heartgoverns the blood” shows the mechanic effects of the heart pump in cardiovascular system.On the other hand, “qi correlates blood” shows the modulation of neruoendocrine incardiovascular system. The abnormal controlling of neuroendocrine system andcardiovascular system leads to the pathological changes of meridian, so that the treatment isbased on regulation of “qi and xue”. According to the theory, we analyzed the cause andpathogenesis of ASO so as to gave the principle of the treatment and the therapeutic method.This experimental study of “WENMAITONG” which is treating on ASO proved thecorrelation between the meridian disease and “heart and qi”, as well as the rationality of thetreatment by “heart and qi”.
“WENMAITONG” is an effective formula to treat early ASO which is summed up byProf. Chen Shouchang. It includes Cassia twig, Membranous milkvetch root, Chineseangelica, Szechwan lovage rhizome. The role of it is war ming Yang and boosting qi,activating the blood circulation and invigorating the meridian. This study applied many kindsof methods to evaluate the mechanism of “WENMAITONG” in preventing ASO andinhibiting the proliferation and migration of VSMC on the levels of entirety and cell.
These rabbits were randomly divided into normal control group, ASO model group and“WENMAITONG” treatment group. The therapeutical effects of “WENMAITONG” wereobserved and the mechanisms were explored in this study.
The objective of this study is the VSMC of rabbit’s aorta by incubation in vitro. Theserum-pharmacological method was applied. The Ang II was to be the induced factor. The eraIV-V VSMC were randomly divided into normal control group, Ang II group,“WENMAITONG” high dosage group, “WENMAITONG” low dosage group, war mingYang and boosting qi group, quicken the blood and free the meridian group. The followingfactors were detected: cell number, cell circle, cell metabolite, protein expression of cytokine.This study clarified the cell biological basement of “WENMAITONG” in prevention andcure ASO from observing its influence on the proliferation and immigration of VSMC.Meanwhile, the science and rationality of this formula were proved by comparing the“WENMAITONG” whole formula with war ming Yang and boosting qi part formula, andquicken the blood and free the meridian part formula.
Results:
1. “WENMAITONG” has the effects on experimental anti ASO which related to inhibit
proliferation and induct apoptosis of VSMC.
The morphological experiments showed that the arterial canal changed narrow,membrane were proliferated and arrangements were disorder, some were moved under theendothelium in ASO rabbits. After using “WENMAITONG”, the inner membrane of theblood vessel were smoothly, middle membrane were no obvious thick, the arrangement ofVSMC was in good order, vessel wall was thinner than model group. The positive cellnumber of PCNA in the treatment group was lower than that of normal control group andmodel group, but the cell number of the apoptosis was double than that of the other twogroups. The effects of this formula to prevention and cure ASO were regulating the balancebetween the rates of speed for cell proliferation and speed for apoptosis, reducing theproliferation of the inner membrane as well as decreasing the procedure of atherosclerosis.
2. “WENMAITONG” has the effects on inhibiting proliferation and immigration ofVSMC which induced by Ang II
The method of MTT shows that the treatment group could inhibit the proliferation ofVSMC which induced by Ang II according to the dosage. The effects of the whole formulawere better than the part of this formula. The effects of “WENMAITONG” were opposingAng II and promoting immigration of VSMC and the effects was obvious in 48 hours.
3. “WENMAITONG” restrain the proliferat
引文
1. 徐淑云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,2002:202～203
2. 司徒镇强,吴军正.细胞培养.西安:世界图书出版公司,1996:68-71
3. Sarkar R,Meinberg EG,Stanley JC,et al.Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cell [J].Circ Res,1996,78:225—230
4. 崔晓兰.中药药理研究的新思路-中药血清药理学.中国中医药科技,1997,4(4): 239
5. Weir MR,Diau VJ.The rennin-angiotensin-aldosterone system:a specific target for hypertension management[J].Am J Hypertens,1999,12(12):205-213
6. Mondrof UF,Geiger H,Herrero M,et al.Involvement of the platelet derived growth factor receptor in angiotensinⅡinduced adtivation of extracellular regulated dinases l and 2 in human mesangial cells[J].FEBSLett,2000,472(1):129-132
7. 6.Thomas WG.Regulation of angiotensin Ⅱ type Ι (AT1) receptor function [J].Regul Pept,1999,79(1):9-23
8. Pramod T Jain,Benjam in F Trump.Human breast cancer cell growth inhibition and deregulation of [Ca2+]i by estradiol.Anti-Cancer Drugs,1997,8:283-287
9. 景涛,何国祥,刘建平,等.大鼠血管平滑肌细胞迁移能力变化与血管紧张素Ⅱ的关系.高血压杂志,2001,9(3):247-249
10. 宋润兰,阮英茆.黄芪甙对体外缺氧培养的人动脉平滑肌细胞增殖和胶原分泌的影响.中国中西医结合杂志,1997,17(7):206-207
11. 李自成,李庚山,黄从新,等.当归提取液对培养的兔主动脉平滑肌细胞增殖的影响.湖北医科大学学报,1997,18(1):26-28
12. 李自成,常青.当归提取液对兔主动脉平滑肌细胞中增殖细胞核抗原表达的影响.暨南大学学报(医学版),1999,20(4):30-33
13. 李琦,温进坤,韩梅.黄芪和当归对血管平滑肌细胞表型标志基因表达和细胞增殖的影响.中国动脉硬化杂志,2004,12(2):147-150
14. 刘虹彬,温进坤,韩梅.丹参对血管平滑肌细胞基质金属蛋白酶和骨桥蛋白基因表达及细胞增殖的影响.中国中西医结合杂志,2002,22(10):764-766
15. 杜先华,杨芳炬,龚应霞,等.丹参对过氧化氢诱导的大鼠血管平滑肌细胞迁移的影响研究.中医药学刊,2003,21(1):124-125
16. 王虹,高秀梅,张伯礼,等.丹参不同组分对大鼠血管平滑肌细胞增殖的影响.天津中医药,2004,21(3):231-233
17. 罗红琳,袁淑兰,肖静,等.川芎嗪抗离体培养动脉平滑肌细胞增殖作用的实验研究.华西医学杂志,1997,12(1):6-7
2. 司徒镇强,吴军正.细胞培养.西安:世界图书出版公司,1996:68-71
3. Sarkar R,Meinberg EG,Stanley JC,et al.Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cell [J].Circ Res,1996,78:225—230
4. 崔晓兰.中药药理研究的新思路-中药血清药理学.中国中医药科技,1997,4(4): 239
5. Weir MR,Diau VJ.The rennin-angiotensin-aldosterone system:a specific target for hypertension management[J].Am J Hypertens,1999,12(12):205-213
6. Mondrof UF,Geiger H,Herrero M,et al.Involvement of the platelet derived growth factor receptor in angiotensinⅡinduced adtivation of extracellular regulated dinases l and 2 in human mesangial cells[J].FEBSLett,2000,472(1):129-132
7. 6.Thomas WG.Regulation of angiotensin Ⅱ type Ι (AT1) receptor function [J].Regul Pept,1999,79(1):9-23
8. Pramod T Jain,Benjam in F Trump.Human breast cancer cell growth inhibition and deregulation of [Ca2+]i by estradiol.Anti-Cancer Drugs,1997,8:283-287
9. 景涛,何国祥,刘建平,等.大鼠血管平滑肌细胞迁移能力变化与血管紧张素Ⅱ的关系.高血压杂志,2001,9(3):247-249
10. 宋润兰,阮英茆.黄芪甙对体外缺氧培养的人动脉平滑肌细胞增殖和胶原分泌的影响.中国中西医结合杂志,1997,17(7):206-207
11. 李自成,李庚山,黄从新,等.当归提取液对培养的兔主动脉平滑肌细胞增殖的影响.湖北医科大学学报,1997,18(1):26-28
12. 李自成,常青.当归提取液对兔主动脉平滑肌细胞中增殖细胞核抗原表达的影响.暨南大学学报(医学版),1999,20(4):30-33
13. 李琦,温进坤,韩梅.黄芪和当归对血管平滑肌细胞表型标志基因表达和细胞增殖的影响.中国动脉硬化杂志,2004,12(2):147-150
14. 刘虹彬,温进坤,韩梅.丹参对血管平滑肌细胞基质金属蛋白酶和骨桥蛋白基因表达及细胞增殖的影响.中国中西医结合杂志,2002,22(10):764-766
15. 杜先华,杨芳炬,龚应霞,等.丹参对过氧化氢诱导的大鼠血管平滑肌细胞迁移的影响研究.中医药学刊,2003,21(1):124-125
16. 王虹,高秀梅,张伯礼,等.丹参不同组分对大鼠血管平滑肌细胞增殖的影响.天津中医药,2004,21(3):231-233
17. 罗红琳,袁淑兰,肖静,等.川芎嗪抗离体培养动脉平滑肌细胞增殖作用的实验研究.华西医学杂志,1997,12(1):6-7