《伤寒论》温阳三方干预心梗后心衰心阳虚证候大鼠心室重构的比较研究
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摘要
慢性心力衰竭是多种心血管疾病的最后归宿和主要死亡原因,心室重构是导致心衰发生的基本机制。近年来随着溶栓及介入技术的发展,心梗后心衰患者比例明显上升
中医在治疗心衰等疾病的历史中留下了大量的有效方剂。但此类方剂数量庞大、治法不同、配伍灵活,需辨证论治,为临床选方用药造成了一定困难。方剂研究的核心问题是方剂配伍规律和方证关系,这也是现代中医药研究的重点和难点。随着现代医学对疾病研究的深入和中西医结合研究的进展,越来越多学者赞同,在中医药理论指导下,西医辨病与中医辨证相结合,借助现代医疗检测手段及理论等病证结合的诊疗研究模式是目前较为成熟的中西医结合模式。病证结合的中医证候模型是中西医结合研究的较为合适的切入点。
目的
1、文献研究从源到流梳理温阳法(方)治疗心力衰竭的研究概况。
2、实验研究从病名诊断、证候及病理演变比较经典著作《伤寒论》中温阳三方对心梗后心衰心阳虚证候模型大鼠在一般情况、相关症状、心功能及心室重构等方面的治疗效果。
3、从分子、基因水平观察促纤维化重要分子TGF-β及其与相关因子ACT-A、AngⅡ的相互作用,从而探讨温阳法(方)治疗心梗后心衰心阳虚证候大鼠的机制与实质。
4、通过以方测证对心梗后心衰心阳虚证候模型大鼠进行评价。
5、通过不同温阳方对心梗后心衰心阳虚证候模型大鼠不同疗效的比较,探讨方证相关性与方剂疗效的关系。
方法
1、用手术冠脉左前降支结扎后第9-12周加用左旋硝基精氨酸(L-NNA)腹腔注射的联合造模方法制作心梗后心衰心阳虚证候大鼠,参照西医心力衰竭诊断标准进行疾病评价；以临床心阳虚辨证标准,参考心气虚标准,作为动物模型的中医辨证依据,收集动物客观指标(心功能、心率、体温、力竭游泳时间等),对该模型进行中医证候分析。
2、对上述证候模型大鼠在术后9-12周分别予桂甘龙牡汤、苓桂术甘汤、真武汤每日灌胃治疗,对其进行
(1)证候相关指标及心功能、血流动力学、心指数变化等指标的收集,观察温阳三方对上述证候大鼠疾病与证候干预治疗的作用。
(2)B超、血流动力学检测、心指数等心室重构相关指标收集,HE染色进行病理学形态观察,Mas son染色法进行胶原染色观察胶原沉积情况,从而观察温阳三方对上述证候模型大鼠心室重构的影响。
(3)酶联免疫吸附法(ELISA)观察温阳三方对上述证候模型大鼠血清中ACT-A、AngⅡ、TGF-β1含量,免疫组化法观察温阳三方对模型大鼠局部心肌组织中ACT-A、AngⅡ、TGF-β1表达的影响,从而观察温阳三方对该TGF-β1与相关作用因子蛋白含量的影响。
(4)逆转录-聚合式酶联反应(RT-PCR)法观察各组大鼠心肌组织中ACT-A、AngⅡ、TGF-β1 mRNA含量的影响,从基因水平探讨温阳三方对上述证候模型大鼠治疗的机制。
结果
1.与假手术组相比,模型组大鼠全身一般情况较差,心率增快,体温降低,夜尿增加,力竭游泳时间减少,心功能评价降低,血流动力学±dp/dtmax、LVSP下降、LVEDP上升,心脏指数及左室指数明显上升,符合中西医关于心力衰竭及心阳虚证的诊断标准,说明造模基本成功。
2.与模型组相比,温阳三方治疗组在一般情况,中医证候指标、心功能、血流动力学及左室指数上有所好转,其中真武汤、苓桂术甘汤疗效较佳,桂甘龙牡汤效果次之；存活率方面以苓桂术甘汤为佳,真武汤、桂甘龙牡汤效果较弱。
3.与模型组相比,温阳三方治疗组对室间隔IVS、E峰、E/A比、LVIDd有不同程度改善作用(P<0.01或0.05),苓桂术甘汤、真武汤能明显降低LVIDs、提高SV(P<0.01或0.05),真武汤能提高EF和FS(P<0.05)；苓桂术甘汤、真武汤能明显降低心指数,真武汤还能显著降低左室指数。在病理学变化和胶原纤维沉积方面的变化,苓桂术甘汤、真武汤疗效较好,三方疗效按桂甘龙牡汤、苓桂术甘汤、真武汤依次递增。
4.苓桂术甘汤、真武汤能使模型大鼠血清中ACT-A、AngⅡ、TGF-β1含量降低,使心肌局部组织中ACT-A、AngⅡ、TGF-β1表达含量减少,三方疗效按桂甘龙牡汤、苓桂术甘汤、真武汤依次递增。
5.《伤寒论》温阳方主要通过下调ACT-A、TGF-β1 mRNA基因,上调AT2 mRNA的表达以达到治疗作用。桂苓术甘汤、真武汤的治疗作用较桂甘龙牡汤明显。除在ACT-A mRNA上桂苓术甘汤、真武汤两组在统计上无明显差异外,真武汤组AT2、TGF-β1 mRNA与苓桂术甘汤组相比均差异明显(P<0.01)
结论
1.《伤寒论》温阳三方中改善各项数据指标(心率、体温、尿量、力竭游泳时间、心功能、血流动力学、心指数)疗效最优者多为真武汤,但结合远期生存率,综合考虑疗效最佳者为苓桂术甘汤。
2、《伤寒论》温阳方治疗心梗后心衰心阳虚证,抑制心室重构,主要是通过下调组织中升高的ACT-A、TGF-β1 mRNA,降低ACT-A、TGF-β1表达；继续上调AT2 mRNA,抑制AngⅡ生成和作用而实现的,这也是温阳方治疗心衰阳虚证的作用靶点和物质基础之一
3、温阳三方中真武汤偏重于改善心室收缩功能；苓桂术甘汤偏重于改善胶原沉积,抑制心室重构而提高生存率；桂甘龙牡汤对部分左室形态学指标心功能(E峰、A峰、E/A比)、胶原沉积、心室重构,降低AngⅡ、TGF-β1表达有一定改善作用。
4、三方中苓桂术甘汤与模型大鼠方证相应程度最高,故总体疗效最佳；也证明了中医治疗疾病,方证对应程度高者疗效较佳,反之则较差。
5、实验结果证明了中医注重脾胃理论的合理性,提示真武汤、桂甘龙牡汤治疗慢性心衰不宜长期单独使用,苓桂术甘汤治疗慢性心衰可长期使用。实验也说明了临床上随证加减的必要性。
6、心梗后心衰心阳虚证候大鼠在证候上兼有脾阳虚水停之证,考虑为脾阳虚兼水停,或心脾两虚兼水停。
Chronic heart failure (CHF) is one of the most serious disease-killers. CHF after cardiac infarction is more serious, of which number continued to increase, with development of thrombolysis and interventional techniques. There are a lot of TCM (Traditional Chniese Medicine) effective prescriptions for CHF, which are some diffcult to choose for doctors.
Studies on law of prescriptions compatibility and prescriptions corresponding to syndromes are the core of researches in TCM prescription, which are also hot and difficult problems now. More and more scholars agree that western medicine disease differentiation combined with TCM syndrome differentiation, under the guide of TCM theory, is relatively appropriate mode of integrated traditional Chinese and western medicine at present.
Objective:
The literature research:trace back to the source, summary the ancient and modern literature of warming yang in treating CHF.
The expriment research:observe the therapeutic effect of ventricle remodeling of heart failure post cardiac infarction rats with heart yang deficiency syndrome respectively treated by three warming yang decoctions in shanghanlun, probe into their mechanism, evaluate the rat model of syndromes by using treatment analyze syndrome, explore the significance of Fang-zheng correlation to the prescriptions and their treatment effect.
Methods:
1. Establishing rat model of integrative medical disease/syndrome. Rat myocardial (heart failure) model was established by anterio-descend ing coronary arterial ligation, and treated by nitric oxide synthase inhibitor 9-12 weeks after operation to induce hypertension and aggravate heart failure. The model rat was observed 12 weeks to collect the information of four-diagnosis, for visceral qi-blood and excess-deficiency syndrome differentiation in combining with the eight-principal syndrome differentiation according to the standard of deficiency syndrome issued by Chinese Association of integrative medicine, assess the disease according to the heart failure diagnosis standard of Framingham.
2. The rat models were respectively treated by three warming yang decoctions in ShangHanLun from 9 to 12 weeks after operation. We observe the rat general condition, heart rate, body temperature, urine volume, exhaustive swimming time, mortality, and cardiac function, blood flow dynamics by heart color ultrasound, heart mass index change in every group, observe pathological morphology changes with H-E staining, myocardial collagen content with masson staining. The expression of activin-a(ACT-A), Angiotensin II (AngⅡ), Transforming growth factorβ(TGF-β1)in serum were detected by ELISA, while those in myocardium detected by immunhischemical staining, and gene expression of those by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR).
Results:
1. Compared with the sham operated(SO) group, the rat models were found with lassitude, cachexia, quickened heart rate, shortened swimming time, lowed cardiac function and body temperature,±dp/dtmax、LVSP drop, LVEDP rise, heart index(HI) and left ventricular index index (LVI) increased obviously, and the establishment of rat models are successful.
2. Compared with the model group, the three warming-yang groups displayed better general condition, TCM Syndromes, parameters of haemodynamics, higher cardiac function, lower HI, LVI, IVS in varying degree, and they could promote the heart function, inhibit the left ventricular remodeling. They can decrease the expression of ACT-A, AngⅡ, TGF-β1 in serum and myocardium, through the machanism of upper the expression of AT2(AngiotensinⅡreceptor, type 2) mRNA, lower the expression of ACT-A mRNA, TGF-β1 mRNA. The Zhen-wu decoction, LingGuiShuGan decoction group are more effientive.
Conclusions:
1. The efficacy of three warming-yang decoctions, zhen-wu decoction is the best in the physical and chemical indicators, and LingGuiShuGan decoction is the best in general.
2. The three warming-yang decoctions cured heart failure post cardiac infarction rats with heart yang deficiency syndrome, inhibit Ventricle Remodeling, The effect of warm-yang decoction mainly through upper the expression of AT2(AngiotensinⅡreceptor, type 2) mRNA, lower the expression of ACT-A mRNA, TGF-β1 mRNA, came into effect.
3. The efficacy of three decoctions, from weak to strong is GuiGanLongmu, LingGuiShuGan, Zhen-wu decoction.
4. A decoction meet the regulation of prescriptions corresponding to syndromes, then its can get good efficacy;
5. The research proved the validity of the spleen and stomach theory of TCM and the necessity of revising decoctions with symptoms, the zhen-wu decoction and GuiGanLongmu decoction had better not be long term used alone.
6. The rat model's TCM syndrome type is much more likely to be related to spleen-deficiency and water retention, based on syndrome detecting from recipe used.
中医在治疗心衰等疾病的历史中留下了大量的有效方剂。但此类方剂数量庞大、治法不同、配伍灵活,需辨证论治,为临床选方用药造成了一定困难。方剂研究的核心问题是方剂配伍规律和方证关系,这也是现代中医药研究的重点和难点。随着现代医学对疾病研究的深入和中西医结合研究的进展,越来越多学者赞同,在中医药理论指导下,西医辨病与中医辨证相结合,借助现代医疗检测手段及理论等病证结合的诊疗研究模式是目前较为成熟的中西医结合模式。病证结合的中医证候模型是中西医结合研究的较为合适的切入点。
目的
1、文献研究从源到流梳理温阳法(方)治疗心力衰竭的研究概况。
2、实验研究从病名诊断、证候及病理演变比较经典著作《伤寒论》中温阳三方对心梗后心衰心阳虚证候模型大鼠在一般情况、相关症状、心功能及心室重构等方面的治疗效果。
3、从分子、基因水平观察促纤维化重要分子TGF-β及其与相关因子ACT-A、AngⅡ的相互作用,从而探讨温阳法(方)治疗心梗后心衰心阳虚证候大鼠的机制与实质。
4、通过以方测证对心梗后心衰心阳虚证候模型大鼠进行评价。
5、通过不同温阳方对心梗后心衰心阳虚证候模型大鼠不同疗效的比较,探讨方证相关性与方剂疗效的关系。
方法
1、用手术冠脉左前降支结扎后第9-12周加用左旋硝基精氨酸(L-NNA)腹腔注射的联合造模方法制作心梗后心衰心阳虚证候大鼠,参照西医心力衰竭诊断标准进行疾病评价；以临床心阳虚辨证标准,参考心气虚标准,作为动物模型的中医辨证依据,收集动物客观指标(心功能、心率、体温、力竭游泳时间等),对该模型进行中医证候分析。
2、对上述证候模型大鼠在术后9-12周分别予桂甘龙牡汤、苓桂术甘汤、真武汤每日灌胃治疗,对其进行
(1)证候相关指标及心功能、血流动力学、心指数变化等指标的收集,观察温阳三方对上述证候大鼠疾病与证候干预治疗的作用。
(2)B超、血流动力学检测、心指数等心室重构相关指标收集,HE染色进行病理学形态观察,Mas son染色法进行胶原染色观察胶原沉积情况,从而观察温阳三方对上述证候模型大鼠心室重构的影响。
(3)酶联免疫吸附法(ELISA)观察温阳三方对上述证候模型大鼠血清中ACT-A、AngⅡ、TGF-β1含量,免疫组化法观察温阳三方对模型大鼠局部心肌组织中ACT-A、AngⅡ、TGF-β1表达的影响,从而观察温阳三方对该TGF-β1与相关作用因子蛋白含量的影响。
(4)逆转录-聚合式酶联反应(RT-PCR)法观察各组大鼠心肌组织中ACT-A、AngⅡ、TGF-β1 mRNA含量的影响,从基因水平探讨温阳三方对上述证候模型大鼠治疗的机制。
结果
1.与假手术组相比,模型组大鼠全身一般情况较差,心率增快,体温降低,夜尿增加,力竭游泳时间减少,心功能评价降低,血流动力学±dp/dtmax、LVSP下降、LVEDP上升,心脏指数及左室指数明显上升,符合中西医关于心力衰竭及心阳虚证的诊断标准,说明造模基本成功。
2.与模型组相比,温阳三方治疗组在一般情况,中医证候指标、心功能、血流动力学及左室指数上有所好转,其中真武汤、苓桂术甘汤疗效较佳,桂甘龙牡汤效果次之；存活率方面以苓桂术甘汤为佳,真武汤、桂甘龙牡汤效果较弱。
3.与模型组相比,温阳三方治疗组对室间隔IVS、E峰、E/A比、LVIDd有不同程度改善作用(P<0.01或0.05),苓桂术甘汤、真武汤能明显降低LVIDs、提高SV(P<0.01或0.05),真武汤能提高EF和FS(P<0.05)；苓桂术甘汤、真武汤能明显降低心指数,真武汤还能显著降低左室指数。在病理学变化和胶原纤维沉积方面的变化,苓桂术甘汤、真武汤疗效较好,三方疗效按桂甘龙牡汤、苓桂术甘汤、真武汤依次递增。
4.苓桂术甘汤、真武汤能使模型大鼠血清中ACT-A、AngⅡ、TGF-β1含量降低,使心肌局部组织中ACT-A、AngⅡ、TGF-β1表达含量减少,三方疗效按桂甘龙牡汤、苓桂术甘汤、真武汤依次递增。
5.《伤寒论》温阳方主要通过下调ACT-A、TGF-β1 mRNA基因,上调AT2 mRNA的表达以达到治疗作用。桂苓术甘汤、真武汤的治疗作用较桂甘龙牡汤明显。除在ACT-A mRNA上桂苓术甘汤、真武汤两组在统计上无明显差异外,真武汤组AT2、TGF-β1 mRNA与苓桂术甘汤组相比均差异明显(P<0.01)
结论
1.《伤寒论》温阳三方中改善各项数据指标(心率、体温、尿量、力竭游泳时间、心功能、血流动力学、心指数)疗效最优者多为真武汤,但结合远期生存率,综合考虑疗效最佳者为苓桂术甘汤。
2、《伤寒论》温阳方治疗心梗后心衰心阳虚证,抑制心室重构,主要是通过下调组织中升高的ACT-A、TGF-β1 mRNA,降低ACT-A、TGF-β1表达；继续上调AT2 mRNA,抑制AngⅡ生成和作用而实现的,这也是温阳方治疗心衰阳虚证的作用靶点和物质基础之一
3、温阳三方中真武汤偏重于改善心室收缩功能；苓桂术甘汤偏重于改善胶原沉积,抑制心室重构而提高生存率；桂甘龙牡汤对部分左室形态学指标心功能(E峰、A峰、E/A比)、胶原沉积、心室重构,降低AngⅡ、TGF-β1表达有一定改善作用。
4、三方中苓桂术甘汤与模型大鼠方证相应程度最高,故总体疗效最佳；也证明了中医治疗疾病,方证对应程度高者疗效较佳,反之则较差。
5、实验结果证明了中医注重脾胃理论的合理性,提示真武汤、桂甘龙牡汤治疗慢性心衰不宜长期单独使用,苓桂术甘汤治疗慢性心衰可长期使用。实验也说明了临床上随证加减的必要性。
6、心梗后心衰心阳虚证候大鼠在证候上兼有脾阳虚水停之证,考虑为脾阳虚兼水停,或心脾两虚兼水停。
Chronic heart failure (CHF) is one of the most serious disease-killers. CHF after cardiac infarction is more serious, of which number continued to increase, with development of thrombolysis and interventional techniques. There are a lot of TCM (Traditional Chniese Medicine) effective prescriptions for CHF, which are some diffcult to choose for doctors.
Studies on law of prescriptions compatibility and prescriptions corresponding to syndromes are the core of researches in TCM prescription, which are also hot and difficult problems now. More and more scholars agree that western medicine disease differentiation combined with TCM syndrome differentiation, under the guide of TCM theory, is relatively appropriate mode of integrated traditional Chinese and western medicine at present.
Objective:
The literature research:trace back to the source, summary the ancient and modern literature of warming yang in treating CHF.
The expriment research:observe the therapeutic effect of ventricle remodeling of heart failure post cardiac infarction rats with heart yang deficiency syndrome respectively treated by three warming yang decoctions in shanghanlun, probe into their mechanism, evaluate the rat model of syndromes by using treatment analyze syndrome, explore the significance of Fang-zheng correlation to the prescriptions and their treatment effect.
Methods:
1. Establishing rat model of integrative medical disease/syndrome. Rat myocardial (heart failure) model was established by anterio-descend ing coronary arterial ligation, and treated by nitric oxide synthase inhibitor 9-12 weeks after operation to induce hypertension and aggravate heart failure. The model rat was observed 12 weeks to collect the information of four-diagnosis, for visceral qi-blood and excess-deficiency syndrome differentiation in combining with the eight-principal syndrome differentiation according to the standard of deficiency syndrome issued by Chinese Association of integrative medicine, assess the disease according to the heart failure diagnosis standard of Framingham.
2. The rat models were respectively treated by three warming yang decoctions in ShangHanLun from 9 to 12 weeks after operation. We observe the rat general condition, heart rate, body temperature, urine volume, exhaustive swimming time, mortality, and cardiac function, blood flow dynamics by heart color ultrasound, heart mass index change in every group, observe pathological morphology changes with H-E staining, myocardial collagen content with masson staining. The expression of activin-a(ACT-A), Angiotensin II (AngⅡ), Transforming growth factorβ(TGF-β1)in serum were detected by ELISA, while those in myocardium detected by immunhischemical staining, and gene expression of those by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR).
Results:
1. Compared with the sham operated(SO) group, the rat models were found with lassitude, cachexia, quickened heart rate, shortened swimming time, lowed cardiac function and body temperature,±dp/dtmax、LVSP drop, LVEDP rise, heart index(HI) and left ventricular index index (LVI) increased obviously, and the establishment of rat models are successful.
2. Compared with the model group, the three warming-yang groups displayed better general condition, TCM Syndromes, parameters of haemodynamics, higher cardiac function, lower HI, LVI, IVS in varying degree, and they could promote the heart function, inhibit the left ventricular remodeling. They can decrease the expression of ACT-A, AngⅡ, TGF-β1 in serum and myocardium, through the machanism of upper the expression of AT2(AngiotensinⅡreceptor, type 2) mRNA, lower the expression of ACT-A mRNA, TGF-β1 mRNA. The Zhen-wu decoction, LingGuiShuGan decoction group are more effientive.
Conclusions:
1. The efficacy of three warming-yang decoctions, zhen-wu decoction is the best in the physical and chemical indicators, and LingGuiShuGan decoction is the best in general.
2. The three warming-yang decoctions cured heart failure post cardiac infarction rats with heart yang deficiency syndrome, inhibit Ventricle Remodeling, The effect of warm-yang decoction mainly through upper the expression of AT2(AngiotensinⅡreceptor, type 2) mRNA, lower the expression of ACT-A mRNA, TGF-β1 mRNA, came into effect.
3. The efficacy of three decoctions, from weak to strong is GuiGanLongmu, LingGuiShuGan, Zhen-wu decoction.
4. A decoction meet the regulation of prescriptions corresponding to syndromes, then its can get good efficacy;
5. The research proved the validity of the spleen and stomach theory of TCM and the necessity of revising decoctions with symptoms, the zhen-wu decoction and GuiGanLongmu decoction had better not be long term used alone.
6. The rat model's TCM syndrome type is much more likely to be related to spleen-deficiency and water retention, based on syndrome detecting from recipe used.
引文
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