从津液代谢角度探讨肺与大肠相表里的理论和实验研究
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摘要
背景
脏腑表里相合理论是中医藏象理论的重要组成部分之一,它强调生理状态下相表里的脏腑之间相互协调、相互制约,病理状态下各脏腑器官之间相互影响、相互传变,治疗中注意“既病防变”,协调相关脏腑功能,恢复脏腑之间阴阳平衡、气血调和。“肺与大肠相表里”是脏腑表里相合理论代表性内容,肺与大肠的相互关系,不仅通过经脉的络属得以体现,同时在生理、病理上形成了一种密不可分的依赖关系,而且在长期的临床实践中得到充分证实。但肺与大肠的结构基础是否具有同源性,活性物质是否具有特异性,调控机制如何发挥作用,这些问题目前均没有满意的答案。科学地解释以上问题,揭示肺与大肠相关性的生物学基础,阐明肺与大肠的内在联系和协调机制,能够更好地指导临床应用。
目的
在“肺与大肠相表里”中医理论指导下,遵循中医病因病机理论,分析津亏肠燥,损伤肺阴的病理过程,并建立肠燥伤肺大鼠模型,以检测“肺系”、“肠系”生理功能为主要切入点,选择反映津亏对肺、大肠生理功能影响的相关指标,并运用增液承气汤对该大鼠模型进行干预,旨在初步阐明肺与大肠生理和病理相关性的生物学基础,揭示在生理和病理状态下肺与大肠相互影响的内在联系和协调机制。
方法
1理论探讨
以“肺与大肠相表里”理论为基础,通过分析津液在肺与大肠生理功能正常发挥的重要作用及津液代谢失常对肺与大肠生理功能的影响,并从津液亏虚导致肠燥便秘、腑气不通,进而引起肺生理功能失常的角度,探讨肺与大肠的内在协调机制。
2实验研究
(1)建立大鼠模型：SD大鼠64只,200±20g,雌雄各半,随机分为4组,即空白组、限水模型组、复方地芬诺酯组、限水+复方地芬诺酯模型组,每组16只。以限水、灌胃复方地芬诺酯、限水+灌胃复方地芬诺酯构建不同的便秘大鼠模型,观察大鼠大便改变情况,并分别在造模第7天、第14天检测肺与大肠含水量,观察肺与大肠病理切片,成功建立津亏肠燥便秘大鼠模型,作为后续研究的基础。
(2)肺与大肠相互影响的内在联系和协调机制研究：SD大鼠60只,200±2 Og,雌雄各半,随机分为5组,即空白组、模型组、增液承气汤组、增液汤组、西药组,每组12只。大鼠限水并灌胃复方地芬诺酯构建津亏肠燥大鼠模型,并运用增液承气汤和增液汤对该大鼠模型干预,给药7天后,观察大鼠大便改变情况、肺与大肠含水量、肺与大肠病理切片、肺与肠组织水通道蛋白1、3的表达及肺、大肠组织中的VIP、P物质的改变。
结果
1建立模型试验
(1)大便质地及排便通畅程度观察
限水组、限水并给药组大鼠大便量减少,粪粒变硬变干,粒形缩短,排出缓慢费力。给药组大鼠大部分粪粒变硬,粪粒表面变干,偶见软而不成形粪便。空白组便质及排便未见异常。
(2)组织病理学观察
光镜下,空白组、给药组、限水组和限水+给药组第7天气管和纤毛未见明显缺损,肺泡未见明显异常,结肠组织结构无明显异常,绒毛丰富。限水组第14天见肺泡扩张,肺间质变宽,支气管有少量炎性细胞浸润。结肠组织结构正常,无明显病变。限水+给药组第14天肺间质增宽,细支气管内炎性细胞浸润,肺泡内少量水肿液形成。结肠肠绒毛,变短,不齐,固有层变薄,嗜酸性粒细胞浸润。
电镜下,空白组、给药组第7天和第14天肺和结肠组织细胞结构正常。限水组第7天肺和结肠组织细胞结构无明显改变,第14天肺胞浆中有板层小体脱落,肠组织绒毛不齐。限水组+给药组第7天肺和结肠组织细胞结构无明显改变,第14天肺泡腔中有大量板层小体脱落。肠组织绒毛变稀,不齐。
(3)排便时间及粒数观察
造模7天和14天时,与空白组相比,限水模型组和限水+给药模型组的首次排便时间延长(P<0.01),排便粒数减少(P<0.01),给药模型组首次排便时间、排便粒数无统计学意义(P>0.05)；到14天时,给药模型组首次排便时间延长、排便粒数减少(P<0.05)；与限水模型组相比,限水+给药模型组7天时首便时间、排便粒数无统计学意义(P>0.05)。到14天时,排便粒数减少(P<0.05)。
(4)便秘大鼠排便重量、粪便含水率检测
造模7天时和14天时,与空白组比,限水模型组和限水+给药模型组的粪便含水率降低(P<0.01),给药模型组粪便含水率无统计学意义(P>0.05)；与限水模型组比,限水+给药模型组粪便含水率无统计学意义(P>0.05)。造模14天,与限水模型组相比,限水+给药模型组含水率较低(P<0.05)。
(5)肺组织、肠组织含水量检测
造模7天和14天时,限水组和限水+给药组大鼠的肺组织含水率与空白组比较均有显著差异(p<0.01),给药组与空白组相比无统计学意义(p>0.05)；限水组和限水+给药组大鼠的肺组织含水率对比无统计学意义(p>0.05)。到14天时,限水组和限水+给药组大鼠的肺组织含水率对比有差异(p<0.05)。大肠组织含水量造模7天和14天时,各组结果与肺组织趋势一致。
2肺与大肠相互影响的内在联系和协调机制研究
(1)大便质地及排便通畅程度观察：模型组大便量减少,粪粒干粒形缩短,排出缓慢费力；增承组粪粒数较模型组多,粒形缩短,有时见粪便较软。增液组和西药组粪粒较硬,粒形短,排出费力；空白组便质及排便未见明显异常。
(2)肺、大肠组织病理观察
肺组织病理切片示：空白组肺泡均匀,肺间质适中。模型组肺泡扩张、水肿,肺间质增宽。西药组肺泡扩张,有大量中性粒细胞浸润,肺间质增宽。增液组肺泡扩张,有少量中性粒细胞聚集。增承组肺泡均匀,肺间质增宽,有少量中性粒细胞聚集。
大肠组织病理切片示：空白组大鼠结肠组织结构正常,绒毛丰富,排列整齐。模型组肠绒毛变短倒伏,排列不齐,固有层变薄,嗜酸性粒细胞浸润。增液组、西药组结肠固有层内均可见大量嗜酸性粒细胞、淋巴细胞。增承组可见少量嗜酸性粒细胞。
(3)大鼠排便变化检测
与空白组相比,模型组的首次排便时间延长(P<0.01),排便粒数减少(P<0.01)；与模型组相比,西药组和增液组首便时间缩短(P<0.05),排便粒数无明显统计学意义(P>0.05),增承组首便时间缩短(P<0.01),排便粒数增加(P<0.05)。模型组粪便较空白组含水率降低(P<0.01),粪便较为干硬；与模型组比,西药组、增液组粪便含水率无统计学意义(P>0.05),增承组粪便含水率增加(P<0.05)。
(4)肺组织、大肠组织含水率检测
模型组肺组织、大肠组织含水率与空白组对比减少,有统计学差异(p<0.01),提示造模成功。与模型组比,肺组织含水率增承组升高(p<0.01),西药组、增液组与模型组相比无统计学意义(p>0.05)；大肠组织含水量增承组、增液组与模型组比较均有显著差异(p<0.01),西药组与模型组相比无统计学意义(p>0.05)。
(5)水通道蛋白(AQP1、AQP3)含量检测
与空白组相比,模型组肺组织AQP1降低(P<0.01)；与模型组比,西药组、增液组肺组织AQP1无统计学意义(P>0.05),增承组肺组织AQP1增加(P<0.05)。与空白组相比,模型组肠组织AQP3降低(P<0.01)；与模型组比,增液组肠组织AQP3增加(P<0.05)。增承组肠组织AQP3增加更加明显(P<0.01)。西药组肠组织AQP3无统计学意义(P>0.05)。
(6)肺、大肠组织VIP、P物质检测
与空白组相比,模型组大鼠肠组织中VIP和SP含量降低(P<0.01)；与模型组相比,增液组VIP和SP含量升高(P<0.05),增承组VIP和SP含量升高(P<0.01),西药组与模型组比较无统计意义(P>0.05)。与空白组相比,模型组大鼠肺组织中VIP含量降低(P<0.01)；与模型组比,增承组VIP含量升高(P<0.05),增液组、西药组VIP含量与模型组比无显著性差异(P>0.05)。
结论
1.肺和大肠在津液的生成、输布和排泄中起了重要作用,津液是维持“肺与大肠相表里”的重要物质之一,津液亏虚是导致病变由肠及肺的主要原因,其病理过程是肠燥津亏、腑气不通,进而加重肺生理功能失常。
2.限水+复方地芬诺酯复合因素模型更接近的模拟肠燥伤肺的基本病理特征,是一种较稳定的动物模型。
3.水通道蛋白-1,3在肺和大肠津液代谢中起重要作用,在津液亏虚状态下,肺与大肠组织中的水通道蛋白含量降低,可能是肺与大肠相关性的生物学基础之一。
4.限水+复方地芬诺酯复合因素引起大肠P物质、VIP表达降低,大鼠肠道濡动减慢,肠燥更甚,进而引起肺脏病理改变,因此,P物质、VIP可能是肠病及肺这一病理过程的启动因子之一。
Backgroud
The theory of entrails being inerior-exteriorly related is one of the important parts of chinese medicine organ picture theory. And it emphasizes on intercoordination and mutual restriction of entrails being inerior-exteriorly related in physiological state, and interaction and transmission of a disease between organs in pathological state. Also it pays attention to correlated organs during the treatment in order to prevent deterioration and recover ying yang balance and qi xue harmony. The theory of lung and the large intestine being inerior-exteriorly related is the representative of entrails theory. The relationship between lung and intesine is not only embodied by meridian and vessels,meanwhile, which are in a symbiosis inseparable relationship in physiological and pathological state, but also it is confirmed in the long time clinic practice. Nevertheless,whether the structure basis of lung and intestine has the same homology, whether the active compound has specificity, how the cotrol mechanisim works, have no satisfied answer now. Explaining above questions scientifically, revealing biological basis of relationship between lung and large intestinal,and elucidating internal connection and concordance mechanism, could better instruct clinical application.
Objective
In the instruction of TCM theory of "the lung and the large intestine being interior-exteriorly related",and following TCM etiology theory, the animal model of intestinal dryness and comsumption of fruid is estabished. Detecting physiological function of pulmonary system and intestine system as the starting point,the related index about the effect of fluid deficiency on physiologic function of the lung and the large intestine were choosed. The study about intervention and mechanism of action of the Fluid-Increasing Decoction for Purgation in rat model was carried out,to elucidate biological basis of physiological and pathological relationship between lung and large intestinal,to reveal their internal connection and concordance mechanism.
Methord
1.Theory approaching:on the basis of theory of the lung and the large intestine being interior-exteriorly related body, by analyzing the important effect of fluid and its metabolism on physiological function of lung and the large intestine, and on the view of lung qi obstruction caused by intestinal dryness and consumption of fluid an obstruction of fu-qi,the internal mechanism of comorbidity of lung and the large intestine was approached, and their biological basis was analyzed
2.Empirical study
(1)Modelling test:64 SD rats,200±20g, male and female with each in half, were randomly divided into 4 groups:blank group, water-limited group, complex diphenoxylate group, water-limited +complex diphenoxylate group, each group has 16 rats.When the rats model were established, the alteration of stool was observed. After 7 and 14 days,the water content of the lung and large intestine and pathological sectionwere tested, to establish and evaluate three constipation model as further study basis.
(2)Observing effect of Fluid-Increasing Decoction for Purgation on intestinal dryness and consumption of fluid in rat model:60 SD rats,200±20g, male and female with each in half, were randomly divided into 5 groups:blank group, model group, Fluid-Increasing Decoction for Purgation group,Fluid-Increasing Decoction group, western medicine group, each group has 12 rats. The model of intestinal dryness and consumption of fluid was constructed by limiting water and lavaging compex diphenoxylate. After 7 days,the water content of the lung and large intestine, pathological section, lung channel protein land 3, the alteration of VIP、P in the large intestine tissue plasma,were tested.
Result
1.Modelling
(1)observing stool texture and easy and smooth degree of defecation:water-limited,and water-limited add medication administration group:the amount of stool was decreased, fecal pellet became hard and dry, the shape was shorten, discarding with great power and slowly. Medication administration group:most fecal pellet became hard, their surface became dry, sometimes the soft and no formed stool could be seen. Blank group:there is no abmormal in stool texture and defecation.
(2)Histopathology observation:blank group were with well-distributed alveoli pulmonis and medium lung interstitium on the 7 and 14th day; the tissue structure of colon was normal,with abundant tomentum and cells lining up in order.
Water-limited group:air tube and cilium could not be seen obviously defect.And alveoli pulmonis could not be seen obviously abnormity,the tissue structure of colon was normal,with abundant tomentum on the 7 th day. On the 14 th day, alveolar ectasia and lung interstitial substance broaden, there were some phlegmasia cell in bronchus,.The tissue structure of colon was normal with no obviously pathological changes.
Medication administration group:alveoli pulmonis and lung interstitial substance could not be seen obviously abnormity. The tissue structure of colon was normal,with abundant tomentum on the 7 th and 14th day.
Water-limited+ medication administration group:alveoli pulmonis and lung interstitial substance could not be seen obviously abnormity and the tissue structure of colon was normal on the 7th day. On the 14 th day, alveolar ectasia and lung interstitial substance broaden, there were some phlegmasia cell in small bronchus, and dropsy cell in alveoli pulmonis.Villus intestinalis were decreased and became short villus intestinalis shape,and lamina propria became thin. And there were eosinophile granulocyte and lympholeukocyte.
Under electron microscope, the blank group:on the 7 and 14th day, the tissue structure of lung and colon were normal.The medication administration group:the tissue structure of lung and colon were with no obviously differece.Water-limited group:the tissue structure of lung and colon were with no obviously differece on the 7 th day. On the 14 th day, lung diaphragm were fallen off, lamellar body in endochylema were fallen off.In intestine tissue collagen fibrils were exist.Water-limited+ medication administration group:lamellar body in lung tissue were partly fallen off on the 7th day. On the 14th day, a great quantity of lamellar body in lung diaphragm were fallen off,In intestine tissue collagen fibrils were hyperplasy.
(3)The water content in lung and intestine tissue:on the 7 th day, water-limited group and water-limited+ medication had significant difference from blank group in lung and intestine tissue (p<0.01),but medication administration group had no significant difference from blank group(p>0.05);Water-limited group had no significant difference from water-limited+ medication administration group in lung tissue (p>0.05),but has significant difference in intestine tissue (p<0.01).It is indicated that the method of water-limited and water-limited+ dose regimen could cut down the water content in lung and intestine tissue. But this two methods had no significant difference.
The water content in lung tissue:on the 14th day, water-limited group and water-limited + medication had significant difference from blank group (p<0.01),but medication administration group had no significant difference from blank group(p>0.05);Water-limited group had difference from water-limited+medication administration group (p<0.05).The water content in intestine tissue:water-limited group and water-limited+medication had significant difference from blank group (p<0.01),but medication administration group had no significant difference from blank group(p>0.05);Water-limited group had no difference from water-limited+medication administration group(p>0.05).It is indicated that the method of water-limited and water-limited+dose regimen could cut down the water content in lung and intestine tissue, but it was more obvious in lung tissue, and there was no significant difference in intestine tissue.
2.Observing effect of Fluid-Increasing Decoction for Purgation on intestinal dryness and consumption of fluid in rat model
(1)observing stool texture and easy and smooth degree of defecation:model group:the amount of stool was decreased, fecal pellet became dry, the shape was shorten, discarding with great power and slowly. Fluid-Increasing Decoction for Purgation group: there were more fecal pellet than model group, the shape was shorten, sometimes soft.Fluid-Increasing Decoction and western medicine group:fecal pellet became hard, he shape was shorten, discarding with great power and slowly. Blank group:there is no abmormal in stool texture and defecation.
(2)Histopathology observation
Lung:blank group were with well-distributed alveoli pulmonis and medium lung interstitium. Model group:pilus in air tube were with lodging shape, adherence, and defecting lamellar.alveoli pulmonis were engorged and dropsy. Western medicine group:alveolar ectasia,there were great amounts of heterophil granulocyte in lung interstitium. Fluid-Increasing Decoction for Purgation group: there were small amounts of heterophil granulocyte.
Enteropathy:blank group:tissue structure of colon was normal, with abundant tomentum. Model group:tomentum were decreased, became short lodging shape,lamina propria became thin, and there were eosinophile granulocyte cells.Fluid-Increasing Decoction and western medicine group:there were eosinophile granulocyte cells and lympholeukocytes.Fluid-Increasing Decoction for Purgation group:alveolar ectasia:only small amounts of eosinophile granulocyte cells.
(3)Water content
Model group had significant difference from blank group (p<0.01),it was indicated that model was successful.Lung: Fluid-Increasing Decoction for Purgation group had significant difference from model group (p<0.01),but western medicine and Fluid-Increasing Decoction group had no significant difference from model group(p>0.05);intestine:Fluid-Increasing Decoction for Purgation group and Fluid-Increasing Decoction group had significant difference from model group (p<0.01),but western medicine group had no significant difference from model group(p >0.05).
(4)Testing the content of channel protein(AQP1、AQP3)
Compared with blank group, the expression of lung AQP1 and intestine AQP3 in model group were decreased(P<0.05).Compared with model group, western medicine and Fluid-Increasing Decoction group AQP1 and AQP3 had no significant difference (p>0.05). Fluid-Increasing Decoction for Purgation group, AQP1 and AQP3 were increased (P<0.05).
(5)Testing VIP. P in tissue
Compared with blank group, the content of VIP in model group were decreased(P<0.01).Compared with model group, Fluid-Increa sing Decoction for Purgation group, VIP were increased(P<0.05), Fluid-Increasing Decoction group were increased(P<0.05).Compared with blank group, the content of SP in model group were decreased(P<0.01).Compared with model group, Fluid-Increasing Decoction for Purgation group, VIP were increased(P<0.05), Fluid-Increasing Decoction group were increased(P<0.05).
Result
1.The model of limited water+complex diphenoxylate is more near the clinical expression and basic pathological feature of intestinal dryness and consumption of fluid, and constipation. It is a reliable animal model.
2.Water channel protein 1,3 play an important role in body fluid metabolism of lung and large intestine. It contains a low contents when body fluid deficient.
3.P, VIP expression in model group is obviously lower than normal control.It maybe the cause of the movement of intestinal tract stepping down.
脏腑表里相合理论是中医藏象理论的重要组成部分之一,它强调生理状态下相表里的脏腑之间相互协调、相互制约,病理状态下各脏腑器官之间相互影响、相互传变,治疗中注意“既病防变”,协调相关脏腑功能,恢复脏腑之间阴阳平衡、气血调和。“肺与大肠相表里”是脏腑表里相合理论代表性内容,肺与大肠的相互关系,不仅通过经脉的络属得以体现,同时在生理、病理上形成了一种密不可分的依赖关系,而且在长期的临床实践中得到充分证实。但肺与大肠的结构基础是否具有同源性,活性物质是否具有特异性,调控机制如何发挥作用,这些问题目前均没有满意的答案。科学地解释以上问题,揭示肺与大肠相关性的生物学基础,阐明肺与大肠的内在联系和协调机制,能够更好地指导临床应用。
目的
在“肺与大肠相表里”中医理论指导下,遵循中医病因病机理论,分析津亏肠燥,损伤肺阴的病理过程,并建立肠燥伤肺大鼠模型,以检测“肺系”、“肠系”生理功能为主要切入点,选择反映津亏对肺、大肠生理功能影响的相关指标,并运用增液承气汤对该大鼠模型进行干预,旨在初步阐明肺与大肠生理和病理相关性的生物学基础,揭示在生理和病理状态下肺与大肠相互影响的内在联系和协调机制。
方法
1理论探讨
以“肺与大肠相表里”理论为基础,通过分析津液在肺与大肠生理功能正常发挥的重要作用及津液代谢失常对肺与大肠生理功能的影响,并从津液亏虚导致肠燥便秘、腑气不通,进而引起肺生理功能失常的角度,探讨肺与大肠的内在协调机制。
2实验研究
(1)建立大鼠模型：SD大鼠64只,200±20g,雌雄各半,随机分为4组,即空白组、限水模型组、复方地芬诺酯组、限水+复方地芬诺酯模型组,每组16只。以限水、灌胃复方地芬诺酯、限水+灌胃复方地芬诺酯构建不同的便秘大鼠模型,观察大鼠大便改变情况,并分别在造模第7天、第14天检测肺与大肠含水量,观察肺与大肠病理切片,成功建立津亏肠燥便秘大鼠模型,作为后续研究的基础。
(2)肺与大肠相互影响的内在联系和协调机制研究：SD大鼠60只,200±2 Og,雌雄各半,随机分为5组,即空白组、模型组、增液承气汤组、增液汤组、西药组,每组12只。大鼠限水并灌胃复方地芬诺酯构建津亏肠燥大鼠模型,并运用增液承气汤和增液汤对该大鼠模型干预,给药7天后,观察大鼠大便改变情况、肺与大肠含水量、肺与大肠病理切片、肺与肠组织水通道蛋白1、3的表达及肺、大肠组织中的VIP、P物质的改变。
结果
1建立模型试验
(1)大便质地及排便通畅程度观察
限水组、限水并给药组大鼠大便量减少,粪粒变硬变干,粒形缩短,排出缓慢费力。给药组大鼠大部分粪粒变硬,粪粒表面变干,偶见软而不成形粪便。空白组便质及排便未见异常。
(2)组织病理学观察
光镜下,空白组、给药组、限水组和限水+给药组第7天气管和纤毛未见明显缺损,肺泡未见明显异常,结肠组织结构无明显异常,绒毛丰富。限水组第14天见肺泡扩张,肺间质变宽,支气管有少量炎性细胞浸润。结肠组织结构正常,无明显病变。限水+给药组第14天肺间质增宽,细支气管内炎性细胞浸润,肺泡内少量水肿液形成。结肠肠绒毛,变短,不齐,固有层变薄,嗜酸性粒细胞浸润。
电镜下,空白组、给药组第7天和第14天肺和结肠组织细胞结构正常。限水组第7天肺和结肠组织细胞结构无明显改变,第14天肺胞浆中有板层小体脱落,肠组织绒毛不齐。限水组+给药组第7天肺和结肠组织细胞结构无明显改变,第14天肺泡腔中有大量板层小体脱落。肠组织绒毛变稀,不齐。
(3)排便时间及粒数观察
造模7天和14天时,与空白组相比,限水模型组和限水+给药模型组的首次排便时间延长(P<0.01),排便粒数减少(P<0.01),给药模型组首次排便时间、排便粒数无统计学意义(P>0.05)；到14天时,给药模型组首次排便时间延长、排便粒数减少(P<0.05)；与限水模型组相比,限水+给药模型组7天时首便时间、排便粒数无统计学意义(P>0.05)。到14天时,排便粒数减少(P<0.05)。
(4)便秘大鼠排便重量、粪便含水率检测
造模7天时和14天时,与空白组比,限水模型组和限水+给药模型组的粪便含水率降低(P<0.01),给药模型组粪便含水率无统计学意义(P>0.05)；与限水模型组比,限水+给药模型组粪便含水率无统计学意义(P>0.05)。造模14天,与限水模型组相比,限水+给药模型组含水率较低(P<0.05)。
(5)肺组织、肠组织含水量检测
造模7天和14天时,限水组和限水+给药组大鼠的肺组织含水率与空白组比较均有显著差异(p<0.01),给药组与空白组相比无统计学意义(p>0.05)；限水组和限水+给药组大鼠的肺组织含水率对比无统计学意义(p>0.05)。到14天时,限水组和限水+给药组大鼠的肺组织含水率对比有差异(p<0.05)。大肠组织含水量造模7天和14天时,各组结果与肺组织趋势一致。
2肺与大肠相互影响的内在联系和协调机制研究
(1)大便质地及排便通畅程度观察：模型组大便量减少,粪粒干粒形缩短,排出缓慢费力；增承组粪粒数较模型组多,粒形缩短,有时见粪便较软。增液组和西药组粪粒较硬,粒形短,排出费力；空白组便质及排便未见明显异常。
(2)肺、大肠组织病理观察
肺组织病理切片示：空白组肺泡均匀,肺间质适中。模型组肺泡扩张、水肿,肺间质增宽。西药组肺泡扩张,有大量中性粒细胞浸润,肺间质增宽。增液组肺泡扩张,有少量中性粒细胞聚集。增承组肺泡均匀,肺间质增宽,有少量中性粒细胞聚集。
大肠组织病理切片示：空白组大鼠结肠组织结构正常,绒毛丰富,排列整齐。模型组肠绒毛变短倒伏,排列不齐,固有层变薄,嗜酸性粒细胞浸润。增液组、西药组结肠固有层内均可见大量嗜酸性粒细胞、淋巴细胞。增承组可见少量嗜酸性粒细胞。
(3)大鼠排便变化检测
与空白组相比,模型组的首次排便时间延长(P<0.01),排便粒数减少(P<0.01)；与模型组相比,西药组和增液组首便时间缩短(P<0.05),排便粒数无明显统计学意义(P>0.05),增承组首便时间缩短(P<0.01),排便粒数增加(P<0.05)。模型组粪便较空白组含水率降低(P<0.01),粪便较为干硬；与模型组比,西药组、增液组粪便含水率无统计学意义(P>0.05),增承组粪便含水率增加(P<0.05)。
(4)肺组织、大肠组织含水率检测
模型组肺组织、大肠组织含水率与空白组对比减少,有统计学差异(p<0.01),提示造模成功。与模型组比,肺组织含水率增承组升高(p<0.01),西药组、增液组与模型组相比无统计学意义(p>0.05)；大肠组织含水量增承组、增液组与模型组比较均有显著差异(p<0.01),西药组与模型组相比无统计学意义(p>0.05)。
(5)水通道蛋白(AQP1、AQP3)含量检测
与空白组相比,模型组肺组织AQP1降低(P<0.01)；与模型组比,西药组、增液组肺组织AQP1无统计学意义(P>0.05),增承组肺组织AQP1增加(P<0.05)。与空白组相比,模型组肠组织AQP3降低(P<0.01)；与模型组比,增液组肠组织AQP3增加(P<0.05)。增承组肠组织AQP3增加更加明显(P<0.01)。西药组肠组织AQP3无统计学意义(P>0.05)。
(6)肺、大肠组织VIP、P物质检测
与空白组相比,模型组大鼠肠组织中VIP和SP含量降低(P<0.01)；与模型组相比,增液组VIP和SP含量升高(P<0.05),增承组VIP和SP含量升高(P<0.01),西药组与模型组比较无统计意义(P>0.05)。与空白组相比,模型组大鼠肺组织中VIP含量降低(P<0.01)；与模型组比,增承组VIP含量升高(P<0.05),增液组、西药组VIP含量与模型组比无显著性差异(P>0.05)。
结论
1.肺和大肠在津液的生成、输布和排泄中起了重要作用,津液是维持“肺与大肠相表里”的重要物质之一,津液亏虚是导致病变由肠及肺的主要原因,其病理过程是肠燥津亏、腑气不通,进而加重肺生理功能失常。
2.限水+复方地芬诺酯复合因素模型更接近的模拟肠燥伤肺的基本病理特征,是一种较稳定的动物模型。
3.水通道蛋白-1,3在肺和大肠津液代谢中起重要作用,在津液亏虚状态下,肺与大肠组织中的水通道蛋白含量降低,可能是肺与大肠相关性的生物学基础之一。
4.限水+复方地芬诺酯复合因素引起大肠P物质、VIP表达降低,大鼠肠道濡动减慢,肠燥更甚,进而引起肺脏病理改变,因此,P物质、VIP可能是肠病及肺这一病理过程的启动因子之一。
Backgroud
The theory of entrails being inerior-exteriorly related is one of the important parts of chinese medicine organ picture theory. And it emphasizes on intercoordination and mutual restriction of entrails being inerior-exteriorly related in physiological state, and interaction and transmission of a disease between organs in pathological state. Also it pays attention to correlated organs during the treatment in order to prevent deterioration and recover ying yang balance and qi xue harmony. The theory of lung and the large intestine being inerior-exteriorly related is the representative of entrails theory. The relationship between lung and intesine is not only embodied by meridian and vessels,meanwhile, which are in a symbiosis inseparable relationship in physiological and pathological state, but also it is confirmed in the long time clinic practice. Nevertheless,whether the structure basis of lung and intestine has the same homology, whether the active compound has specificity, how the cotrol mechanisim works, have no satisfied answer now. Explaining above questions scientifically, revealing biological basis of relationship between lung and large intestinal,and elucidating internal connection and concordance mechanism, could better instruct clinical application.
Objective
In the instruction of TCM theory of "the lung and the large intestine being interior-exteriorly related",and following TCM etiology theory, the animal model of intestinal dryness and comsumption of fruid is estabished. Detecting physiological function of pulmonary system and intestine system as the starting point,the related index about the effect of fluid deficiency on physiologic function of the lung and the large intestine were choosed. The study about intervention and mechanism of action of the Fluid-Increasing Decoction for Purgation in rat model was carried out,to elucidate biological basis of physiological and pathological relationship between lung and large intestinal,to reveal their internal connection and concordance mechanism.
Methord
1.Theory approaching:on the basis of theory of the lung and the large intestine being interior-exteriorly related body, by analyzing the important effect of fluid and its metabolism on physiological function of lung and the large intestine, and on the view of lung qi obstruction caused by intestinal dryness and consumption of fluid an obstruction of fu-qi,the internal mechanism of comorbidity of lung and the large intestine was approached, and their biological basis was analyzed
2.Empirical study
(1)Modelling test:64 SD rats,200±20g, male and female with each in half, were randomly divided into 4 groups:blank group, water-limited group, complex diphenoxylate group, water-limited +complex diphenoxylate group, each group has 16 rats.When the rats model were established, the alteration of stool was observed. After 7 and 14 days,the water content of the lung and large intestine and pathological sectionwere tested, to establish and evaluate three constipation model as further study basis.
(2)Observing effect of Fluid-Increasing Decoction for Purgation on intestinal dryness and consumption of fluid in rat model:60 SD rats,200±20g, male and female with each in half, were randomly divided into 5 groups:blank group, model group, Fluid-Increasing Decoction for Purgation group,Fluid-Increasing Decoction group, western medicine group, each group has 12 rats. The model of intestinal dryness and consumption of fluid was constructed by limiting water and lavaging compex diphenoxylate. After 7 days,the water content of the lung and large intestine, pathological section, lung channel protein land 3, the alteration of VIP、P in the large intestine tissue plasma,were tested.
Result
1.Modelling
(1)observing stool texture and easy and smooth degree of defecation:water-limited,and water-limited add medication administration group:the amount of stool was decreased, fecal pellet became hard and dry, the shape was shorten, discarding with great power and slowly. Medication administration group:most fecal pellet became hard, their surface became dry, sometimes the soft and no formed stool could be seen. Blank group:there is no abmormal in stool texture and defecation.
(2)Histopathology observation:blank group were with well-distributed alveoli pulmonis and medium lung interstitium on the 7 and 14th day; the tissue structure of colon was normal,with abundant tomentum and cells lining up in order.
Water-limited group:air tube and cilium could not be seen obviously defect.And alveoli pulmonis could not be seen obviously abnormity,the tissue structure of colon was normal,with abundant tomentum on the 7 th day. On the 14 th day, alveolar ectasia and lung interstitial substance broaden, there were some phlegmasia cell in bronchus,.The tissue structure of colon was normal with no obviously pathological changes.
Medication administration group:alveoli pulmonis and lung interstitial substance could not be seen obviously abnormity. The tissue structure of colon was normal,with abundant tomentum on the 7 th and 14th day.
Water-limited+ medication administration group:alveoli pulmonis and lung interstitial substance could not be seen obviously abnormity and the tissue structure of colon was normal on the 7th day. On the 14 th day, alveolar ectasia and lung interstitial substance broaden, there were some phlegmasia cell in small bronchus, and dropsy cell in alveoli pulmonis.Villus intestinalis were decreased and became short villus intestinalis shape,and lamina propria became thin. And there were eosinophile granulocyte and lympholeukocyte.
Under electron microscope, the blank group:on the 7 and 14th day, the tissue structure of lung and colon were normal.The medication administration group:the tissue structure of lung and colon were with no obviously differece.Water-limited group:the tissue structure of lung and colon were with no obviously differece on the 7 th day. On the 14 th day, lung diaphragm were fallen off, lamellar body in endochylema were fallen off.In intestine tissue collagen fibrils were exist.Water-limited+ medication administration group:lamellar body in lung tissue were partly fallen off on the 7th day. On the 14th day, a great quantity of lamellar body in lung diaphragm were fallen off,In intestine tissue collagen fibrils were hyperplasy.
(3)The water content in lung and intestine tissue:on the 7 th day, water-limited group and water-limited+ medication had significant difference from blank group in lung and intestine tissue (p<0.01),but medication administration group had no significant difference from blank group(p>0.05);Water-limited group had no significant difference from water-limited+ medication administration group in lung tissue (p>0.05),but has significant difference in intestine tissue (p<0.01).It is indicated that the method of water-limited and water-limited+ dose regimen could cut down the water content in lung and intestine tissue. But this two methods had no significant difference.
The water content in lung tissue:on the 14th day, water-limited group and water-limited + medication had significant difference from blank group (p<0.01),but medication administration group had no significant difference from blank group(p>0.05);Water-limited group had difference from water-limited+medication administration group (p<0.05).The water content in intestine tissue:water-limited group and water-limited+medication had significant difference from blank group (p<0.01),but medication administration group had no significant difference from blank group(p>0.05);Water-limited group had no difference from water-limited+medication administration group(p>0.05).It is indicated that the method of water-limited and water-limited+dose regimen could cut down the water content in lung and intestine tissue, but it was more obvious in lung tissue, and there was no significant difference in intestine tissue.
2.Observing effect of Fluid-Increasing Decoction for Purgation on intestinal dryness and consumption of fluid in rat model
(1)observing stool texture and easy and smooth degree of defecation:model group:the amount of stool was decreased, fecal pellet became dry, the shape was shorten, discarding with great power and slowly. Fluid-Increasing Decoction for Purgation group: there were more fecal pellet than model group, the shape was shorten, sometimes soft.Fluid-Increasing Decoction and western medicine group:fecal pellet became hard, he shape was shorten, discarding with great power and slowly. Blank group:there is no abmormal in stool texture and defecation.
(2)Histopathology observation
Lung:blank group were with well-distributed alveoli pulmonis and medium lung interstitium. Model group:pilus in air tube were with lodging shape, adherence, and defecting lamellar.alveoli pulmonis were engorged and dropsy. Western medicine group:alveolar ectasia,there were great amounts of heterophil granulocyte in lung interstitium. Fluid-Increasing Decoction for Purgation group: there were small amounts of heterophil granulocyte.
Enteropathy:blank group:tissue structure of colon was normal, with abundant tomentum. Model group:tomentum were decreased, became short lodging shape,lamina propria became thin, and there were eosinophile granulocyte cells.Fluid-Increasing Decoction and western medicine group:there were eosinophile granulocyte cells and lympholeukocytes.Fluid-Increasing Decoction for Purgation group:alveolar ectasia:only small amounts of eosinophile granulocyte cells.
(3)Water content
Model group had significant difference from blank group (p<0.01),it was indicated that model was successful.Lung: Fluid-Increasing Decoction for Purgation group had significant difference from model group (p<0.01),but western medicine and Fluid-Increasing Decoction group had no significant difference from model group(p>0.05);intestine:Fluid-Increasing Decoction for Purgation group and Fluid-Increasing Decoction group had significant difference from model group (p<0.01),but western medicine group had no significant difference from model group(p >0.05).
(4)Testing the content of channel protein(AQP1、AQP3)
Compared with blank group, the expression of lung AQP1 and intestine AQP3 in model group were decreased(P<0.05).Compared with model group, western medicine and Fluid-Increasing Decoction group AQP1 and AQP3 had no significant difference (p>0.05). Fluid-Increasing Decoction for Purgation group, AQP1 and AQP3 were increased (P<0.05).
(5)Testing VIP. P in tissue
Compared with blank group, the content of VIP in model group were decreased(P<0.01).Compared with model group, Fluid-Increa sing Decoction for Purgation group, VIP were increased(P<0.05), Fluid-Increasing Decoction group were increased(P<0.05).Compared with blank group, the content of SP in model group were decreased(P<0.01).Compared with model group, Fluid-Increasing Decoction for Purgation group, VIP were increased(P<0.05), Fluid-Increasing Decoction group were increased(P<0.05).
Result
1.The model of limited water+complex diphenoxylate is more near the clinical expression and basic pathological feature of intestinal dryness and consumption of fluid, and constipation. It is a reliable animal model.
2.Water channel protein 1,3 play an important role in body fluid metabolism of lung and large intestine. It contains a low contents when body fluid deficient.
3.P, VIP expression in model group is obviously lower than normal control.It maybe the cause of the movement of intestinal tract stepping down.
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[1]靳文学,杨宇.从粘膜免疫系统看“肺与大肠相表里”[J].四川中医,2005,23(12)：123.
[2]哈木拉提·吾甫尔,李风森,艾买江·吐尼亚孜.“肺与大肠相表里”的科学性及其对哮喘治疗的指导意义[J].新疆医科大学学报,2000,23(2)：151-152.
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[13]张余森,彭代国,白朝勇,等.“肺与大肠相表里’’动物模型的制作及其机理初探[J].中兽医医药杂志,1987；(2)：1.
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