【作者】 张焱；

【导师】 张庆荣；

【作者基本信息】 辽宁中医药大学 ， 中医基础理论， 2009， 博士

【摘要】 目的:本实验通过研究脾虚哮喘及哮喘大鼠肺、回肠组织神经肽及受体表达水平的改变,探讨脾虚哮喘及哮喘动物机体内的神经内分泌免疫调节失衡状态;通过对脾虚哮喘及哮喘大鼠肺、回肠组织神经肽及受体表达水平改变的比较研究,探讨脾虚对哮喘的影响,为“脾虚为哮喘之关键病机”的论点提供新的依据,以进一步验证和深化哮喘的中医病机理论;通过研究培土生金中药对脾虚哮喘及哮喘大鼠肺、回肠组织神经肽及受体表达水平的影响,探讨培土生金法治疗哮喘的作用机制;通过对脾虚哮喘及哮喘大鼠肺、回肠组织神经肽及受体表达水平相关性的研究,探讨培土生金理论的现代科学内涵和肺脾二脏的密切关系。材料与方法:以中医理论为指导,复合利用中医脾虚动物模型和西医哮喘动物模型的造模方法,建立大鼠脾虚哮喘病证结合的模型。脾虚动物造模采用饮食不节加疲劳过度的方法。造模结束后,比较两模型组动物造模前后体重、肛温变化情况,以脾虚模型评估标准评估两模型组动物症状。以动物定量指标的变化有统计学意义、症状符合脾虚模型评估标准来判定脾虚动物模型建立成功。哮喘动物造模采用以卵蛋白为致敏原,氢氧化铝、百日咳灭活杆菌为免疫佐剂,腹腔注射致敏后以卵蛋白液雾化吸入激发建立动物哮喘模型。以大鼠出现呼吸加深加快,点头运动明显,腹肌强烈收缩,偶可闻及痰鸣音等类似人类哮喘的症状为造模成功。造模成功后,采用培土生金中药进行干预,共12天。干预结束后,采用血液白细胞及嗜酸粒细胞绝对计数方法测定大鼠末梢血及支气管肺泡灌洗液(BALF)中白细胞(WBC)、嗜酸性粒细胞(EOS)计数;采用双抗体夹心酶联免疫法(ELISA)测定大鼠血清及BALF中自介素-4(IL-4)、干扰素-γ(IFN-γ)的浓度;采用免疫组化方法半定量分析肺及回肠组织中P物质(SP)、血管活性肠肽(VIP)含量;采用逆转录聚合酶链反应(RT-PCR)方法半定量分析肺组织中SP及其NK1受体mRNA的表达;并应用光镜对肺、回肠组织形态学进行观测。结果:1.同对照(C)组比较,疾病加证候(A)组和疾病(B)组大鼠均表现为血中及BALF中白细胞计数、嗜酸性粒细胞计数显著升高(P＜0.01);疾病加证候(A)组和疾病(B)组大鼠均表现为血中及BALF中IL-4的浓度显著升高(P＜0.01),IFN-γ的浓度显著降低(P＜0.01),IFN-γ/IL-4比值显著降低(P＜0.01);疾病加证候(A)组和疾病(B)组大鼠均表现为肺组织中SP及其NK1受体mRNA表达显著增强(P＜0.01);疾病加证候(A)组和疾病(B)组大鼠均表现为肺、回肠组织中SP含量显著增加(P＜0.01),VIP含量显著减少(P＜0.01)。2.与疾病(B)组比较,疾病加证候(A)组外周血白细胞计数、BALF中白细胞计数、嗜酸性粒细胞计数、血清IL-4浓度、BALF中IL-4浓度、肺组织SP及其NK1受体mRNA表达、肺组织及回肠组织SP含量均显著高于疾病(B)组(P＜0.05);BALF中IFN-γ浓度及IFN-γ/IL-4比值、肺组织及回肠组织VIP含量均显著低于疾病(B)组(P＜0.01或P＜0.05)。3.与相应的疾病组比较,两中药(A1,B1)组血中及BALF中嗜酸性粒细胞计数显著降低(P＜0.01或P＜0.05)。其中,疾病加证候中药(A1)组BALF中嗜酸性粒细胞计数与对照组比较,已无显著差异(P＞0.05);疾病加证候中药(A1)组血中及BALF中白细胞计数显著降低(P＜0.01),疾病中药(B1)组BALF中白细胞计数显著降低(P＜0.01)。与相应的疾病组比较,两中药组(A1,B1)血中及BALF中IL-4的浓度显著降低(P＜0.01),IFN-γ的浓度显著升高(P＜0.01或P＜0.05),IFN-γ/IL-4比值显著升高(P＜0.01或P＜0.05)。其中,疾病加证候中药(A1)组血中IFN-γ浓度、BALF中IL-4的浓度及BALF中IFN-γ/IL-4比值与对照(C)组比较,已无显著差异(P＞0.05)。与相应的疾病组比较,两中药(A1,B1)组肺、回肠组织中SP含量显著减少(P＜0.01),VIP含量显著增加(P＜0.01),其中,疾病加证候中药(A1)组肺及回肠组织SP含量、疾病中药(B1)组回肠组织SP含量与对照(C)组比较,已无显著差异(P＞0.05)。与相应的疾病组比较,两中药(A1,B1)组肺组织中SP及其NK1受体mRNA表达显著减少(P＜0.01或P＜0.05)。其中,疾病加证候中药(A1)组肺组织SPmRNA表达水平、疾病中药(B1)组肺组织NK1受体mRNA表达水平与对照(C)组比较,已无显著差异(P＞0.05)。4.各组大鼠血中IFN-γ/IL-4比值与肺组织SP含量间存在着明显的负相关(r=-0.698,P＜0.01);各组大鼠血中IFN-γ/IL-4比值与肺组织VIP含量间存在着明显的正相关(r=0.741,P＜0.01)。各组大鼠血中IFN-γ/IL-4比值与回肠组织SP含量间存在着明显的负相关(r=-0.627,P＜0.01);各组大鼠血中IFN-γ/IL-4比值与回肠组织VIP含量间存在着明显的正相关(r=0.766,P＜0.01)。各组大鼠BALF中IFN-γ/IL-4比值与肺组织SP含量间存在着明显的负相关(r=-0.688,P＜0.01);各组大鼠BALF中IFN-γ/IL-4比值与肺组织VIP含量间存在着明显的正相关(r=0.324,P＜0.01)。各组大鼠BALF中IFN-γ/IL-4比值与回肠组织SP含量间存在着明显的负相关(r=-0.615,P＜0.01);各组大鼠BALF中IFN-γ/IL-4比值与回肠组织VIP含量间存在着明显的正相关(r=0.679,P＜0.01)。各组大鼠肺组织中SP与VIP含量之间存在着明显的负相关(r=-0.678,P＜0.01);各组大鼠回肠组织中SP与VIP含量之间存在着明显的负相关(r=-0.620,P＜0.01)。各组大鼠肺组织与回肠组织中SP含量之间存在着明显的正相关(r=0.602,P＜0.01);各组大鼠肺组织与回肠组织中VIP含量之间存在着明显的正相关(r=0.608,P＜0.01)。5.病理结果显示:疾病(B)组与疾病加证候(A)组均有不同程度的支气管及肺组织炎性病变,两中药(A1,B1)组的支气管及肺组织的炎性病变均有所减轻;疾病(B)组与疾病加证候(A)组均有不同程度的回肠组织炎性病变,两中药(A1,B1)组的回肠组织炎性病变程度有所减轻,但以疾病加证候治疗(A1)组的减轻程度明显。结论:1.哮喘及脾虚哮喘大鼠均存在着明显的气道局部或/和全身炎症性改变;哮喘及脾虚哮喘大鼠均存在着全身及气道局部免疫细胞因子IFN-γ、IL-4表达失衡;哮喘及脾虚哮喘大鼠均存在着肺、回肠组织内神经肽VIP、SP表达水平失衡;哮喘及脾虚哮喘大鼠均存在着肺组织内神经肽SP及其NK1受体mRNA表达水平失衡。2.脾虚可以加重哮喘机体气道内炎性细胞聚集及外周血炎性细胞聚集,加重哮喘机体肺、回肠组织炎性病变,从而加重哮喘机体的局部及全身炎症反应;脾虚可以加重哮喘机体血清及BALF中免疫细胞因子IFN-γ、IL-4表达失衡,加重哮喘机体肺、回肠组织内神经肽VIP、SP表达水平失衡,加重肺组织内神经肽SP及其NK1受体mRNA表达水平失衡,从而加重哮喘机体的神经内分泌免疫调节失衡,成为哮喘反复发作的内在原因之一。3.培土生金中药可以减轻模型大鼠气道局部及全身的炎性细胞浸润,下调血中及BALF中白细胞及嗜酸性粒细胞计数;培土生金中药可以调整全身及气道局部免疫细胞因子IFN-γ、IL-4表达失衡状态,上调血清及BALF中IFN-γ浓度,下调IL-4浓度,上调IFN-γ/IL-4比值;培土生金中药可以减轻肺、回肠组织内神经肽VIP、SP及NK1受体表达水平的失衡状态,下调肺组织中SP及其NK1受体mRNA表达水平,下调肺、回肠组织中SP含量表达水平,上调肺、回肠组织中VIP含量表达水平;培土生金中药能够减轻模型大鼠的大、小气道及肺、回肠组织的炎性病变。4.肺、回肠组织神经肽VIP、SP表达异常及肺组织SP及其NK1受体mRNA表达的异常均与哮喘慢性气道炎症的形成与持续存在明显相关性,与气道局部及全身的炎症状态呈明显相关性,与气道局部及全身的免疫细胞因子IFN-γ、IL-4表达失衡状态亦呈明显相关性。这进一步证实了神经肽VIP、SP在哮喘发病过程中的重要作用,特别是介导哮喘的神经内分泌免疫调节失衡。肺组织与回肠组织的神经肽VIP、SP的表达水平存在着明显的相关性,从神经肽VIP、SP及神经内分泌免疫调节的角度进一步证实了肺脾二脏的密切关系。更多还原

【Abstract】 OBJECTIVE:We attempted to investigate the imbalanced states of neuro-endocrine-immuno-modulation through study of the changes of neurikins and their receptors of the tissues of the lung and ileum in experimental rates with spleen-deficiency asthma and simple asthma;to investigate the influence of spleen deficiency on asthma by comparison of the changes of expressive levels between the two kinds of rat models,and thus to provide a new evidence for the view points of "the spleen deficiency as the key patho-mechanism".We attempted the effective mechanism of the therapy of "strengthening the earth and promoting the metal(SEPM)" in treatment of asthma through study of the influence of Chinese herbal remedy of SEPM to the expressive levels of neurikins and their receptors of the tissues of the lung and ileum in experimental rates with spleen-deficiency asthma and simple asthma;to investigate the modern connotation of SEPM theory and the close relationship between the lung and the spleen by studying the correlation between the expressive levels of neurikins and their receptors of the tissues of the lung and ileum in experimental rates with spleen-deficiency asthma and simple asthma.MATERIAL AND METHODS,Taking the theory of traditional Chinese medicine (TCM) as the guide,we set up compound rat models with asthma of spleen deficiency by combination of establishing spleen deficiency animal models in TCM with establishing asthmatic animal models in Western medicine.The spleen models were set up by using the method of improper diet plus physical overstrain.The body weight and temperature of the anus of the animals were compared before and after the model establishment;and the symptoms of the animals were assessed by the standards for evaluation of spleen deficiency models in TCM.The successful animal models were determined by the statistical significance in the changes of their quantitative indexes and their symptomatologic accordance with the standards for evaluation of spleen deficiency.Asthmatic animal models were set up by using ovalbumin as the antigen,and sodium hydroxide and pertusis vaccine as the adjuvant.The asthmatic animals were set up by nebulization following the intraperitoneal injection of valbumin.The successful animal models were evaluated by the appearance of similar symptoms of human asthma in rats such as tachypnea,remarkable motion of nodding,strong construction of abdominal muscle,and occasional pulmonary rale and grunting.The successfully established animal models were given Chinese herbal remedy of SEPM for 12 days.Then the following determinations were conducted:white blood cell count(WBC) and eosinophil count(EOS) in peripheral blood and bronchial alveolar lavage fluid (BALF) by absolute counting;the contents of interleuki-4(IL-4) and interferon-γ(IFN-γ) in BALF by enzyme-linked immunoabsorbent assay(ELISA); the contents of substance P(SP) vasoactive intestinal peptide(VIP) by semi-quantitative analysis of immune-chemical assay;the expressions of SPmRNA and neurokinin 1(NK1) receptor mRNA by reverse transcription-polymerase chain reaction(RT-PCR).RESULTS:1.Compared with the control(C) group,the number of WBC and EOS of the peripheral blood and BALF in the compound(A) group and disease(B) group increased(P＜0.01);the content of IL-4 of BALF in group A and B decreased (P＜0.01),and the content of IFN-γof BALF decreased(P＜0.01),and the ratio of IFN-γ/IL-4 was remarkably decreased(P＜0.01).The mRNA expressions of SPmRNA and NK1 receptor mRNA in group A and B enhanced(P＜0.01);the content of SP in group A and B increased(P＜0.01) and the content of VIP obviously decreased(P＜0.01).2.Compared with group B,the WBC in peripheral blood;the WBC and EOS in BALF;serum IL-4 level,IL-4 level in BALF,the expressions of SPmRNA and NK1 receptor mRNA,and the contents of SP of the lung and ileum tissues in group A all increased remarkably(P＜0.01 or P＜0.05).The level of IFN-γand ratio of IFN-γ/IL-4 in BALF,and the content of VIP in the lung and ileum tissues in group A all decreased remarkably(P＜0.05).3.Compared with the correspondent disease groups,the EOS in blood and BALF in the two Chinese herbal groups(A1,B1) remarkably decreased(P＜0.01 or P＜0.05).And the EOS of BALF in group A1 had showed no significant difference from that in the control group(P＞0.05).The WBC and EOS of blood and BALF in group A1 obviously decreased(P＜0.01);the WBC of BALF in group B1 obviously decreased(P＜0.01).The levels of IL-4 of blood and BALF in both group A1 and B1 remarkably decreased(P＜0.01),but the levels of IFN-γobviously increased (P＜0.01 or P＜0.05);and the ratio of IFN-γ/IL-4 increased(P＜0.01 or P＜0.05).And the level of IFN-γof blood,the level of IL-4 of BALF,and the ratio of IFN-γ/IL-4 of BALF in group A1 had showed no significant difference from those in group C(P＞0.05).The expressions of SPmRNA and NK1 receptor mRNA in group A1 and B1 were remarkably decreased(P＜0.01 or P＜0.05).And the expressive levels of SPmRNA in group A1 and NK1 receptor mRNA in group B1 had showed no significant difference from those in group C(P＞0.05).The contents of SP in the lung and ileum in both group A1 and B1 obviously decreased(P＜0.01),but the content of VIP remarkably increased(P＜0.01).And the contents of SP in the lung and ileum in group A1,and the content of SP in the ileum in group B1 had showed no significant difference from those in group C(P＞0.05).4.There was an obviously negative correlation between the ratio of IFN-γ/IL-4 of blood and the content of SP of the lung tissue in each group(r =-0.698,P＜0.01);there was an obviously positive correlation between the ratio of IFN-γ/IL-4 of blood and the content of VIP of the lung tissue(r=0.741, P＜0.01).There was an obviously negative correlation between the ratio of IFN-γ/IL-4 of blood and the content of SP of the ileum tissue in each group (r=-0.627,P＜0.01);there was an obviously positive correlation between the ratio of IFN-γ/IL-4 of blood and the content of VIP of the ileum tissue(r =0.766,P＜0.01).There was an obviously negative correlation between the ratio of IFN-γ/IL-4 of BALF and the content of SP of the lung tissue in each group (r=-0.688,P＜0.01);there was an obviously positive correlation between the ratio of IFN-γ/IL-4 of BALF and the content of VIP of the lung tissue(r= 0.324,P＜0.01).There was an obviously negative correlation between the ratio of IFN-γ/IL-4 of BALF and the content of SP of the ileum tissue in each group (r=-0.615,P＜0.01);there was an obviously positive correlation between the ratio of IFN-γ/IL-4 of BALF and the content of VIP of the ileum tissue(r= 0.679,P＜0.01).There was an obviously negative correlation between the contents of SP and VIP of the lung tissue in each group(r=-0.678,P＜0.01);there was an obviously positive correlation between the contents of SP and VIP of the lung tissue(r=-0.620,P＜0.01).There was an obviously negative correlation between the contents of SP and VIP of the ileum tissue in each group(r=0.602,P＜0.01);there was an obviously positive correlation between the contents of SP and VIP of the ileum tissue(r=0.608,P＜0.01).5.The pathological results showed that there were bronchial and pulmonary inflammation at different degrees in both group B and A,and the inflammation in both group A1 and B1 had been reduced;there were ileal inflammation at different degrees in both group B and A,and the inflammation in both group A1 and B1 had been reduced,with the inflammation in group A1 was more obvious.CONCLUSION:1.There are,in asthmatic and spleen deficiency asthmatic rats,evident airway or/and general inflammation,expressive imbalance of general and airway immune cytokin IFN-γand IL-4,expressive imbalance of neurikins VIP and SP, and expressive imbalance of the SPmRNA and NK1mRNA receptors of the tissues of the lung and ileum,and the imbalance of neuro-endocrine-immuno-modulation.2.Spleen deficiency can deteriorate the aggregation of inflammatory cells in the peripheral blood and airway of asthmatic rat models,and aggravate the pulmonary and ileal inflammation,and thus exacerbate the general and local inflammatory reaction of the asthmatic body.It can also exacerbate the imbalance of immune cytokins IFN-γand IL-4 in serum and BALF,and aggravate the expressive imbalance of neurikins VIP and SP and the SPmRNA and NK1mRNA receptors of the tissues of the lung and ileum,and thus make the imbalance of neuro-endocrine-immuno-modulation worse,being one of the inner causes for repeated attack of asthma.3.Chinese remedy of SEPM can reduce the airway and general infiltration of inflammatory cells in rat models,down-regulate WBC and EOS in peripheral blood and BALF.It can also regulate the imbalanced state of general and local airway immune cytokins IFN-γand IL-4,up-regulare the content of IFN-γ, down-regulate the content of IL-4,and up-regulate the ratio of IFN-γ/IL-4. It can relieve the expressive imbalance of neurikins VIP,SP and NK1 receptors of the tissues of the lung and ileum,and down-regulate the expressive levels of SPmRNA and NK1mRNA receptors;down-regulate the expressive levels of SP and up-regulate the expressive levels of VIP in both the lung and ileum tissues. And the Chinese remedy of SEPM can reduce the tissue inflammation of the airways, lungs and ileum of the rat models.4.The expressive imbalance of neurikins VIP and SP,and the SPmRNA and NK1mRNA receptors of the tissues of the lung and ileum is obviously correlated with the development and persistence of chronic airway inflammation in asthma, with general and local airway inflammatory state,and with the imbalanced state of general and local airway immune cytokins IFN-γand IL-4.This further reveals the important role of neurikins VIP and SP in development of asthma,especially in inducing imbalance of neuro-endocrine-immuno-modulation.There is evident correlation between the expressive levels of VIP and SP in the tissues of the lung and ileum,which further expounds the close relationship between the lung and the spleen from the angle of neurikins VIP and SP and neuro-endocrine-immuno-modulation.更多还原