从HPA轴水平初步探讨缺血性中风急性期补肾护脑法的生物学基础

【作者】 唐璐；

【导师】 孙塑伦；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2011， 博士

【摘要】 中风病急性期的治疗一直是医学界关注的重点,它直接关系到中风病的预后转归和功能恢复。“扶正”与“祛邪”在中风病的治疗中相辅相成,贯穿始终,急性期治疗多采用偏重祛邪的方法,但在临床实践中我们发现,早期应用“扶正”的方法对促进功能恢复、改善预后转归具有重要的意义。本研究在病证结合模式指导下,从理论、实验、临床多方面入手,从脑肾相关的角度探讨了缺血性中风急性期应用补肾法对促进功能恢复、改善预后转归的积极作用,以期完善中医脑病理论,并为中风病疗效评价体系提供生物学的效应指标。理论研究部分：利用脑病文献库对650余本古代医籍的电子图书进行整理,将其中与脑肾理论相关的论述内容梳理分类,从组织结构络属、生理功能联系、病因病机影响、理法方药应用多层次论述了脑肾关系。肾藏精,精生髓,髓通于脑,脑为髓会聚之处,二者密切相关,组织结构、生理功能等方面的内在联系,构成了它们在病因病机上相互影响的重要基础,也是临床中运用补肾法治疗中风病的理论依据。动物实验部分：目的：通过观察MCAO模型大鼠缺血后不同时点ACTH、CRH和COR的动态变化,地黄饮子加减方对ACTH、CRH和COR表达的干预作用及其对神经细胞凋亡、HSP70表达水平的影响,探讨其在脑缺血后纠正HPA轴功能紊乱、促进神经保护的作用效应,以揭示缺血性中风病早期应用补肾中药对脑组织的保护机制,也为临床应用补肾护脑法减轻神经功能缺损和神经元损伤、改善预后转归提供了理论依据。方法：健康雄性SD大鼠,体重280g±10g。第一部分实验中随机分为正常组、假手术组、模型组,每组分为两个时点,每个时点8只,手术组采用改良的线栓法建立MCAO模型,分别于造模后6h、48h取材,检测血ACTH、CRH、COR含量,以多聚甲醛灌注固定取脑,于视交叉处将大脑前后分开,将脑组织石蜡包埋切片,切片厚度约为4μm,做HE染色观察神经细胞形态的变化,用免疫组织化学染色方法观察不同时间点脑组织细胞凋亡的表达。第二部分实验中随机分为模型组、中药组、假手术组,每组10只,造模后第2天开始灌胃,地黄饮子组每日灌胃中药1ml·kg-1,模型组和假手术组灌胃等体积的蒸馏水,连续7天,于最后一次灌胃结束后1h取材,检测血ACTH、CRH、COR含量,以多聚甲醛灌注固定取脑,将脑组织石蜡切片做HE染色观察神经细胞形态的变化,并用免疫组织化学染色方法观察细胞凋亡和HSP70的表达。结果：1.血浆ACTH、CRH、COR含量的测定结果在脑缺血发生后6h,模型组和假手术组的CRH、ACTH、COR含量明显均低于正常组,统计学结果有显著差异；在48h时模型组ACTH、COR的含量明显高于正常组和6h模型组,统计学结果有显著差异；CRH的含量较6h时进一步降低,明显低于正常组,统计学结果有显著差异。2.MCAO大鼠不同缺血时点脑组织细胞凋亡表达模型组大鼠造模后6h即可见TUNEL染色阳性细胞,48h仍可见,与正常组和假手术组比较,有极显著差异(P<0.01),但6h与48h组之间统计学无明显差异。3.地黄饮子加减方对MCAO大鼠血浆HPA轴的干预作用脑缺血发生后7天,模型组的ACTH、CRH含量均明显低于正常组,COR含量明显高于正常组,统计学有显著差异；地黄饮子组的COR明显低于正常组、模型组,统计学有显著差异；地黄饮子组ACTH、CRH含量均明显高于模型组,统计学有显著差异；ACTH、CRH含量略低于正常组,统计学无差异。COR与ACTH和CRH呈反向性变化。4.地黄饮子对MCAO大鼠缺血后脑组织细胞凋亡表达的干预效应灌胃7天后的MCAO大鼠,模型组、中药组均可见脑组织有细胞凋亡表达,二者与正常组相比,统计学结果有显著差异(P<0.01)；模型组脑组织细胞凋亡表达较中药组明显,模型组与中药组相比,统计学结果有显著差异(P<0.01)。5.MCAO大鼠缺血后HPA轴与脑组织细胞凋亡表达的相关性分析模型组ACTH含量与脑组织细胞凋亡表达的TUNEL染色结果存在显著负相关关系,但CRH、COR则与凋亡表达无明显相关性。中药组ACTH、CRH、COR三者与脑组织细胞凋亡表达无明显相关性。6.地黄饮子对MCAO模型大鼠脑组织HSP70表达的干预效应同正常组比较,模型组和中药组脑组织均有HSP70表达,且统计学有显著差异；同模型组相比,中药组脑组织的HSP70表达更为明显,统计学有显著差异。7.MCAO大鼠缺血后HPA轴与脑组织HSP70表达的相关性分析模型组COR含量与脑组织HSP70表达存在显著正相关关系,但CRH、ACTH则与HSP70表达无明显相关性。中药组HPA轴各指标与脑组织HSP70表达无明显相关性。结论：1.脑缺血后6h,HPA轴呈抑制状态；脑缺血后48h,HPA轴明显紊乱,可能是该时点症状加重、死亡率增高的一个原因。2.地黄饮子加减方可以改善MCAO模型大鼠术后的整体状态,减轻动物体重下降率。3.地黄饮子加减方可以通过提高CRH的含量来抑制大鼠体内COR的释放,从而改善HPA轴的紊乱状态。4.地黄饮子加减方可以明显抑制脑组织细胞凋亡的表达,这种作用可能与其改善HPA轴的紊乱状态有关。5.地黄饮子加减方可以通过促进缺血区脑组织HSP70的表达发挥神经保护作用。临床研究部分：以病证结合模式为指导,探讨缺血性中风急性期证候与生物学指标的相关性,本研究病例的数据来源于国家重点基础研究发展计划(973计划)课题-缺血性中风病证结合的诊断标准与疗效评价体系研究(课题编号：2003CB517102)。使用《缺血性中风证候要素诊断量表》,纳入211例急性期缺血性中风病患者,动态采集四诊信息以判断证候要素,采集血清标本进行生物学指标ACTH、COR、HSP70的检测,初步探讨了它与中医证候、病机在生物学基础层面的关系,从一定程度上完善了缺血性中风病证结合的诊断标准,为建立早期运用补肾中药对缺血性中风患者预后影响的临床疗效评价体系提供了生物学指标依据,同时也为构建完整的中医脑病理论体系奠定了基础。更多还原

【Abstract】 In recent, there has been growing concerning about treatment of acute stroke which has implications for prognosis and rehabil itat ion. In Tranditional Chinese medicine (TCM), "reinforcing the healthy qi" and "eliminating the pathogenic factors" are the main methods of treatment throughout the pathological process of stroke. Current trends in treatment of acute stroke was with an emphasis on "eliminating the pathogenic factors", whereas through summarizing the clinical experience, "reinforcing the healthy qi" were found exerting significant effect to improve prognosis and rehabilitation. With the integrating disease and syndrome pattern, this study is to explore positive function of "reinforcing the healthy qi " treatment in acute phase on improving prognosis and rehabilitation from the perspective of "combination with kindey and brain" in order to provide the biomark of evaluting the effecacy of acute stroke. Theory research:The theory about "combination with kindey and brain" was screened and catagoried with respects to organizational structure, Physiological function, Pathogenesis and application of herbs from 650 ancient medical books involved in an e-book archive. The kidney stores the essence, essence produces marrow, marrow connects to brain, and brain is the convergence of marrow, the brain and kidney have close relationship. Organizational structure and physiological function not only composes an important basis of interaction on the pathogenesis, but also provides the theoretical basis of clinical treatment for acute stroke. Animal experiment:Objective:To investigate dynamic level changes of ACTH, CRH and COR of MCAO rats at different time point, expression levels of ACTH, CRH, COR were detected by ELISA at 6h and 48h after cerebral ischemia. To determine if Dihuangyinzi decoction interferes with the HPA axis, apoptosis process and the expression of HSP70 in MCAO rats and to explore the role of Dihuangyinzi decoction in neuroprotection effect and improving the disturbed condition of HPA axis, the morphological changes of neural cell and apoptosis in cerebral histiocyte were detected by staining brains slices with hematoxylin-eosin(HE) and imunohistochemical (IHC) method. In summary, this study was with the aim of revealing neuroprotection mechanism and basis theory with application of reinforcing kidney herbs in acute phase of stroke.Method:Male Sprague-Dawley (SD) rats of body weight 280±10g were used for this test.Part one:48 SD rats were randomly divided into 3 groups:normal group, sham operatedgroup and MCAO model group, each group was 16. MCAO was induced by inserting a nylon monofilament through the external carotid artery into theintracranial circulation to occlude the middle cerebral artery at its origin. SD rats were killed 6 or 48h after cerebral ischemia. The expression levels of ACTH, CRH, COR were detected by ELISA at 6h and 48h after cerebral ischemia.Furthemore, the brain specimens were fixed in paraformaldehyde, embedded in paraffin, and then cut into Sagittal slices of 4μm thickness.To detect the morphological changes of neural cell and apoptosis in cerebr al histiocytes, brain slices were staind with hematoxylin-eosin (HE) and Ini-munohistochemical(IHC) method, respectively.Part two:30 SD rats were randomly divided into 3 groups:MCAO model group, herb formula group,and sham operated group,each group was 10. For drug tre atment, herb formula group were given Dihuangyinzi decoction from 2days to 8days after ischemia (1ml/kg body mass) by intragastric administration met hod, whereas the MCAO model group and sham operated group received equivale nt volumes of distilled water. SD rats were killed 1h after the last intrag astric administration. The expression levels of ACTH, CRH, COR were detecte d by ELISA. Furthemore, the brain specimens were fixed in paraformaldehyde, embedded in paraffin, and then cut into Sagittal slices of 4μm thickness. To detect the morphological changes of neural cell, apoptosis in cerebral histiocytes and the expression of HSP70, brain slices were staind with hema toxylin-eosin(HE) and imunohistochemical (IHC) method, respectively.Result:1. Expression levels of ACTH, CRH and COR in serumThe levels of ACTH, CRH and COR at 6h after ischemia were significantly lower in MCAO model group and sham operated group than in normal group(P<0.05); the levels of ACTH, COR were significantly greater in MCAO model group at 48h after ischemia than that in normal group and MCAO model group at 6h after ischemia(P<0.05); the level of CRH at 48h after ischemia in MCAO model group was significantly lower in MCAO model group at 6h after ischemia and in normal group (P<0.05).2. Apoptosis in cerebral histiocytes of MCAO rats at different time point.TUNEL-positive cerebral histiocytes were found by light microscope in MCAO model group at 6h and 48h after ischemia specimens (vs normal group and sham operated group, P<0.01), however, there was no significant difference between MCAO model group at 6h after ischemia and MCAO model group at 48h after ischemia. 3 Dihuangyinzi decoction interferes with the HPA axis serum reaction of MCAO At 8days after ischemia, the levels of ACTH, CRH were significantly lower in MCAO model group than in normal group (P<0.05), the level of COR in MCAO model group were significantly higher than that in normal group; The level of COR was significantly lower in herb formula group than in normal and MCAO model group; The levels of ACTH, CRH were significantly higher in herb formula group than in MCAO model group, the levels of ACTH, CRH were lower in herb formula group than in normal group withou significant difference.A reverse change was observed between the level of ACTH, CRH and the level of COR.4. Dihuangyinzi decoction interferes with apoptosis in cerebral histiocytes of MCAO rats.At 7days after intragastric administration, TUNEL-positive cerebral histiocytes were found in MCAO model group and herb formula group (vs normal group, P<0.01); The level of apoptosis in cerebral histiocytes was significantly greater in MCAO model group than in herb formula group (P<0.01).5. Correlation analysis between apoptosis in cerebral histiocytes and HPA axis serum reaction of MCAO rats.In MCAO model group, there was significantly negative correlation between the level of ACTH and apoptosis in cerebral histiocytes by TUNEL staining, while there were no correlations between the levels of CRH, COR and the apoptosis in cerebral histiocytes. In herb formula group, there were no correlations between the levels of ACTH,CRH, COR and the apoptosis in cerebral histiocytes.6. Dihuangyinzi decoction interferes with the expression of HSP70 in cerebral tissu of MCAO rats.The expression of HSP70 were found in MCAO model group and herb formula group (vs normal group, P<0.05); The expression of HSP70 was significantly greater in herb formula group than in MCAO model group (P<0.05).7. Correlation analysis between the expression of HSP70 in cerebral tissu and HPA axis serum reaction of MCAO rats.In MCAO model group, there was significantly positive correlation between the level of COR and the expression of HSP70, while there were no correlations between the levels of CRH, ACTH and the expression of HSP70. In herb formula group, there were no correlations between the levels of ACTH,CRH, COR and the expression of HSP70 in cerebral tissue.Conclusion:1. At 6h after ischemia, HPA axis serum reaction was in an inhibited state. At 48h after ischemia, HPA axis serum reaction was in an obviously disturbed condition which might lead to more complicated syptoms and higher mortality. 2. Dihuangyinzi decoction can improve the holistic state of MCAO rats and prevent from weight loss.3. Dihuangyinzi decoction can inhibit the release of COR by increasing the level of CRH, which can improve the disturbed condition of HPA axis serum reaction.4. Dihuangyinzi decoction can obviously inhibit apoptosis in cerebral histiocytes, which may have implication for improving the disturbed condition of HPA axis serum reaction5. Dihuangyinzi decoction can exert neuroprotection effect by enhancing the expression of HSP70 in the ischemia cerebral tissue.Clinical reseach:Under the guidance of "integrating disease and syndrome" research model, this study revealed the correlations between TCM syndrome and biomarker in acute phase of ischemic stroke. All the cases of this study have been chosen from National Point Foundation Research Development Plan (973 plan)---Ischemic Stroke Diagnosis Combining Symptoms and Signs of Disease and Effect Evaluation System Research. (No.2003CB517102). In this study, to explore the relationship between biomarker and TCM syndrome and to develop the diagnosic standards for ischemic stroke under the "integrating disease and syndrome" pattern, " Ischemic Stroke TCM Syndrome Factor Diagnostic Scale" (ISTSFDS) was collected for Syndrome diagnosis and serum samples were collected for detecting the levels of ACTH, COR and HSP70. In summary, this study may provide the theoretical basis of TCM neurology.更多还原