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脑缺血病理过程中ADAM17-TNF-α通路的变化及通络救脑方药的干预机制
【作者】 王润俊；
【导师】 李澎涛；
【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2011， 硕士
【摘要】 随着现代医学的发展,传统中医络病学说被赋予了新的含义。许多中枢神经系统疾病如缺血性脑卒中、阿尔茨海默病等均属于传统中医络病的范畴,“毒损脑络”是其关键病机,并且在络病学说的指导下,取得了行之有效的治疗效果。传统中医络病学说与微循环关系极为密切,特别是神经血管单元概念的提出为传统中医络病学说提供了现代医学基础。众所周知,TNF-α作为一种炎症因子在许多疾病,如缺血性脑卒中、中枢神经变性疾病等的发病过程中发挥关键作用。ADAM17是一种在TNF-α分泌成熟过程中起关键作用的脱落酶。在中枢神经系统中,脑微血管内皮细胞、神经元及胶质细胞均可分泌ADAM17和TNF-α。ADAM17-TNF-a通路在中枢神经系统疾病尤其是缺血性脑卒中的发病过程中发挥极其关键的作用。因此,在传统中医络病学说的指导下,我们观察拟缺血状态(脑缺血及细胞氧糖剥夺)下脑内ADAM17-TNF-a通路的变化特点,并且用通络救脑注射液(TLJN)干预来探讨TLJN抗神经损伤机制,进而为“病从络治”提供了现代生物学基础。目的：观察ADAM17、TNF-a在MCAO脑缺血大鼠模型脑组织及血清中的时序性表达特征,探索其与缺血性脑损伤的相关性；并且采用TLJN干预,探索该药抗缺血性脑损伤的途径,进而研究TLJN的抗神经损伤机制是否与ADAM17-TNF-a通路有关。此外,通过体外实验来观察ADAM17、TNF-a在正常、缺氧及通络救脑注射液干预下脑微血管内皮细胞条件培养液中表达的时序特征,进一步为传统中医络病学说提供现代医学依据。方法：1.对MCAO大鼠模型进行神经功能评分后,采用TTC染色观察MCAO大鼠模型脑损伤的程度、脑组织切片HE染色观察脑组织形态结构的变化,探讨通络救脑注射液在不同时间点的抗神经损伤作用。2.采用ELISA和免疫组织化学的方法观察MCAO大鼠模型脑组织及血清中ADAM17、TNF-α在不同时间点的变化特征,探讨ADAM17-TNF-a通路在缺血性脑疾病的发病过程中的作用,以及通络救脑注射液是否通过该通路发挥其神经保护作用。3.观察脑微血管内皮细胞条件培养液(CMEC-CM)中ADAM17、TNF-a的表达情况及通络救脑注射液对CMEC-CM中ADAM17、TNF-a表达的影响。在成功培养并鉴别脑微血管内皮细胞(CMEC)的基础上,制备OGD CMEC模型,采用TLJN干预OGD CMEC后,采用ELISA方法分析CMEC-CM中ADAM17、TNF-a时序变化情况。结果：1.通过对MCAO大鼠模型进行神经功能评分以及观察MCAO大鼠模型脑组织脑损伤水平和形态结构状况,我们发现随着脑缺血的进展,脑梗死体积增加,脑组织形态结构损伤恶化。光镜显示,脑梗死灶及梗死灶周围的神经元数量明显减少、细胞水肿、细胞核碎裂、溶解及间质疏松呈水肿样,血管间隙增宽,内皮细胞和胶质细胞水肿,炎性细胞浸润及继发性出血。通络救脑注射液能显著缩减脑梗死面积、减轻脑水肿和上述病理学变化,并能显著恢复神经功能。阳性对照药依达拉奉注射液具有相似的药效但其疗效不如通络救脑注射液。2. MCAO大鼠模型血清及脑组织中ADAM17、TNF-a在脑缺血早期均显著升高,并具有时序性特征,通络救脑注射液对MCAO大鼠模型各时间点血清及脑组织中均能ADAM17、TNF-a的表达均有显著的降低作用。3.通过观察脑微血管内皮细胞条件培养液(CMEC-CM)中AD AM17、TNF-a表达时序特征及通络救脑注射液对CMEC-CM中ADAM17、TNF-a含量的影响,我们发现OGD可促进CMEC在不同时间点分泌更多的ADAM17、TNF-α; TLJN能够减少不同时间点OGD脑微血管内皮细胞分泌ADAM17、TNF-α。结论：1. TLJN能够改善神经功能、显著缩小脑梗死体积、减轻脑组织病理损害,表明TLJN具有显著的抗脑缺血损伤作用。阳性对照药依达拉奉注射液也具有相似的作用但作用弱于TLJN.2.脑缺血后,受损脑组织中ADAM 17、TNF-a表达显著增高且呈现同步性ADAM17-TNF-a通路在脑缺血病理过程中起关键作用,TLJN的抗脑缺血损伤机制与抑制缺血后脑组织中ADAM17-TNF-a通路有关。3. TLJN抑制缺血后脑组织中ADAM17-TNF-a通路抗缺血性脑损伤作用的实现途径,与影响脑微血管内皮细胞分泌ADAM17和TNF-α密切相关。
【Abstract】 With the development of modern medicine, Collateral disease in traditional Chinese medicine boasts a new meaning.A great many central nervous system(CNS) disease such as ischemic stroke, AD belong to the category of Collateral disease in trditional Chinese medicine,’toxin attacks brain collaterals’is the key point in its progressive pathogenesis, and in the guide of collateral, effective clinical curative outcome has been acquired. The relationship between Collateral in trditional Chinese medicine and microcirculation is extremely close, especially, the concept of neurovascular unit (NVU) coming up provides Collateral in trditional Chinese medicine with morden medical basis.It is well known that TNF-αas a imflemation factor plays a important role in the pathgenesis of a great many disease such as ischemic stroke and so on. ADAM17/TACE is a key shadase that play a key role in release of TNF-α. ADAM17 and TNF-αcan be secreted by cerebral microvascular endothelial cells(CMEC), neuron and glial in CNS. ADAM17-TNF-αpathway is extremely immportant for the pathgenesis of CNS disease especially ischemic stroke.As a result, under guide of’collateral theory’in traditional Chinese medicine, we observe change characters of the ADAM17-TNF-αpathway in brain after hypoxia(cerebral ischemia and OGD) and intervention by injection of Tongluojiunao (TLJN) to explore the mechanism of neuroprotection by TLJN, and provide the modern biological basis for’cure from collateral disease’.Objective:Observe characteristic changes in time series of ADAM17, TNF-αin serum and brain tissue of middle cerebral artery occlusion (MCAO) rat model to explore the relationship between ADAM17, TNF-αand brain injury,in addition, through intervention by TLJN observe the neuroprotection of TLJN after cerebral ischemia, furthermore, to explore weather there is the relationship between the mechanism neuroprotection of TLJN and the pathway of ADAM17-TNF-αIn addition, in vitro observe characteristic changes in time series of ADAM17,TNF-αin normal, oxygen and gclucose deprivation (OGD) and TLJN intervention CMEC condition medium (CMEC-CM). The researchs above furthermore provide Collateral disease in traditional Chinese medicine with modern medical basis.Method:1. Appraise the neurofunction rate of MCAO rat model,and observe morphological stractural changes and information about brain injury of MCAO rat brain tissue to study neuroprotection of TLJN in different time, using brain tissue slice with HE staining and TTC staining2. Observe characteristic changes in different time of ADAM17, TNF-a of MCAO rat serum and brain tissue using ELISA and immunohistochemistry to explore the role of ADAM17-TNF-a pathway played in pathgenesis of ischemic stroke in vivo and weather TLJN plays its neuroprotective role through ADAM17-TNF-a pathway in ischamic stroke in vivo.3. Observe expression of ADAM17,TNF-a in CMEC-CM and influence on expression of ADAM 17,TNF-a in CMEC-CM intervented by TLJN.First, successfully cultivate and identify CMEC. Second,make OGD CMEC model. Third, add TLJN to intervene OGD CMEC. Finally, using ELISA to detect changes in time series of ADAM17, TNF-a in CMEC-CM.Result:1. Through appraising the neurofunction rate of MCAO rat model,and observing morphological stractural changes and information about brain injury of MCAO rat brain tissue, we found that with cerebral ischemia span extending, neuro function morphological stracture of cerebral tissue and cerebral infarct size become deteriorated. Through light microscope (LM), we found that neuron obviously decrease, cellular swell, karyorrhexis, and matrix swell in ischemic infarct focus. It also can be observed that interspace of blood vessel become large; endothelial cells obviously decrease, cell deflate and karyorrhexis; glia become swell and dropsy and inflaming cell infiltrating around ischemic infarct focus. Hemorrhagic focus also can be observed. TLJN can significantly extenuate cerebral infarct size, brain edema and obviously restore neuro function. Through light microscope, we found that TLJN can significantly extenuate pathological changes. Positive control drug injection of Edaravone has the similar effect but its effect is less than TLJN’s.2. ADAM17 and TNF-a in serum and brain tissue of MCAO rat model significantly elevate in the earlier stage after cerebral ischemia and vary with time extending. In addition.TLJN can significantly reduce expression of ADAM17, TNF-a in brain in different time.3. Through observing characteristic expression in different time of AD AM17, TNF-a in CMEC-CM and influence on expression of ADAM17, TNF-a in CMEC-CM by TLJN, we found that OGD can promote CMEC to secrete more AD AM17, TNF-a in different time and TLJN can promote OGD cerebral microvascular endothelial cell to secrete less ADAM17, TNF-αin different time.Conclusions:1. TLJN can obviously extenuate cerebral edema, cerebral infarct area, pathological injury of cerebral tissue and restore neuro function. As a result, the founding above can indicate that TLJN boasts a better anti-neuroinjury on brain after cerebral ischemia. Positive control drug injection of Edaravone has the similar effect but its effect is less than TLJN’s.2. After cerebral ischemia, ADAM17 and TNF-αin injured brain coherently and significantly elevate. ADAM17-TNF-αpathway may play a key role in pathgenesis of cerebral ischemia and the mechanism of anti-neuroinjury by TLJN may be related to inhibiting ADAM17-TNF-αpathway in injuried brain after cerebral ischemia.3. The mechanism of anti-neuroinjury after cerebral ischemia by TLJN through inhibition of ADAM17-TNF-αpathway in cerebral ischemic tissue may be closely related to both ADAM17 and TNF-αsecretion of CMEC.