“瘀血生风”假说检验
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摘要
目的:探讨缺血性中风与瘀血的关系以及活血熄风法对缺血性中风大鼠的干预作用和作用机理。
方法:采用光化学法诱导制备缺血性中风大鼠模型,观察具有活血熄风作用的活血熄风方对模型大鼠的拮抗作用以及在不同水平层面上的影响,用评分法观察神经功能情况,TTC染色法测脑梗塞体积,称重法测脑组织水肿,激光多普勒测量仪测局部脑血流,放免法测IL-1β、TNF-α和ET的变化,分光光度法测脑组织中SOD、GSH-PX、ATPase及LDH的活性以及MDA、NO含量,HE染色观察脑组织病理形态学变化,TUNEL法观察神经细胞凋亡情况,免疫组化法测凋亡相关基因Bax、Bcl-2、Caspase-3蛋白表达,RT-PCR法测脑组织中Caspase-3mRNA及VEGFmRNA表达。
结果:活血熄风方能明显改善缺血性中风大鼠神经症状,减小脑梗塞范围,减轻脑水肿,增加局部脑血流,降低IL-1β、TNF-α的含量,调节ET/NO比值;增强脑组织中SOD、GSH-PX、Na~+-K~+-ATPase、Ca~(2+)-ATPase及LDH的活性,降低MDA含量,减少TUNEL阳性细胞数,上调抑凋亡基因Bcl-2蛋白的表达,抑制促凋亡基因Bax、Caspase-3蛋白表达及Caspase-3mRNA表达,上调VEGFmRNA的表达。
结论:光化学法诱导的缺血性中风大鼠模型行为学改变以及导致这些行为改变的内在病理变化与瘀血具有相关性,活血熄风法治疗具有明显的脑保护作用,其作用机制可能与增加脑血流量,抑制炎症反应,调节血管舒缩状态,改善能量代谢障碍,抗自由基损伤,调节凋亡相关基因表达,促进血管新生等有关。
Objective: Research the relationship between focal cerebral infarction and blood stasis, and study the effect and its mechanism on the focal cerebral infarction rats by promoting blood flow and stopping endogenous wind.
Method: The model rats of focal cerebral infarction was established based on the principle of photochemistry initiation of thrombosis. Through observing the antagonistic effect and influence in different level on model rats by treat with Huoxue Xifeng Decoction (HXD) which can promote blood flow and stop endogenous wind. Observe the neurological symptoms through scoring, measure the cerebral infarction volume use TTC staining method, measure the water content of brain tissue with weighting method and Laser Doppler flowmetry was used to detect the regional cerebral blood flow(rCBF) in the ischemic core. Radio-immunity method was used to detect the contents of IL-1β、TNF-αand ET. Spectrophotometric method was used to detect the activities of SOD,GSH-PX, ATPase, LDH and the contents of MDA and NO. Pathomorphological changes was observed by HE staining method. Apoptosis of nerve cells was studied by TUNEL. The protein express of apoptosis related gene Bax、Bcl-2、Caspase-3 was observed by immunohistochemical method, the express of Caspase-3mRNA and VEGFmRNA was studied by RT-PCR.
Result: Contrast to the model, HXD could obviously promote neurological deficit, decrease the water content of brain tissue and the volume of cerebral infarction zone,increase the rCBF, decrease the contents of IL-1βand TNF-α, regulate the ratio of ET/NO, promote the activities of SOD, GSH-PX, Na~+-K~+-ATPase, Ca~(2+)-ATPase and LDH, decrease MDA content, decrease numbers of TUNEL masculine cells, up-regulation the express of Bcl-2 protein which can restrain apoptosis, down-regulation the express of Bax、Caspase-3 protein and Caspase-3mRNA which can hasten apoptosis and up-regulation the express of VEGFmRNA.
Conclusion: The model rats of focal cerebral infarction which induced by photochemistry had stagnated blood signs, and the treatment of promoting blood flow and stopping endogenous wind has significant brain protective effect, the mechanism may be related to that it can increase the rCBF,restrain inflammatory reaction , regulate the blood vessel relax-contract condition ,improve energy metabolism and antagonize lesion of free radicals,regulate the express of apoptosis related gene and promote the blood vessel born newly.
方法:采用光化学法诱导制备缺血性中风大鼠模型,观察具有活血熄风作用的活血熄风方对模型大鼠的拮抗作用以及在不同水平层面上的影响,用评分法观察神经功能情况,TTC染色法测脑梗塞体积,称重法测脑组织水肿,激光多普勒测量仪测局部脑血流,放免法测IL-1β、TNF-α和ET的变化,分光光度法测脑组织中SOD、GSH-PX、ATPase及LDH的活性以及MDA、NO含量,HE染色观察脑组织病理形态学变化,TUNEL法观察神经细胞凋亡情况,免疫组化法测凋亡相关基因Bax、Bcl-2、Caspase-3蛋白表达,RT-PCR法测脑组织中Caspase-3mRNA及VEGFmRNA表达。
结果:活血熄风方能明显改善缺血性中风大鼠神经症状,减小脑梗塞范围,减轻脑水肿,增加局部脑血流,降低IL-1β、TNF-α的含量,调节ET/NO比值;增强脑组织中SOD、GSH-PX、Na~+-K~+-ATPase、Ca~(2+)-ATPase及LDH的活性,降低MDA含量,减少TUNEL阳性细胞数,上调抑凋亡基因Bcl-2蛋白的表达,抑制促凋亡基因Bax、Caspase-3蛋白表达及Caspase-3mRNA表达,上调VEGFmRNA的表达。
结论:光化学法诱导的缺血性中风大鼠模型行为学改变以及导致这些行为改变的内在病理变化与瘀血具有相关性,活血熄风法治疗具有明显的脑保护作用,其作用机制可能与增加脑血流量,抑制炎症反应,调节血管舒缩状态,改善能量代谢障碍,抗自由基损伤,调节凋亡相关基因表达,促进血管新生等有关。
Objective: Research the relationship between focal cerebral infarction and blood stasis, and study the effect and its mechanism on the focal cerebral infarction rats by promoting blood flow and stopping endogenous wind.
Method: The model rats of focal cerebral infarction was established based on the principle of photochemistry initiation of thrombosis. Through observing the antagonistic effect and influence in different level on model rats by treat with Huoxue Xifeng Decoction (HXD) which can promote blood flow and stop endogenous wind. Observe the neurological symptoms through scoring, measure the cerebral infarction volume use TTC staining method, measure the water content of brain tissue with weighting method and Laser Doppler flowmetry was used to detect the regional cerebral blood flow(rCBF) in the ischemic core. Radio-immunity method was used to detect the contents of IL-1β、TNF-αand ET. Spectrophotometric method was used to detect the activities of SOD,GSH-PX, ATPase, LDH and the contents of MDA and NO. Pathomorphological changes was observed by HE staining method. Apoptosis of nerve cells was studied by TUNEL. The protein express of apoptosis related gene Bax、Bcl-2、Caspase-3 was observed by immunohistochemical method, the express of Caspase-3mRNA and VEGFmRNA was studied by RT-PCR.
Result: Contrast to the model, HXD could obviously promote neurological deficit, decrease the water content of brain tissue and the volume of cerebral infarction zone,increase the rCBF, decrease the contents of IL-1βand TNF-α, regulate the ratio of ET/NO, promote the activities of SOD, GSH-PX, Na~+-K~+-ATPase, Ca~(2+)-ATPase and LDH, decrease MDA content, decrease numbers of TUNEL masculine cells, up-regulation the express of Bcl-2 protein which can restrain apoptosis, down-regulation the express of Bax、Caspase-3 protein and Caspase-3mRNA which can hasten apoptosis and up-regulation the express of VEGFmRNA.
Conclusion: The model rats of focal cerebral infarction which induced by photochemistry had stagnated blood signs, and the treatment of promoting blood flow and stopping endogenous wind has significant brain protective effect, the mechanism may be related to that it can increase the rCBF,restrain inflammatory reaction , regulate the blood vessel relax-contract condition ,improve energy metabolism and antagonize lesion of free radicals,regulate the express of apoptosis related gene and promote the blood vessel born newly.
引文
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