针刺调控基质金属蛋白酶网络延长脑梗死溶栓时间窗的研究

发布时间：2018-06-04 09:53

本文选题：脑缺血再灌注 + 醒脑开窍针刺法　；参考：《南京中医药大学》2016年博士论文

【摘要】：目的：观察醒脑开窍针刺法延长脑梗死溶栓时间窗的效应；探讨醒脑开窍针刺法对缺血再灌注损伤的脑保护作用的可能机制。方法：将SD大鼠分为假手术组、模型组、溶栓组、针刺+溶栓组(针刺组)、药物+溶栓组(对照组),以自体血栓栓塞法制作局灶性脑缺血大鼠模型,rt-PA为溶栓手段,以“醒脑开窍”针刺法为干预手段,观察针刺干预对大鼠神经行为学评分、脑梗死容积百分比、脑含水量、脑出血量以及血脑屏障通透性的影响；在此基础上,同时运用Real-time PCR和Western blot方法分别检测各组大鼠皮层中炎性因子TNF-α、 IL-1β、ICAM-1的mRNA及蛋白表达情况；并运用明胶酶谱法检测MMP-2、MMP-9活性变化,最后运用Western blot方法检测紧密连接相关蛋白ZO-1、occludin的表达,以探讨针刺调控基质金属蛋白酶网络的作用机制。结果：1、针刺延长脑梗死溶栓时间窗的效应研究溶栓治疗后脑梗死大鼠神经行为学评分、脑含水量、脑出血量以及血脑屏障通透性随着溶栓时间的延长而增加(P0.05)；溶栓治疗后脑梗死容积百分比与治疗前相比并无明显变化(P0.05)；针刺以及抑制剂干预均能降低神经行为学评分,减少脑梗死体积、脑含水量以及脑出血量,稳定血脑屏障通透性(P0.05)；相比抑制剂,针刺在降低神经行为学评分、减少脑出血量以及稳定血脑屏障通透性上更具优势(P0.05)；在减少脑梗死体积以及脑含水量上两者无统计学差异(P0.05)。2、针刺延长脑梗死溶栓时间窗的机制研究(1)炎性因子的mRNA及蛋白表达：溶栓治疗后脑梗死大鼠TNF-α、IL-1β以及ICAM-1的mRNA及蛋白表达均升高(P0.05)；针刺和抑制剂干预均能降低这三种炎性因子的mRNA及蛋白表达(P0.05)；相比抑制剂,针刺在降低ICAM-1的mRNA及蛋白表达上更具优势(P0.05)；而在降低TNF-α、IL-1β的mRNA及蛋白表达上二者无统计学差异(P0.05)。(2)基质金属蛋白酶活性：溶栓前后比较MMP-2和MMP-9差异具有统计学意义(P0.05)；针刺和抑制剂能降低溶栓后MMP-9活性,且针刺效应大于抑制剂效应(P0.05)；针刺和抑制剂在降低MMP-2活性上与溶栓组比较差异无统计学意义(P0.05)(3)紧密连接蛋白表达：溶栓治疗后脑梗死大鼠ZO-1和occludin的蛋白表达随着溶栓时间的延长而降低(P0.05)；针刺和抑制剂均能提高溶栓后脑梗死大鼠ZO-1和occludin的蛋白表达(P0.05)；相比抑制剂,针刺提高溶栓后脑梗死大鼠ZO-1和occludin的蛋白表达更为显著(P0.05)。结论：大鼠脑缺血后溶栓治疗受到时间窗的严格限制,超过时间窗溶栓会导致神经功能损伤、血脑屏障改变以及出血性转化,且这些影响随着溶栓时间的延长而加重。醒脑开窍针刺法和MMPs广谱抑制剂强力霉素均可通过改善脑缺血再灌注大鼠神经功能损伤、减少溶栓治疗后血脑屏障通透性改变以及出血性转化来实现延长脑梗死溶栓时间窗的效应,且前者神经保护作用较后者更明显。此外,醒脑开窍针刺法较MMPs广谱抑制剂强力霉素能更显著的抑制脑缺血再灌注大鼠皮层相关炎性因子的mRNA及蛋白表达,继而抑制MMP-2、MMP-9过度激活,从而上调紧密连接相关蛋白的表达,从调控MMPs网络途径更好的保护脑组织。
[Abstract]:Objective: To observe the effect of Xingnao Kai Qiao acupuncture on prolonging the time window of thrombolytic thrombolysis in cerebral infarction and to explore the possible mechanism of the protective effect of the acupuncture on cerebral ischemia and reperfusion injury. Methods: the SD rats were divided into sham operation group, model group, thrombolytic group, acupuncture + thrombolytic group (acupuncture group), drug + thrombolytic group (control group) and autologous thromboembolism. The rat model of focal cerebral ischemia was made in the legal system. Rt-PA was the means of thrombolytic therapy. The effects of acupuncture intervention on the neurobehavioral score of the rats, the percentage of cerebral infarction, the volume of cerebral infarction, the amount of brain water content, the amount of cerebral hemorrhage and the permeability of blood brain barrier were observed. On the basis of this, Real-time PCR and Western BL were used at the same time. Ot method was used to detect the mRNA and protein expression of inflammatory factors TNF- a, IL-1 beta, ICAM-1, and MMP-2 and MMP-9 activity by gelatin zymography. Finally, the Western blot method was used to detect the expression of ZO-1 and occludin of closely linked proteins, in order to explore the effect of acupuncture on the regulation of matrix metalloproteinase network. Results: 1, the effect of acupuncture prolonging thrombolytic time window of cerebral infarction study on neurobehavioral score of rats with cerebral infarction after thrombolytic therapy, brain water content, cerebral hemorrhage and blood brain barrier permeability increased with the prolongation of thrombolytic time (P0.05); the percentage of infarct volume after thrombolytic treatment was not significantly different from that before treatment (P0.0 5) Acupuncture and inhibitor intervention can reduce neurobehavioral score, reduce cerebral infarction volume, brain water content and cerebral hemorrhage, and stabilize blood brain barrier permeability (P0.05). Compared with inhibitors, acupuncture is more advantageous in reducing neurobehavioral score, reducing cerebral hemorrhage and stabilizing blood brain barrier permeability (P0.05); in reducing the brain There was no statistical difference between the infarct volume and the brain water content (P0.05).2. The mechanism of prolonging the thrombolytic time window of cerebral infarction (1) the mRNA and protein expression of inflammatory factors: the expression of mRNA and protein in TNF- a, IL-1 beta and ICAM-1 in rats after thrombolytic therapy increased (P0.05), and the intervention of acupuncture and inhibitor could reduce these three species. MRNA and protein expression (P0.05) of inflammatory factors; compared with inhibitors, acupuncture was more advantageous in reducing mRNA and protein expression in ICAM-1 (P0.05), but there was no statistical difference in the mRNA and protein expression of IL-1 beta in the decrease of TNF- alpha (P0.05). (2) the activity of matrix metalloproteinase: the difference of MMP-2 and MMP-9 before and after thrombolytic was statistically significant. Meaning (P0.05); acupuncture and inhibitor can reduce MMP-9 activity after thrombolytic, and acupuncture effect is greater than inhibitor effect (P0.05); acupuncture and inhibitors have no significant difference (P0.05) (3) close connexin expression in reducing the activity of MMP-2 with thrombolytic group (P0.05): the protein expression of ZO-1 and occludin in cerebral infarction rats after thrombolytic treatment with thrombolytic thrombolysis Prolongation of time (P0.05); acupuncture and inhibitors can improve the protein expression of ZO-1 and occludin (P0.05) in rats with cerebral infarction after thrombolytic infarction (P0.05). Compared with the inhibitor, the protein expression of ZO-1 and occludin in rats with cerebral infarction after thrombolytic treatment is more significant (P0.05). Conclusion: Thrombolytic therapy in rats after cerebral ischemia is strictly limited by the time window. More than the time window thrombolytic can cause nerve function damage, blood brain barrier change and hemorrhagic transformation, and these effects aggravate with the prolongation of thrombolytic time. The wake brain opening acupuncture method and the MMPs broad-spectrum inhibitor, doxycycline can improve the nerve function of cerebral ischemia reperfusion rats and reduce the permeability of blood brain barrier after thrombolytic therapy. The effect of sexual change and hemorrhagic transformation on prolonging the time window of thrombolytic thrombolysis was achieved, and the former was more obvious than the latter. In addition, the effect of Xingnao opening and orifices acupuncture was more significant than MMPs broad-spectrum inhibitor, doxycycline, to inhibit the mRNA and protein expression of cortical related inflammatory factors in cerebral ischemia reperfusion rats, and then inhibit MMP-2, MM P-9 overactivates the expression of tight junction proteins, which can better protect brain tissue by regulating MMPs network.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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