CORM-2对大鼠脑出血后炎症反应的抑制作用及分子机制

发布时间：2018-06-08 18:34

本文选题：脑出血 + CORM-2　；参考：《南昌大学》2017年硕士论文

【摘要】：目的与背景:脑出血是一种常见的、致命的卒中亚型,出血后灶周组织小胶质细胞被激活,释放大量炎症因子和趋化因子,启动继发性脑损伤。因此调控灶周炎症反应对减轻脑出血继发性损伤具有重要意义。内源性CO产生的主要途径是HO-1催化血红素降解过程,目前已有众多学者应用CORM-2来研究CO的生物学效应,在体和离体研究均发现低浓度的CO具有抗炎性损伤的作用。本研究采用CORM-2干预,提高体内CO含量,通过建立脑出血动物模型,进行在体实验研究,观察CORM-2对脑出血后灶周炎性损伤的影响并探讨其相关机制。方法:采用自体股动脉血注入大鼠基底节区建立脑出血模型,随机将SD大鼠分为以下4个实验组:正常对照组、单纯ICH组、CORM-2+ICH组和iCORM-2+ICH组,每组各5只,分别在ICH术后6h、24h、48h和5d采用Gareia18分测评法对大鼠进行神经功能评分,评分后收集大鼠脑组织标本,采用Western Blot检测脑组织中HO-1、IKKβ、NF-κB p65以及TNF-α蛋白表达情况,免疫荧光双标染色方法检测灶周小胶质细胞活化情况以及炎症因子TNF-α与小胶质细胞共表达情况。结果:(1)大鼠脑出血术后神经功能评分在术后各时间点均有所下降,且在48h评分最低(P0.05);CORM-2干预可改善大鼠神经缺损症状。(2)与正常对照组相比,ICH组术后6h灶周IKKβ及炎性因子NF-κB p65和TNF-α蛋白表达均开始升高(P0.05),48h达高峰(P0.05),且持续到第5天表达量仍高于正常对照组(P0.05);抗氧化蛋白HO-1表达趋势与TNF-α蛋白基本一致(P0.05)。(3)CORM-2干预组与ICH组比较,IKKβ、NF-κB p65及TNF-α蛋白表达水平均有不同程度的减少,且在48h下降最明显(P0.05);同时HO-1表达明显增加(P0.05)。(4)大鼠脑出血后小胶质细胞活化,表现为术后6h灶周大量Iba-1标记的阳性细胞,且荧光双标染色发现大多数为TNF-α与Iba-1标记共表达阳性细胞,CORM-2干预组术后5天TNF-α与Iba-1标记共表达阳性细胞比例较ICH组明显减少(P0.05)。(5)iCORM-2干预组较ICH组各蛋白表达及神经功能评分无明显差异(P0.05)。结论:(1)大鼠脑出血后6h灶周IKK/NF-κB通路开始被激活;(2)CORM-2干预可有效地增加HO-1表达,抑制IKKβ及炎性因子NF-κB p65和TNF-α的表达;(3)大鼠脑出血后CORM-2可能通过抑制IKK/NF-κB信号通路,抑制炎症因子TNF-α的表达,从而减轻脑损伤,改善大鼠神经功能损伤。(4)CORM-2的抗炎作用可能与灶周小胶质细胞有关。
[Abstract]:Objective and background: intracerebral hemorrhage (ICH) is a common and fatal subtype of stroke. After intracerebral hemorrhage microglia are activated release a large number of inflammatory factors and chemokines and initiate secondary brain injury. Therefore, the regulation of perifocal inflammation is of great significance in alleviating secondary injury of intracerebral hemorrhage. The main pathway of endogenous CO production is HO-1 catalyzing heme degradation. At present, many researchers have applied CorM-2 to study the biological effect of CO. In vitro and in vitro, it has been found that low concentration of CO has anti-inflammatory effect. In this study, CorM-2 intervention was used to increase CO content in vivo. By establishing animal model of intracerebral hemorrhage, in vivo experimental study was carried out to observe the effect of CorM-2 on perifocal inflammatory injury after intracerebral hemorrhage and to explore its related mechanism. Methods: intracerebral hemorrhage model was established by injecting autologous femoral arterial blood into the basal ganglia of rats. SD rats were randomly divided into four groups: normal control group, ICH group (ICH group), CorM-2 ICH group and iCorM-2 ICH group (5 rats in each group). The neurological function of rats was evaluated with Gareia 18 score at 6 h, 24 h, 48 h and 5 d after ICH. The brain tissue samples were collected. Western blot was used to detect the expression of HO-1IKK 尾 -NF- 魏 B p65 and TNF- 伪 protein in brain tissue. The activation of microglia and the co-expression of inflammatory factor TNF- 伪 with microglia were detected by double immunofluorescence staining. Results the neurological function score of rats with intracerebral hemorrhage decreased at all time points after ICH. And the lowest P0.05CorM-2 intervention at 48h could improve the symptom of nerve defect in rats.) compared with the normal control group, the expression of IKK 尾 and inflammatory factors NF- 魏 B p65 and TNF- 伪 began to increase in ICH group at 6h after operation, and reached the peak at 48h, and lasted until the 5th day. The expression trend of HO-1 and TNF- 伪 protein was similar to that of TNF- 伪 protein, and the expression levels of Ike 尾 -NF- 魏 B p65 and TNF- 伪 protein in ICH group and ICH group were decreased in different degrees. The expression trend of HO-1 protein was similar to that of TNF- 伪 protein in ICH group and ICH group, and the expression trend of HO-1 protein was similar to that of TNF- 伪 protein in ICH group and ICH group. The expression of HO-1 significantly increased the activation of microglia after intracerebral hemorrhage in rats, which was characterized by a large number of Iba-1 labeled positive cells around the focal area 6 h after operation. Fluorescence double labeling staining showed that the proportion of TNF- 伪 and Iba-1 co-expressed cells in CorM-2 intervention group was significantly lower than that in ICH group on the 5th day after operation compared with ICH group. The expression of each protein and neuronal work in ICH group was significantly lower than that in ICH group, and the ratio of TNF- 伪 and Iba-1 co-expressed positive cells in ICH group was significantly lower than that in ICH group. There was no significant difference in energy score (P 0.05). Conclusion the activation of IKK / NF- 魏 B pathway at 6 h after intracerebral hemorrhage can effectively increase the expression of HO-1, and inhibit the expression of IKK 尾 and inflammatory factors NF- 魏 B p65 and TNF- 伪. CorM-2 may inhibit IKK- / NF- 魏 B signal pathway after intracerebral hemorrhage in rats. Inhibiting the expression of inflammatory factor TNF- 伪, thus reducing brain injury, and improving the anti-inflammatory effect of Corm-2 in rats with nerve function injury may be related to the microglia around the focal area.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

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