炎症反应和氧化应激在创伤性颅脑损伤中的作用及CHPG和葛根素对其干预效果的研究

发布时间：2018-05-31 22:16

本文选题：炎症 + 应和　；参考：《南京大学》2013年博士论文

【摘要】：第一部分:绪论本部分重点介绍了论文相关的研究背景,包括创伤性颅脑损伤后的继发性脑损伤发生机制;代谢性谷氨酸受体的基本类型和药理学研究进展;mGluR5及其激动剂CHPG在神经胶质细胞中作用研究;葛根素的神经保护作用及抗氧化应激通路等。第二部分:大鼠创伤性颅脑损伤后代谢性谷氨酸受体5(mGluR5)的表达及细胞分布类型背景与目的:创伤性颅脑损伤(TBI)后大量的谷氨酸被释放,从而可以激活一系列的谷氨酸受体,其中包括代谢性谷氨酸受体5(mGluR5),进一步发生相关的胞内反应。本研究旨在描述TBI后mGluR5蛋白及mRNA水平的时间表达情况,同时明确mGluR5在脑内的细胞分布类型。方法:采用改良的Feeney自由落体模型制作中度局灶性颅脑损伤模型(40g*15cm)。在伤后不同的时间点(0.5h,1h,6h,12h,24h,3d)留取伤灶周围皮层组织用于Western Blotting及RT-qPCR检测,在mGluR5蛋白水平表达高峰留取脑组织用于免疫组化及荧光双染检测。结果:(1)mGluR5蛋白水平在伤后明显增加,伤后24h时达到高峰,在伤后3d mGluR5的表达仍较空白组明显增高。(2)mGluR5mRNA水平在伤后呈现双相改变,早期明显升高(0.5h,1h,),后期则明显降低(24h,3d)。(3)常规免疫组化及荧光双染显示TBI后伤灶周围神经元明显丢失,小胶质细胞活化,星形胶质细胞缠结,神经元及胶质细胞均表达mGluR5。结论:创伤性颅脑损伤后,mGluR5无论是蛋白水平还是mRNA水平,时间或者空间上均发生改变,这些改变提示mGluR5可能参与了 TBI后的继发性脑损伤及自身保护调节。第三部分:大鼠创伤性颅脑损伤后应用mGluR5激动剂CHPG对TBI诱导的炎症反应的影响背景与目的:大量文献已经证实以小胶质细胞为主导的炎症反应是创伤性颅脑损伤后继发性脑损伤的重要因素之一。体外研究发现mGluR5激动剂CHPG可以抑制小胶质细胞的活化及其炎症因子的释放。本研究旨在探讨mGluR5激动剂CHPG能否通过减轻TBI后的炎症反应来发挥神经保护作用。方法:采用改良的Feeney自由落体模型制作中度局灶性颅脑损伤模型(40g*15cm)。TBI或者空白损伤30min后通过侧脑室注射给予250nmol的CHPG或者溶剂。全部大鼠于伤后24小时处理,留取伤灶周围皮层标本,用于检测脑水肿、Fluoro-Jade C染色、ED-1染色、炎症因子含量(ELISA,RT-qPCR)。结果:(1)mGluR5激动剂CHPG可以显著减少TBI后伤灶周围皮层脑水肿及神经元退行性改变。(2)mGluR5激动剂CHPG可以显著减少TBI后伤灶周围皮层ED-1的表达。(3)mGluR5激动剂CHPG在蛋白及mRNA水平两个方面均能显著减少TBI后伤灶周围皮层相关炎症因子(IL-1beta,IL-6,TNF-alpha)的表达。结论:创伤性颅脑损伤后,mGluR5激动剂CHPG可以通过抑制小胶质细胞的活化及其炎症因子的释放来减少伤灶周围皮层神经元的退变,从而发挥其神经保护作用。第四部分:葛根素对大鼠创伤性颅脑损伤诱导的氧化应激性脑损伤的影响及其对PI3K/Akt信号通路的作用背景与目的:相关研究已经证实氧化应激反应是创伤性颅脑损伤后继发性脑损伤的重要因素之一。近期,大量体内及体外文献均报导了葛根素能通过激活PI3K/Akt通路发挥其抗氧化应激的作用。但关于葛根素在TBI中的作用尚属未知。因此本研究主要探讨了葛根素对TBI诱导的氧化应激性脑损伤的影响及可能的作用机制。方法:采用改良的Feeney自由落体模型制作中度局灶性颅脑损伤模型(40g*15cm)。TBI或者空白损伤10min前通过腹腔注射给予200mg/kg的葛根素或者溶剂。其中一个亚组在给予葛根素前通过侧脑室注射给予PI3K/Akt通路抑制剂LY294002。全部大鼠于伤后24小时处理,留取伤灶周围皮层标本,用于检测Phospho-Akt表达,Fluoro-JadeC染色、氧化应激标志物(MDA,GSH,Na+-K+-ATPase活性,MPO活性)。结果:(1)葛根素能够显著减轻TBI后伤灶周围皮层神经元退行性改变。(2)葛根素能够显著升高GSH水平及Na+-K+-ATPase活性,降低MDA含量及MPO活性。(3)葛根素能够明显增强phospho-Akt的表达,同时给予PI3K/Akt通路抑制剂LY294002后葛根素的保护作用被明显减弱。结论:创伤性颅脑损伤后,葛根素可以通过激活PI3K/Akt通路,进而发挥其抗氧化应激作用,最终能够减少伤灶周围皮层神经元的退变,从而发挥其神经保护作用。
[Abstract]:The first part: the introduction focuses on the research background of the paper, including the mechanism of secondary brain injury after traumatic brain injury, the basic types of the metabolic glutamate receptor and the progress of pharmacology, the study on the role of mGluR5 and its agonist CHPG in Neuroglia; the neuroprotective effect of puerarin and its resistance to Neuroglia The second part: the second part: the expression of glutamate receptor 5 (mGluR5) in the offspring of traumatic brain injury in rats and the background and objective of the type of cell distribution: a large number of glutamic acid is released after traumatic brain injury (TBI), which can activate a series of glutamate receptors, including the metabolic glutamate receptor 5 (mGluR5), and the following This study was designed to describe the time expression of mGluR5 protein and mRNA levels after TBI, and to identify the type of cellular distribution of mGluR5 in the brain. Methods: a modified Feeney free falling body model was used to make moderate focal brain injury model (40g*15cm). At different time points after injury (0.5h, 1H, 6h, 12h, 24h, 3D) The surrounding cortex of the wound was used for the detection of Western Blotting and RT-qPCR, and the peak of the expression of the mGluR5 protein was used for immunohistochemistry and fluorescence double staining. Results: (1) the level of mGluR5 protein increased obviously after injury, the peak of 24h after injury, and the expression of 3D mGluR5 after injury was still higher than that in the blank group. (2) mGluR5mR The level of NA was changed in two phases after injury (0.5h, 1H, 1H), and later obviously decreased (24h, 3D). (3) conventional immunohistochemistry and fluorescent double staining showed that the neurons around the injured area were obviously lost, the microglia activation, astrocyte tangles, the Shen Jing Yuan and glial cells all expressed mGluR5. conclusion: traumatic brain injury, mGl UR5, whether it is protein level or mRNA level, changes in time or space, these changes suggest that mGluR5 may be involved in secondary brain injury and self-protection regulation after TBI. Third part: the background and purpose of the effect of mGluR5 agonist CHPG on TBI induced inflammatory response after traumatic brain injury in rats: a large number of literature It has been confirmed that the microglia led inflammatory response is one of the important factors for secondary brain damage after traumatic brain injury. In vitro studies have found that mGluR5 agonist CHPG can inhibit the activation of microglia and the release of inflammatory factors. The purpose of this study was to explore whether mGluR5 agonist CHPG could reduce inflammation after TBI. Methods: the modified Feeney free falling body model was used to make the moderate focal brain injury model (40g*15cm).TBI or the blank injury 30min after the injection of CHPG or solvent to 250nmol by injection of the lateral ventricle. All rats were treated at 24 hours after injury and left the surrounding cortex specimens of the wound, for the detection of brain edema. Fluoro-Jade C staining, ED-1 staining, and inflammatory factor content (ELISA, RT-qPCR). Results: (1) mGluR5 agonist CHPG can significantly reduce the cerebral edema and neuronal degeneration around the injured focal lesion after TBI. (2) mGluR5 agonist CHPG can significantly reduce the expression of ED-1 in the cortex around the injured focal lesion. (3) the agonist is at the protein and the level of two Conclusion: after traumatic brain injury, the mGluR5 agonist CHPG can reduce the activation of microglia and the release of inflammatory factors to reduce the degeneration of the peripheral cortical neurons in the wound, thus exerting its neuroprotection after traumatic brain injury. Conclusion: after traumatic brain injury, the mGluR5 agonist CHPG can reduce the degeneration of the periteno cortical neurons in the wound. The fourth part: the fourth part: the effect of Puerarin on oxidative stress brain injury induced by traumatic brain injury in rats and the background and purpose of its effect on the PI3K/Akt signaling pathway: the related study has confirmed that the oxidative stress reaction is one of the important factors of secondary brain injury after traumatic brain injury. The effect of Puerarin on antioxidant stress by activating PI3K/Akt pathway is reported. However, the role of Puerarin in TBI is unknown. Therefore, this study mainly discussed the effect and possible mechanism of Puerarin on TBI induced oxidative stress brain injury. Methods: a modified Feeney free falling body model was used. The moderate focal craniocerebral injury model (40g*15cm).TBI or blank injury before 10min was injected into the puerarin or solvent given to 200mg/kg by intraperitoneal injection. One of the subgroups was given the PI3K/Akt pathway inhibitor LY294002. in the lateral ventricle before granting puerarin, and all the rats were treated 24 hours after the injury, and the surrounding cortex specimens were left for the injury. Phospho-Akt expression, Fluoro-JadeC staining, oxidative stress markers (MDA, GSH, Na+-K+-ATPase activity, MPO activity). Results: (1) puerarin can significantly reduce the degeneration of peripheral cortical neurons after TBI injury. (2) puerarin can significantly increase the level of GSH water level and Na+-K+-ATPase activity, reduce MDA content and MPO activity. (3) puerarin The expression of phospho-Akt was obviously enhanced and the protective effect of Puerarin was obviously weakened after the PI3K/Akt pathway inhibitor LY294002. Conclusion: after traumatic brain injury, puerarin can activate the PI3K/Akt pathway and then exert its antioxidation stress. The protective effect of the nerve.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R651.15

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