脂多糖诱导的小胶质细胞M1极化对小鼠焦虑样行为的影响

发布时间：2018-10-31 16:33

【摘要】：目的观察脑内注射脂多糖(LPS)引起的脑内M1型小胶质细胞极化对实验动物焦虑样行为的影响,并试图通过Myd88抑制剂MIP逆转LPS带来的炎症反应。探究实验动物焦虑样行为的改变与中枢小胶质细胞极化的关系。材料与方法本实验将C57雄性小鼠随机分为4组,第一天每两组分别单侧侧脑室注射Myd88抑制剂(MIP)和Myd88对照肽(CP),第二天第一组再次注射生理盐水、第二组注射LPS、第三组注射生理盐水、第四组注射LPS,即四个实验组分别为CP-Con、MIP-Con、CP-LPS、MIP-LPS。最后一次脑内注射48 h后观察实验动物焦虑样行为的改变,用免疫荧光染色观察内侧前额叶皮质和海马区i NOS、Arg-1的表达,用Western Blot的方法检测内侧前额叶皮质和海马区IL-1β、TNF-α、iNOS、Arg-1、Myd88、P-IRF3等分子的表达。结果1.模型建立后行为学的改变模型建立48 h后,旷场实验显示CP-LPS组相较于其他三组小鼠在中央活动的距离最短,中央活动的时间最少,这提示CP-LPS组相较于其他三组小鼠表现出更明显的焦虑样行为;MIP-LPS组的中央活动距离和时间显著高于CP-LPS,这提示MIP能够逆转LPS引起的焦虑样行为。高架十字迷宫实验中CP-LPS组相较于其他三组小鼠在开臂活动的时间最少,活动的距离最短,这提示CP-LPS相较于其他三组小鼠表现出更明显的焦虑样行为;MIP-LPS组在开臂活动的时间和距离显著高于CP-LPS,这再次提示MIP能够逆转LPS引起的焦虑样行为。2.不同处理组实验动物的海马和内侧前额叶皮质小胶质细胞的活化情况免疫荧光结果显示,在与焦虑样情绪相关的脑区,包括海马的DG、CA1、CA2区及m PFC内CP-LPS组有更多的M1型小胶质细胞的激活,而MIP-Con组和MIP-LPS组有更多的M2型小胶质细胞激活。3.不同处理组实验动物的关键脑区的重要分子蛋白表达量的变化Western Blot的结果显示,在与焦虑样情绪相关的脑区包括:海马的DG、CA1、CA2区及mPFC内CP-LPS组有更多的M1小胶质细胞蛋白标记物iNOS的表达,而MIP-Con和MIP-LPS组有更多的M2型小胶质细胞蛋白标记物Arg-1的表达。CP-LPS组有更多的促炎因子IL-1β、TNF-α的表达。MIP-Con组和MIP-LPS组Myd88非依赖通路关键分子P-IRF3的表达显著增高。结论LPS能够诱导小胶质细胞向M1型极化,并且可能引起实验动物的焦虑样行为。MIP可以逆转LPS的作用,缓解LPS引发的实验动物的焦虑样行为。这种调控可能通过Myd88非依赖通路实现且与小胶质细胞向M2型极化密切相关。
[Abstract]:Objective to observe the effect of intrabrain injection of lipopolysaccharide (LPS) on the anxiety like behavior of M1 microglia in rats and to reverse the inflammatory response induced by LPS by Myd88 inhibitor MIP. To explore the relationship between the changes of anxiety-like behavior and the polarization of central microglia in experimental animals. Materials and methods the C57 male mice were randomly divided into 4 groups. On the first day, each group was given intraventricular injection of Myd88 inhibitor (MIP) and Myd88 control peptide (CP),. The first group was injected with normal saline again on the second day, and the second group was injected with LPS,. The third group was injected with normal saline, the fourth group was injected with LPS, and the four experimental groups were treated with CP-Con,MIP-Con,CP-LPS,MIP-LPS.. The changes of anxiety like behavior were observed 48 hours after the last intracerebral injection, and the expression of I NOS,Arg-1 in medial prefrontal cortex and hippocampus was observed by immunofluorescence staining. The expressions of IL-1 尾, TNF- 伪 and iNOS,Arg-1,Myd88,P-IRF3 in medial prefrontal cortex and hippocampus were detected by Western Blot. Result 1. After the model was established, the open field experiment showed that the distance of central activity was the shortest and the time of central activity was the least in CP-LPS group compared with the other three groups. This suggests that the CP-LPS group showed more obvious anxiety behavior than the other three groups. The distance and time of central activity in MIP-LPS group was significantly higher than that in CP-LPS, which suggested that MIP could reverse the anxiety behavior induced by LPS. In the elevated cross maze test, the CP-LPS group had the least time and the shortest distance in the open arm activity compared with the other three groups, which indicated that the CP-LPS group had more obvious anxiety behavior than the other three groups. The time and distance of open arm activity in MIP-LPS group was significantly higher than that in CP-LPS, suggesting that MIP could reverse the anxious behavior induced by LPS. 2. Activation of microglia in the hippocampus and medial prefrontal cortex of experimental animals in different treatment groups showed that DG,CA1, was found in the brain regions associated with anxiety like emotions, including the hippocampus. M 1 microglia were activated in CA2 region and m PFC in CP-LPS group, while M 2 microglia were activated in MIP-Con group and MIP-LPS group. Changes in the expression of important Molecular proteins in key brain regions of Experimental Animals in different treatment groups the results of Western Blot showed that DG,CA1, in the hippocampus was involved in the brain regions associated with anxiety. In CA2 region and mPFC, the expression of M1 microglial protein marker iNOS was more in CP-LPS group, while in MIP-Con and MIP-LPS group there was more Arg-1 expression of M2 microglial protein marker, while in CP-LPS group, the expression of inflammatory factor IL-1 尾 was higher than that in CP-LPS group. Expression of TNF- 伪. The expression of P-IRF3, a key molecule of Myd88 independent pathway, was significantly increased in MIP-Con group and MIP-LPS group. Conclusion LPS can induce M1 polarization of microglia and may induce anxiety like behavior in experimental animals. MIP can reverse the effect of LPS and alleviate the anxiety like behavior induced by LPS in experimental animals. This regulation may be achieved through Myd88 independent pathway and is closely related to microglial M 2 polarization.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749

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