雷帕霉素对大鼠脊髓损伤的保护作用及机制研究

发布时间：2018-05-08 11:21

本文选题：脊髓损伤 + 雷帕霉素　；参考：《吉林大学》2015年博士论文

【摘要】：脊髓损伤（spinal cord injury, SCI）的病理过程包括原发性和继发性损伤，前者指暴力发生时脊髓的不可逆性损伤，后者是在此基础上发生的局部缺血、水肿、电解质紊乱等一系列生理生化反应。缺血缺氧和炎症反应均能引起活性氧(reactive oxgen specieces，ROS)的产生和释放，ROS大量堆积导致局部组织氧化损伤，神经细胞发生凋亡和坏死。寻找有效药物减少脊髓神经元继发性死亡，一直以来都是人们研究的热点。雷帕霉素（Rapamycin, Rap）是肾移植术后抗排斥反应药物，是一种高效的免疫抑制剂。近期发现，雷帕霉素又具有上调自噬的作用。中枢神经系统创伤性疾病研究显示，雷帕霉素会随动物种类和疾病模型不同而发挥截然相反的作用，脊髓损伤时，雷帕霉素是否具有神经保护作用，目前尚无定论。本研究建立大鼠脊髓损伤模型，观察雷帕霉素对损伤脊髓是否具有保护作用；选择H2O2氧化损伤PC12细胞模型，进一步探讨雷帕霉素对神经细胞的保护作用机制，通过NF-kB和NLRP3炎症小体表达情况，初步探索自噬和炎症之间相互作用的关系，论文分以下两部分阐述。第一部分雷帕霉素对脊髓撞击损伤大鼠的神经保护作用目的：建立大鼠脊髓损伤模型，观察雷帕霉素是否具有上调自噬、下调炎症以及抑制凋亡的神经保护作用。方法：Wistar雌性大鼠45只。开椎板假手术组3只，42只大鼠制备脊髓撞击损伤模型，随机分成雷帕霉素治疗组和损伤对照组。术后3d、7d、14d取材，进行H-E染色、ED-1染色、GFAP免疫荧光染色，观察脊髓损伤病理学变化；酶组织化学法检测组织髓过氧化物酶(MPO)浓度；Western blot法检测炎性因子TNF-α、IL-1β、自噬相关蛋白Beclin-1的表达；TUNEL法检测脊髓组织细胞凋亡指数；NF免疫组化染色观察脊髓组织中存活神经元。结果：成功建立大鼠脊髓撞击损伤模型，术后3d雷帕霉素治疗组MPO值低于损伤组(P0.05)，Western blot结果显示雷帕霉素组炎性因子IL-lβ，TNF-α 表达低于损伤组(P0.05)，自噬相关蛋白Beclin-1表达高于损伤组。术后7d ED-1阳性小胶质细胞在雷帕霉素组明显少于损伤对照组(P0.05)，术后14d GFAP荧光染色阳性星型胶质细胞，雷帕霉素组有减少趋势，但与损伤组之间无显著性差异。H-E染色结果显示，在损伤区与正常脊髓交界处，损伤组炎症细胞数目较多。TUNEL结果显示，雷帕霉素组凋亡细胞数目明显减少(P0.05)。NF染色显示，雷帕霉素组神经元数目明显多于单纯损伤组(P0.05)。结论：雷帕霉素作用于大鼠脊髓撞击损伤，不仅可以上调自噬；而且能够抑制小胶质细胞活化和增殖、减少中性粒细胞及淋巴细胞浸润，减少炎性因子表达，减轻了神经组织继发性炎症反应；雷帕霉素最终减少凋亡细胞数量，增加神经元存活，对损伤脊髓具有保护作用。第二部分雷帕霉素对氧化应激损伤PC12细胞的保护作用及机制研究目的：观察雷帕霉素预处理对氧化应激损伤PC12细胞的保护作用，从自噬和炎症之间的关联信号通路探讨雷帕霉素保护作用的机制。方法：实验共分五组：正常PC12细胞组；H2O2+PC12组；RAP+H2O2+PC12组；RAP+SN50+H2O2+PC12组；3-MA+H2O2+PC12组，制备H2O2氧化损伤PC12细胞模型。MTT法检测各组细胞存活率；光学显微镜观察细胞形态学变化；MDC染色观察细胞自噬囊泡变化；ROS染色观察细胞活性氧变化；ELISA法检测培养上清和细胞内炎性因子TNF-α、IL-1β变化；Western blot法检测自噬相关蛋白Beclin-1、LC3，促凋亡蛋白Bax、Cleave-caspase3，炎症信号通路NF-kB、炎症小体NLRP3、炎性因子IL-1β表达变化。结果：200μM H2O2处理PC12细胞24h，成功构建氧化损伤细胞模型。MTT法结果提示Rap和Rap+SN50组与H2O2组比较，细胞存活率增加；3-MA组与H2O2组比较，细胞存活率减少。ROS染色显示H2O2组和3-MA组的平均荧光强度与正常组比较明显增高，Rap和Rap+SN50组与H2O2组比较荧光强度明显减少。MDC染色后发现H2O2组自噬囊泡数量较多；Rap和Rap+SN50组细胞内自噬囊泡荧光颗粒明显增强；3-MA组细胞自噬囊泡数量较少。ELISA结果显示H2O2组细胞损伤后培养上清和细胞内TNF-α、IL-1β分泌水平明显增高；而Rap和Rap+SN50组TNF-α、IL-1β分泌水平降低。Western blot结果显示Rap和Rap+SN50组自噬相关蛋白LC3和Beclin-1的表达明显高于H2O2组，而3-MA组LC3和Beclin-1的表达明显低于H2O2组；炎症通路蛋白NF-кB和NLRP3表达在Rap和Rap+SN50组明显低于H2O2组，而3-MA组NF-кB的表达明显高于H2O2组；促凋亡蛋白Bax和Cleave-caspase3表达在Rap和Rap+SN50组低于H2O2组，而3-MA组Bax和Cleave-caspase3的表达则明显高于H2O2组。结论：在H2O2氧化损伤PC12细胞模型中，雷帕霉素通过上调自噬相关蛋白LC3和Beclin-1表达，减少损伤线粒体ROS释放，下调NF-kB信号通路和NLRP3炎症小体活化，，减少炎性因子TNF-α、IL-1β表达，雷帕霉素下调促凋亡蛋白Bax和Cleave-caspase3表达，增加PC12细胞存活，具有神经细胞保护作用。综上所述，我们在大鼠体内和体外培养PC12细胞实验证实，雷帕霉素具有神经保护作用。初步探讨其可能存在的机制：脊髓损伤后，雷帕霉素上调自噬水平，减少ROS产生，通过NF-kB和NLRP3信号通路减少炎性因子的产生和释放，雷帕霉素下调促凋亡信号，减少死亡细胞数量；在脊髓损伤区域，由于死亡细胞炎性因子释放减少，下调了周边炎性细胞活化和增殖迁移，减小局部炎症反应强度，雷帕霉素减少凋亡细胞数量，保护了残存神经细胞。
[Abstract]:The pathological process of spinal cord injury (SCI) includes primary and secondary injuries. The former refers to the irreversible injury of the spinal cord at the time of violence. The latter is a series of physiological and biochemical reactions, such as local ischemia, edema, and electrolyte disturbance on this basis. The oxygen deficiency and the inflammatory response can cause the reactive oxygen species (reactive oxge). The production and release of n Specieces, ROS, a large accumulation of ROS causes oxidative damage in local tissues and apoptosis and necrosis of nerve cells. Finding effective drugs to reduce secondary death of spinal neurons has always been a hot spot of research.
Rapamycin (Rap) is an anti rejection drug after renal transplantation and is an effective immunosuppressant. Recently, rapamycin has the effect of increasing autophagy. The central nervous system traumatic disease study shows that rapamycin will play a very opposite effect on the animal species and disease model, spinal cord injury. Whether rapamycin has neuroprotective effects is uncertain.
In this study, the rat spinal cord injury model was established to observe the protective effect of rapamycin on the injured spinal cord, and to select the H2O2 oxidative damage PC12 cell model to further explore the protective mechanism of rapamycin on the nerve cells, and to explore the interaction between autophagy and inflammation through the expression of NF-kB and NLRP3 corpuscles. The thesis is divided into two parts.
Part 1 the neuroprotective effect of rapamycin on spinal cord impact injury in rats
Objective: to establish a rat spinal cord injury model and observe whether rapamycin has the neuroprotective effect of upregulated autophagy, down-regulation of inflammation and inhibition of apoptosis.
Methods: 45 Wistar female rats. 3 rats in the sham operation group and 42 rats were prepared for the spinal cord impact injury model, which were randomly divided into rapamycin treatment group and injury control group. After operation, 3D, 7d, 14d were obtained, H-E staining, ED-1 staining, and GFAP immunofluorescence staining were used to observe the pathological changes of spinal cord injury; enzyme histochemical method was used to detect tissue medullary tissue. The concentration of oxide enzyme (MPO) and Western blot method were used to detect the expression of inflammatory factor TNF- alpha, IL-1 beta, autophagy related protein Beclin-1; TUNEL method was used to detect the apoptosis index of spinal cord tissue; NF immunohistochemical staining was used to observe the survival neurons in the spinal cord tissue.
Results: the spinal cord impact injury model was successfully established, and the MPO value of rapamycin treated group 3D after operation.
Lower than the injury group (P0.05), Western blot results showed rapamycin inflammatory factor IL-l beta, TNF- alpha
The expression of autophagy related protein Beclin-1 was higher than that of injury group (P0.05), and the 7d ED-1 positive microglia in rapamycin group after operation was significantly less than that of the injury control group (P0.05), and 14d GFAP fluorescence after operation.
In the light staining positive astrocytes, there was a decreasing trend in the rapamycin group, but there was no significant difference between the injured group and the injured group. The results of.H-E staining showed that the number of inflammatory cells in the injured group and normal spinal cord was more.TUNEL. The number of apoptotic cells in the rapamycin group decreased significantly (P0.05).NF staining, and the rapamycin group The number of neurons was significantly more than that of the simple injury group (P0.05).
Conclusion: rapamycin can not only increase autophagy, but also inhibit the activation and proliferation of microglia, reduce the infiltration of neutrophils and lymphocytes, reduce the expression of inflammatory factors and reduce the secondary inflammatory response in the nerve tissue, and reparamycin can eventually reduce the number of apoptotic cells and increase the nerve. Yuan Cunhuo has a protective effect on the injury of the spinal cord.
The second part is the protective effect and mechanism of rapamycin on PC12 cells injured by oxidative stress.
Objective: To observe the protective effect of rapamycin preconditioning on PC12 cells damaged by oxidative stress, and to explore the mechanism of the protective effect of rapamycin from the correlation signal pathway between autophagy and inflammation.
Methods: the experiment was divided into five groups: normal PC12 cell group, group H2O2+PC12, group RAP+H2O2+PC12, group RAP+SN50+H2O2+PC12, group RAP+SN50+H2O2+PC12; 3-MA+H2O2+PC12 group, H2O2 oxidative damage PC12 cell model.MTT method was used to detect the cell survival rate of each group; optical microscope observation of cell morphological changes; MDC staining observation of cell autophagic vesicles; ROS staining The changes of active oxygen in cells were observed. ELISA assay was used to detect TNF- alpha and IL-1 beta in cultured supernatant and intracellular inflammatory factors, and Western blot was used to detect autophagy related protein Beclin-1, LC3, apoptotic protein Bax, Cleave-caspase3, NF-kB of inflammatory signaling pathway, NLRP3 inflammatory body, and changes in the expression of inflammatory cell IL-1 beta.
Results: the PC12 cell 24h was treated with 200 M H2O2, and the.MTT method of the oxidative damage cell model was successfully constructed. The cell survival rate of Rap and Rap+SN50 groups was increased compared with that of H2O2 group, and the 3-MA group was compared with the H2O2 group, and the cell survival rate reduced by.ROS staining showed the average fluorescence of the H2O2 group and the group.
The intensity was significantly higher than that in the normal group. The fluorescence intensity of Rap and Rap+SN50 groups was significantly lower than that in the H2O2 group. The number of autophagic vesicles in the H2O2 group was more than that in the H2O2 group. The intracellular autophagic vesicles in the Rap and Rap+SN50 groups were obviously enhanced, and the less.ELISA results of the autophagic vesicles in the 3-MA group showed that the cells in the H2O2 group were damaged after the cell injury. The secretion of TNF- alpha and IL-1 beta in both the clear and the cells increased significantly, while the secretion level of TNF- alpha and IL-1 beta in the Rap and Rap+SN50 groups decreased by.Western blot and showed that the expression of LC3 and Beclin-1 were significantly higher in Rap and Rap+SN50 groups than those in the group. The expression of NF- and B in group 3-MA was significantly higher than that in group H2O2, and the expression of Bax and Cleave-caspase3 in 3-MA group was significantly higher than that in group H2O2, and the expression of Bax and Cleave-caspase3 in Rap and Rap+SN50 groups was lower than that of the H2O2 group, while the expression of NF- and B was significantly higher than that of the group of H2O2. The expression of autophagy related protein LC3 and Beclin-1 reduces the release of ROS in damaged mitochondria, reduces the activation of NF-kB signaling pathway and NLRP3 inflammatory corpuscle, reduces the expression of inflammatory factor TNF- alpha and IL-1 beta, reduces the expression of Bax and Cleave-caspase3, and increases the survival of PC12 cells, and has the protective effect of nerve cells.
To sum up, our experiment in rat and in vitro culture of PC12 cells confirmed that rapamycin has a neuroprotective effect. Preliminary study of its possible mechanism: after spinal cord injury, rapamycin up-regulated the autophagy level, reduced ROS production, reduced the production and release of inflammatory factors through the NF-kB and NLRP3 signaling pathways, and down regulation of rapamycin The apoptotic signal reduces the number of dead cells; in the region of spinal cord injury, the reduction of inflammatory factors in the dead cells reduces the activation and proliferation of peripheral inflammatory cells, reduces the intensity of local inflammatory reaction, reduces the number of apoptotic cells by rapamycin, and protects the remnants of the deity cells.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.2

【参考文献】