外源性硫化氢对丙烯腈诱导的大鼠星形胶质细胞毒性的影响及其机制研究

发布时间：2018-06-13 01:43

  本文选题：硫化氢 + 丙烯腈　； 参考：《江苏大学》2017年硕士论文

【摘要】：目的:硫化氢(hydrogen sulfide,H_2S)作为继CO、NO后的第三种气体信号分子,有着重要的神经保护性和神经调节作用,在保护化学物和药物的神经损伤的应用中有着广阔前景。丙烯腈(acrylonitrile,AN)是一种高毒的有机腈,易经呼吸道、皮肤和胃肠道侵入人体,主要侵害神经系统。我们前期研究已经发现AN神经毒性与硫化氢、谷胱甘肽等含硫化合物代谢密切相关,因此,本实验以大鼠原代星形胶质细胞为研究对象,硫氢化钠(NaHS)为外源性H_2S供体:1.初步探索外源性H_2S对AN诱导的星形胶质细胞毒性的影响;2.进一步探索外源性H_2S对AN诱导的细胞毒性影响的调控机制;3.为研发新的丙烯腈预防和治疗药物提供科学依据,以期寻找新的解毒方向。方法:1.采用AN处理原代培养的星形胶质细胞,NaHS为外源性H_2S供体预处理细胞1 h。MTT法检测细胞活力和LDH漏出率检测细胞毒性评价NaHS对AN诱导的细胞毒性的影响。检测GSH和ROS含量的变化评价细胞氧化应激水平的改变。2.单纯应用NaHS处理细胞,检测自噬相关蛋白Beclin1、SQSTM1/P62、Atg5表达变化,免疫荧光方法观察星形胶质细胞中自噬颗粒变化,吖啶橙染色检测细胞酸性自噬泡,自噬双标腺病毒检测自噬流等方法检测NaHS对原代星形胶质细胞自噬水平的影响。3.单独应用NaHS处理细胞,采用细胞核质蛋白分离,免疫印迹法检测Nrf2、HO-1、γ-GCS的表达以及免疫荧光标记Nrf2观察其核转位情况来综合评价NaHS对细胞Nrf2/ARE信号通路的影响。4.通过自噬抑制剂和siRNA转染的方法抑制自噬或Nrf2的表达,MTT法和LDH漏出率法检测NaHS对AN诱导的细胞毒性效应的改变,探索调控自噬或Nrf2/ARE信号通路对NaHS诱导的保护效应的影响。结果:1.采用NaHS(0、200、400、800μM)预处理细胞1 h,与AN单独处理组相比,细胞活力明显升高,LDH渗出率下降。同时,细胞抗氧化物还原性GSH含量升高,氧化应激产物ROS水平降低。2.NaHS单独处理细胞1h后,自噬相关蛋白Beclin1,Atg5表达明显升高,而SQSTM1/P62表达下降,并具有浓度依赖性。同时单独NaHS处理组星形胶质细胞内LC3绿色荧光斑点明显增多,AO染色结果显示酸性自噬泡(红色荧光斑点)也明显增多,采用自噬双标腺病毒检测结果提示自噬流增强,说明NaHS可诱导星形胶质细胞自噬。3.单纯NaHS处理细胞,与对照组相比,细胞核内Nrf2分布增多,Nrf2/ARE通路下游蛋白HO-1、γ-GCS表达升高,说明外源性NaHS可引起Nrf2发生核转位。4.分别用药理学和基因学手段抑制自噬或Nrf2信号后,NaHS对AN毒性的拮抗效应减弱。结论:1.NaHS预处理对AN诱导的星形胶质细胞毒性有拮抗效应,并可改善AN诱导的氧化应激损伤。2.单纯NaHS处理可诱导细胞自噬的活化,同时也可诱导Nrf2发生核转位,激活Nrf2/ARE信号通路。3.抑制自噬或Nrf2表达,NaHS对AN毒性的拮抗效应均减弱,说明NaHS对AN毒性的保护效应与其对自噬以及Nrf2/ARE信号通路的激活均有一定的关联。这提示H_2S可能成为一种新的丙烯腈中毒治疗药物。
[Abstract]:Objective: hydrogen sulfide (H_2S), a third gas signal molecule following CO and NO, has an important neuroprotective and neuromodulation effect. It has a broad prospect in the application of protecting chemicals and drugs for nerve damage. Acrylonitrile (acrylonitrile, AN) is a highly toxic organic nitrile, easy to pass through the respiratory tract, skin, and gastrointestinal tract. We have found that AN neurotoxicity is closely related to the metabolism of sulfur compounds, such as hydrogen sulfide, glutathione, and so on. Therefore, this experiment takes the primary astrocytes of rats as the research object, sodium thiohydrate (NaHS) is a exogenous H_2S donor: 1. preliminary exploration of exogenous H_2S to AN induced stars The effect of the toxicity of glial cells; 2. further explore the regulatory mechanism of exogenous H_2S on the cytotoxicity induced by AN; 3. to provide scientific basis for the development of new prophylaxis and treatment of drugs, in order to find new detoxification directions. Method: 1. using AN to treat primary cultured astrocytes, and NaHS as a exogenous H_2S donor Detection of cell viability and LDH leakage rate by cell 1 h.MTT assay the effect of NaHS on the cytotoxicity of AN induced cytotoxicity. Detection of changes in GSH and ROS content and evaluation of changes in oxidative stress levels of cells.2. simply applied NaHS treated cells to detect the changes of Beclin1, SQSTM1/P62, Atg5 expression of autophagy related proteins and immunofluorescence methods The changes of autophagic particles in astrocytes, acridine orange staining and detection of autophagic vesicles, autophagy detection of autophagy by autophagy double labeled adenovirus, the effect of NaHS on the autophagy level of primary astrocytes was detected by.3. alone with NaHS cells, cell nuclear protein separation, and Western blot to detect the expression of Nrf2, HO-1, and gamma -GCS And immunofluorescent labeling Nrf2 observation of its nuclear transposition to evaluate the effect of NaHS on cell Nrf2/ARE signaling pathway,.4. inhibits autophagy or Nrf2 expression through autophagy inhibitors and siRNA transfection. MTT and LDH leaks detect the changes in the cytotoxic effects of NaHS on AN induced by AN, and explore the regulation of autophagy or Nrf2/ARE signaling pathway. The effects on the protective effect induced by NaHS. Results: 1. the cells were pretreated with NaHS (0200400800 M) for 1 h, compared with the AN alone treatment group, the cell viability was significantly increased and the LDH exudation rate decreased. At the same time, the reduced GSH content of the cell antioxidants was increased, the ROS level of the oxidative stress products was reduced by.2.NaHS alone, and the autophagy associated protein Be was processed. The expression of clin1, Atg5 was significantly increased, while the expression of SQSTM1/P62 decreased and was dependent on concentration. At the same time, the LC3 green fluorescence spots in astrocytes were significantly increased in the NaHS treatment group, and the results of AO staining showed that the acid autophagic vesicles (red fluorescent spots) were also significantly increased. The autophagic double standard adenovirus detection results suggested the enhancement of autophagic flow, indicating N. AHS can induce the autophagy of astrocytes to autophagy.3. NaHS cells. Compared with the control group, the distribution of Nrf2 in the nucleus increased, the downstream protein HO-1, and the expression of gamma -GCS in the Nrf2/ARE pathway increased, indicating that the exogenous NaHS could cause the Nrf2 occurrence of nuclear transposition.4. to inhibit the autophagy or Nrf2 signal by pharmacological and genetic hand segments, and the antagonism of NaHS to toxicity. The effect is weakened. Conclusion: 1.NaHS pretreatment can antagonize the toxicity of AN induced astrocytes, and improve the oxidative stress induced by AN, which can induce the activation of autophagy in.2. alone, and also induce the nuclear transposition of Nrf2, and activate the Nrf2/ARE signaling pathway to inhibit autophagy or Nrf2 expression by.3., and the antagonism of NaHS to AN toxicity. The effects were weakened, indicating that the protective effect of NaHS on the toxicity of AN and the activation of the autophagy and the Nrf2/ARE signaling pathway were related to a certain extent. This suggests that H_2S may be a new drug for the treatment of acrylonitrile poisoning.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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