内质网应激反应在异丙酚神经保护中的作用

发布时间：2018-04-22 18:28

本文选题：异丙酚 + 内质网应激　；参考：《安徽医科大学》2014年博士论文

【摘要】：内质网(endoplasmic reticulum ER)是真核细胞中的重要细胞器,其基本生理功能包括Ca2+调节、生物大分子的合成、加工等。内质网非常敏感,葡萄糖或营养素缺乏、蛋白质糖基化抑制、二硫键形成障碍、蛋白质转运异常、Ca2+耗竭等刺激均可导致ER功能失调,导致ER应激反应(ER stress,ERS)。ERS持续存在或过强时将诱导ER相关性死亡(endoplasmic reticulum associated death,ERAD),造成细胞损伤。有研究表明,当脑组织缺血时,大量ATP耗竭、Ca2+超载及自由基生成等因素,均可以诱导过度ERS而导致神经细胞损伤。异丙酚(2,6-diisopropylphenol)是一种非巴比妥类静脉麻醉药,具有快速、可靠的镇静催眠作用,临床广泛应用于麻醉诱导、麻醉维持和ICU镇静。因其化学结构类似于内源性抗氧化剂维生素E和外源性抗氧化剂丁羟基甲苯(BHT),因而具有清除自由基作用及抗氧化作用。诸多研究表明异丙酚具有神经细胞保护作用,但其神经保护机制仍不清楚。本研究拟探讨ERS在异丙酚发挥神经保护中的作用及机制。目的:观察异丙酚是否具有抑制ERS引起的细胞损伤作用,并探讨ERS相关蛋白在其保护中的作用。使用ERS的诱导剂依霉素(tunicamycin Tm)诱导SH-SY5Y和neuro-2a神经细胞出现ERS,MTT法检测SH-SY5Y细胞活力,流式细胞技术(Flow cytometry FCM)验证Tm引起的neuro-2a神经细胞凋亡;逆转录聚合酶联反应(Reverse Transcription Polymerase Chain Reaction,RT-PCR)和免疫印迹(Western Blot,WB)检测ERS引起Bi P和CHOP的m RNA及蛋白水平的变化;免疫荧光(Immunofluorescence IF)观察细胞形态和两种蛋白的表达和胞内定位;免疫印迹法检测ERS三条通路相关蛋白的表达;用si RNA沉默内源性的Bi P的表达,观察neuro-2a神经细胞活力变化,同时观察凋亡蛋白的表达。结果:1.异丙酚能防止ERS导致的神经细胞损伤我们首先利用MTT法检测不同浓度的异丙酚对SH-SY5Y细胞增殖活性的影响,结果显示,与对照组相比,10μM的异丙酚能提高SH-SY5Y细胞增殖活性。预先在SH-SY5Y细胞培养液中加入不同浓度的异丙酚6小时后,再用Tm诱导6小时。结果显示当异丙酚浓度在1、10、20μM和40μM时,异丙酚能不同程度的抑制Tm引起的细胞活力下降,10μM浓度作用最为显著,DAPI/PI双染法也验证这一结果。流式细胞技术亦显示异丙酚预先给药可显著降低Tm引起的neuro-2a细胞凋亡率。2.异丙酚提高Bi P的表达Bi P被认为是ERS标志性蛋白,为了解异丙酚对Tm引起的细胞损伤保护机制,我们检测了该药与Bi P表达的量效关系。将不同浓度异丙酚共孵育对数生长期的SH-SY5Y细胞后,提取总蛋白,WB结果显示1、10、20、40、80μM异丙酚均可不同程度的上调Bi P的表达,且浓度为10μM时,Bi P的表达水平最高。同时我们还检测了ERS凋亡途径重要的信号分子CHOP,发现CHOP没有明显表达。异丙酚导致Bi P表达上调还具有时间依赖性,共孵育3小时后Bi P表达开始上调,6小时达峰值,持续到24小时。IF实验同时证实10μM的异丙酚上调Bi P的表达。3.异丙酚抑制Tm诱导的CHOP蛋白的表达在了解单纯异丙酚不影响SH-SY5Y细胞系中的CHOP表达之后,我们试图知晓该药对Tm引起的CHOP表达是否有影响。用不同浓度(0.01、0.1、1、10、20、40、80μM)异丙酚共孵育SH-SY5Y细胞6小时后,再用Tm(5μg/ml)刺激细胞6小时后提取总蛋白,WB显示0.1、1、10、20μM异丙酚均能显著抑制Tm诱导的CHOP蛋白的表达,且以10μM效果最为显著。选取10μM为异丙酚终浓度,同样方法处理不同浓度的Tm(2.5、5、10μg/ml)刺激SH-SY5Y细胞,WB结果显示CHOP蛋白均显著下降。为进一步了解异丙酚预处理对于两种ERS相关蛋白表达的影响,我们还选用了小鼠神经瘤neuro-2a细胞,用异丙酚(10μM)和Tm(5μg/ml)处理,分别提取总RNA和总蛋白,进一步验证异丙酚对Bi P和CHOP的影响,结果与前类似。IF实验也证实异丙酚不仅能抑制Tm诱导的CHOP的表达,而且抑制CHOP蛋白从胞浆向胞核转移。4.异丙酚对UPR三种通路蛋白的影响鉴于异丙酚差异性地调节ERS标志性蛋白Bi P和CHOP,我们继续观察了异丙酚对UPR通路中几个关键蛋白表达水平的影响。用异丙酚(10μM)和Tm(5μg/ml)处理neuro-2a细胞,方法同前,提取总蛋白做WB分析。结果显示,异丙酚能显著上调s-XBP1和cleaved ATF6蛋白水平,对磷酸化的PERK和e IFα水平无明显影响。而用Tm刺激后,异丙酚能抑制磷酸化PERK和e IFα的的升高,而对升高的s-XBP1和cleaved ATF6蛋白水平无明显影响。5.异丙酚对ERS相关凋亡蛋白表达的影响前面实验结果表明异丙酚能抑制Tm引起的神经细胞凋亡,我们进一步用WB法验证ERS途径中的凋亡前蛋白ATF4表达和凋亡通路共有的终末效应蛋白caspase3的活化水平。结果显示Tm诱导的ATF4的高表达和活化的caspase 3水平均被异丙酚明显抑制。6.内源性Bi P敲低抑制异丙酚对CHOP和caspase 3的影响为了解异丙酚抑制ERS导致的细胞损伤的潜在机制,我们应用RNAi技术对neuro-2a细胞中内源性Bi P进行敲低。转染三条Bi P-si RNA 24小时后提取neuro-2a细胞总蛋白,WB法检测Bi P-si RNA的敲低效果。结果显示其中有一条si RNA对Bi P的沉默效果明显,Bi P蛋白质表达水平下降,遂选取此条si RNA进行后续实验。将验证有效的Bi P-si RNA转染neuro-2a细胞24小时后,加入10μM异丙酚,6小时后予5μg/ml Tm处共孵育,提取总蛋白后检测CHOP和活化的caspase 3表达水平。结果显示内源性Bi P被敲除后,异丙酚不能有效抑制Tm引起的CHOP表达和caspase 3活化。7.Bi P基因敲低后异丙酚的神经细胞保护作用消失为进一步明确异丙酚引起的Bi P高表达是否具有抗ERS引起的凋亡作用,我们在neuro-2a细胞中瞬时转染si RNA-Bi P后,给予异丙酚处理,MTT结果显示在转染si RNA-Bi P后,异丙酚所导致的细胞增殖活性增加的作用消失,流式细胞技术亦显示无论是否预先予以异丙酚处理,两组的细胞凋亡率基本一致。提示在瞬时转染si RNA-Bi P后,异丙酚的神经保护作用消失。结论:以上研究结果表明,临床应用浓度的异丙酚神经保护作用可能与其上调Bi P表达、抑制PERK/e IFα/ATF4/CHOP通路引起的细胞凋亡有关。
[Abstract]:Endoplasmic reticulum ER is an important organelle in eukaryotic cells. Its basic physiological functions include Ca2+ regulation, biosynthesis of biological macromolecules, processing and so on. Endoplasmic reticulum is very sensitive, glucose or nutrient deficiency, protein glycosylation inhibition, two sulfur bond formation obstacle, protein transport abnormal, Ca2+ exhaustion and so on can lead to ER Dysfunction of the ER stress response (ER stress, ERS).ERS will induce ER related death (endoplasmic reticulum associated death, ERAD) and cause cell damage when the.ERS is persistent or too strong. Cell injury. Propofol (2,6-diisopropylphenol) is a non barbiturate intravenous anesthetic. It has a rapid and reliable sedative hypnotic effect. It is widely used in anesthesia induction, anesthesia maintenance and ICU sedative. Its chemical structure is similar to endogenous antioxidant vitamin E and exogenous antioxidant butyl hydroxy toluene (BHT). A number of studies have shown that propofol has neuroprotective effects, but its neuroprotective mechanism is still unclear. This study intends to explore the role and mechanism of ERS in the protection of propofol. Objective: To observe whether propofol can inhibit cell damage induced by ERS and to explore ERS phase. SH-SY5Y and Neuro-2a neurons were induced by ERS inducer (tunicamycin Tm), and SH-SY5Y cell viability was detected by MTT method. Flow cytometry (Flow cytometry FCM) was used to verify the apoptosis of neuronal cells caused by Tm, and reverse transcription polymerase chain reaction (RT PCR). Se Chain Reaction, RT-PCR) and immunoblotting (Western Blot, WB) detected the changes in ERS causing Bi P and CHOP m and protein levels; immunofluorescence observed cell morphology and the expression of two proteins and intracellular localization; the expression of three pathway related proteins was detected by immunoblotting; The expression of Neuro-2a nerve cell activity was observed and the expression of apoptotic protein was observed. Results: 1. propofol could prevent ERS induced nerve cell damage. First, we detected the effect of propofol on the proliferation of SH-SY5Y cells with different concentrations of propofol by MTT method. The results showed that, compared with the control group, 10 M of propofol could improve SH-SY5Y. Cell proliferation activity. After adding different concentrations of propofol in SH-SY5Y cell culture for 6 hours, Tm was induced for 6 hours. The results showed that when the concentration of propofol at 1,10,20 mu M and 40 mu M, the propofol could decrease the cell vitality of Tm in varying degrees, and the concentration of 10 mu M was the most significant. The DAPI/PI double staining method also verified this knot. Flow cytometry also showed that propofol could significantly reduce the apoptosis rate of Neuro-2a cells induced by Tm,.2. propofol increased Bi P expression, Bi P was considered as a ERS marker protein, to understand the protective mechanism of cell damage caused by propofol on Tm. We detected the dose effect relationship between the drug and Bi P expression. After incubation of the SH-SY5Y cells in the logarithmic growth period, the total protein was extracted, and the results of WB showed that 1,10,20,40,80 uh M could increase the expression of Bi P in varying degrees. When the concentration was 10 mu M, the expression level of Bi P was the highest. At the same time, we also detected the important signal molecule CHOP of ERS apoptosis pathway, and found that CHOP was not obviously expressed. The up-regulated expression of P was also time dependent. After 3 hours of incubation, the expression of Bi P began to rise, the peak value reached 6 hours, and the.IF test lasted to 24 hours. The expression of 10 mu M was confirmed by the expression of.3. propofol and the expression of CHOP protein inhibited by Tm induced CHOP protein after understanding the CHOP expression in the pure propofol SH-SY5Y cell line. We tried to know whether the drug had an effect on the expression of CHOP induced by Tm. After incubating SH-SY5Y cells with different concentration (0.01,0.1,1,10,20,40,80 M) propofol for 6 hours, the total protein was extracted after 6 hours by Tm (5 g/ml), and WB showed that 0.1,1,10,20 micron M propofol could significantly inhibit the Tm induced CHOP protein expression, and the maximum effect was 10 micron. 10 M was selected as the final concentration of propofol, and the same method was used to treat SH-SY5Y cells with different concentrations of Tm (2.5,5,10 mu g/ml). WB results showed that CHOP protein decreased significantly. In order to further understand the effect of propofol pretreatment on the expression of two ERS related proteins, we also selected the mice neuroma Neuro-2a cells, using propofol (10 micron M). And Tm (5 mu g/ml) treatment, the total RNA and total protein were extracted, and the effect of propofol on Bi P and CHOP was further verified. The results similar to the previous.IF test also proved that the propofol could not only inhibit the expression of CHOP induced by Tm, but also inhibit the effect of.4. Isoprophenol on the three pathway proteins from the cytoplasm to the nucleus in view of the difference of propofol. The effect of propofol on the expression level of several key proteins in the ERS pathway was observed. We continued to observe the effect of propofol on the expression level of several key proteins in the UPR pathway. Using propofol (10 mu M) and Tm (5 mu g/ml) to treat Neuro-2a cells, the method was used before and extracted the total protein for WB analysis. The results showed that isopropofol could significantly increase s-XBP1 and cleaved protein levels, and phosphorus. The levels of acidified PERK and E IF alpha were not significantly affected, and propofol could inhibit the increase of phosphorylated PERK and E IF alpha after Tm stimulation, but there was no significant effect on the elevated s-XBP1 and cleaved ATF6 protein levels. The previous experimental results of.5. propofol on the expression of ERS related apoptotic proteins showed that propofol could inhibit the neuronal decay caused by the inhibitory effect of propofol. WB method was further used to verify the activation level of terminal effector protein Caspase3 in the expression of pre apoptotic protein ATF4 and apoptosis pathway in ERS pathway. The results showed that the high expression of Tm induced ATF4 and the level of activated caspase 3 were all obviously inhibited by propofol and the effect of propofol on CHOP and caspase 3 In order to understand the potential mechanism of propofol inhibiting cell damage caused by ERS, we used RNAi technology to knock down endogenous Bi P in Neuro-2a cells. The total protein of Neuro-2a cells was extracted after transfection of three Bi P-si RNA, and WB method was used to detect the low effect of Bi P-si. The expression level of P protein was decreased, then the Si RNA was selected for the follow-up experiment. The effective Bi P-si RNA transfected to Neuro-2a cells for 24 hours, 10 u M propofol was added, and 5 mu g/ml Tm was reincubated after 6 hours, and the CHOP and activated caspase 3 was detected after extracting the total protein. No effective inhibition of Tm induced CHOP expression and caspase 3 activation of.7.Bi P gene knockout, the neuroprotective effect of propofol disappeared to further clarify whether the Bi P high expression induced by propofol has the effect of anti ERS induced apoptosis. We transiently transfect Si RNA-Bi P in Neuro-2a cells and give propofol treatment. After transfection of Si RNA-Bi P, the increase of cell proliferation activity caused by propofol disappeared. Flow cytometry also showed that the rate of apoptosis in the two groups was basically the same. It suggested that the neuroprotective effect of propofol disappeared after transient transfection of Si RNA-Bi P. Conclusion: the above results showed that The neuroprotective effect of propofol in clinical application may be related to its up regulation of Bi P expression and inhibition of apoptosis induced by PERK/e IF alpha /ATF4/CHOP pathway.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R614

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