【作者】 陈曦；

【导师】 赵国庆；

【作者基本信息】 吉林大学 ， 麻醉学， 2018， 博士

【摘要】 近年的研究表明,丙泊酚不仅在动物的脓毒症休克中有保护作用,在器官和细胞的缺血/再灌注（I/R）、氧化应激损伤中同样有保护作用。丙泊酚对心肌细胞和海马细胞的保护作用就表现为保护心肌细胞和海马细胞对抗I/R的自噬性细胞死亡。包括丙泊酚、七氟烷在内的全麻药都会上调骨骼肌的自噬行为,这种自噬现象在全麻过程中呈时间依赖性逐渐上调,并在六小时时达到高峰,且去神经支配与全麻状态缺一不可。但是,关于丙泊酚发挥对自噬的促进作用是通过何种更具体的分子机制,以及是否能够对实际的临床用药有指导意义的相关研究很少。自噬是胞质溶胶和细胞器被隔离到双层膜的小泡中后运送到溶酶体/空泡中降解,并使形成的大分子进行再循环的一个过程。自噬过程发生时,细胞会消化掉自身的一部分,即self-eating,这个过程初一看似乎是不利于细胞的。但事实上,如果没有诱发因素存在,正常情况下细胞只维持较低程度的自噬以维持自稳。而所谓的诱发因素,既有来自于细胞外的诸如外界中的营养成分不足、缺血、缺氧、生长因子的浓度的变化等,也有来自于细胞内的,比如细胞内的代谢压力、衰老或破损的细胞器堆积、蛋白质折叠错误或异常聚集等。但是在细胞存活和生长的过程中,这些因素是经常存在的,因此,正常情况下,细胞保持了一种很低的、基础的自噬活性以维持自稳,同时,自噬在机体的细胞成分更新,组织代谢,生长和发育等一系列重要的生命基本进程中发挥着非常重要的生物学作用。在疾病形成的过程中自噬同样扮演着非常重要的角色,包括神经退行性疾病,Danon肌病等许多疾病的发生、发展都与自噬关系密切。自噬本身是一种保护性的生理过程,确保细胞稳态以及细胞内功能协调,然而自噬过度激活则会造成严重的细胞结构和功能的破坏,因此我们不能简单地定义自噬对于生物体的利弊。有研究表明,内质网应激和自噬相互影响的关系对于骨骼肌疾病中肌纤维损伤的发生发展非常重要。内质网应激依赖性的肌细胞死亡和肌炎以及dysferlin相关的营养不良肌病的肌细胞损伤有关。内质网应激是由于蛋白合成过度增加,错误折叠蛋白增多,细胞内钙含量不平衡,糖、能量缺乏,局部缺血等潜在的病理生理干扰素增加,内质网稳态遭到破坏失衡,进而产生的一种细胞对于外界不良因素的保护性应答机制。目前的研究主要集中在胰腺和脂肪组织中,而在骨骼肌中研究并不多。内质网适应性的应答机制有:第一unfolded protein response（UPR）。UPR激活可以上调3 ER membrance-associated protein、PERK、IRE1以及ATF6蛋白表达,使之合成增加,同时伴侣蛋白的基因和蛋白表达也会增加,例如BIP/Grp78以及Grp94,在内质网应激发生的过程中表达会上调。内质网应激激活之后释放内质网腔内的钙离子到细胞质中,激活钙依赖性细胞通路。细胞内钙离子增加,钙离子激活CaMKKβ,激活MAPK通路激活自噬。同时,细胞内钙离子增加,增加的钙离子会激活PKCs磷酸化,进而诱导LC3转移对TG、TN刺激的内质网应激产生应答。也就是说,细胞内钙离子增加与内质网应激在激活自噬的过程中有协同作用。此外,内质网将大量钙离子释放到细胞质内,会引起线粒体肿胀,破坏呼吸链功能,也会增强自噬的进程。第二,ER overload response（EOR）。EOR可以通过上调NF-κb通路相关蛋白的表达,以及调节炎症应答来发挥其保护作用。如果EOR和UPR都没有被激活,则细胞在不良的外界环境中将进入程序性细胞死亡的阶段。丙泊酚在不同的组织器官中以及肿瘤细胞中发挥的保护作用,这种保护作用也与MAPK通路,NF-κB等有密切联系。丙泊酚可能是通过调节细胞内钙离子流量调节线粒体功能和脑内代谢以发挥其保护作用。包括丙泊酚在内的全麻药在大鼠的麻醉中会促进骨骼肌自噬。基于以上的事实,丙泊酚对骨骼肌细胞自噬的促进作用,是否是通过内质网应激来发挥作用值得探讨,需要深入研究。本文以小鼠成肌细胞（C2C12）为研究对象,探讨了丙泊酚对真核生物正常细胞自噬的作用及其机制,为丙泊酚的作用机制提供新的研究思路。方法:（1）分别用不同浓度的丙泊酚（0μM、25μM、50μM、100μM、150μM、250μM、300μM、400μM、500μM、600μM、700μM、800μM、900μM）处理小鼠成肌细胞（C2C12）48h。CCK8法检测不同浓度丙泊酚对分化的小鼠成肌细胞（C2C12）生存率的影响。分别用不同浓度的丙泊酚（400μM、900μM）处理分化的小鼠成肌细胞（C2C12）3h,通过流式细胞仪用流式细胞术检测细胞凋亡和细胞内ROS的变化。（2）为了检测丙泊酚对自噬的作用,分别用不同浓度的丙泊酚（50μM、100μM、200μM、400μM）处理分化的小鼠成肌细胞（C2C12）不同时间段:3h,6h,24h。通过RT-PCR法、Western Blot方法和免疫组织化学进行检测,分别检测自噬相关蛋白LC3、p62、Beclin-1基因和蛋白表达水平的变化,同时检测mTOR、p-mTOR、AMPK、p-AMPK的蛋白表达水平的变化。自噬抑制剂氯喹二磷酸盐,可以抑制蛋白水解酶的作用,从而抑制自噬自噬的后期阶段,即抑制自噬溶酶体降解,为了进一步确认丙泊酚的自噬的作用及对自噬流的影响,用丙泊酚与自噬抑制剂氯喹二磷酸盐联合处理分化的小鼠成肌细胞（C2C12）,Western Blot方法检测LC3和p62的表达以确定自噬激活情况,通过流式细胞仪用流式细胞术检测细胞凋亡水平的变化确定自噬在丙泊酚对细胞存活率的作用的影响。（3）为了检测丙泊酚促进自噬的作用是否通过内质网应激来发挥作用,用丙泊酚（400μM）和内质网应激抑制剂牛磺酸去氧熊胆酸（TUDCA）1mM联合处理小鼠成肌细胞（C2C12）3h,用荧光染料H2-DCFDA标记活性氧簇（ROS）,用流式细胞术检测细胞内ROS水平的变化,并通过高内涵成像分析系统分析细胞内ROS水平的变化;用荧光染料Rhod-2am标记细胞内的钙离子,用高内涵成像分析系统分析药物处理后三个小时内不同时间段,细胞内Ca²⁺水平的变化;用Western Blot方法检测自噬相关蛋白LC3、p62表达情况的变化,以及内质网应激标志蛋白Bip、CHOP的变化。（4）为了检测丙泊酚促进自噬的作用与活性氧簇（ROS）的水平升高是否有相关性,丙泊酚（400μM）和ROS抑制剂N-乙酰半胱氨酸（NAC）3mM处理小鼠成肌细胞（C2C12）3h,通过流式细胞仪检测细胞凋亡和细胞内ROS的变化。用Western Blot方法检测自噬标记蛋白LC3、p62的变化。结果1.丙泊酚对细胞增殖能力的影响低浓度（小于300μM）表现为促进,而高浓度（>300μM时）则表现为抑制,900μM的丙泊酚会引起细胞凋亡,但不是通过活性氧簇（ROS）增加发挥作用。2.丙泊酚上调自噬相关蛋白LC3、p62、Beclin-1的基因表达水平和蛋白表达水平,上调AMPK/p-AMPK比率,同时下调mTOR/p-mTOR比率,且这种作用呈现出浓度依赖性。丙泊酚对自噬的促进作用与对照组相比,3小时检测时比较显著,6小时基本没有差别,24小时没有检测到明显的变化。3.丙泊酚与自噬抑制剂氯喹二磷酸盐（CQ）共同孵育细胞时,自噬标记蛋白LC3-II和p62的表达相较于丙泊酚单独处理或CQ单独处理时增加更为显著。4.丙泊酚与内质网应激抑制剂牛磺酸去氧熊胆酸（TUDCA）或者ROS抑制剂N-乙酰半胱氨酸（NAC）共同孵育时,都会减弱丙泊酚对自噬的促进作用。并且在丙泊酚单独处理细胞时显著上升的自噬标记蛋白Bip、CHOP,在丙泊酚与内质网应激抑制剂TUDCA共同作用时,表达水平也会显著下调。在对钙离子进行不同时间段的检测时发现,丙泊酚刚处理细胞两个小时之内,相对于对照组钙离子显著增加,两个小时到三个小时之间,钙离子水平显著下调,内质网应激抑制剂单独处理或者与丙泊酚共处理的细胞的钙离子水平,在检测的三个小时小时之内变化不明显;相对应的,丙泊酚单独处理细胞时,ROS水平显著增加,而内质网应激抑制剂TUDCA或者自噬抑制剂3-MA和丙泊酚共处理细胞时,则可以解除丙泊酚对ROS水平的促进作用。结论1.丙泊酚细胞毒性较低,一般情况下不会引起凋亡,浓度极高（900μM）时会引起细胞凋亡,但是并不通过ROS增加来发挥作用。2.丙泊酚可以激活自噬,且对自噬的促进作用呈浓度依赖性,与作用时间有关系。3.丙泊酚对自噬的促进作用是通过内质网应激来发挥作用的,同时细胞内的钙离子增加,ROS生成增加,增加的ROS不仅产生于内质网应激激活后,激活自噬也会增加ROS生成,ROS增加对自噬同样有促进作用。更多还原

【Abstract】 Propofol protects rat cardiomyocytes and hippocampal neurons against ischemia/reperfusion-induced autophagic cell death.Autophagy is an important regulatory mechanism,and its inhibition may either result in direct cell death,or sensitize cells to stimuli-induced damage.Intriguingly,recent advances suggest a possible link between autophagy and anesthetic-induced cytotoxicity.For example,Morissette et al.reported that smooth muscle cell death due to the local anesthetics bupivacaine and lidocaine was associated with increased autophagy.Others also demonstrated in vitro that autophagosomes accumulate in cells exposed to the local anesthetic dibucaine.General anesthetics,including propofol,and sevoflurane will all increase autophagy in skeletal muscle,and this process of autophagy in general anesthesia in a time-dependent manner gradually increase and peak in six hours,also denervation and general anesthesia are needed.However,propofol played in accelerating Autophagy is a more specific mechanism by which,and whether real clinical significance,is still unknown.Autophagy is the cytosol and organelles are isolated in the bilayer vesicles,and transported to the Lysosomes/vacuoles in the degradation and thus creating a process for recycling of macromolecules.Autophagy appears in many eukaryotic cells,but autophagic cell death is not only features in multicellular organisms.Autophagy is part of cells digest itself,namely self-eating.It seems to be bad for the cell.In fact,Autophagy occurs seldom under normal circumstances,unless there is a presence of predisposing factors.Both from outside,such as malnutrition,ischemia and hypoxia,the concentration of growth factors and other changes also have intracellular metabolic stress,aging or damaged organelles,protein folding error,or gather together,and so on.These factors are always there,therefore,cells maintained a low,underlying autophagic activity in order to maintain homeostasis,and Autophagy in the cellular component updates,tissue metabolism,growth and development of a series of important basic life process plays a very important role in biology.In addition,Autophagy play an important role in several diseases including neurodegenerative diseases,Danon myopathy and many closely related to the development of disease.Autophagy is a protective physiological process,ensuring cell homeostasis and coordination functions within the cell,whereas excessive activation of Autophagy can cause serious cell structure and function of the damage,because we simply cannot define Autophagy for pros and cons of living organisms.Studies have shown that,endoplasmic reticulum stress dependence of muscle cell death and muscle inflammation and malnutrition associated dysferlin myopathy is related to muscle cell damage,endoplasmic reticulum stress and Autophagy plays an important role in skeletal muscle diseases affect each muscle fiber damage.Endoplasmic reticulum stress is due to excessive protein synthesis increased,misfolded proteins increased and intracellular calcium imbalances,sugar,energy deficiency and ischemia and potential pathophysiological interferon increased.All these factors would cause destruction of endoplasmic reticulum steady-state.Endoplasmic reticulum stress is a kind of cell protective response to adverse external factors.Most researches is about the pancreas and adipose tissue,research on skeletal muscle is little.Endoplasmic reticulum has two adaptive responseive mechanism,the first is unfolded protein response(UPR).UPR activation can be raised by 3 ER membrance-associated protein,PERK,IRE1 and ATF6 protein synthesis and can increase gene expression of chaperone,BIP/Grp78 and Grp94,following released endoplasmic reticulum calcium into the cytoplasm of the cavity,activating calcium-dependent pathways.Intracellular calcium increasing activated CaMKK β and MAPK pathway then activated autophagy.Mmeanwhile,intracellular calcium increasing,activate phosphorylation of PKCs,introduce LC3 transferred to TG,TN and stimulate the endoplasmic reticulum stress response.Increaseed in intracellular calcium,and endoplasmic reticulum stress in the activation process of Autophagy is in synergy.Meanwhile,endoplasmic reticulum releasing high levels of calcium into the cytoplasm,caused mitochondrial swelling and destruction of the respiratory chain function,also enhanced autophagic processes.The other adaptive responseive mechanism is ER overload response(EOR).EOR upregulate NF-Kappa b pathway related protein expression,and modulate inflammatory responses to achieve its protective effect,if the UPR and EOR had not been activated,the cells will enter a phase of programmed cell death in adverse environment.Propofol play a protective effect of tumor cells,and MAPK pathways,NF-Kappa b in different tissues and organs,which are closely based on the above facts,whether propofol on the promoting role of Autophagy in skeletal muscle played by the endoplasmic reticulum stress-provoking.Our research is based on mice myoblasts(C2C12)study discussed propofol on normal eukaryotic cell and mechanism of Autophagy,provide new ideas for clinical medicine.Method:(1)Propofol of different concentrations(0μM、25μM、50μM、100μM、150μM、250μM、300μM、400μM、500μM、600μM、700μM、800μM、900μM)-treated with mice myoblasts(C2C12)24h.CCK8 assay for detection of propofol with different concentration on differentiation of Myoblast cells in mice(C2C12)affect the survival rate.Propofol of different concentrations(400μm,900μm)treated with differentiation of Myoblast cells in mice(C2C12)3h,useing flow cytometry detected cell apoptosis and changes of ROS in cells.(2)As to detection whether propofol could induced autophagy,different concentration of propofol(50 μ m,and 100 μ m,and 200 μ m,and 400 μ m)processing differentiation of small rat into muscle cell(C2C12)3h,6h,24 h,respectively.RT-PCR,Western Blot method and immune histology to detect related protein of autophagy,LC3,and p62,and Beclin-1 gene and protein expression level of changes,while detection m TOR,and p-m TOR,AMPK,p-AMPK protein expression level changes respectively.Autophagy inhibitors chloroquine diphosphate,can inhibit proteolytic enzyme.To determine whether the effect of propofol induced autophagy,propofol and chloroquine diphosphate co-treated with differentiation C2C12.Flow cytometry detectedcell apoptosis to describe the role of propofol inducing apoptosis,Western Blot detect the alteration of LC3 and p62 protein expression level.(3)Propofol(400 μ M)and ER stress inhibitors TUDCA 1mM co-treated with differentiated(C2C12)3h,to clarify whether propofol induced autophagy through ER stress.Flow cytometry detected fluorescent dye H2-DCFDA marked ROS.Cells imaged on the high-throughput optical imager Operetta using the integrated Columbus image data storage and analysis systems.Calcium marked by fluorescent dye Rhod-2AM and detected the concentration of calcium in three hours.Detectation method is the same as ROS.Western blot detected LC3 and p62,ER-stress activative maker Bip and CHOP also detected.(4)Finally,to introduce the relationship between propofol induced autophagy upregulation and ROS increasing,treated differentiated C2C12 with propofol and ROS inhibitor NAC for 3h.Flow cytometry detected apoptosis and the alteration of ROS.Western blot detect LC3 and p62.Result1.CCK8,flow cytometry shows that low concentration(less than 300 μM)of propofol promote cell proliferation of,while higher concentrations(>300 μM when)showed inhibition.900 μM propofol induced cell apoptosis,but not through reactive oxygen species(ROS)increased.2.Western blot,RT-PCR and immunohistochemistry results shows that propofol increases Autophagy-related protein LC3,p62,Beclin-1,p-AMPK,p-m TOR,gene expression and protein expression levels in a concentration-dependent manner,peaking at three hours,6 hours disappeared,will not be activated again within 24 hours.3.Propofol and autophagy inhibitors chloroquine diphosphate(CQ),ER stress inhibitors or ROS inhibitors NAC co-treated with differentiated C2C12,will all weakened the effect of propofol induced autophagy.Meanwhile,Bip and CHOP increased when propofol treated C2C12,also decreased significantly if co-treated with propfol and TUDCA.Integrated Columbus image data storage and analysis systems results showed that calcium increased in two hours when cells treated by propofol,decreased in following one hour.When C2C12 co-treated with propofol and TUDCA,the alteration of calcium level is inapparent in 3 hours.Relevantly,ROS level significantly increased if differentiated C2C12 treated by propofol only.ROS inhibitor n-acetyl cysteine(NAC)and propofol co-treated cells,Autophagy-related protein expression in cells declined compared to propofol treated alone.Conclusion1.Propofol,normally do not cause apoptosis,extremely high concentration(900 m)can cause apoptosis,but not through increased ROS pathway.2.Propofol activates autophagy in a concentration-dependent manner,has a relationship with exposure time.Incubating for long time does not enhance the effect of propofol on Autophagy.3.Propofol induced autophagy is related with endoplasmic reticulum stress.Endoplasmic reticulum stress activation release calcium into the cytoplasm,increased intracellular calcium transfer to the mitochondria,mitochondrial release of ROS,increaseed ROS also helpful for autophagy.更多还原