Study Of Propofol Induced Autophagy And Its Mechanism

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 ? 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(0?M?25?M?50?M?100?M?150?M?250?M?300?M?400?M?500?M?600?M?700?M?800?M?900?M)-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(400?m,900?m)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 ? m,and 100 ? m,and 200 ? m,and 400 ? 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 ? 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 ?M)of propofol promote cell proliferation of,while higher concentrations(>300 ?M when)showed inhibition.900 ?M 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.