英文版病理学论文范文-《Melatonin Protects N2a against Ischemia/Reperfusion Injury through AutophagyEnhancement》

cell survival under extreme condi-y f f [14] Iy ftions and provides energ or cell unctioning . n-creased autophag has been shown to promote neuronal survival ina number o diseasemodels includingcerebral ischemia[15,16].Thisisparticularly important for terminally differentiated cells likeneurons. On theother hand,over- production of ROS results in impaired mitochondria,which in turn releases pro-apoptotic factor and activatesapoptosis. Mitochondrial apoptosis pathway is generally acknowledged to play an important role in neuron death resultedfromI/R,which hasbeenprovedboth in vitro and in vivo[17, 18]. Thus, timely scavenge of impaired mito-chondria should avoid, or at least attenuate mitochondria apoptosis,thereby increasing theresistance of neurons to adverse stimuli and prolonging cell survival. Autophagic
deliveryto lysosomesis themajor degradativepathwayin mmitochondrial turnover[19]. Autophagy contributes to the removal of damaged mitochondria that would otherwise
activatecaspasesandapoptosis. Here we put forward that melatonin may protect neurons against I/R-mediated injury via autophagy en- hancement in addition to antioxidation. Our observation on the elevation of autophagy following melatonin treatmenton N2acells should improveourunderstanding of the mechanisms for neuroprotective effect of mela-tonin, and help to lay a foundation for informed treat-mentandpotential drugs.

 

1MATERIALS AND METHODS
1.1 Reagents
Melatonin, Rapamycin, 3-methyladenine (3-MA),
Lyso Tracker Red and Mito Tracker Green were pur-chased fromSigma-Aldrich Company Ltd (USA).Mela-tonin was dissolved in methanol and distilled water (3:7,
v/v). Fresh drug solution was prepared in a darkened
hood shortly before its application. Rapamycin was dis-solved in ethanol.3-MA wasdissolved indistilled water. Fetal bovine serum (FBS), Ham F12 and Dulbecco’s modified Eagle’s medium (DMEM/F12 medium) were purchased from GIBCO Life Technologies Ltd (UK). Primary antibodies wererabbit anti-LC3B (Sigma, USA); rabbit anti-Beclin1 and rabbit anti-p-PKB (Santa Cruz Biotechnology,Inc.SantaCruz,USA);andanti-GAPDH (Proteintech Group, Inc., USA). Secondary antibody, alkaline phosphatase-conjugated affinipure anti-rabbit IgG wasfromProteintechGroup,Inc.(USA).
1.2 Cell Lineand CultureConditions
N2a cells (mouse neuroblastoma cells) were main-tained in DMEM/F12 supplemented with 10% FBS and 100 U/mL penicillin/streptomycin at 37ºC in 5% CO2. Serial subcultivation wasperformedeveryother day.
1.3 I/R Modeland Experimental Groups
Experimental groups included group of normally cultured N2a (Nor), group of N2a undergoing I/R (I/R), group of melatonin treatment upon I/R (I/R+Mel), group of rapamycin treatment upon I/R (I/R+Rap), group of 3-MA and melatoninco-treatmentupon I/R(I/R+Mel+3-MA), group of melatonin administration on normal N2a (Nor+Mel),and group of rapamycintreatment on normal N2a (Nor+Rap). The experimental model was estab-lished by90 min of ischemia, followed by 24 h of reper-fusion,according to themethoddescribedby Goldberg etal[20]. To simulate ischemia, N2a cells exposed to DMEM deprived of serum and glucose were put into an anaerobic chamber containing a gas mixture 5% O2 and95% N2. Oxy (OGSD) gen-glucose-serum deprivation
was terminated by exposing the treated cells back into fresh medium containing the serum, and the cultures were then incubated in the normal incubator for 24 h to simulate reperfusion. Melatonin was given in the fresh medium at the commencement of reperfusion in the group of melatonin treatment at a final concentration of 100 µmol/L. Similarly, rapamycin was given at a final concentration of 100 nmol/L, 3-MA of 10 mmol/L. The cells were harvested at the end of reperfusion or 24 hafter theadministrationof drugs.
1.4Assessment of Cell Viabilityby MTT
Cells wereseeded