病理学论文

Melatonin Protects N2a against Ischemia/Reperfusion Injury through AutophagyEnhancement*YanchunGUO (国艳春)1△, Jianfei WANG(王剑飞)1△, Zhongqiang WANG (王忠强)2,Yi YANG (杨易)1,XimingWANG (王西明)1,Qiuhong DUAN (段秋红)1#1Department ofBiochemistryand Molecular Biology,School ofBasicMedical Sciences,Tongji Medical College, Huazhong Univer-sity ofScience andTechnology,Wuhan 430030,China2Department ofEmergencyMedicine,Wuhan General Hospital ofGuangzhou Command, Wuhan 430070,China©Huazhong University ofScienceand Technologyand Springer-VerlagBerlin Heidelberg2010 Summary:Researches have shown that melatonin is neuroprotectant inischemia/reperfusion-media-ted injury. Although melatonin is known as an effective antioxidant, the mechanism of the protectioncannot be explained merely by antioxidation. This study was devoted to exploreother existing mecha-nisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through ele-vating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron sur-vival.To find it out, an ischemia/reperfusion model in N2acells was established for examinations. Theresults showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reper-fusiononset following ischemia and greatly promoted cell survival, whileautophagy blockageby 3-MAled to theshortened N2a cell survival as assessed by MTT,transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably in-creased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression ofp-PKB, key kinase in PI3K/PKBsignaling pathway,showed adecrease when compared with untreatedsubjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possiblyoneof themechanisms underlying neuroprotection ofmelatonin.Keywords:melatonin; autophagy; ischemia/reperfusion; rapamycin; 3-MA;LC3; Beclin1; PKB;N2aCerebral ischemia is the most common type of cerebrovascular diseases[1, 2]. Cerebra l ischemia caused by multiple reasons results in neuronal death involving many patho physiologic mechanisms including severe failureof metabolicenergy support,elevated intr acellular Ca2+, damage to macromolecules and cytoskeleton, mi- tochondrial injury an d activation of inflammatory reac- tion[3]. Reperfusion and reoxygenation of the ische mic tissue re-established by prompt medical treatment in an effort to prevent severe n eurologic damage and favor survival of individuals, also may provide chemical sub- s trates for further increasing cellular alterations,neuronal death and neurologic deficits[4]. Overproduction of freeradicalsduringcerebralischemiaandreperfusion,among other pat hophysiologic mechanisms, is known to con- tribute to functional disruption and neur onal death[3]. Additionally, a cascade of pro-apoptotic phenomena perpetrates abnorma l cell conditions leading to perma- nentfunctional and structuraldamageand cell death[5, 6].Yanchun GUO,E-mail:toelinor@,△Theauthors contributed equally to this wore.#Corresponding author, E-mail: duanqhwz@*This project was supportedby agrant from thePhD Programs F fM y f f (N)oundatio no inistr o Educationo China o.20070487101.Experimental data from numerous studies clearly showed that melatonin, a biogeni c amine produced by the pineal gland, is a highly effective agent in reducing neuron al loss and neurophysiologic deficits associated with brain ischemiaand reperfusion (I/ R) both in animal models and at cell level[7-12].Yet thereal mechanism for this neuro nal protection is not fully elucidated. Re- searches concerning the mechanism mainly focused on melatonin’s antioxidation, which, however, cannot pro- vide sufficient expl anation, suggesting many other mechanisms maycontributetotheneuronalprotectionof mel atonin as well. In particular, our previous study showed that melatonin did not suppre ss reactive oxygen species (ROS) immediately after its application but raised ROSleve l,whereasa stronger antioxidation effect of 6-OH melatonin was observed. However, th e neuro- protectiveeffectof 6-OH melatoninispoorer than thatof melatonin[13].Autophagy is a process in which eukaryotic cells self-digest part of their cytosoli ccomponents so as to de-grade proteins and organelles to survive starvation and elimi nate oxidatively damaged, aberrant macromolecules andorganellesto keep homeostasisin responsetostress.Itis acrucial process for cell survival under extreme condi-y f f [14] I y ftions and provides energ or cell unctioning . n-creased autophag has been shown t o promote neuronal survival ina number o diseasemodels includingcerebral ischemia[15, 16].Thisisparticularly important for terminally differentiated cells likeneurons. On theot her hand,over- production of ROS results in impaired mitochondria,which in turn relea ses pro-apoptotic factor and activatesapoptosis. Mitochondrial apoptosis pathway is gen erally 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 m ito-chondria should avoid, or at least attenuate mitochondria apoptosis,thereby increasin g theresistance of neurons to adverse stimuli and prolonging cell survival. Autophagic deliveryto lysosomesis themajor degradativepathwayin mmitochondrial turnover[19]. Aut ophagy 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 N2ac ells should improveourunderstanding of the mechanisms for neuroprotective effect of mela-tonin, and help to lay a foundation for informed treat-mentandpotential drugs.1MATERIALS AND METHODS1.1 ReagentsMelatonin, 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 darkenedhood shortly before its application. Rapamycin was dis-solved in ethanol.3-MA wasdis solved indistilled water. Fetal bovine serum (FBS), Ham F12 and Dulbecco’s modified Eagle’s medium (DMEM/F12 medium) were purchased from GIBCO Life Technologi es Ltd (UK). Primary antibodies wererabbit anti-LC3B (Sigma, USA); rabbit anti-Becli n1 and rabbit anti-p-PKB (Santa Cruz Biotechnology,Inc.SantaCruz,USA);andanti-GAP DH (Proteintech Group, Inc., USA). Secondary antibody, alkaline phosphatase-conjugat ed affinipure anti-rabbit IgG wasfromProteintechGroup,Inc.(USA).1.2 Cell Lineand CultureConditionsN2a cells (mouse neuroblastoma cells) were main-tained in DMEM/F12 supplemen ted with 10% FBS and 100 U/mL penicillin/streptomycin at 37ºC in 5% CO2. Serial subcultivation wasperformedeveryother day.1.3 I/R Modeland Experimental GroupsExperimental 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 melat onin 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 depri vationwas terminated by exposing the treated cells back into fresh medium containing th e serum, and the cultures were then incubated in the normal incubator for 24 h to si mulate reperfusion. Melatonin was given in the fresh medium at the commencement o f reperfusion in the group of melatonin treatment at a final concentration of 100 µmo l/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 theadmi nistrationof drugs.1.4Assessment of Cell Viabilityby MTTCells wereseeded in 96-well plates and weregrown to 80% confluencebeforefollow-up procedures.Thecell viability of N2a cells subjected to different treatments was analyze d by using MTT assay. Specifically, N2a cells were incubated with MTT (5 mg/mL) for 3 h at 37°C. Subsequently, the formazan crystals were dis- solved in dimethyl sul foxide (DMSO). Absorbance (A) value was determined at 570 nm in a microplate rea der within30 min.1.5Transmission Electron Microscopy (TEM)N2a cells from different experimental groups were washed with ice-cold PBS and then collected at 1500 r/min for 10 min by centrifugation. The cell pellets were fixed with 2.5% glutaraldehyde in 100 mmol/L sodium phosphate(pH 7.4) overnightatroom t emperaturebefore preparation of specimens for TEM. Images were ob- tained using el ectron microscope (FEI Tecnai G2 12,Netherlands).1.6Laser ConfocalScanning Microscopy(LCSM)Laser confocal scanning was performed as follows.N2a cells were seeded on cover slips fixed in culture dishes to 70%–80% confluence before incubation at37ºC for 30 min with 100 nmol/L prewarmed Lyso Tracker Red and 45 min in the presence of 100 nmol/L prewarmed Mito Tracker Green, according to our pre-liminary experimen ts and the manufacturer’s instruction.Cells were then washed with PBS and incubate d with fresh medium for microscopy. Images were collected using a confocal microsc ope(OLYMPUS, FLUOVIEW,FV1000, Japan) at a magnification of 100. The excita-tion/emission wavelengths for Lyso Tracker Red and Mito Tracker Green were 490/51 6 and 577/590 nm, re-spectively.2RESULTS2.1 Melatonin Administration Significantly IncreasedCellViabilityas Assessed by MTT To evaluate the cell viability of N2a cells expos ed to different drugs under I/R condition, N2a cells were deprived of oxygen-glucose-serum (OGSD) for 90 min before treated with different drugs when replaced with fre sh medium for 24 h, and MTT was performed at the end of 24 h.Normally cultured N2acells withor without drugs wereset for control. Theviability of N2a cellswas signif icantly increased in I/R+Mel groups as compared with I/R group (P<0.01), while that was slightly de- creased in Nor+Rap group. Melatonin and 3-MA (auto- phagy inhib itor) co-treatment also increased the cell vi- ability, but it was not as strong as melat onin treatment. Moreover,melatonin did not show marked effects on the viability of n ormally cultured N2a cells as measured by MTT(fig.1).Fig. 1 Evaluation ofcell viability by MTT After undergoing ischemia for 90 min, N2a cells were treated with different drugs at reperfusion onset for 24 h and the cell viability was measured by MTT. Normally cultured N2a cells with or without drugs were set for control. Cell viability was significantly increased in I/R+Mel group. ** P<0.012.2The Increased Number of Autophagolysosomes in N2a Cells in the Presence of Melatonin under the TCMTo observe the morphologic changes of cellular ul- trastructure, attempt was made to examine theN2a cells with different treatments under TEM. Autophagic vacu-oles (morphological features typical for autophagy) were occasional in the cytoplasm of no rmally cultured N2a cells(fig.2A).A lotof autophagicvacuoles werepresent in the cytopl asm of normal N2a cells treated with rapa- mycin, while few autophagic vacuoles we re found in those treated with melatonin (fig. 2B, and C). Lipiddroplets were frequent in the cytoplasm of I/R injured N2a cells, and more autophagi c vacuoles were found than in normally cultured ones (fig. 2E, and F). Apop-tosisand necrosiswere frequent in I/Rinjured N2a cells, and the former was morecommon (fig. 2D,G andH).In I/R+Rap group, lesslipid droplets and a large number of autolysosome s containing swelling mitochondria and other cellular components were seen in the cy toplasm (fig. 2I, J and K). In I/R+Mel+3-MA group, no auto-phagosome was observe d, but necrosis and apoptosis were present (fig. 2L). Abundant autophagosomes and a utolysosomes containing disintegrated cellular struc-tures were present in the cytoplasm of I/R+Mel group,whileno lipid droplets were found(fig.2M,N,O andP).An autophagos ome with distinctive double-layered membrane (ultrastructural feature of autophagy) co n- tained cytoplasmic components including some pre-served organelles likeRER, dens ebodies,ribosomes(fig.2P).2.3 Co-localization of Large Number of Lysosomeswith Mitochondriain N2a Cells of I/R+MelGroup Laser confocal scanning microscop y revealed an autophagic morphology in cells where lysosomes were stained with Lys o Tracker Red and mitochondria were labeled with Mito Tracker Green. Yellow show n in merged pictures represented the occurrence of co-localization of mitochondria an d lysosomes (Red and green makes yellow). In theimages of normal N2a cells,co-loca lization could be visualized (fig. 3, Nor group). Normal N2a cells treated with melato nin showed similar results (fig. 3, Nor+Mel group). In response to I/R the occurrence of co-localization was increased slightly as compared with the normally cultured N2a cells (fig. 3, I/R group).In contrast to other groups,co-localizationof mitochondria and lysosomes displayed a prominent in-crease in the presence of melatonin in I/R-injured N2a cells(fig.3,I/R+Melgroup)2.4 Effects of Melatonin on LC3-I, LC3-II, Beclin1and phosphorylated PKB Levels in N2a Cells Fol-lowingOGSD To determine the activation of autophagy, cells from four experimental groups were subjected to im-m unoblot assay for detection of the protein levels of the two forms ofmicrotubule-assoc iated protein 1lightchain 3 (MAP1-LC3), Beclin1. The results showed that the protein level of LC3-II was significantly increased in I/R+Mel group as compared with othe r groups, while LC3-I showed a slight increase simultaneously (fig. 4), suggesting an increase in autophagosomal formation.Similar result was observed with eclin1 a critic alcomponent in autophag as detected with anti- eclin1 antibod ig.4 .Fig. 2 TCMshowing ultrastructural features ofcontrol N2acells and existenceof morphologicalfeatures ofautophagy,apoptosis and necrosis inI/RN2acells exposed tomelatonin orrapamycin or melatonin+3-MA A: The electron micrograph exhibits the ultrastructural morphology ofa normally cultured N2acell. Scarceautolysosomes (ar-row) are present in the cytoplasm; B: Autophagic vacuoles (arrow)are frequent in the cytoplasm of normal N2a cells treated with rapamycin; C: In the cytoplasm of normal N2a cells treatedwith melatonin few autophagic vacuoles (arrow) are present and the cells are rich in mitochondria; D, E, F, G, and H: N2a cells undergoing I/R. Lipid droplets are frequent and moreauto-phagicvacuoles (arrow) arepresent in thecytoplasm (E). Higher-power magnification photomicrograph demonstrates different stage of autolysosome as indicated by arrow and arrowhead respectively (F). An apoptotic body (black arrow) containing or-ganelles and nuclear is engulfed by an adjacent cell. Another apoptotic body of less electron density (white arrow) could be underdegradation (D).Thenucleus oftheapoptoticcell appears strongly rearrangedand condensationofchromatin is observed. Thenucleus also generates many compact electron densemicronuclei, released in the extracellularspace (arrowheads). Apop-toticbodies (arrows) containing fragments ofcytoplasmicorganelles were seen (G). The micrograph shows an apoptotic body (arrowhead) and amorphous debris of a necrotic cell (arrow) (H); I, J, and K: I/R-injured N2a cells treated with rapamycin. Autolysosomes and liposomes are present in the cytoplasma (I). Higher-power magnification photomicrographs reveal that abundant degradative autolysosomes ofvarious sizes (arrows)distribute throughout thecytoplasm. Engulfed organelles in the autophagosome display degenerative alterations. Autolysosomes contain some swelling mitochondria (arrowheads) and other cytoplasmic components (J and K); L: I/R-injured N2acells co-treated with melatonin and 3-MA. Thecell indicated by arrow-head shows a morphology characteristic for apoptosis: shrinkage of cell, aggregation of organelles in perinuclear area with amorphous cytoplasm on the peripheral area, high condensation ofchromatin in nucleus and intact plasmamembrane. An N2a cell displays thefeatureofnecrosis including disruption ofplasmamembrane and nuclearenvelope(downarrow). Another N2a cell shows amorphous debris ofnecrosis (up arrow); M, N, O, and P: I/R-injured N2a cells treated with melatonin. Plenty of autophagic vacuoles and no lipid droplets are present in the cytoplasma (M). Higher-power magnification photomicrograph shows autolysosomes ofvarious sizes (arrow) containing deformed mitochondria (arrowhead) and other intracellular contents (N and O). A giant autophagosome (arrowhead) with double-membrane contains a large part ofthe intracellular components.Two other autolysosomes (whitearrows)are present in thecytoplasma(P)N ; Nucleus L ; Lipid drople M: MitochondrionHuazhongUnivSciTechnol［Med Sci］30 (1):2010Fig. 3 Monitoring theactivity change ofmitochondria autophagy underlaser confocal scanning microscopy(×100). Scalebar: 10 µmMitochondria and lysosomes were probed with Mito Tracker Green, and Lyso Tracker Red, respectively. Merged images are the overlapping ones of mitochondria staining and lysosome staining, where yellow represents co-localization of lysosomes and mitochondria. Compared with normal N2a cells treated with or without melatonin, more mitochondria autophagy was seen in cells undergoing I/R.Autophagosomes were increased remarkablyin I/R+Mel groupas compared withothergroupsFig. 4 Expression levels ofLC3-I, LC3-II,Beclin1and p-PKBdetectedby immunoblotting N2a cells in four experimental groups (Nor, I/R, I/R+Mel, Nor+Mel) were assessed for LC3-I and LC3-II levels by im-munoblotting with anti-LC3B (A,B, C, D). So did Beclin1 (E,F) and p-PKB (G, H) with antibodies. To ensure equal protein loading, samples were also blotted for GAPDH. LC3-I (A, B) showed an increase and LC3-II (A, C, D) was significantly in-creased in thepresenceof melatonin upon I/R injury. Theexpression level of Beclin1 (E, F) was dramatically increased upon the employment of melatonin on I/R-injured N2a cells while p-PKB (G, H)was decreased simultaneously. Similar result was observed inat least threeindependent experiments.*P<0.05，**P<0.01Phosphoinositide3-kinase/proteinkinaseB/mam-maliantargeto rapam cin P /P /m signaling pathwa is engaged in negative control o autophag .As shown in ig. 4 2a cells undergoing / displa ed a slightincreaseof phosphorylated PKBas compared with normal 2a cells. he e pression level o phosphor -lated P was signi icantl decreased upon administra-tiono melatonin whencomparedtoother groups.3DISCUSSIONMelatonin is a potentfreeradical scavenger and an-tioxidant[21]. Accordingly attention has been paid to me-latonin as a neuroprotective drug against I/R injury inview of its antioxidant actions opposite to the harmfulcellular actions of freeradicals [22-24]. Nevertheless, sug-gestions have been put forth that the neuroprotective effect of melatonin might involve other unknown mechanisms apart from antioxidation[13, 25]. As evidence accumulates that autophagy plays a crucial role in many neuronal diseases, here we report experimental findings in thisstudy showing thatautophagy canbe enhanced by melatonin and possibly serve as a mechanism in neuro-protection of melatoninagainstI/R-mediated injury.In our research,weemployedsimulated I/Rof mouse neuroblastoma cells (N2a) as an in vitro model of I/R injury to the neurons. According to our preliminary studies,90 minof ischemiafollowed by 24 h of reperfu- sion showed the occurrence of apoptosis quite visible, while60 min of ischemia did not change much and 120 min of ischemia displayed plenty of necrotic neurons.Therefore in present study I/R model was set up on 90 min of ischemia. The occurrence of autophagy was as-sessed by revealing morphologic changeof ultrastructure by TEM, observing labeled-N2a cells using LCSM and investigating the protein levels of the molecules closely related to autophagyby using immunoblotting.The results of MTT showed the administration of melatonin significantly prolonged N2acell survival after I/R injury,whereas co-treatment of melatonin and 3-MA was not as strong as melatonin in this regard. TEM ex-hibited that autophagy was seldom in normal N2a cells, butincreasedremarkably in thepresenceof rapamycin ormelatonin, and the former was more dramatic than the latter. Besides, I/R injured N2a cells exposed to mela- tonin did not show the existence of lipid droplets in the cytoplasmas thoseexposed torapamycin. Similar results were observed by LCSM. The combined administration of melatonin and autophagy inhibitor, 3-MA, results in more neuron death than melatonin alone as detected by MTTandTEM.The mammalian homologue of autophagy-related gene8 (Atg8) inyeast,LC3,exists in cells in two forms. When autophagy is induced, LC3-I (18 kD), cytosolic form, is modified to the membrane-bound form, LC3-II (16 kD), which is then recruited to autophagosomal membranes. Therefore, the amount of LC3-II directly correlates with autophagosome numbers[26,27] and serves as a specific marker of autophagy in mammalian. The mammalian homolog of yeast Atg6,Beclin1, is aprinci- pal regulator in autophagosome formation and in initia-tion of autophagy through class III PI3K pathway. Re-cruitment ofPI3K-Beclin1 complexes together withAtg12-Atg5 is an initial step in autophagosome forma-tion[28]. Beclin1 is also essential for the recruitment ofother Atg proteins involved in expansion step in auto-phagy[29]. It has been reported that over-expression of nduces autophag in east and mammalian cells 0 . As eclin1 promotes autophag enhanced e-clin1 e pression has been usedas amarker o autophag to estimate dynamic change on activation of autophagy.The significant increase of LC3 and Beclin1 was ob-served in ischemia N2a cells exposed to melatonin by using immunoblotting. PI3K/PKB/mTOR signaling pathway is engaged in negativecontrol of autophagy. In responseto PI3K acti- vation, PKB is activated by phosphorylation and then regulates theactivityof anumber of targets includingthe serine/threonine kinase mTOR. PKB activates mTOR through multiple steps including phosphorylating and inhibiting the tuberous sclerosis complex 2 (TSC2) which negatively regulates mTOR by acting as a GTPase-activating protein (GAP), leading to downregu- lation of autophagy [31-33]. Therefore, autophagy is upregulated upon mTOR inhibition when phosphoryla-tion of its upstream kinase PKB reduced, for activation of mTOR suppresses autophagy [34-37]. The results also suggested PI3K/PKB signaling pathway was partially inactivated uponmelatonin treatment.According to our findings,conclusion can bemade that in addition to antioxidation, the activation of auto-phagy may be a component of the survival mechanisms turned on and enhanced by melatonin after neuronalI/R. A reasonable explanation for decreased cell viability by rapamycin isthat excessiveautophagicactivity/overacti-vation of autophagycan bedetrimentaland results incell death. Therefore, melatonin-induced autophagy is cyto-protective until overpassing the threshold of cellular constituents degradation leading to autophagic cell death termed as a PCD type II. Besides, we found that mela-tonin application on normally cultured N2acells did not activate autophagy. It is due to that the occurrence of autophagy requires notonly thepresenceof melatonin in this case,but also the activation of key molecules by the signal of injury.Specifically, the transformationof LC3II from LC3I requires the activation of Atg5 which is en- abled by ROS. The results also reconfirmed that auto- phagy is a constitutive cellular event; it is enhanced un-der certain conditions such as starvation and drug treat- ments[38].Presumably,moderately increased autophagy serves as a mechanism to protect I/R injured neurons from apoptosis and necrosis through following aspects. First, autophagy should provide energy for cell functioning during starvation (self-digestion) by degrading intracel-lular macromolecules and organelles. Second, autophagy can timely remove damaged organelles including mito-chondriawhich may otherwise initiate apoptosisthrough mitochondria apoptosis pathway. And third, autophagy can facilitate theclearanceof impaired protease, protein aggregation, protecting cells from further damage and contributing tocytoplasmicremodeling. In conclusion these data all contribute, from differ-ent perspectives, to that autophagy might be one of the mechanisms underlying neuroprotection of melatonin against I/Rinjury. Still, itawaits further study on animal model.REFERENCES1O G, B , M S , M yy x y x β ylivieri rack C uller- pahn et al. ercur inducescell c toto icit and o idativestressandincreases -am loid。