Ethanol induced mitochondria injury and permeability transition pore opening: Role of mitochondria i

PO Box 2345, Beijing 100023, ChinaWorld /Gastroenterol 2007 April 28: 13(16): 2352-2356orld Journal of Gastroenterology ISSN 1007-9327wig@wignet.comc 2007 The w]G Press. All rights reservedRAPID COMMUNICATIONEthanol induced mitochondria injury and permeabilitytransition pore opening: Role of mitochondria in alcoholic liverdiiseaseMing Yan, Ping Zhu, Hui-Min Liu, Hai-Tao Zhang, Li LiuMing Yan, Ping Zhu, Hui-min Liu, Hai-tao Zhang, Department CONCLUSION: Chronic alcohol intake might leadof Digestive Diseases, Qilu Hospital, Shandong University, Jinan to broken mitochondria Ptp, decreased mitochondria250012, Shandong Province, Chinamembrane potential and injury, and elevated intracellularLi Liu, Jinan Infectious Disease Hospital, Jinan 250012, ShandongCa*production. Ethanol-induced chondriosome injuryProvince. ChinSupported by Natural Science Foundation of Shandong Province,may be an important mechanism of alcoholic diseasesNo.032050113Correspondence to: Ming Yan, Department of Digestive 2007 The WJG Press. All rights resDiseases, Qilu Hospital, Shandong University, Jinan 250012handong Province, China. ymylh @163Key words: Alcoholic liver disease; Chondriosome;Telephone:+86531-8216917lFax:+86-531-82169025Apoptosis; Ultra microstructure; Membrane potentialsReceived:2007-02-24Accepted: 2007-03-15Permeability transition pore; Transmembrane potentialchondriosome mass: caYan M, Zhu P, Liu HM, Zhang HT, Liu L. Ethanol inducedAbstrmitochondria injury and permeability transition poreopening: Role of mitochondria in alcoholic liver diseaseAIM: To observe changes of mitochondria and World Gastroentero/2007: 13(16):2352-2356investigate the effect of ethanol on mitochondrial perme-abilitytransitionpore(ptp),mitochOndRiaLmembranehttp://www.wjgnet.com/1007-9327/13/2352.asppotential(MMP, AYm) and intracellular calciumconcentration in hepatocytes by establishing an animalmodel of alcoholic liver disease(ALD)METHODS: Fourty adult male Wistar rats were INTRODUCTIONrandomly divided into two groups, the model group Alcohol abuse used to be a principal factor for the cause(20)was administered alcohol intragastrically plus an of serious liver disease in western countries, In China theOliver oil diet to establish an ALD model, and the control incidence of alcoholic liver disease(ALD) has increasedgroup(20)was given an equal amount of normal saline Alcohol abuse has been considered the second leadingThe ultramicrostructural changes of mitochondria were cause of hepatic lesion after virus hepatitis. The accurateobserved under electron microscopy. Mitochondria ofmechanism of ethanol-induced hepatic cells injury isliver was extracted, and patency of PTp, mitochondrial still not completely clear. Some researchers found thatmembrane potential(A%m), mitochondrial mass and ethanol could activate lipid peroxidation, leading to liverwere detected by flow cytometry using rhodamine123 injury. Since the mitochondrion is one of the fundamental(Rh123), Nonyl-Acridine Orange and calcium fluorescent parts of reactive oxygen species(ROS) formation, weprobe Fluo-3/AM, respectively.investigated mitochondrial changes in ALD in order toprove the correlation between mitochondrion injury andRESULTS: Membrane and cristae were broken or hepatocytes apoptosis induced by ethanol and revealdisappeared in mitochondria in different shapes under the role of ethanol-induced mitochondrion injury in theelectron microscopy. Some mitochondria showed U pathogenesis of AL.D, hoping that by this study we couldnegamitochondrion. In the model group, liver improve the prevention and treatment of ALDmitochondria PTP was broken and mitochondria swelledthe absorbance at 450 nm, A540 decreased (0.0136+000254500321±0.003, model vs control,P<001MA1中国煤化工mitochondria transmembrane potential(239 4638+ Mater127263ws377.5850±16.8119,P<0.01) was lowered;HeaCNMHGng150±5g,weremitochondrial mass(174350+ 1.9880 Vs 31.6738* purchased from the Experimental Animal Cente3.4930, P<0.01); and [Ca" ]i was increased in liver cells of Shandong University. Olive oil(unsaturated fatt(7.0020±0.5008w10.2050±0.4701,P<0.01)acid content>90%)was a product of Algemesi Cowww而克据Yan M et a/ Role of mitochondria in alcoholic liver disease2353pain.Erguotou(a liquor of 60 )was a product of two-step perfusion of Seglen. After anesthetizing withBeijing Hongxing Erguotou Alcohol Factorypentobarbital sodium, the abdominal cavity of the rats wasRhodamin123 and Fluo-3/AM were purchased from opened from the middle abdominal wall Plastic duct wasSigma Co, USA. DMEM was obtained from GIBCO. inserted into the portal vein after exposure. The livers wereFBS was purchased from Hangzhou Sijiqing NAO was perfused first with Ca"-and Mg-free D-Hanks solutionobtained from GENMEDfor 20 min(20-40 mL/min) at 37C to remove blood andCa in the liver, and next with 0.01%(w/v) collagenaseAnimals and protocolsolution at 37 C when the fluid Rowed from the inferiorForty adult Wistar rats(4 wk old), weighing (150 5)g, vena cava became clear. The membrane of gently fullywere fed in separate cages with 5 in each. After feeding digested liver was removed with curved forceps, thennormally for a week, these rats were randomly divided into gently the hepatocytes and sinusoid endothelial cellstwo groups. Twenty were given intragastric alcohol as the were dispersed in Dulbecco's modified Eagle's mediumALD model group whose food was composed of ethanol After filtering through 200-screen mesh to remove cell46%(intragastric administration twice a day at an eight- aggregates and tissue fibers, the cell suspension washour interval), protein 18%o, carbohydrate 12%o, fat 14% washed thrice in DMEM/H with low-speed centrifugation(mainly olive oil, unsaturated fatty acid 9%) by energy as (50 X &, 5 min X 3)to obtain purified hepatocyteswell as vitamin complex and essential minerals at the sametime. In the first four weeks, the rats were lavaged with Detection of mitochondrial membrane potential of30% ethanol (4 g/kg per day), 40% ethanol (5 g/kg per hepatocytesday)in the following four weeks, 50% ethanol(6 g/kg per Rh123 powder was prepared with methanol stored atday )from wk 9 to 12. Another twenty rats in the control -20C as a 1 g/L solution. The storing solution wasgroup were given an equal volume of normal saline and diluted to 5 mg/L with PBS when used. After incubatingthe composition of food was the same as the model group in Rhodamin123(final concentration 2.5 mg/L) forintil the end of the 12 wk1 h at 37C, hepatocytes(1 x 10) were washed thrice inpreheating PBS. After resuspending in 300 uL PBS, filteredPreparation and observation of samples under electron through 200-screen mesh, 10" cells were measured withflow cytometry using the CellQuest software(maximumLiver tissue(1 mm)from the right lobe of the liveer nearbsorbing wave length 590 nm, excitation wave lengththe porta hepatis was removed and pre-fixed for 2 h at 488 nm)4C in 4% paraformaldehyde solution with 0. 1 mol/L1% osmium tetroxide in 0. 1 mol/L phosphate buffer(pH for 20 min, the hepatocytes(1 x 10) were centrifuged at7.3). Post-fixation was followed by dehydration in ethanol, 300 x g for 5 min. The precooling GENMED preservingembedding in Epon 812 and polymerization. Tissues fluid was added into the supernatant, mixed with cellwere cut using an LKB 2088 ultramicrotome, stained with granulations and analyzed with fow cytometry using thetransmission electron microscope (EM-2000 EXIL, 80 nm, and excitation wave length 495 nm wave length 5191%uranyl acetate and lead citrate, and examined under a CellQuest software(maximum absorbingKV, Japan)Detection of intracellular calcium concentrationPatency of mitochondrial PTPAfter incubating in1mFuo3/AM⑤μmol/n)at37℃Isolation and purification of mitochondria: Approxi- for 40 min, the hepatocytes( 1 x 10) were washed 3 timesmately 5 g of the liver harvested from sacrificed rats was with PBS, resuspended in 300 HL PBS and filtered throughhomogenized in a ten-fold volume of isolation buffer(0. 33 a 200-screen mesh, and 10" cells were collected andmmol/L sucrose, 0.025 mmol/L EDTA). After filtering detected with flow cytometry using the CellQuest softwarewith double-ply gauze to remove tissue dregs, the liver (maximum absorbing wave length 506 nm, and excitationhomogenate was centrifuged for 10 min at 800 x &. The wave length 490 nm)supernatant was further centrifuged for 10 min at 8200 x gThe second supernatant was discarded and the sediment Statistical analysiswas resuspended with washing buffer (0.33 mmol/L All values were expressed as mean SD and analyzedsucrose). The suspension obtained was centrifuged for with the SPSS Version. All experiments were repeatedanother 10 min at 8200 g to get the purified mitochondria. at least three times. P<0.05 was considered statisticallyDetection of mitochondrial PTP opening: The purifiedsignificant.mitochondria were diluted to 0.3 mg/mL with test solution(230 mmol manicol, 70 mmol sucrose, 3 mmol Hepes, pH7. 4). The absorbance of mitochondria was detected at 570REV凵中国煤化工nm using an ultraviolet spectrophotometer at 20C to find UltrC MH Gyte mitochondria underhe state of mitochondrial PTP openingelectron microscopyThererich organelles with lots of ribosomes,Isolation and purification of hepatocytesendoplasmic reticulum, mitochondria and few lipidHepatocytes were isolated with the procedure of in situ droplets in hepatocytes in the control group under2354 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol April 28, 2007 Volume 13 Number 16Table 1 Absorbance comparison of liver mitochondria in two groups of rats20.03050.03040.033100320.03140.032500300136±00025001670,016900161001230011900120.0113001160.0321±00013up<0.01 vs control group.Figure 1 MitochondriaControlappearance under6000): A: Mitochondriain normal group: B人非人Mitochondria in modelgroup. M: mi chondriaG: glycogen, N nucleusreticulum, LD: lipiddroplet. The long arrowshows abnormallydistributed chromatin in505050I5 megaU-type mitochondriaFigure 3 Mitochondna mass detected by flow cytometrymegamitochondrion. The glycometabolism was abnormal,and glycogen increased with various densities. Manymyelin figures of metabolites and few lysosomes couldControlbe obscrvcd. Nuclcar material prcscntcd irregular profileswith a pseudo-inclusion body found within Nuclearchromatin was abnormal in distribution and occasionally10"10 10"10 gathered at the perimeter. The number of plasmocytesRh123Rho123incrcascd(Figure 1B).We had established the ALD model success fully,nd confirmed our view point that alcohol could causeModelstructural and morphological damage in mitochondriaPatency of PTP in hepatocytes mitochondriaThe results of A540 detected in model group(0.0136+0.0025)are obviously lower than in the control group(0.0321 +0.0013), P<0.01, which demonstrated thatthe PTP in liver mitochondria of rats were opened in theRho123model group(Table 1)The data indicated that ethanol could incrcaseFigure 2 Mitochondnial membrane potential detected by flow cytometrymitochondria membrane permeability, resulting inmitochondria swelling and damageelectron microscopy. Nuclei of hepatocytes were round Mitochondrial membrane potential of hepatocytes detewith clcar mcmbrancs and well-distributcd nuclear cted by Rh123hromatin. Thc perinuclear cisterna and nucleopore were The mean fluorescence intensity(MFD) of Rh123 inclcar(Figurc 1A)the model group was 239464%+ 12.726%o while inMany lipid droplets with cqual size and electron the control group was 377.585%+16.812%,whichdensity and chromaffin lipid droplets could be seen in demlhe model group. The endoplasmic reticulum became (中国煤化工 al membrane potentialased evidently with aswollen. The membrane was ruptured and cristae were statiCNMHGSO.O1)(Figure 2)broken or disappeared in the mitochondria resulting indifferent shapes, and the electron density of their ground Changes of liver mitochondria masssubstance decreased. Some mitochondria were U-type or The MFI of diphosphatidyl glycerol in the model groupwww.yyg数据Yan M et al. Role of mitochondria in alcoholic liver disease23558ControlModel by oxidative stress is an early event that plays an importantrole in the pathogenesis of ethanol induced apoptosis.In this study, we found that the mitochondria of themoand membrane1010101010 10 10 10, 10 10 and cristac were broken or disappeared under electronmicroscopy. We also observed U-form mitochondri(deformed mitochondria) in different sizes and megamito-Modelchondria. In hepatocytes, there are some myelin figures,chromatins distribute abnormally and some can be edgegathering, indicating the existence of apoptosis. All ofthese further confirmed the damage in the structure andfunction of mitochondria. Meanwhile, the structural andfunctional damage of mitochondria play a very importantrole in accelerating hepatocyte aging and apoptosis inducedby ethanolFigure 4 Intracellular calcium concentrations in hepatocytes detected by flow Mitochondria PTP opening influenced by ethanolcytometryIn recent years, the knowledge of apoptosis has shiftedgradually from the mode of nucleolus-central tomitochondria-central adjustment. In apoptosis, the basicwas 31.6738%+ 3.4930% while the control group was change in mitochondria is mitochondrial permeability74350%±19882%,P<0.01( Figure3)transition(MPT) and decreased transmembrane potentialand other transformation of apoptosis, leading finallyIntracellular calcium concentration in hepatocytescell apoptosis.The MFI of Fluo-3/AM in the model group was 7.0020% PTP is a nonselective passage with a high conductance0.5008% while the control group was 10.2050%+ which stretches across the outer and inner membrane0.4701%o, which demonstrated that the intracellular calcium of mitochondria. Physiologically, the diameter of PPT isconcentration in hepatocytes in the model group increased 0.2-0.3large enough to allow micromolecule soluteevidently with a statistically significant difference(P <0.01) to pass. By the effect of Ca, ADP, ATP and so on.Figure 4PTP switches alternately. This condition may play a role inmaterial exchange between mitochondria and cytoplasmDISCUSSIONBut pathologically, the opening diameter is obviously large(1.8-2. 6 Hm)so that the solute smaller than 1.5 kDa canExcessive alcohol consumption can result in multiple organ pass through PPT to the cytoplasm, causing mitochondrialry, of which alcoholic liver disease(ALD) is the most swelling and transmembrane potential disappearancecommon With economic development and improvement PTp opening has a self-enlarging phenomenon that afterf living conditions of the people, the incidence of liver reveiving the death signal, can perform positive feedback indiseases caused by alcohol abuse has been increasing in the same cell. It means that once PTP maintains opening,China, although its pathogenesis remains obscure. In the it will no longer depend on the previous stimulant signal.past, most researches about ALD focused on oxidative and the cell will die irreversibly. Our experiment indicatcdtress,cytokines unbalance, level alteration of proteins and that long-term alcohol abuse of ethanol can make PTPgenes and so on. Recently, more attention has been on the open, the mechanism is possibly because in hepatocytefunction of mitochondria in the pathogenesis of ALD, but anti-oxidation materials such as materials containinghe mode of mitochondrial damage has not been clarified. sulfydryl are reduced, and/or lipid peroxidation intensifies,In our study, we presented data showing structure and causing injury to the mitochondrial membrane. Thus, thefunction damage, membrane permeability transition as well permeability of the membrane increases, mitochondrias membrane potential alteration of mitochondria, hoping swell, and finally the mitochondria are injuredto reveal the correlation between mitochondrial day stage of ALDRelationship between ethanol and mitochondriatransmembrane potentialFigure and structure alteration of liver mitochondria The permeability of the mitochondrial outer membrane isinduced by ethanolcomparatively large, material smaller than 15 kDa can passMitochondria have long been known to be very important through it freely, so substances of the outer compartmentorganelles of the eukaryocyte. They produce ATP to of the mitochondria are similar to cytoplasm. Thesupply energy directly for life activity of the mammala…*灬灬n1 al inner membrane isells. Moreover, mitochondria are considered the key small中国煤化工 ller th1.kDaorganelle in the initiation of apoptosis since mitochondrial to paCN MH Ghorylation to supplydisintegration not only leads to a loss of their function but energIn this process, energyalso causes the release of proapoptotic factors. Emerging produced by the tricarboxylic acid cycle is transmitted toevidence suggests that mitochondrial dysfurnction caused electrons, which shuttles along respiratory enzymes ofwww.wjgnet.com2356 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol April 28, 2007 Volume 13 Number 16generation of ATE The proton pump is localized on the swelling of mitochondria, and finally apoptosi 1(Ay m),the inner mitochondrial membrane, thus manifesting the prompt decrease of the membrane potentiinner membrane, it pumps protons from groundmass ton our study, we found that the Ca level of the modelthe intermembrane in order to form the transmembrane group is significantly higher than that of the control grouppotential between outer and inner membrane of the It indicates that Ca plays an important role in the processmitochondria Aym 5l. When protons return, they pass of ALD. The Ca signal may induce mitochondrialenergy to ADP and Pi to generate ATP. So Aym plays damage through several possible ways as follows:(1)thea crucial role in keeping the function of mitochondria. elevation of Ca" density in hepatocytes makes PTP open,Further studies of apoptosis indicate that there is causing Ca" to transfer from cytoplasm to mitochondriamitochondrial polarization and Avm disruption in injured This damage is reversible;(2)the over-loading of Caincells induced by apoptosis repulsion, moreover these mitochondria maintains PTP open, leading to a decreasehanges take place before alteration due to apoptosis in of Avm, swelling of mitochondria, and finally apoptosiscells. All of these indicate that Aym alteration is the early So our study indicats that the Ca signal transductionstage of apoptosissystem plays an crucial role in the pathogenesis of ALDIn our experiment, we measured mitochondrialIn summary, our study demonstrates that intransmembrane potential(Aym) changes during culture process of hepatocvte injury, ethanol metabolismusing Rhodamine 123(specific fluorescence mark) induce mitochondrial membrane depolarization andstaining and Aow cytometry. Results reveal that long-term permeability changes in cultured hepatocytes. It indicatesalcohol consumption can cause transmembrane potential that mitochondrial damage must be of pathophysiologicalsignificantly lower than the control group, indicating that importance in the process of ALD. The pathway ismitochondrial depolarization and Aym alteration must be possibly Ca transferring from cytoplasm to mitochondria,induced by ethanolmitochondrial membrane potential, finally leading tohepatocyte injuryMechanism of mitochondrial quality degression inducedby ethanolREFERENCESThe mitochondrial quality is the amount of all the intrcellular mitochondria and their content, and it is an 1 Alpini G, Phillips JO, Vroman B, LaRusso NF. Recentimportant index of mitochondrial injury. Nonyl-Acridineadvances in the isolation of liver cells. Hepatology 1994; 20494-514Orange(NAO) is fluorescent can bind specifically 2 Loeffler M, Kroemer G. The mitochondrion in cell deathto unoxidizable phospholipids. The inner membranecontrol: certainties and incognita. Exp Cell Res 2000: 256: 19-26of mitochondria, and the fluorescence intensity3Crompton M. Mitochondrial intermembrane junctionalproportional to the quality of phospholipids in thecomplexes and their role in cell death. 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