线粒体靶向抗氧化剂Mitoquinone对人精子冻融氧化应激损伤的保护作用

目的:探讨线粒体靶向抗氧化剂Mitoquinone(Mito Q)对冻融人精子的保护作用。方法:选取60份健康生育男性精液标本,每份精液一式6份,不含Mito Q者设为对照组(G0),而G1、G2、G3、G4、G5实验组混合液中分别含有2 nmol/L、20 nmol/L、200 nmol/L、2μmol/L、20μmol/L Mito Q,37℃孵育1 h后检测活性氧(ROS)、丙二醛(MDA)和线粒体膜电位(MMP)变化。选取合适Mito Q浓度B1、B2组用于精子冷冻保存,B0组未添加Mito Q,B1、B2组在精子冷冻保护液中分别含有200 nmol/L和2μmol/L Mito Q,进行冷冻保存,检测冷冻复苏后的ROS水平、MDA含量和MMP改变。结果:新鲜精液添加Mito Q孵育后,G3组和G4组前向运动精子百分率[(30.8±10.2)%和(32.7±13.5)%]和总活动率[(70.6±9.0)%和(70.3±11.9)%]显著高于G0组[(17.6±5.0)%、(54.9±11.5)%](P0.05);随着Mito Q浓度的增加,ROS水平呈下降趋势,G3、G4、G5组(分别为86.5±31.6、93.6±42.0、45.1±15.0)显著低于G0组(160.8±39.7)(P0.05);MDA含量G3、G4组[分别为(0.9±0.5)、(0.9±0.5)μmol/mg]明显低于G0组[(1.9±1.1)μmol/mg](P0.05),而G5组[(1.7±0.7)μmol/mg]不但没有降低,反而显著高于G3、G4组(P0.05);与G0组MMP(1 701±251)相比,G5组(1 156±216)显著降低(P0.05),而G1、G2、G3、G4组(分别为1 810±298、1 995±437、1 950±334、1 582±314)无明显变化。冷冻复苏后各组前向运动精子百分率和总活动率均较新鲜精液明显下降(P0.01),B1组前向运动精子百分率[(3.2±2.3)%]较B0组[(0.8±0.6)%]明显改善(P0.05);B1组精子总活动率[(43.0±9.5)%]较B0组[(26.5±11.4)%]明显改善(P0.05);B1组ROS[(34.6±12.3)]和B2组ROS[(37.0±10.5)]均较B0组[(56.9±14.3)]显著下降(P0.05),B1组MDA[(1.4±0.5)μmol/mg]和B2组MDA[(1.4±0.6)μmol/mg]均较B0组[(2.6±1.0)μmol/mg]显著下降(P0.05),B1组MMP[(1 010.0±131.5)]和B2组MMP[(880.6±128.6)]均显著高于B0组[(721.1±24.8)](P0.05)。结论:在精液冻存液中添加200 nmol/L的Mito Q能有效提高人精子质量,可作为精液冷冻保护添加剂用于精液的冷冻保存。     

LonP1表达与线粒体稳态以及精子活力的关联性研究

目的探究线粒体lon蛋白水解酶(LonP1)对精子线粒体稳态的维持调节作用及其可能存在的调节机制。方法收集2016年6月至2017年6月于温州医科大学附属第一医院生殖中心进行精液常规检查的120名男性精液标本,根据精子活力分为正常活力精子(NS)和弱精子症精子(AS)两组,采用计算机辅助精液分析系统(CASA)检测各组精子运动参数,通过免疫荧光法和免疫蛋白印迹法检测精子中LonP1的蛋白表达水平,流式细胞术检测各组精子的线粒体膜电位强度(MMP,以绿色荧光强度/红色荧光强度比值表示,比值越小代表膜电位越高)和线粒体内活性氧(mROS)水平,比较两组各指标参数差异,并分析LonP1表达水平和精子活力及精子线粒体功能相关指标(MMP,mROS)的关联性。另外,NS组精液经密度梯度离心法处理后,沉淀精子加入培养液重悬,以LonP1功能抑制剂CDDO-ME 2.5μmoL/L和5μmol/L为终浓度进行体外培养,同时设置空白对照组(0μmol/L),比较各组精子运动参数、LonP1表达水平以及精子线粒体功能相关指标。结果 (1)人类成熟精子中存在LonP1的表达,且稳定表达于精子中段的线粒体鞘部;(2)NS组和AS组精子LonP1表达水平(0.52vs.0.22)与精子活力(69.89%vs.26.10%)、MMP(4.72%vs.12.73%)呈正相关(P0.05),与mROS含量呈负相关(418.85vs.460.52)(P0.05);(3)经LonP1功能抑制剂CDDO-ME处理后,与空白对照组比较,CDDO-ME药物处理组的LonP1蛋白表达水平随CDDO-ME作用浓度增加而显著降低(P0.05),5μmol/L浓度组的MMP水平显著下降(78.81%vs.7.34%)、mROS含量增加(640.68vs.462.26),差异均有统计学意义(P0.05);(4)CDDO-ME处理后,除精子活率(PR+NP)外,精子运动参数平均路径(VAP)和曲线速率(VSL)均随CDDO-ME作用浓度增加而下降(P0.05),与空白对照组相比,5μmol/L浓度组的精子活力(PR)(52.08%vs.65.15%)、曲线速率(VCL)(48.28μm/s vs.78.78μm/s)、侧摆幅度(ALH)(1.28μm vs.1.93μm)、直线性(LIN)(38.83%vs.56.05%)和前向性(STR)(45.53%vs.81.25%)均显著下降,差异有统计学意义(P0.05)。结论精子线粒体鞘部存在LonP1的稳定表达,LonP1能够通过精子线粒体稳态维持改善精子活力。     

POMHEX可抑制小鼠精子运动和获能

目的 探究抗肿瘤药物POMHEX对成熟小鼠精子功能的影响。方法 将小鼠精子在体外暴露于POMHEX(0.1～100μmol/L),通过计算机辅助精液分析系统评估精子活动率和前向运动精子百分率；伊红-苯胺黑染色法检测精子存活率；荧光素-荧光素酶系统检测精子ATP水平；蛋白质印迹实验检测精子获能相关蛋白质酪氨酸磷酸化；考马斯亮蓝G250染色检测精子顶体反应。结果 在不影响存活率的条件下，0.1μmol/L及以上浓度POMHEX可降低小鼠精子活动率(P<0.01)和前向运动精子百分率(P<0.05);1μmol/L及以上浓度可抑制小鼠精子ATP水平(P<0.01)和获能过程蛋白质酪氨酸磷酸化的增加。POMHEX虽然不影响小鼠精子的自发顶体反应，但在5μmol/L及以上浓度会抑制由钙离子载体A23187诱导的顶体反应(P<0.01)。结论 抗肿瘤药物POMHEX在体外可抑制小鼠精子运动和获能，对小鼠精子造成生殖毒性。提示接受POMHEX治疗的患者可提前进行生育力保存。     

异丙酚对乳鼠心肌细胞氧化损伤时线粒体功能的影响

目的 评价异丙酚对乳鼠心肌细胞氧化损伤时线粒体功能的影响.方法 SD乳鼠20只,分离乳鼠心肌细胞,接种于96孔培养板原代培养48 h,随机分为5组,每组32孔,对照组(C组):继续培养4 h;氧化损伤组(OI组):加入叔丁基过氧化氢,终浓度为100 μmol/L,孵育4 h;异丙酚1 μmol/L组、10 μmol/L组和30 μmol/L组(P1-3组):加入叔丁基过氧化氢,终浓度为100 μmol/L,同时加入异丙酚,终浓度分别为1、10、30,μmol/L,孵育4 h.于细胞培养或孵育4 h时,测定上清液乳酸脱氢酶(LDH)活性,细胞谷胱甘肽(GSH)、丙二醛(MDA)含量和超氧化物岐化酶(SOD)活性、心肌细胞线粒体活力、线粒体膜电位和心肌细胞凋亡率.结果 与C组比较,其余4组上清液LDH活性、心肌细胞MDA含量、凋亡率升高,心肌细胞线粒体活力、膜电位降低、心肌细胞GSH含量、SOD活性降低(P<0.05);与OI组比较,P2,3组上清液LDH活性、心肌细胞MDA含量、凋亡率降低,心肌细胞线粒体活力、膜电位升高,P3组心肌细胞GSH含量、SOD活性升高(P<0.05),P1组上述指标差异无统计学意义(P>0.05);P2组与P3组上述指标比较差异无统计学意义(P>0.05).结论 异丙酚减轻心肌细胞氧化损伤的机制与改善线粒体功能、抑制细胞凋亡有关.     

钙调蛋白抑制剂对小鼠精子获能影响的研究

目的:探讨钙调蛋白是否参与小鼠精子获能过程。方法:用50、100、200μmol/L浓度的钙调蛋白抑制剂W7和10、20、30μmol/L浓度的钙调蛋白抑制剂卡米达佐(CZ)分别与小鼠精子孵育2h,通过金霉素染色法计算出B型精子百分率。并用100μmol/L钙调蛋白抑制剂W7和10μmol/LCZ分别与小鼠精子孵育2h后,再加入5μmol/L孕酮诱发顶体反应,计算精子顶体反应百分率。结果:不同浓度的W7和CZ作用小鼠精子,B型精子百分率呈浓度依赖性方式下降,与对照组比较,均有显著性差异(P<0.05)。顶体反应率与对照组相比均有极显著差异(P<0.01)。结论:钙调蛋白参与小鼠精子获能,是精子获能过程中的关键蛋白。     

白藜芦醇对人精子冷冻损伤的保护作用

目的:通过在人精子冷冻保护液中添加白藜芦醇,研究其对冻融后精子质量和功能的影响。方法:选择正常精液与少弱精子症样本各50例,液化后的精液样本分别与甘油-卵黄-柠檬酸盐(GEYC)冷冻保护液或含有30μmol/L白藜芦醇的GEYC冷冻保护液混匀。冷冻复苏前后,进行精子活力、存活率及顶体反应分析。采用丙二醛(MDA)及活性氧(ROS)检测试剂盒评估精子脂质过氧化程度及ROS水平。通过罗丹明123(Rh123)染色法及TUNEL试验检测精子线粒体膜电位及DNA损伤。结果:在正常精液和少弱精子症样本组中,与各组冷冻前新鲜精液相比,冻融后前向运动精子百分率、总活力、存活率、线粒体膜电位及顶体反应率均显著下降(P0.05),而精子ROS、MDA水平和DNA碎片指数(DFI)均显著升高(P0.05)。冷冻保护液中添加30μmol/L白藜芦醇后,正常精液组前向运动精子百分率[(43.1±6.3)%]、总活力[(56.9±7.4)%]、存活率[(67.5±5.6)%]、线粒体膜电位[(63.4±7.5)%]及顶体反应百分率[(26.3±4.7)%]较冷冻对照(未加白藜芦醇)的前向运动精子百分率[(32.7±4.8)%]、总活力[(44.8±6.9)%]、存活率[(52.3±6.1)%]、线粒体膜电位[(56.5±7.0)%]及顶体反应百分率[(16.6±3.8)%]均显著提高(P0.05),而精子ROS、MDA水平和DFI较冷冻对照均显著降低(P0.05)。在少弱精子症组中,添加白藜芦醇也均显著地提高了冷冻后精子前向运动百分率、总活力百分率、存活率、线粒体膜电位及顶体反应百分率,尤其是DFI[28.5±4.8)%]较冷冻对照[(36.3±5.7)%]显著降低(P0.01)。结论:在精液冷冻保护液中添加白藜芦醇可以通过降低精子内ROS水平减少精子冷冻损伤,从而改善解冻后精子质量和功能。     

钙通道拮抗剂体外对人精子运动参数的影响

目的　探讨钙通道拮抗剂在体外对人精子运动参数的影响。方法　将不同类型的钙通道拮抗剂在体外与生育男性经上游优化处理的精子分别共同孵育 10、2 0、3 0、60min ,与正常组进行对照研究。采用计算机辅助精子分析系统检测人精子运动参数 (精子活率、精子前向运动百分率、畸形率、平均路径速度、精子侧摆幅度和摆动幅度 )。结果　硝苯地平 (10 μmol/L)在体外对精子活率 (P <0 .0 1)、精子前向运动百分率 (P <0 .0 1)、精子平均路径速度 (P <0 .0 0 1)、精子侧摆幅度 (P <0 .0 1)和摆动幅度 (P <0 .0 1)等指标的差异有非常显著性。结论　人精子细胞中存在L 型电压依赖性钙通道 ,且L 型钙通道拮抗剂可导致男性不育。     

地塞米松通过线粒体途径诱导成骨细胞凋亡的研究

目的观察地塞米松对离体成骨细胞凋亡的影响,探讨地塞米松对成骨细胞凋亡的分子作用机制。方法采用不同浓度地塞米松(10~(-8)、10~(-6)、10~(-4)mol/L)干预SD大鼠离体成骨细胞,DAPI染色观察细胞核形态,透射电镜观察细胞核及线粒体形态,流式细胞术检测细胞凋亡率,JC-1荧光探针检测线粒体跨膜电位。结果 10~(-8)、10~(-6)、10~(-4)mol/L地塞米松干预24 h后,DAPI染色发现,10~(-8)mol/L地塞米松组的成骨细胞很少发生凋亡,10~(-6)mol/L和10~(-4)mol/L地塞米松组的成骨细胞凋亡明显,地塞米松的浓度越高,核固缩、核裂解等凋亡现象越明显;透射电镜观察发现,随着地塞米松浓度增加,细胞核固缩明显,线粒体肿胀、空泡样变化现象增多;成骨细胞的凋亡率随着地塞米松浓度的增加而逐渐增高,与空白对照组相比,10~(-8)mol/L地塞米松组细胞凋亡率无明显升高(P0.05),10~(-6) mol/L和10~(-4)mol/L地塞米松组的成骨细胞凋亡率分别较空白对照组增加7.240%和31.173%(P0.05);随着地塞米松浓度的增加,线粒体膜电位逐渐减低,与空白对照组相比较,10~(-8)mol/L地塞米松组细胞膜电位下降不明显(P0.05),10~(-6) mol/L和10~(-4)mol/L地塞米松组膜电位下降分别较空白对照组降低6.814%和17.846%(P0.05)。结论地塞米松通过激活线粒体途径诱导成骨细胞凋亡,存在浓度依赖性。     

还少胶囊联合左卡尼汀治疗少、弱、畸精子症的疗效观察

目的:观察还少胶囊及其联合左卡尼汀口服液治疗少、弱、畸精子症的临床疗效及对精子参数的影响。方法:采用随机对照的临床研究,将确诊的186例脾肾虚损型男性不育患者患者随机分为治疗组、对照组和联合治疗组,每组62例。治疗组给予还少胶囊治疗,每日3次,每次3粒;对照组给予左卡尼汀口服液治疗,每日3次,每次1支(10 ml);联合用药组给予还少胶囊联合左卡尼汀口服液治疗,用法同上。3组均用药12周,随访3次,治疗后第4周、第8周、第12周分别检测两组精液质量(精液量、精子浓度、精子活率、前向运动精子百分率)及精子畸形率。结果:180例患者完成了临床研究,治疗12周后,联合用药组精液量提高42.77%,精子浓度提高142.37%,精子活动率增加28.61%,前向运动精子百分率增加24.39%,精子畸形率下降6.27%,与用药前相比都有显著提高,差异有统计学意义(P0.05)。联合用药组在提高精液量、精子浓度、精子活率、前向运动精子百分率上优于对照组(P0.05),在提高精子活率、前向运动精子百分率上优于治疗组(P0.05)。治疗组用药后精液量、精子浓度、精子活率、前向运动精子百分率、精子畸形率与用药前相比都有显著提高,差异有统计学意义(P0.05),治疗组在提高精液量和精子浓度上显著优于对照组(P0.05)。结论:还少胶囊联合左卡尼汀治疗少、弱、畸形精子症安全有效,值得临床推广。     

重组人睾丸精子结合蛋白对人精子运动参数的影响

目的:探讨重组人睾丸精子结合蛋白(TSBP)对体外培养人精子运动参数的影响。方法:将22例生育男性的精液经Percoll密度梯度离心后,分别与0.01mg/ml及0.1mg/ml的重组His6-TSBP在体外共同孵育1h或3h,同时设立对照组,Western印迹检测重组His6-TSBP与精子膜的结合情况,计算机辅助精液分析(CASA)系统测定重组His6-TSBP对精子运动参数的影响。将12例弱精子症患者的精液按同样方法处理,检测重组His6-TSBP对弱精子症患者精子运动参数的影响。结果:0.1mg/ml重组His6-TSBP与生育男性精子作用1h可以提高体外培养精子的前向运动百分率(a+b级精子百分率),培养3h后前向运动百分率和活率均有所提高,差异具有显著性(P<0.05);0.01mg/ml重组His6-TSBP对检测各指标均无显著性影响。0.1mg/ml重组His6-TSBP与弱精子症患者精子作用3h可以提高精子前向运动百分率(P<0.05),但对活率无显著影响。结论:0.1mg/ml的重组His6-TSBP在体外可以提高生育男性精子的前向运动百分率和活率及弱精子症患者精子的前向运动百分率。     

Oxidative capacity, lipotoxicity, and mitochondrial damage in type 2 diabetes

Recent evidence points toward decreased oxidative capacity and mitochondrial aberrations as a major contributor to the development of insulin resistance and type 2 diabetes. In this article we will provide an integrative view on the interrelation between decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations in type 2 diabetes. Type 2 diabetes is characterized by disturbances in fatty acid metabolism and is accompanied by accumulation of fatty acids in nonadipose tissues. In metabolically active tissues, such as skeletal muscle, fatty acids are prone to so-called oxidative damage. In addition to producing energy, mitochondria are also a major source of reactive oxygen species, which can lead to lipid peroxidation. In particular, the mitochondrial matrix, which contains DNA, RNA, and numerous enzymes necessary for substrate oxidation, is sensitive to peroxide-induced oxidative damage and needs to be protected against the formation and accumulation of lipids and lipid peroxides. Recent evidence reports that mitochondrial uncoupling is involved in the protection of the mitochondrial matrix against lipid-induced mitochondrial damage. Disturbances in this protection mechanism can contribute to the development of type 2 diabetes.     

Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis

The aim of this study was to evaluate conventional semen parameters (density, morphology, and progressive motility) and the flow-cytometric parameters of DNA fragmentation, mitochondrial membrane potential, phosphatidylserine externalization, and chromatin compactness in patients with varicocele before and after varicocelectomy. Thirty men (26.5 ± 3.2 years old, range 20-32 years) with oligoasthenoteratozoospermia and grade 3 left varicocele were selected (without other causes of male infertility). Each of them underwent sperm analysis and flow cytometric evaluation before and 4 months after subinguinal microsurgical varicocelectomy (SMV). After varicocelectomy, men had significantly higher sperm density, progressive motility, and normal forms compared with baseline. They also had a significantly lower percentage of spermatozoa with low mitochondrial membrane potential. After SMV, they showed a significantly lower percentage of spermatozoa with phosphatidylserine externalization, an early sign of apoptosis. Significantly decreased percentages of spermatozoa with abnormal chromatin compactness and spermatozoa with DNA fragmentation were found after SMV compared with baseline. Subinguinal microsurgical varicocelectomy improves sperm function in oligoasthenoteratozoospermia secondary to grade 3 left varicocele. Improvements are seen in conventional parameters and biofunctional parameters not routinely evaluated.     

The mitochondrial uncoupling agent 2,4-dinitrophenol improves mitochondrial function, attenuates oxidative damage, and increases white matter sparing in the contused spinal cord

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.     

Relationship between sperm mitochondrial membrane potential,sperm motility,and fertility potential

AIM:To analyze the relationship between sperm mitochondrial membrane potential and sperm motility parameters by means of a computer-assisted sperm analyzer (CASA) and in-vitro fertilization rate(%FR). METHODS: Semen samples were obtained from 26 men undergoing in vitro fertilization-embryo transfer (IVF-ET). Informed consent was obtained from all men prior to the study. Samples were prepared using wash and swim-up method in HEPES-HTF medium. The sperm motility (%MOT), progressive motility (%PMOT), average path velocity (VAP) microm/s), straight line velocity (VSL) (micro m/s), curvilinear velocity (VCL) (microm/s) and %hyperactivated sperm (%HA), and the %FR were assessed. The samples were incubated in the presence of 2.0 mciromol/L of 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37 degrees C in air and washed in PBS before flow cytometry (FACSCalibur: Becton Dickinson) analysis. The mitochondrial probe JC-1 was used to identify the mitochondrial membrane potential. The sperm was divided into three populations according to the fluorescence pattern as follows: the high mitochondrial membrane potential group (n=8), the moderate group (n=5), and the low group (n=13). Statistical analysis was performed using unpaired t-test. RESULTS:Significant differences were found between the high and the low groups in %MOT (91.1+/-8.5 vs 63.0+/-32.7, mean+/-SD), VAP (73.0+/-14.2 vs 52.1+/-12.5), VCL (127.0+/-28.1 vs 87.0+/-22.6), %HA (27.3+/-23.6 vs 7.2+/-9.0) and %FR [73.2 (48/56) vs 59.0 (69/117)]. No significant differences were found in other CASA parameters. CONCLUSION: When the sperm mitochondrial membrane potential increases, sperm motility parameters and fertility potential will also increase. The JC-1 dye method is useful to predict sperm fertility potential.     

Proximal tubular cytochrome c efflux: determinant, and potential marker, of mitochondrial injury

BACKGROUND: Cytochrome c (cyt c) is released from mitochondria after tissue injury, but little is known of its subsequent fate. This study was undertaken to ascertain: (1) does cyt c readily gain access to the extracellular space; (2) if so, what are some determinants of this process; and (3) might cyt c release be a potentially useful marker of in vivo tissue damage. METHODS: Isolated mouse proximal tubules (PT) were subjected to site 1 (rotenone; Rot), site 2 (antimycin A, AA), or site 3 (hypoxic) respiratory chain blockade (+/- 2 mmol/L glycine, to prevent plasma membrane disruption/cell death). Alternatively, oxidant injury was imposed (Fe(2+) or cholesterol oxidase). Extra- and intracellular cyt c levels were quantified by Western blot. Plasma or urine cyt c levels were also determined after rhabdomyolysis or ischemic acute renal failure (ARF) (in mice), or clinical ARF. RESULTS: AA, Rot, and hypoxia caused variable degrees of PT cyt c release (AA > rot approximately hypoxia), but at most, <20% of total cell content was involved. In contrast, Fe(2+) evoked approximately 65% cyt c efflux, and cholesterol oxidation caused approximately 100% cyt c release. Glycine did not block cyt c efflux, dissociating this process from plasma membrane disruption/necrotic cell death. After rhabdomyolysis, plasma cyt c levels rose and correlated with the severity of ARF (r, 0.93 vs. BUNs). Cyt c was detected in urine after both experimental and clinical ARF. CONCLUSION: Cell cyt c release is dependent on the site and the type of mitochondrial injury sustained. Oxidative injury, in general, and cholesterol oxidation, in particular, seem particularly relevant in this regard. After mitochondrial release, cyt c traverses plasma membranes, eventuating in the extracellular space. The data suggest that plasma and/or urine cyt c appearance might function as a clinically useful in vivo marker of mitochondrial stress and the tissue injury sustained.     

乙醇诱导肝细胞线粒体质量和跨膜电位变化的研究

目的：探讨酒精性肝病过程中乙醇诱导肝细胞线粒体质量和跨膜电位变化.方法：将20只健康雄性大鼠随机分为2组：酒精性肝病模型组10只,用高度白酒灌胃建立动物模型;对照组10只,给予相同体积的生理盐水代替白酒灌胃.分离大鼠肝细胞用Rhodamine123和NAO标记,以流式细胞仪测定线粒体质量和跨膜电位.结果：与对照组相比,模型组的线粒体跨膜电位（△ψm）显著降低（P＜0.01）;模型组的线粒体膜中心磷脂含量显著减少（P＜0.01）.结论：长期摄入酒精可导致肝脏线粒体跨膜电位降低以及线粒体膜损伤,从而引发肝细胞的破坏.