补肾健脾活血方调控Apaf-1/CHOP-Caspase-9/Bcl-2通路抑制成骨细胞氧化应激

目的 探讨补肾健脾活血方对减轻大鼠成骨细胞氧化应激损伤和调控线粒体介导的细胞凋亡的机制。方法 提取大鼠原代成骨细胞,通过CCK8实验检测补肾健脾活血方干预下对成骨细胞增殖活性的影响、碱性磷酸酶(alkaline phosphatase, ALP)染色检测成骨细胞ALP活性、茜素红染色检测成骨细胞的矿化情况、实时定量PCR检测Apaf-1和CHOP基因的mRNA表达、蛋白质印迹检测Caspase-9和Bcl-2蛋白表达量。结果 CKK8实验和碱性磷酸酶染色表明氧化应激损伤的大鼠成骨细胞增殖活性显著降低,成骨分化受抑制,同时Apaf-1和CHOP的mRNA表达以及Caspase-9的蛋白表达明显增加(P≤0.01),Bcl-2蛋白表达则显著减少(P≤0.01);经补肾健脾活血方干预后,成骨细胞增殖活性得到明显的增强、成骨分化增加,Apaf-1和CHOP的mRNA及Caspase-9的蛋白表达均显著减少(P<0.05),而Bcl-2的蛋白表达则显著升高(P≤0.000 1)。结论 补肾健脾活血方可以促进成骨细胞增殖和成骨分化,显著降低Apaf-1和CHOP的mRNA及Caspase-9...     

自吞噬在脂多糖诱导HL-1心肌细胞损伤中的作用

目的 评价自吞噬在脂多糖(LPS)诱导HL-1心肌细胞损伤中的作用.方法 采用随机数字表法,将培养的HL-1细胞随机分为4组(n=15):正常对照组(C组)不予任何处理,继续培养24h;LPS组在细胞培养液中加入LPS(终浓度1 μg/ml);自吞噬诱导剂纳巴霉素组(R组)在细胞培养液中加入纳巴霉素(终浓度0.2 μg/ml),孵育48 h时加入LPS(终浓度1 μg/ml);自吞噬抑制剂三甲基嘌呤组(3-MA组)在细胞培养液中加入加入3-MA(终浓度10 mmol/L),孵育48 h时加入LPS(终浓度1μg/ml).各组孵育4h时测定自吞噬蛋白微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)表达水平,并观察线粒体超微结构,计算自吞噬体数量,测定线粒体光密度值、线粒体膜电位(JC-1).于孵育24h时测定细胞凋亡率和Caspase-3活性.结果 与C组比较,LPS组LC3Ⅱ表达水平、线粒体光密度值、细胞凋亡率和Caspase-3活性升高,自吞噬体数量增加,JC-1降低(P＜0.05);与LPS组比较,R组LC3Ⅱ表达水平和JC-1升高,自吞噬体数量增加,线粒体光密度值、细胞凋亡率和Caspase-3活性降低,3-MA组LC3Ⅱ表达水平和JC-1降低,自吞噬体数量减少,线粒体光密度值、细胞凋亡率和Caspase-3活性升高(P＜0.05).R组线粒体超微结构损伤较LPS组减轻,3-MA组较LPS组加重.结论 自吞噬可减轻LPS诱导的HL-1心肌细胞损伤,机制可能与清除受损线粒体,改善线粒体功能,抑制细胞凋亡有关.     

二甲双胍调控PI3K/Akt/mTOR通路减轻糖皮质激素诱导的成骨细胞凋亡

目的探讨二甲双胍(Met)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对糖皮质激素地塞米松(Dex)诱导的成骨细胞凋亡的影响。方法将体外培养的小鼠胚胎成骨细胞前体细胞MC3T3-E1分为对照组(正常培养)、Dex组(以Dex处理)、Met组(以Dex和Met共同处理)、Met+IGF-1组(以Dex、Met和PI3K/Akt/m TOR通路活化剂IGF-1共同处理)、Met+NVP-BEZ235组(以Dex、Met和PI3K/Akt/m TOR通路抑制剂NVP-BEZ235共同处理),采用免疫印迹法(WB)检测MC3T3-E1细胞中PI3K、Akt、磷酸化(p)-Akt、m TOR和p-mTOR蛋白表达水平,通过噻唑蓝(MTT)法检测MC3T3-E1细胞存活率、流式细胞术检测MC3T3-E1细胞凋亡率、实时荧光定量PCR检测MC3T3-E1细胞中Bcl-2和Bax mRNA表达水平、Caspase-3活性测定试剂盒检测MC3T3-E1细胞Caspase-3活性、JC-1探针检测MC3T3-E1细胞线粒体膜电位变化。结果与对照组比较,Dex组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显降低,而细胞凋亡率、Bax mRNA表达水平和Caspase-3活性均明显升高(P0.05);与Dex组比较,Met组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显升高,而细胞凋亡率、Bax mRNA表达水平、Caspase-3活性明显降低(P0.05);给予IGF-1作用后Met对MC3T3-E1细胞的作用效果明显增强,而给予NVP-BEZ235作用后Met对MC3T3-E1细胞的作用效果明显减弱(P0.05)。结论 Met可通过激活PI3K/Akt/m TOR通路抑制线粒体凋亡途径,减轻糖皮质激素Dex诱导的成骨细胞凋亡。     

利多卡因对缺氧/复氧后肝细胞Bcl-2、Caspase-3蛋白表达及细胞凋亡的影响

目的探讨利多卡因预处理对肝细胞缺氧/复氧后Bcl-2、Caspase-3蛋白表达及细胞凋亡的影响。方法将体外培养的肝细胞分为三组:缺氧/复氧组(Ⅰ组)、利多卡因预处理组(Ⅱ组)和正常对照组(Ⅲ组),每组10份。检测肝细胞缺氧/复氧培养后细胞培养液中谷丙转氨酶(ALT)、谷草转氨酶(AST)浓度,肝细胞Bcl-2、Caspase-3蛋白表达、肝细胞凋亡率及超微结构变化。结果Ⅰ、Ⅱ组细胞培养液中ALT、AST浓度、肝细胞Caspase-3蛋白表达和肝细胞凋亡率较Ⅲ组均升高(P<0.05),透射电镜示肝细胞损伤,可见凋亡细胞;Ⅱ组细胞复氧后培养液中ALT、AST浓度、肝细胞Caspase-3蛋白表达和细胞凋亡率均低于Ⅰ组(P<0.05),而肝细胞Bcl-2蛋白表达较Ⅰ组升高(P<0.05),透射电镜观察也显示Ⅱ组肝细胞比Ⅰ组损伤轻微。结论利多卡因预处理可以在一定程度上减轻缺氧/复氧后肝细胞损伤,降低细胞凋亡率,保护机制可能与Bcl-2、Caspase-3蛋白表达有关。     

沉默信息调节因子1对高糖诱导的足细胞凋亡的影响及机制研究

目的研究沉默信息调节因子1(silence information regulator 1,SIRT1)对高糖诱导的足细胞凋亡的影响和机制。方法取足细胞分别给予不同处理,分为5组:Control组(5.6 mmol/L葡萄糖培养液培养)、Osmotic-NC组(葡萄糖5.6 mmol/L+甘露醇25 mmol/L培养液培养)、HG组(30 mmol/L葡萄糖培养液培养)、HG+SIRT1组[SIRT1过表达载体(pcDNA3.1-SIRT1)转染+30 mmol/L葡萄糖培养液培养]、HG+NC组[阴性对照载体(pcDNA3.1)转染+30 mmol/L葡萄糖培养液培养],采用qRT-PCR和Western blot技术检测各组SIRT1表达效果,以试剂盒分别检测各组细胞中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)、活性氧簇(ROS)水平,采用流式细胞术检测各组细胞凋亡,采用Western blot检测各组细胞中Cleaved Caspase-3和Cleaved Caspase-9蛋白表达变化。结果高糖培养后的足细胞中SIRT1 mRNA及蛋白水平均降低。转染SIRT1过表达载体后的足细胞经过高糖处理以后,细胞中的SIRT1 mRNA及蛋白水平升高。高糖处理后的足细胞中SOD、CAT、GSH-Px活性降低,MDA和ROS水平升高,细胞凋亡率升高,细胞中Cleaved Caspase-3和Cleaved Caspase-9蛋白水平升高。上调SIRT1可以提高高糖条件下足细胞SOD、CAT、GSH-Px活性,减少细胞中MDA和ROS水平,减少细胞凋亡率和Cleaved Caspase-3、Cleaved Caspase-9蛋白表达。结论上调SIRT1可以降低高糖诱导的足细胞凋亡,其作用机制可能与降低氧化损伤有关。     

Bcl-2过表达对TNF-α诱导大鼠成骨细胞凋亡的影响

目的 探讨凋亡相关基因Bcl-2过表达对TNF-α诱导大鼠成骨细胞凋亡的影响及相关机制。方法将含有Bcl-2cDNA的逆转录病毒表达载体pDOR-SB质粒转染原代培养的大鼠成骨细胞并且进行阳性克隆筛选。用30 ng/ml TNF-α刺激转染组和对照组细胞24 h,流式细胞术、透射电镜检测细胞凋亡,应用免疫组化结合计算机图像分析技术检测转染组和对照组细胞Bcl-2蛋白及TNF-α刺激前后Caspase-3蛋白表达。结果 Bcl-2蛋白表达在转染加压筛选组显著高于对照组(P<0.05)。TNF-α刺激后转染组细胞亚二倍体凋亡峰比例为6.6％,对照组为16.1％。TNF-α刺激后转染组细胞.超微结构大多正常,对照组细胞出现明显凋亡征象。TNF-α刺激后两组细胞 Ciaspase-3蛋白表达均升高;TNF-α刺激前Caspase-3蛋白表达转染组与对照组比较差异无显著性(P>0.05),TNF-α刺激后Cas-pase-3表达对照组较转染组显著高(P<0.05)。结论 Bcl-2过表达对TNF-α诱导的成骨细胞凋亡有抵抗作用,Bcl-2过表达的抗凋亡作用可能部分通过抑制TNF-α刺激引起的Caspase-3蛋白表达升高实现,有望作为抑制成骨细胞凋亡的侯选基因。     

褪黑素通过Nrf-2途径抑制高铁诱导成骨细胞的凋亡

目的 探讨Nrf-2途径在褪黑素抑制高铁诱导成骨细胞凋亡中的作用。方法 用细胞贴壁法培养成骨细胞 (hFOB1. 19)后，将枸橼酸铁铵(400μmol/ L)和褪黑素（100μmol/L)加人细胞培养基，培养24 h后，Annexin V-FITC/PI流式细胞学检测细胞凋亡水平;DCFH-DA荧光探针检测细胞内ROS水平；Western blot检测凋亡相关蛋白线粒体内Bax和caspase- 3，以及Nrf-2途径相关蛋白Nrf-2和HO-1。结果 高浓度枸橼酸铁铵干预成骨细胞后，与对照组相比，细胞内ROS水平增高，枸橼酸铁铵处理后凋亡增加，凋亡相关蛋白，线粒体内Bax表达增加，caspase-3表达增加，同时，Nrf-2途径相关蛋白Nrf-2及 HO-1表达增加。而褪黑素可以进一步增加细胞内的Nrf-2及HO-1水平，同时降低细胞内ROS水平，细胞凋亡率和凋亡相关蛋白表达明显降低。结论 褪黑素可以通过Nrf-2途径抑制成骨细胞的氧化应激水平，从而降低高铁引起的成骨细胞凋亡。     

褪黑素对H2O2诱导的成骨细胞氧化应激损伤的保护作用

摘要：目的 探讨褪黑素对H2O2诱导的成骨细胞氧化应激损伤的保护作用机制。方法 取24 h内新生SD大鼠10只，采用组织块贴壁法提取原代成骨细胞并行差速贴壁提纯细胞。细胞传至第2代时行碱性磷酸酶及茜素红染色进行鉴定。将鉴定后的成骨细胞用不同浓度的H2O2作用不同时间并行CCK8试验检测细胞增殖情况。选择合适的浓度和时间建立氧化应激损伤模型，并用褪黑素及EX527进行干预。实验分为对照组、H2O2组、褪黑素组及EX527组。分别对四组细胞行碱性磷酸酶染色、茜素红染色，检测四组细胞活性氧、丙二醛、超氧化物歧化酶含量，同时用流式细胞仪检测四组细胞凋亡率，免疫印迹法检测凋亡相关蛋白Bax、Bcl2和成骨相关蛋白BMP2、RUNX2以及SIRT1和p66SHC的表达量。结果 经鉴定，成功提取原代成骨细胞。CCK8结果显示，400 μmol/L H2O2作用4 h成骨细胞活性降至52 %，适宜用于建立氧化应激损伤模型。H2O2作用后成骨细胞活性及矿化能力降低，ROS含量、MDA含量升高，SOD活性降低，细胞凋亡率升高，伴随有SIRT1、BMP2、RUNX2、Bcl2表达降低，p66SHC和Bax表达升高。褪黑素预处理后缓解了H2O2对成骨细胞的氧化应激损伤作用，部分恢复了成骨细胞的活性及矿化能力，SIRT1抑制剂EX527能够逆转褪黑素的上述作用。结论 褪黑素通过调节SIRT1/p66SHC通路来抑制H2O2诱导的成骨细胞的氧化应激损伤并促进成骨。     

牵张性脊髓损伤后神经细胞中Caspase-3表达及其作用的实验研究

目的:观察大鼠牵张性脊髓损伤后脊髓神经细胞中半胱氨酸天冬氨酶3(Caspase-3)的表达及其在神经细胞凋亡中的作用。方法:大鼠脊髓T13~L2经牵张损伤,皮层体感诱发电位监测P1-N1波幅下降至术前波幅70%后维持10m in,分别于术后6h、1、4、7、14、21d处死取材。采用流式细胞仪、原位末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法(TUNEL法)、免疫组织化学检测等方法观察大鼠脊髓神经细胞中Caspase-3表达变化及神经细胞凋亡情况,测定Caspase-3活性。结果:脊髓损伤后神经细胞凋亡率、TUNEL阳性细胞数、Caspase-3免疫组织化学阳性表达及Caspase-3活性测定均较空白对照组及椎板切除组显著升高(P<0.05或0.01),前三项指标改变趋势大致相同,均为术后7d达高峰,而Caspase-3活性则术后4d达高峰。结论:大鼠牵张性脊髓损伤后神经细胞中Caspase-3表达增高、Caspase-3活性增强,是检测神经细胞凋亡的早期生物学指标,对认识脊髓损伤机制具有一定的意义。     

益肾方对大鼠肾小管上皮细胞氧化应激的保护作用及其机制研究

目的:探究益肾方对NRK-52E细胞氧化损伤的保护作用。方法:建立H_2O_2诱导NRK-52E氧化损伤模型,分为正常对照组,H_2O_2模型组,益肾方组。流式细胞术测定细胞凋亡率,运用Western-blot技术检测Caspase-3和BCL-2的蛋白表达。结果:与正常对照组比较,H_2O_2造模组的细胞凋亡率明显增高(P 0. 01);与H_2O_2模型组比较,益肾方组在益肾方浓度100μg/ml、200μg/ml、400μg/ml时,细胞的凋亡率出现明显的下降(P 0. 05)。与H_2O_2模型组相比,益肾方组在益肾方浓度100μg/ml、200μg/ml时,Caspase-3的蛋白表达明显降低,BCL-2的蛋白表达增加(P 0. 05)。结论:益肾方对NRK-52E细胞氧化损伤的保护机制,可能与其降低细胞凋亡,同时下调Caspase-3和上调BCL-2的蛋白表达有关。     

Nitric oxide protects osteoblasts from oxidative stress-induced apoptotic insults via a mitochondria-dependent mechanism.

Nitric oxide (NO) contributes to the regulation of osteoblast activities. In this study, we evaluated the protective effects of NO pretreatment on oxidative stress-induced osteoblast apoptosis and its possible mechanism using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP) at a low concentration of 0.3 mM significantly increased cellular NO levels without affecting cell viability. However, when the concentration reached a high concentration of 2 mM, SNP increased the levels of intracellular reactive oxygen species and induced osteoblast injuries. Thus, administration of 0.3 and 2 mM SNP in osteoblasts were respectively used as sources of NO and oxidative stress. Pretreatment with NO for 24 h significantly ameliorated the oxidative stress-caused morphological alterations and decreases in alkaline phosphatase activity, and reduced cell death. Oxidative stress induced osteoblast death via an apoptotic mechanism, but NO pretreatment protected osteoblasts against the toxic effects. The mitochondrial membrane potential was significantly reduced following exposure to the oxidative stress. However, pretreatment with NO significantly lowered the suppressive effects. Oxidative stress increased cellular Bax protein production and cytochrome c release from mitochondria. Pretreatment with NO significantly decreased oxidative stress-caused augmentation of Bax and cytochrome c protein levels. In parallel with cytochrome c release, oxidative stress induced caspase-3 activation and DNA fragmentation. Pretreatment with NO significantly reduced the oxidative stress-enhanced caspase-3 activation and DNA damage. Results of this study show that NO pretreatment can protect osteoblasts from oxidative stress-induced apoptotic insults. The protective action involves a mitochondria-dependent mechanism.     

西罗莫司诱导人肝癌细胞株BEL-7402凋亡及其机制

目的探讨西罗莫司（SRL）在体外诱导人肝癌细胞株BEL-7402的凋亡作用及其机制。方法以不同浓度的SRL（5、10、20、30、40和50nmol／L）作用于体外培养的人肝癌细胞株BEL-7402，应用流式细胞仪检测培养24、48和72h时的细胞凋亡情况；Hoechst 33258荧光染色法观察细胞凋亡时的形态学变化；Western Blot法观察BEL-7402细胞中Caspase-3、Bcl-2、Bcl-xl和Bax基因的表达变化；采用Caspase-3试剂盒检测Caspase-3酶的活性；JCl染色法检测细胞线粒体膜电位（△ψbm）。结果SRL可诱导BEL-7402细胞凋亡，并与药物浓度和作用时间呈正相关。SRL作用BEL-7402细胞后48h，在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征，凋亡过程中线粒体膜电位下降及Caspase-3酶原蛋白激活，伴有Bcl-2、Bcl-xl蛋白表达的降低和Bax蛋白上调。结论SRL能使抗凋亡蛋白Bcl-2、gcl-xl表达降低，促凋亡蛋白Bax表达上调，线粒体膜电位下降，激活Caspase-3酶原蛋白，从而诱导细胞凋亡。     

SATB2 participates in regulation of menadione‐induced apoptotic insults to osteoblasts

Special AT‐rich sequence binding protein 2 (SATB2), a nuclear matrix attachment region‐binding protein, can regulate embryonic development, cell differentiation, and cell survival. Previous studies showed that SATB2 is involved in osteoblast differentiation and skeletal development. In this study, we evaluated the role of SATB2 in oxidative stress‐induced apoptotic insults to human osteoblast‐like MG63 cells and mouse MC3T3‐E1 cells. Exposure of MG63 cells to menadione increased intracellular reactive oxygen species levels in a concentration‐ and time‐dependent manner. Simultaneously, menadione‐induced oxidative stress triggered cell shrinkage and decreased cell viability. In addition, treatment of MG63 cells with menadione time‐dependently decreased the mitochondrial membrane potential but enhanced caspase‐3 activity. As a result, menadione‐induced DNA fragmentation and cell apoptosis. As to the mechanism, exposure of MG63 cells to menadione amplified SATB2 messenger (m)RNA and protein expression in a time‐dependent manner. Knockdown of translation of SATB2 mRNA using RNA interference led to chromatin disruption and nuclear damage. When MG63 cells and MC3T3‐E1 cells were treated with SATB2 small interfering RNA, menadione‐induced cell apoptosis was increased. We conclude that menadione causes oxidative stress in human osteoblasts and induces cellular apoptosis via a mitochondrion‐caspase protease pathway. In addition, SATB2 may play a crucial role in protecting against oxidative stress‐induced osteoblast apoptosis. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1058–1066, 2012     

Caspase-dependent cleavage of cadherins and catenins during osteoblast apoptosis.

As transmembrane, Ca2+-dependent cell-cell adhesion molecules, cadherins play a central role in tissue morphogenesis and homeostasis. Stable adhesion is dependent on interactions of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins. In the present study, we have detected the expression of alpha-, beta-, and gamma-catenins in human osteoblasts, which assemble with cadherins to form two distinct complexes containing cadherin and alpha-catenin, with either beta- or gamma-catenin. In osteoblasts undergoing apoptosis, proteolytic cleavage of N-cadherin and beta- and gamma- catenins but not alpha-catenin was associated with the activation of caspase-3 and prevented by the caspase inhibitor Z-VAD-fmk. The pattern of cadherin/catenin cleavage detected in apoptotic osteoblasts was reproduced in vitro by recombinant caspase-3. The presence of a 90-kDa extracellular domain fragment of N-cadherin in conditioned medium from apoptotic cells indicates that additional extracellular or membrane-associated proteases also are activated. Disruption of N-cadherin-mediated cell-cell adhesion with function-blocking antibodies induced osteoblast apoptosis, activation of caspases, and cleavage of beta-catenin. These findings provide compelling evidence that N-cadherin-mediated cell-cell adhesion promotes osteoblast survival and suggest that the underlying mechanism may involve activation of beta-catenin signaling.     

Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis

In periradicular lesions and periodontal disease, bacterial invasion leads to chronic inflammation resulting in disruption of the structural integrity of the periodontal ligament and progressive alveolar bone destruction. The pathogenesis of these conditions has been attributed not only to bacterial-induced tissue destruction but also to a defect in periodontal tissue repair. Accumulated data have also shown that lipopolysaccharide (LPS) can directly induce cell death or apoptosis in many cell types, including macrophages, osteoblasts, vascular endothelial cells, hepatocytes and myocytes. The present study hypothesized that bacterial LPS-induced apoptosis in osteoblasts and periodontal ligament fibroblasts (PDL cells) is an important contributing factor to the defect in periodontal tissue repair in periodontal and periapical disease. Macrophages have been shown to respond to bacterial LPS by increasing the production of proinflammatory cytokines. In addition, large numbers of macrophages are present in inflamed periodontal tissue. We speculated that macrophages were a potential candidate cell for mediating apoptosis in osteoblasts and PDL cells in response to bacteria-derived LPS. The macrophage-like cell line, RAW 264.7, was stimulated with LPS, and the conditioned medium was used to treat osteoblasts and PDL cells. Bacterial LPS had no direct apoptotic effect on mouse osteoblasts or PDL cells, whereas the conditioned medium from LPS-activated macrophages was able to induce apoptosis in these cells. To evaluate the contribution of tumor necrosis factor-alpha (TNF-) released from macrophages on osteoblast and PDL cell apoptosis, cells were incubated with conditioned medium from LPS-treated macrophages in the presence and absence of anti-TNF- neutralizing antibodies. TNF- neutralizing antibody pretreatment inhibited the effect of conditioned medium from LPS-treated macrophages on osteoblast and PDL cell apoptosis in a dose-dependent manner. These results suggest that LPS could indirectly induce apoptosis in osteoblasts and PDL cells through the induction of TNF- release from macrophages. These studies provide insight into a potential mechanism by which bacterial-derived LPS could contribute to defective periodontal and bone tissue repair in periodontal and periapical disease.     

Molecular mechanism of nitric oxide-induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. Our previous study showed that NO induced osteoblast apoptosis [Chen RM, Liu HC, Lin YL, Jean WC, Chen JS, Wang JH. Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein. J Orthop Res 2002;20:295-302]. This study was further aimed to evaluate the mechanism of NO-induced osteoblast apoptosis from the viewpoints of mitochondrial functions, intracellular oxidative stress, and the anti-apoptotic Bcl-2 protein using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP), an NO donor, significantly increased amounts of lactate dehydrogenase in the culture medium, and decreased cell viability in concentration- and time-dependent manners. Administration of SNP in osteoblasts time-dependently led to DNA fragmentation. The mitochondrial membrane potential was significantly reduced following SNP administration. SNP decreased complex I NADH dehydrogenase activity in a time-dependent manner. Levels of cellular adenosine triphosphate (ATP) were suppressed by SNP. In parallel with the mitochondrial dysfunction, SNP time-dependently increased levels of intracellular reactive oxygen species. Immunoblotting analysis revealed that SNP reduced Bcl-2 protein levels. Exposure to lipopolysaccharide (LPS) and IFN-γ significant increased endogenous nitrite production. In parallel with the increase in endogenous NO, administration of LPS and IFN-γ suppressed cell viability, mitochondrial membrane potential, and ATP synthesis. Results of this study show that NO released from SNP can induce osteoblast insults and apoptosis, and the mechanism may involve the modulation of mitochondrial functions, intracellular reactive oxygen species, and Bcl-2 protein.