阿托伐他汀的肝毒性机制研究

目的 研究阿托伐他汀肝毒性损伤作用及机制。方法 将24只Wistar han雄鼠分为对照组和阿托伐他汀低（68.5mg/kg）、高剂量组（205.5 mg/kg）,按照10 mL/kg的药液体积给药,溶媒对照组ig等体积5% CMC-Na,连续ig 28 d。检测血清中天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）、尿素氮（BUN）和血肌酐（CRE）的含量,HE染色观察肝组织病理。在体外,HepG2细胞经传代培养后,给予阿托伐他汀干预24 h,检测细胞存活率,丙二醛（MDA）水平、Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性及线粒体膜电位。结果 与对照组比较,阿托伐他汀高剂量组大鼠肝细胞弥漫性肿胀,核分裂多见,部分肝细胞极性消失,排列紊乱（P<0.05）。与对照组比较,阿托伐他汀高剂量组给药后血清中ALT和AST显著升高（P<0.05、0.01）。在体外,与对照组比较,阿托伐他汀125、250、500 μmol/L能明显抑制细胞存活率（P<0.05、0.001）。与对照组比较,阿托伐他汀500 μmol/L HepG2细胞MDA含量明显升高（P<0.01）。与对照组比较,阿托伐他汀125 μmol/L能使Na⁺-K⁺-ATP酶活性增强,500 μmol/L使Na⁺-K⁺-ATP酶活性降低（P<0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能使能使Ca²⁺-Mg²⁺-ATP酶活性降低（P<0.01,0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能降低线粒体膜电位（P<0.001）。结论 阿托伐他汀高剂量可导致肝组织损伤,其毒性作用通过破坏细胞的线粒体膜电位,抑制Na⁺-K⁺-ATP酶、Ca²⁺-Mg²⁺-ATP酶活性,细胞膜脂质过氧化,从而破坏细胞内微环境的平衡,导致细胞凋亡和坏死。     

注射用益气复脉(冻干)对阿霉素诱导H9c2(2-1)心肌细胞毒性的保护作用

目的 探讨注射用益气复脉（冻干,YQFM）对阿霉素（doxorubicin,DOX）诱导H9c2（2-1）心肌细胞毒性的保护作用。方法 H9c2（2-1）心肌细胞随机分为对照组,模型组（终浓度为0.3 μmol/L的DOX处理细胞48 h）,YQFM低、中、高剂量（125、625、3 125 μg/mL）组（提前2 h加入药物预处理,然后加入终浓度为0.3 μmol/L的DOX处理48 h）,采用CCK-8法检测细胞活力;使用乳酸脱氢酶（LDH）和ATP试剂盒检测细胞LDH和ATP水平;应用Hoechst 33258染色法检测细胞凋亡;JC-1法检测细胞线粒体膜电位;Western blotting法检测caspase-3蛋白的表达水平。结果 与模型组比较,YQFM中、高剂量组显著增加细胞存活率（P<0.05、0.01）,低、高剂量组明显改善细胞凋亡;低、中、高剂量组LDH活性显著降低（P<0.05、0.01）,ATP含量显著增加（P<0.05、0.01）,线粒体膜电位明显恢复。结论 YQFM抑制DOX诱导H9c2（2-1）的细胞毒性,其作用机制可能与抑制线粒体凋亡信号通路的激活有关。     

芍药苷对醋酸铅诱导海马神经元凋亡及Bcl-2/Bax蛋白表达的影响

目的 探讨芍药苷(paeoniflorin,PF)对醋酸铅诱导海马神经元凋亡及Bcl-2/Bax蛋白表达的影响。方法 分离培养胎鼠海马神经元细胞,细胞免疫荧光染色鉴定纯度。MTT测定海马神经元细胞活力以确定醋酸铅最适造模浓度及时间,同时筛选合适剂量PF干预海马神经元凋亡。依据MTT测定结果,分为空白组、模型组和20,40,80 μmol·L^-1 PF组干预海马神经元细胞,作用24 h后,加醋酸铅染毒,检测细胞色素C (cytochrome C,Cyt-C)含量、线粒体膜电位及细胞内Ca²⁺浓度。流式细胞仪检测细胞凋亡情况,Western blotting测定海马神经元细胞中caspase-3、cleaved-caspase-3、caspase-8、cleaved-caspase-8、caspase-9、cleaved-caspase-9、Bax、Bcl-2蛋白表达水平。结果 细胞免疫荧光染色鉴定结果显示分离培养的细胞为海马神经元细胞,且纯度较高。MTT测定结果显示醋酸铅最适造模浓度及时间为25 μmol·L^-1染毒24 h;PF剂量为20,40,80 μmol·L-1可显著改善海马神经元细胞活性,呈剂量依赖性。与空白组相比,模型组Cyt-C含量、凋亡率、细胞内Ca²⁺浓度显著升高(P<0.01),线粒体膜电位显著降低(P<0.01)。与模型组相比,40 μmol·L^-1 PF组和80 μmol·L^-1 PF组可降低Cyt-C含量、凋亡率、细胞内Ca²⁺浓度(P<0.05或P<0.01),升高线粒体膜电位(P<0.01),20 μmol·L^-1PF组可显著升高线粒体膜电位(P<0.05)。此外,一定剂量的PF可下调cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9、Bax蛋白表达,上调Bcl-2蛋白表达。结论 PF可抑制醋酸铅诱导的海马神经元凋亡,可通过调控Bcl-2/Bax蛋白表达发挥神经保护作用。     

青天葵甲醇提取物通过ERK通路抑制人肝癌细胞增殖并诱导其凋亡

目的 研究青天葵甲醇提取物（Nervilia fordii methanol extracts，NFME）体外对人肝癌SMMC7721和HepG2细胞的凋亡作用及其作用机制。方法 紫外-可见分光光度法测定青天葵中总黄酮和总多酚的含量；MTT法检测不同浓度（0，0.25，0.5，1，1.5，2 mg·mL^-1）NFME处理24 h对SMMC7721、HepG2和LO2细胞生长抑制率的影响；克隆形成试验观察NFME对SMMC7721和HepG2细胞克隆形成率的影响；Hoechst凋亡染色观察NFME对SMMC7721细胞凋亡的影响；流式细胞术检测NFME对SMMC7721细胞凋亡和细胞周期的影响；Western blotting测试NFME作用下caspase3、PARP、ERK1/2和c-Raf蛋白磷酸化水平的变化。结果 NFME中总多酚含量为（4.25±0.46）mg·g^-1，总黄酮含量为（4.72±0.13）mg·g^-1；MTT试验结果显示，与对照组相比，在给药24 h时0.5 mg·mL^-1的NFME就能抑制SMMC7721和HepG2细胞的增殖（P<0.001）；克隆形成试验表明，0.125 mg·mL^-1的NFME能够抑制SMMC7721和HepG2细胞集落的形成（P<0.001）；Hoechst凋亡染色观察到0.25 mg·mL^-1的NFME作用24 h能够观察到SMMC7721细胞发生凋亡（P<0.05）；流式细胞试验结果证实0.5 mg·mL^-1的NFME能够诱导SMMC7721细胞的凋亡（P<0.01），凋亡率14.43%，阻滞细胞周期于S期；Western blotting结果表明NFME能使SMMC7721细胞中caspase3发生剪切，随着给药浓度增加其剪切作用越明显（P<0.05），活化的caspase3剪切下游PARP的表达；同时NFME能够降低SMMC7721细胞中ERK1/2和c-Raf蛋白的磷酸化水平（P<0.05）。结论 NFME能抑制人肝癌SMMC7721和HepG2细胞的活性并诱导其发生凋亡，其作用机制可能与抑制ERK信号通路从而诱导细胞凋亡有关。     

nNOS抑制剂亚胺基烯丁基-L-鸟氨酸对心肌缺血再灌注损伤的影响及机制

目的 探讨nNOS选择性抑制剂亚胺基烯丁基-L-鸟氨酸（L-VNIO）对心肌缺血再灌注（I/R）损伤的影响及机制。方法 构建SD大鼠离体心脏I/R模型和H9c2细胞缺氧/复氧（H/R）模型；nNOS抑制剂L-VNIO（10 μmol·L^-1）持续给药整个再灌注或复氧过程。TTC染色测定心肌梗死面积；流式细胞术检测H9c2细胞凋亡率；Fluo-3/AM Ca²⁺荧光探针通过流式细胞仪检测H9c2细胞内Ca²⁺浓度；试剂盒法测定离体心脏灌流液乳酸脱氢酶（LDH）、丙二醛（MDA）水平以及H9c2细胞MDA水平和超氧化物歧化酶（SOD）活性；离体心脏提取肌浆网，试剂盒法检测肌浆网Ca²⁺-ATP酶（SERCA）活性，Western blotting检测肌浆网SERCA蛋白表达；Western blotting检测离体心脏中受磷蛋白（PLB）和兰尼碱受体2（RyR2）蛋白表达水平和磷酸化水平。结果 与I/R或H/R模型组相比，L-VNIO显著降低细胞凋亡率，减少心肌梗死面积，降低LDH、MDA水平，提高SOD活性，差异均有统计学意义（P<0.05）；此外，与I/R或H/R模型组相比，L-VNIO组明显降低细胞内Ca²⁺超载，增高PLB磷酸化水平，降低RyR2磷酸化水平，增强SERCA活性（P<0.05）。结论 nNOS抑制剂L-VNIO可以减轻I/R损伤，机制与调节Ca²⁺转运相关蛋白而降低I/R引起的Ca²⁺超载相关。     

人源HepaRG肝细胞毒性与遗传毒性高通量筛选方法的初步建立

目的 利用正常人源肝细胞（HepaRG）和高内涵技术检测肝毒性标志物,并结合微核试验和单细胞凝胶电泳试验建立体外细胞毒性和遗传毒性的快速筛选平台。方法 选取适当的荧光探针Hoechst33342、DCFH-DA、Fluo4-AM、MitoTracker^® Red CMX Ros联合高内涵技术研究不同大黄蒽醌类单体（AQs）对HepaRG细胞活性氧簇（ROS）、胞内Ca²⁺含量及线粒体膜完整性等肝毒性标志物的影响,并开展高内涵法胞质分裂阻断法微核试验和高通量彗星电泳试验,综合评价AQs致肝细胞毒性及染色体、DNA损伤情况。结果 与对照组比较,HepaRG细胞经25.0 μg/mL大黄素、12.5和25.0 μg/mL芦荟大黄素、50和25.0 μg/mL大黄酚处理24 h后,胞内ROS含量显著增多;12.5和25.0 μg/mL芦荟大黄素和50.0 μg/mL大黄酸可引起胞内Ca²⁺含量显著增多;大黄素25.0 μg/mL、芦荟大黄素25.0 μg/mL、大黄酚50.0和25.0 μg/mL、大黄酸50.0和25.0 μg/mL组导致线粒体明显损伤（P<0.05、0.01）。与对照组比较,25.0 μg/mL大黄素诱导微核率、尾DNA含量和彗星尾距（OTM）数值均显著升高（P<0.05、0.01）;50.0 μg/mL大黄酚给药72 h后微核率显著升高（P<0.01）。结论 AQs的研究结果与现有文献报道基本相符。本研究成功建立肝细胞毒性和遗传毒性的联合快速筛选模型,有助于药物研发早期的毒性筛选。     

五味子乙素通过ROS介导内质网应激诱导人乳腺癌MDA-MB-231细胞凋亡的研究

目的 研究五味子乙素对人乳腺癌MDA-MB-231细胞凋亡的影响及其作用机制。方法 用细胞计数试剂(CCK-8)检测不同浓度五味子乙素对MDA-MB-231细胞存活率的影响;五味子乙素（10、20、40 μmol/L）作用 MDA-MB-231 细胞 24 h,分别用Annexin V-FITC/PI检测细胞凋亡情况;用DCFA-DA荧光探针检测细胞内活性氧（ROS）水平;用Western blot法检测细胞凋亡及内质网应激相关蛋白（Bcl-2、Bax、CHOP、GPR78、PERK、p-PERK、p-eIF2α、eIF2）的表达。结果 与空白组比较,随着五味子乙素浓度增大,细胞存活率明显降低,其IC₅₀为19.16 μmol/L;与对照组比较,五味子乙素（10、20、40 μmol/L）均能抑制细胞克隆形成(P<0.05),且呈剂量依赖;五味子乙素（10、20、40 μmol/L）均可诱导细胞凋亡（P<0.05）,使抗凋亡蛋白BCL-2的表达显著降低,促凋亡蛋白Bax的表达显著升高(P<0.05);五味子乙素（10、20、40 μmol/L）显著升高细胞内ROS水平(P<0.05),且呈剂量依赖;五味子乙素（10、20、40 μmol/L）能够激发内质网应激,使内质网应激相关蛋白CHOP、GPR78、p-eIF2α表达增多(P<0.05),且呈剂量依赖。结论 五味子乙素可能通过ROS介导内质网应激诱导MDA-MB-231细胞凋亡。     

基于Nrf2/HO-1信号通路研究橄榄苦苷的体外抗炎作用

目的 探讨橄榄苦苷对脂多糖（LPS）诱导的小鼠腹腔巨噬细胞（RAW264.7）炎症的保护作用及机制。方法 MTT法检测橄榄苦苷（0、10、20、40 μmol/L）对RAW264.7细胞活性的影响;用橄榄苦苷（10、20、40 μmol/L）预处理细胞1 h后,LPS诱导炎症模型,Griess试剂检测细胞内Nitrite释放;Western blotting方法检测细胞iNOS、COX-2、Nrf2、Keap-1、HO-1、NQO1蛋白表达水平;流式细胞仪检测细胞内NO、ROS、Ca²⁺的水平;荧光显微镜检测线粒体膜电位（MMP）水平;ELISA法检测细胞上清液中肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）的释放。结果 与模型组比较,橄榄苦苷组Nitrite释放水平显著降低（P<0.05、0.01、0.001）,iNOS、COX-2蛋白的表达显著降低（P<0.001）,NO的产生显著减少（P<0.05、0.01）;TNF-α、IL-6的释放受到显著抑制（P<0.001）;Ca²⁺释放受到显著抑制（P<0.01）;ROS生成受到显著抑制（P<0.01）;JC-1单体降低,恢复聚合物状态（红色荧光变多）,MMP稳定性增强;Keap1蛋白表达显著降低, Nrf2、HO-1、NQO1蛋白的表达均显著升高（P<0.05、0.01、0.001）。结论 橄榄苦苷对LPS诱导的RAW264.7细胞炎症具有抑制作用,其作用机制可能与激活Nrf2/HO-1信号通路相关。     

壳聚糖纳米颗粒对K562细胞增殖的影响

目的 评价壳聚糖纳米颗粒对K562细胞增殖的影响,并探讨相关机制。方法 采用MTT法对壳聚糖纳米颗粒的最佳作用时间和作用浓度进行筛选,免疫荧光技术和透射电镜技术考察壳聚糖纳米颗粒对K562细胞形态的影响,流式细胞仪检测壳聚糖纳米颗粒对线粒体膜电位、细胞内ROS含量和Ca²⁺浓度的影响。结果 壳聚糖纳米颗粒(15 μg·mL^-1)可以明显诱导K562细胞出现凋亡改变,表现为：细胞核变小,出现核聚缩等现象。同时线粒体膜电位崩解,ROS含量增加,Ca²⁺浓度增加。结论 壳聚糖纳米颗粒对K562细胞的增殖有抑制作用,其机制可能与诱导细胞凋亡有关。     

槐耳颗粒联合DC-CIK细胞对乳腺癌MDA-MB-231干细胞体内外杀伤作用研究

目的 探究白屈菜红碱（CHE）对腺样囊性癌细胞（ACC2）生长的抑制作用及机制。方法 利用CCK8法、EdU法、Hoechst33342/PI双染色法、试剂盒法检测CHE对ACC2细胞活力、细胞增殖、细胞凋亡和活性氧（ROS）水平的影响;通过Western blotting技术检测CHE对Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达的影响;利用斑马鱼移植瘤模型检测CHE对斑马鱼体内ACC2细胞生长的抑制作用。结果 CCK-8结果显示：与对照组比较,2、3、4、5、6、7、8、9、10 μmol/L的CHE显著降低ACC2细胞的存活率（P<0.05、0.01）,且呈浓度相关性; ROS检测结果显示：与对照组比较,5、8 μmol/L的CHE导致ACC2细胞内的ROS水平显著上升（P<0.05、0.01）; EdU增殖检测结果表明：与对照组比较,5、8 μmol/L的CHE致使ACC2细胞的增殖能力显著下降（P<0.01）;Hoechst/PI染色结果显示：与对照组比较,CHE 5、8 μmol/L组ACC2细胞凋亡率显著上升（P<0.01）。抗氧化剂N-乙酰半胱氨酸（NAC）显著抑制CHE诱导的ROS水平升高、细胞凋亡增加（P<0.01）;Western blotting结果显示：2、5、8 μmol/L的CHE能够显著上调Cleaved-Caspase 3、PARP、NF-κB蛋白的表达（P<0.01）,且呈现浓度相关性,5、8 μmol/L的CHE能够显著上调p-JNK的蛋白表达（P<0.01）,8 μmol/L的CHE能够显著上调p-p38的蛋白表达（P<0.01）;NAC显著降低由CHE导致的Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达增加（P<0.01）,5、8 μmol/L CHE能够有效抑制斑马鱼体内肿瘤的生长（P<0.01）。结论 体外及斑马鱼移植瘤模型证明,CHE可以有效抑制ACC2细胞生长,其机制与提高细胞ROS水平,上调NF-κB、p-JNK、p-p38表达,从而抑制细胞增殖、诱导细胞凋亡相关。     

Caffeine-inducible ATP release is mediated by Ca2+-signal transducing system from the endoplasmic reticulum to mitochondria

Adenosine triphosphate (ATP) is released as an autocrine/paracrine signal from a variety of cells. The present study was undertaken to clarify the Ca(2+)-signal pathway involved in the caffeine-inducible release of ATP from cultured smooth muscle cells (SMC). The release of ATP induced by caffeine (3 mM) was almost completely inhibited by ryanodine and tetracaine, but not by 2-APB, thus being mediated by ryanodine receptors (RyR). The expression of messenger RNA from only RyR-2 was detected in the cells. Furthermore, the induced release was attenuated by mitochondrial inhibitors, rotenone and oligomycin and by Cl(-) channel blockers, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Increase in Ca(2+)-signals with fluo 4 and rhod-2 caused by caffeine were reduced by tetracaine and oligomycin plus carbonyl cyanide m-chlorophenylhydrazone, respectively. A close spatial relation between the endoplasmic reticulum (ER) and mitochondria was electromicroscopically observed in the SMC, supporting the existence of a Ca(2+)-signaling bridge on both the organelli. These results suggest that caffeine stimulates ryanodine receptor (RyR-2) and facilitates a Ca(2+)-signal transducing system from ER to mitochondria, and then, the signal appears to accelerate the ATP synthesis in mitochondria. In addition, the mitochondrial event may lead further cell signaling to the cell membrane and activates Cl(-) channels, resulting in the extracellular release of cytosolic ATP.     

WJ9708012 exerts anticancer activity through PKC-α related crosstalk of mitochondrial and endoplasmic reticulum stresses in human hormone-refractory prostate cancer cells

Aim:

To investigate the anticancer mechanism of a methoxyflavanone derivative, WJ9708012, highlighting its role on a crosstalk between endoplasmic reticulum (ER) and mitochondrial stress.

Methods:

Cell proliferation was examined using sulforhodamine B assay. Cell-cycle progression, Ca²⁺ mobilization and mitochondrial membrane potential (ΔΨ_m) were detected using flow cytometric analysis. Protein expression was detected using Western blot.

Results:

WJ9708012 displayed an antiproliferative and apoptotic activity in human hormone-refractory prostate cancer cells with IC₅₀ values of 6.4 and 5.3 μmol/L in PC-3 and DU-145 cells. WJ9708012 induced a prompt increase of cytosolic Ca²⁺ level and activation of protein kinase C (PKC)-α. The cleavage of μ-calpain was also induced by WJ9708012. Furthermore, WJ9708012 induced cell-cycle arrest at G₁-phase associated with down-regulation of cyclin D1, cyclin E and cyclin-dependent kinase-4 expressions. It also caused a rapid and time-dependent decrease of phosphorylation level of mTOR (Ser²⁴⁴⁸), 4E-BP1 (Thr³⁷/Thr⁴⁶/Thr⁷⁰) and p70S6K (Thr³⁸⁹), indicating the inhibition of mTOR-mediated translational pathways. The ER stress was activated by the identification of up-regulated GADD153 and glucose-regulated protein-78 protein levels. The subsequent mitochondrial stress was also identified by the observation of a decreased Bcl-2 and Bcl-xL expressions, an increased truncated Bid and Bad and a loss of ΔΨ_m.

Conclusion:

WJ9708012 induces an increase of cytosolic Ca²⁺ concentration and activation of PKC-α. Subsequently, a crosstalk between ER stress and mitochondrial insult is induced, leading to the inhibition of mTOR pathways and arrest of the cell-cycle at G₁ phase. The apoptosis is ultimately induced by a severe damage of mitochondrial function.     

KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

BACKGROUND AND PURPOSE

An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na⁺/Ca²⁺ exchanger (NCX_rev), was instrumental in establishing the role of NCX_rev in glutamate-induced Ca²⁺ deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested.

EXPERIMENTAL APPROACH

Fluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca²⁺ flux measurements were made in isolated rat brain mitochondria.

KEY RESULTS

KB-R7943 inhibited NCX_rev with IC₅₀= 5.7 ± 2.1 µM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca²⁺ with IC₅₀= 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca²⁺-independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca²⁺ in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC₅₀= 11.4 ± 2.4 µM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca²⁺ uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.

CONCLUSIONS AND IMPLICATIONS

KB-R7943, in addition to NCX_rev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action.     

线粒体动力学与2型糖尿病和糖尿病并发症关系的研究进展

线粒体动力学即线粒体融合和分裂保持动态平衡的过程,线粒体的融合与分裂过程决定线粒体形态的可塑性。这种动态平衡的稳定是维持线粒体功能和机体功能的前提和基础,并且受多种化学酶及蛋白等因素调节。因线粒体为细胞能量代谢中心,故线粒体动力学与人类代谢性疾病的关联日益受到重视。近年来,大量研究表明,线粒体动力学失衡可以引起胰岛素抵抗和β细胞功能障碍,并且与糖尿病并发症发生密切相关。     

Mitochondrial fusion and fission proteins: novel therapeutic targets for combating cardiovascular disease

Mitochondria are no longer considered to be solely the static powerhouses of the cell. While they are undoubtedly essential to sustaining life and meeting the energy requirements of the cell through oxidative phosphorylation, they are now regarded as highly dynamic organelles with multiple funtions, playing key roles in cell survival and death. In this review, we discuss the emerging role of mitochondrial fusion and fission proteins, as novel therapeutic targets for treating a wide range of cardiovascular diseases.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Mechanism of primary Cd2+-induced rat liver mitochondria dysfunction: discrete modes of Cd2+ action on calcium and thiol-dependent domains

We attempted to discern discrete sites of Cd²⁺ deleterious action on rat liver mitochondrial function. In particular, EGTA, ADP, and cyclosporin A (potent mitochondrial permeability transition antagonists) affected mainly Cd²⁺ -induced changes in resting state respiration, eliminating its stimulation in KCl medium, while dithiothreitol (DTT, a dithiol reductant) produced its effect both on Cd²⁺ activation of the basal respiration and Cd²⁺ depression of uncoupler-stimulated respiration, evoking its restoration. Substantial differences in DTT influence on mitochondrial respiration at low and high [Cd²⁺] were revealed, namely, an enhanced mitochondrial permeabilization in the presence of saturated [DTT] at high [Cd²⁺] took place. Besides, DTT only partially reversed Cd²⁺ -induced swelling in NH₄NO₃ medium when glutamate plus malate or succinate without rotenone was used. Contrarily, DTT produced complete reversal of the swelling of succinate-energized mitochondria when rotenone was present in the medium. In addition, in the presence of rotenone both Cd²⁺ -produced activation of the resting state respiration in KCl medium and Cd²⁺-induced swelling in sucrose medium of succinate-energized mitochondria were more sensitive to cyclosporin A than the same Cd²⁺ effects obtained on mitochondria oxidizing succinate (without rotenone) or glutamate plus malate. We have concluded that Cd²⁺, producing primary mitochondrial dysfunction, acts both as a thiol and Me²⁺ binding site reagent. Suppositions about possible localization of separate sites of direct Cd²⁺ effects on mitochondrial function were made.     

The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells)

Mitochondrial DNA-deficient (rho(0)) cells were generated following a 26-day incubation of MOLT-4 lymphoblastoid T cells in ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide). The absence of mitochondrial DNA (mtDNA) in the resultant MOLT-4 rho(0) cells was confirmed by Southern analysis and quantitative polymerase chain reaction (PCR). MOLT-4 rho(0) cells proliferated more slowly than parental cells (wild type) and produced significantly more lactate (approximately fourfold increase; P < 0.001) with concomitantly reduced oxygen consumption (12.3% vs. 100%; P < 0.001) compared with the wild type. MOLT-4 rho(0) cells also showed reduced cytochrome c oxidase activity and a reduced cytochrome c oxidase/citrate synthase activity ratio compared to parental wild-type MOLT-4 cells (P < 10(-11)). Electron microscopy showed elongated mitochondria with parallel cristae in MOLT-4 cells although the mitochondria in MOLT-4 rho(0) cells appeared enlarged, some were vacuolated with either an absent or a grossly distorted cristae pattern. Vital staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was used to image mitochondria in intact cells and study mitochondrial transmembrane potential (Deltapsi(m)). Flow cytometry using JC-1 indicated that MOLT-4 rho(0) had a lower Deltapsi(m) than MOLT-4. Sodium fluoride (an inhibitor of the glycolytic pathway) at a concentration of 20 mM further reduced the Deltapsi(m) in MOLT-4-rho(0) cells. This data suggested that a glycolytic pathway product, possibly ATP, was required for the maintenance of Deltapsi(m) in MOLT-4 rho(0) cells.     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。     

Effects of interactions of EGCG and Cd(2+) on the growth of PC-3 cells and their mechanisms.

The preventive and therapeutic effects of a major component of catechins of green tea, epigallocatechin-3-gallate (EGCG), on prostate cancer have been demonstrated in many studies. It is well known that metal ions are necessary for human health, but an imbalance in metal ions metabolism can lead to many diseases including prostate cancer. Understanding the interactions of EGCG with metal ions might elucidate its mechanism in preventing and curing prostate cancer. The present study focused on the effects of Cd(2+) and EGCG on the growth of androgen-insensitive prostate cancer cell PC-3 investigated by MTT assay, the effects of EGCG and Cd(2+) on absorption of Cd(2+) and Zn(2+) by PC-3 cells were detected by atomic absorption spectroscopy (AAS), and the interactions of EGCG with Cd(2+) were determined by distribution coefficient and UV-Vis spectroscopy detection. The results showed that Cd(2+) suppressed viability of PC-3 cells in concentration- and time-dependent manner, and EGCG enhanced the effect of Cd(2+) on PC-3 cells. EGCG was shown to decrease the absorption Cd(2+) and increase the absorption of Zn(2+) by PC-3 cells, while the effects of Cd(2+) on the absorption of Cd(2+) and Zn(2+) were opposite to that of EGCG. In the presence of both EGCG and Cd(2+), absorption of Cd(2+) and Zn(2+) by PC-3 cells was dependent on concentrations of EGCG, Cd(2+) and its order of addition. Results from the distribution coefficient determination and UV-Vis spectroscopy analysis indicated that Cd(2+) might affect conformation of EGCG, while no complex of EGCG with Cd(2+) was observed in the system.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address