Recent studies have shown that microRNA (miRNAs) can play important roles in the regulation of endothelial cell (EC) function. However, the expression profile of miRNAs and their effects on the apoptosis of ECs under microgravity conditions remains unclear. In this study, the apoptosis of human pulmonary microvascular endothelial cells (HPMECs) under simulated microgravity was identified by Annexin V and propidium iodide double staining and transmission electron microscopy. miRNA microarray assay was used to screen the differentially expressed miRNAs in HPMECs under simulated microgravity, and eight differentially expressed miRNAs were identified. Specifically, miR-503-5p, which was found to be most significantly upregulated in both microarray and quantitative reverse-transcription polymerase chain reaction assays, was selected for further functional investigation. Overexpression of miR-503-5p induced apoptosis of HPMECs under normal gravity and aggravated the negative effects of simulated microgravity on HPMECs. Furthermore, silencing of miR-503-5p expression effectively attenuated the negative effects of simulated microgravity on HPMECs. Further experiments showed that the mRNA and protein expression of anti-apoptotic factor B-cell lymphoma-2 (Bcl-2), which has been confirmed as a direct target of miR-503-5p, was inhibited by the upregulation of miR-503-5p and increased by the downregulation of miR-503-5p. Taken together, our findings demonstrate, for the first time, that miR-503-5p can induce apoptosis of HPMECs under simulated microgravity through, at least in part, inhibiting the expression of Bcl-2.

目的 评价氯胺酮的线粒体毒性,从而探讨氯胺酮可能的毒性机制.方法 HepG2细胞培养基中分别加入齐多夫定10～2000 μmol·L-1培养7d,或加入氯胺酮50～3000 μmol·L-1培养24 h,CCK8细胞计数法测定细胞存活率.HepG2细胞培养基中分别加入齐多夫定100 μmol· L-1培养7d后,或加入氯胺酮100和1200 μmol·L-1培养24 h,化学发光法检测胞内ATP合成水平,荧光探针法检测胞内钙离子浓度、活性氧及线粒体膜电位(△Ψm)的变化,同时设齐多夫定1000 μmol· L-1用于实时荧光定量PCR检测线粒体DNA(mtDNA)表达水平的变化.结果 与正常对照组相比,齐多夫定10～ 2000 μmol· L-1可以抑制细胞存活,IC50为100 μmol·L-1;氯胺酮1000～ 3000 μmol· L-1抑制细胞存活,IC50为1200 μmol·L-1.与正常对照组相比,齐多夫定100 μmol·L-1组线粒体ATP合成水平显著下降(P＜0.05),胞内钙离子浓度明显升高(P＜0.05),活性氧水平显著升高(P＜0.05),△Ψm显著下降(P＜0.05),而1000 μmol· L-1可以明显干扰mtDNA表达水平(P＜0.05).氯胺酮100 μmol· L-1组ATP合成水平明显下降(P＜0.05),其他指标均无显著性差异;氯胺酮1200 μmol·L-1组ATP合成水平显著下降(P＜0.01),活性氧水平和胞内钙离子浓度显著升高(P＜0.01),△Ψm显著下降(P＜0.05),mtDNA水平无明显改变.结论 氯胺酮可以通过干扰线粒体代谢功能而诱导线粒体损伤.

目的 评价马兜铃酸的线粒体毒性,从而探讨马兜铃酸的可能毒性机制.方法 HepG2细胞培养基中分别加入齐多夫定8 000～20 000 μmol/L,或加入马兜铃酸25～500μmol/L,均培养24 h,以CCK8细胞计数法测定细胞存活率.同时比较不同浓度齐多夫定(8 000、16 000和20 000μmol/L)和马兜铃酸(25、200和500 μmol/L)培养24 h的胞内ATP合成水平和胞内钙离子浓度、活性氧及线粒体膜通透性转换孔 (MPTP)变化,透射电镜观察线粒体超微结构.结果 齐多夫定8 000～20 000 μmol/L可抑制细胞存活,IC50为12 713μmol/L;马兜铃酸25～500 μmol/L可抑制细胞存活,IG0为214.6 μmol/L.与溶媒对照组(DMEM)相比,齐多夫定≥8 000 μmol/L细胞内活性氧水平显著升高(P＜0.01);≥16 000 μmol/L线粒体ATP显著下降(P＜0.01)、钙离子浓度明显升高(P＜0.01),并可见线粒体结构发生病理改变;20 000 μmol/L组MPTP开放水平显著升高(P＜ 0.01).与溶媒对照组(DMSO)相比,马兜铃酸≥25 μmol/L,ATP合成水平明显下降(P＜0.01)、MPTP开放水平显著升高(P＜0.01);≥200 μmol/L细胞内钙离子浓度明显升高(P＜0.01);500 μmol/L细胞内活性氧水平显著升高(P＜0.01)、并可见线粒体结构发生病变.结论 马兜铃酸可以通过干扰破坏线粒体代谢功能和结构而诱导线粒体损伤.

目的:评价DPP-Ⅳ抑制剂类口服降糖药A化合物的线粒体毒性,从而探讨A化合物的可能毒性机制.方法:HepG2细胞培养基中分别加入曲格列酮50～300μmol/L,培养24h,或加入A化合物溶液100～300 μmol/L,培养24h,CCK8细胞计数法测定细胞存活率.HepG2细胞培养基中分别加入曲格列酮100、200和225 μmol/L培养24 h,或加入A化合物溶液100、150和200μmol/L培养24h,化学发光法检测胞内ATP合成水平,荧光探针法检测胞内钙离子浓度、活性氧、线粒体膜电位(△ψm)变化,透射电镜观察线粒体超微结构.结果:与溶媒对照组相比,曲格列酮50～300 μmol/L可以抑制细胞存活率,IC50为178μmol/;A化合物100～300 μmol/L对细胞存活率有抑制作用,IC50为159 μmol/L.与溶媒对照组相比,曲格列酮≥200 μmol/L时导致线粒体ATP显著下降(P＜0.01)、△ψm显著性下降(P＜0.01),≥100μmol/L时可致活性氧水平和钙离子浓度显著升高(P＜ 0.05或P＜ 0.01).A化合物≥100 μmol/L时ATP合成水平显著降低、钙离子浓度显著升高(P＜0.01),≥150μmol/L时活性氧水平明显升高(P＜0.01),≥200μ,mol/L时△ψm显著性下降(P＜0.05),并可导致线粒体结构发生病变.结论:DPP-Ⅳ抑制剂类口服降糖药A化合物可以通过干扰线粒体代谢功能和结构而诱导线粒体损伤.

OBJECTlVE To establish a real-time cell analysis system ( RTCA) for early drug car-diotoxicity evaluation. METHODS An in vitro drug cardiotoxicity evaluation method was established using RTCA Cardio system and primary cultured cardiomyocytes of neonatal rats. The beating rate, am-plitude and beating rhythm irregularity ( BRl ) of cardiomyocytes were observered after antiarrhythmic drugs, such as quinidine and lidocaine were added, to assess the effect of the above method on cardio-toxicity evaluation. RESULTS RTCA Cardo E-Plate 96 was inoculated with primary cultured cardiomyo-cytes that began to beat after 24 h and beat regularly after 48 h. The stable beating was maintained for a minimum of three days. The beating of cardiomyocytes decreased rapidly from 155±5 to 0 after incuba-tion with quinidine. The beating recovered gradually after 6 h. Quinidine at 3.1μmol·L-1 caused the beat-ing rate to return to 124±16. Quinidine allowed the beating rate to return to normal when the concentra-tion was less than 100.0μmol·L-1 . The beating rate, amplitude and BRl of cardiomyocytes changed in a concentration-dependent manner when incubating with lidocaine. The higher the concentration, the more significant the inhibition of lidocaine on cardiomyocytes. CONCLUSlON The cardiotoxicity of quinidine and lidocaine can be detected accurately using RTCA Cardio system, suggesting that this system can be used in early evaluation of drug cardiotoxicity.

OBJECTlVE To investigate the action mechanism of antidepressant fluoxetine on hERG ( ether-a-go-go-related gene ) potassium channel, and the effect of protein kinase C ( PKC ) agonist phorbol-12-myristate-13-acetate ( PMA) on fluoxetine inhibition. METHODS The whole cell patch clamp technique was used to record the change in hERG potassium current ( IKr ) on HEK293 cells that stably expressed hERG potassium channel ( hERG-HEK293 steady-state cells) , which was treated with fluoxe-tine 0.01, 0.1, 1 and 10μmol·L-1 , to study the concentration-and voltage-dependence of the effects on IKr, and to observe the changes in activation, inactivation and recovery dynamics of hERG potassium channel treated with fluoxetine 1μmol·L-1 . On this basis, the effect of PMA of 1μmol·L-1 on inhibition of fluoxetine 1 μmol·L-1 was explored. RESULTS Fluoxetine 0.01, 0.1, 1 and 10 μmol·L-1 inhibited IKr on hERG-HEK293 steady-state cells in a concentration- and voltage-dependent manner. The half maximal inhibitory concentration ( lC50 ) was about 0. 8 mmol·L-1 , and the Hill coefficient was about 1. 1. Fluoxetine 1 μmol·L-1 could reduce the activation, deactivation and recovery currents of IKr and affect the activation and recovery of hERG potassium channel. After fluoxetine inhibition of IKr became stable, PMA 1 μmol·L-1 could inhibit the blocking effect of fluoxetine on hERG potassium channels. CONCLUSlON Fluoxetine has obvious inhibitory effect on IKr of hERG-HEK293 steady-state cells, but the effect could be inhibited by PKC agonist PMA.

Circulating microRNA (miRNA) expression profiles have been reported to be promising biomarkers for drug-induced liver injury in preclinical and clinical practice. Proper normalization is critical for accurate miRNAs expression analysis. Herein, using SYBR green quantitative real-time PCR (RT-qPCR), we evaluated the expression stability of six candidate reference genes including two commonly used small RNAs (U6, 5S) and four miRNAs (let-7a, miR-92a, miR-103 and miR-16) in plasma of rats with acetaminophen-induced hepatotoxicity. Data were analysed using geNorm, Normfinder, BestKeeper and comparative delta-Ct statistical models, and the results consistently show that miR-103 is the most stably expressed reference gene. Whereas the commonly used housekeeping genes 5S or U6 are all not suitable normalizers, because 5S exhibits extensive variability in expression and U6 has a low expression level across the plasma samples. Then the effect of reference genes on normalization of plasma miR-122 was assessed; when normalized to the most stable reference gene there were significant differences between the acetaminophen-treated group and the vehicle group. However, when the data were normalized to a less stably expressed gene, miR-16, a biased result was obtained. Therefore, we recommend that miR-103 as suitable reference gene for plasma miRNAs analysis for acetaminophen-induced liver injury. Data presented in this paper are crucial to successful biomarker discovery and validation for the diagnosis of the early stage of acetaminophen hepatotoxicity.