Mechanism of adrenocortical toxicity induced by quinocetone and its bidesoxy-quinocetone metabolite in porcine adrenocortical cells in vitro

Quinocetone (QCT) is a new feeding antibacterial agent in the QdNOs family. The mechanism of its adrenal toxicity is far from clear. This study was conducted to estimate the adrenal cell damage induced by QCT and its bidesoxy-quinocetone (B-QCT) metabolite and to further investigate their mechanisms. Following doses of QCT increasing from 5 to 50 μM, cell apoptosis and necrosis, mitochondrial dysfunction and redox imbalance were observed in porcine adrenocortical cells. The mRNA levels of the six components of intermediary enzymes and the adrenal renin-angiotensin-aldosterone system (RAAS) displayed a dysregulation induced by QCT, indicating that QCT might influence aldosterone secretion not only through the upstream of the production but also through the downstream of the adrenal RAAS pathway. In contrast, B-QCT had few toxic effects on the cell apoptosis, mitochondrial dysfunction and redox imbalance. Moreover, LCMS-IT-TOF analysis showed that no desoxy metabolites of QCT were found in either cell lysate or supernatant samples. In conclusion, we reported on the cytotoxicity in porcine adrenocortical cells exposed to QCT via oxidative stress, which raised awareness that its toxic effects resulted from N→O groups, and its toxic mechanism might involve the interference of the steroid hormone biosynthesis pathway.     

The effect of Fe2O3 and ZnO nanoparticles on cytotoxicity and glucose metabolism in lung epithelial cells

Metallic nanoparticles (NPs) have potential applications in industry and medicine, but they also have the potential to cause many chronic pulmonary diseases. Mechanisms for their cytotoxicity, glucose and energy metabolism responses need to be fully explained in lung epithelial cells after treatment with metallic nanoparticles. In our study, two different metallic nanoparticles (Fe₂O₃ and ZnO) and two cell‐based assays (BEAS‐2B and A549 cell lines) were used. Our findings demonstrate that ZnO nanoparticles, but not Fe₂O₃ nanoparticles, induce cell cycle arrest, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial dysfunction and glucose metabolism perturbation, which are responsible for cytotoxicity. These results also suggest that the glucose metabolism and bioenergetics had a great potential in evaluating the cytotoxicity and thus were very helpful in understanding their underlying molecular mechanisms. Copyright © 2015 John Wiley & Sons, Ltd.     

Silibinin prevents dopaminergic neuronal loss in a mouse model of Parkinson's disease via mitochondrial stabilization

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the nigrostriatal pathway. The lipophile 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) can cross the blood–brain barrier and is subsequently metabolized into toxic1‐methyl‐4‐phenylpyridine (MPP⁺), which causes mitochondrial dysfunction and the selective cell death of dopaminergic neurons. The present article reports the neuroprotective effects of silibinin in a murine MPTP model of PD. The flavonoid silibinin is the major active constituent of silymarin, an extract of milk thistle seeds, and is known to have hepatoprotective, anticancer, antioxidative, and neuroprotective effects. In the present study, silibinin effectively attenuated motor deficit and dopaminergic neuronal loss caused by MPTP. Furthermore, in vitro study confirmed that silibinin protects primary cultured neurons against MPP⁺‐induced cell death and mitochondrial membrane disruption. The findings of the present study indicate that silibinin has neuroprotective effects in MPTP‐induced models of PD rather than antioxidative or anti‐inflammatory effects and that the neuroprotection afforded might be mediated by the stabilization of mitochondrial membrane potential. Furthermore, these findings suggest that silibinin protects mitochondria in MPTP‐induced PD models and that it offers a starting point for the development of treatments that ameliorate the symptoms of PD. © 2015 Wiley Periodicals, Inc.     

慢性阻塞性肺疾病合并外周骨骼肌功能障碍患者炎性因子和代谢水平及线粒体功能的变化

目的观察慢性阻塞性肺疾病(COPD)合并外周骨骼肌功能障碍(PSMD)患者炎性因子白细胞介素(IL)-6、IL-8和IL-10水平、代谢指标及线粒体功能的变化。方法选择2012年7月至2013年7月在泸州市人民医院接受治疗的COPD患者86例作为研究对象,根据是否合并PSMD分为COPD合并PSMD组43例和COPD组43例,另选择健康人43例作为对照组,检测并对比各组受试者炎症细胞因子水平、代谢指标水平及线粒体功能。结果COPD合并PSMD组和COPD组患者血清IL-6、IL-8水平显著高于对照组(P<0.05),但IL-10水平显著低于对照组(P<0.05);COPD合并PSMD组患者血清IL-6、IL-8水平显著高于COPD组(P<0.05),但IL-10水平显著低于COPD组(P<0.05)。COPD合并PSMD组和COPD组患者空腹胰岛素(FINS)、瘦素(LP)水平显著高于对照组(P<0.05),而胰岛素样生长因子-1(IGF-1)水平显著低于对照组(P<0.05);COPD合并PSMD组患者FINS、LP水平显著高于COPD组(P<0.05),而IGF-1水平低于COPD组(P<0.05)。COPD合并PSMD组和COPD组患者线粒体Ⅲ态呼吸速率(ST3)、Ⅳ态呼吸速率(ST4)、呼吸控制率(RCR)、线粒体DNA(mt DNA)表达水平均显著低于对照组(P<0.05),COPD合并PSMD组患者ST3、ST4、RCR及mt DNA表达水平均显著低于COPD组(P<0.05)。线粒体功能与mt DNA表达水平呈正相关(r=0.825,P<0.05)。结论 COPD患者合并PSMD与机体的炎症作用及代谢异常有紧密联系,PSMD的发生机制与线粒体的功能有一定关系。     

胆囊癌组织中线粒体DNA含量变化的研究

目的探讨线粒体DNA(mt DNA)含量的改变与胆囊癌的关系。方法通过实时荧光定量PCR检测30例胆囊癌组织、30例胆囊癌旁组织和30例胆囊良性病变组织标本中mt DNA的含量。结果胆囊癌组织中mt DNA的平均含量为(766±143)×10~6拷贝/L,胆囊癌旁组织中mt DNA的平均含量为(343±94)×10~6拷贝/L,胆囊良性病变组织中mt DNA的平均含量为(386±104)×10~6拷贝/L,胆囊癌组织中mt DNA平均含量分别高于胆囊癌旁组织(t=11.583,P<0.001)和胆囊良性病变组织(t=13.320,P<0.001)。胆囊癌组织中mt DNA含量的改变与患者性别、年龄及肿瘤分化程度无关(P>0.05)。胆囊良性病变组织与胆囊癌旁组织中mt DNA的平均含量比较,差异无统计学意义(t=1.673,P=0.107)。结论胆囊癌发生与mtDNA含量增加可能相关,mtDNA含量的检测是胆囊癌诊断的一种方法。     

3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-one (CHP) inhibits human ovarian cell proliferation by inducing apoptosis

Coumarins induce apoptosis by activating mitochondrial pathway and caspase-3-dependent apoptotic pathway. In the present study, we first time investigated the effect of 3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-one (CHP) on induction of apoptosis in human ovarian carcinoma cells. The data from MTT assay revealed a significant inhibitory effect on cell viability at 30 (87%) and 50 μM (74%) concentration of CHP in OVCAR-3 and OVCAR-420 cells, respectively after 72 h. Apoptosis analysis using annexin V/PI double staining followed by flow cytometry showed 59 and 52% binding to annexin V-FITC in OVCAR-3 and OVCAR-420 cells respectively. propidium iodide (PI) staining and flow cytometry examination indicated a significant increase in percentage of cells in G2/M phase after treatment with CHP compared to DMSO control group. Reactive oxygen species (ROS) assay kit showed increase in levels of ROS. We used rhodamine-123 (Rh-123) staining and flow cytometry assay to determine changes in mitochondrial membrane potential (ΛΨm). The results revealed that CHP significantly decreased MMP to 85.65 ± 1.2443% & 49.78 ± 1.6554% at 10 and 30 μM respectively in OVCAR-3 compared to 95.97 ± 2.1243% in control group. Western blot analysis clearly indicated a significant increase in the expression of Caspase-3, Bax, and release of Cytochrome c and decrease in Bcl-2, CDK1 and Cyclin B1 expression on treatment with CHP. Therefore, CHP may become a potential candidate for the treatment of human ovarian cancers.     

Mitochondrial Potassium Channels as Pharmacological Target for Cardioprotective Drugs

Brief periods of ischemia are known to confer to the myocardium an increased resistance to the injury due to a later and more prolonged ischemic episode. This phenomenon, known as ischemic preconditioning (IPreC), is ensured by different biological mechanisms. Although an exhaustive comprehension of them has not been reached yet, it is widely accepted that mitochondria are pivotally involved in controlling cell life and death, and thus in IPreC. Among the several signaling pathways involved, as triggers and/or end effectors, in the mitochondrial mechanisms of cardioprotection, an important role is played by the activation of potassium channels located in the mitochondrial inner membrane (mitoK) of cardiomyocytes. Presently, different types of mitoK channels have been recognized in the heart, such as ATP‐sensitive (mitoK_ATP) and calcium‐activated (mitoBK_Ca and mitoSK_Ca) potassium channels. Consistently, drugs modulating mitoK, on one hand, have been employed as useful experimental tools for early basic studies on IPreC. On the other hand, activators of mitoK are promising and innovative therapeutic agents for limiting the myocardial injury due to ischemic episodes. In this review, we report the experimental evidence supporting the role of mitoK in signaling pathways in the mechanisms of cardioprotection and an overview on the most important molecules acting as modulators of these channels, with their profiles of selectivity. Some innovative pharmaceutical strategies for mitochondriotropic drugs have been also reported. Finally, an appendix describing the main experimental approaches usually employed to study mitoK in isolated mitochondria or in intact cells has been added.     

Mppa介导的光动力疗法诱导人卵巢癌细胞凋亡

目的：探讨Mppa（methyl pyropheophorbide-a）介导的光动力疗法(Mppa-PDT)抑制人卵巢癌SKOV3细胞活性、触发其凋亡的机制。方法：Mppa-PDT作用于人卵巢癌SKOV3细胞后,CCK-8法检测细胞活性;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率;DAPI染色观察细胞凋亡的核的形态学改变;DCFH-DA染色观察细胞内活性氧（ROS）的产生;单细胞凝胶电泳观察DNA损伤情况;Western blot检测P53、Caspase-3、Bax、Bcl-2蛋白的表达变化。结果：1.Mppa-PDT能显著抑制人卵巢癌SKOV3细胞的活性,其抑制作用具有一定的剂量依赖性（P<0.05）;2. Mppa-PDT诱导的人卵巢癌SKOV3细胞凋亡率显著高于对照组（空白对照、单药对照、单光对照）（P<0.05）,且空白、单药、单光三组对照组间凋亡率无明显差异（P>0.05）;3.LD50剂量的Mppa-PDT作用于人卵巢癌SKOV3细胞后,DAPI细胞核荧光染色可见到细胞核深染的凋亡细胞;DCFH-DA荧光染色发现Mppa-PDT组细胞内ROS水平高于三组对照组;单细胞凝胶电泳观察到Mppa-PDT组的DNA损伤情况高于三组对照组;Western blot检测发现P53、caspase-3、Bax蛋白升高,Bcl蛋白降低。结论：Mppa介导的光动力疗法能够显著抑制人卵巢癌SKOV3细胞活性并诱发其凋亡,且伴随有DNA损伤及线粒体凋亡途径的激活。     

Role of Plasma Amino Acids and Urinary Organic Acids in Diagnosis of Mitochondrial Diseases in Children

BackgroundDiagnostic difficulty in mitochondrial diseases (MD) results not only from the wide spectrum of symptoms and signs but also from the absence of a reliable screening or diagnostic biomarker.AimTo investigate the likelihood of MD in patients with symptoms and signs impressive of MD through quantitative measurement of plasma amino acids, and urinary organic acids.MethodsTwenty patients with symptoms and signs suggestive of MD were further evaluated by quantitative plasma amino acids and urinary organic acids assay and neuroimaging.ResultsPlasma amino acid results revealed elevation of alanine in 11, glycine in five, and proline in two patients. Abnormal urinary organic acid analysis was present in six patients; increased urinary lactate (20%), dicarboxylicaciduria (15%), and urinary ketone bodies (10%). Upon enrollment our patients scored as possible MD according to the MD scoring system. At the end of the study, five patients still scored as possible MD, eight patients as probable MD, and seven patients as definite MD. All patients with definite MD had elevated serum lactate. In three patients, elevated urinary lactate was the only abnormality. Alanine was elevated in all patients with definite MD, whereas proline was elevated in only one. Magnetic resonance imaging of the brain showed atrophic changes in one patient and bilateral basal ganglia hyperintensity in another.ConclusionUrinary organic acids and quantitative plasma amino acids can help in the diagnosis of MD, especially when the economic burden and absence of specialized centers limits the diagnosis.     

Increased mitochondrial fission and volume density by blocking glutamate excitotoxicity protect glaucomatous optic nerve head astrocytes

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity‐mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N‐methyl‐d ‐aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin‐related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1‐CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation. GLIA 2015;63:736–753