Utilizing engineered extracellular vesicles as delivery vectors in the management of ischemic stroke:a special outlook on mitochondrial delivery

Ischemic stroke is a secondary cause of mortality worldwide,imposing considerable medical and economic burdens on society.Extracellular vesicles,serving as natural nanocarriers for drug delivery,exhibit excellent biocompatibility in vivo and have significant advantages in the management of ischemic stroke.However,the uncertain distribution and rapid clearance of extracellular vesicles impede their delivery efficiency.By utilizing membrane decoration or by encapsulating therapeutic cargo within extracellular vesicles,their delivery efficacy may be greatly improved.Furthermore,previous studies have indicated that microvesicles,a subset of large-sized extracellular vesicles,can transport mitochondria to neighboring cells,thereby aiding in the restoration of mitochondrial function post-ischemic stroke.Small extracellular vesicles have also demonstrated the capability to transfer mitochondrial components,such as proteins or deoxyribonucleic acid,or their sub-components,for extracellular vesicle-based ischemic stroke therapy.In this review,we undertake a comparative analysis of the isolation techniques employed for extracellular vesicles and present an overview of the current dominant extracellular vesicle modification methodologies.Given the complex facets of treating ischemic stroke,we also delineate various extracellular vesicle modification approaches which are suited to different facets of the treatment process.Moreover,given the burgeoning interest in mitochondrial delivery,we delved into the feasibility and existing research findings on the transportation of mitochondrial fractions or intact mitochondria through small extracellular vesicles and microvesicles to offer a fresh perspective on ischemic stroke therapy.

Unlocking the future:Mitochondrial genes and neural networks in predicting ovarian cancer prognosis and immunotherapy response

BACKGROUND Mitochondrial genes are involved in tumor metabolism in ovarian cancer(OC)and affect immune cell infiltration and treatment responses.AIM To predict prognosis and immunotherapy response in patients diagnosed with OC using mitochondrial genes and neural networks.METHODS Prognosis,immunotherapy efficacy,and next-generation sequencing data of patients with OC were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus.Mitochondrial genes were sourced from the MitoCarta3.0 database.The discovery cohort for model construction was created from 70% of the patients,whereas the remaining 30% constituted the validation cohort.Using the expression of mitochondrial genes as the predictor variable and based on neural network algorithm,the overall survival time and immunotherapy efficacy(complete or partial response)of patients were predicted.RESULTS In total,375 patients with OC were included to construct the prognostic model,and 26 patients were included to construct the immune efficacy model.The average area under the receiver operating characteristic curve of the prognostic model was 0.7268[95% confidence interval(CI):0.7258-0.7278]in the discovery cohort and 0.6475(95%CI:0.6466-0.6484)in the validation cohort.The average area under the receiver operating characteristic curve of the immunotherapy efficacy model was 0.9444(95%CI:0.8333-1.0000)in the discovery cohort and 0.9167(95%CI:0.6667-1.0000)in the validation cohort.CONCLUSION The application of mitochondrial genes and neural networks has the potential to predict prognosis and immunotherapy response in patients with OC,providing valuable insights into personalized treatment strategies.

Diabetes mellitus and glymphatic dysfunction:Roles for oxidative stress,mitochondria,circadian rhythm,artificial intelligence,and imaging

Diabetes mellitus(DM)is a debilitating disorder that impacts all systems of the body and has been increasing in prevalence throughout the globe.DM represents a significant clinical challenge to care for individuals and prevent the onset of chronic disability and ultimately death.Underlying cellular mechanisms for the onset and development of DM are multi-factorial in origin and involve pathways associated with the production of reactive oxygen species and the generation of oxidative stress as well as the dysfunction of mitochondrial cellular organelles,programmed cell death,and circadian rhythm impairments.These pathways can ultimately involve failure in the glymphatic pathway of the brain that is linked to circadian rhythms disorders during the loss of metabolic homeostasis.New studies incorporate a number of promising techniques to examine patients with metabolic disorders that can include machine learning and artificial intelligence pathways to potentially predict the onset of metabolic dysfunction.

Mitochondrial transplantation confers protection against the effects of ischemic stroke by repressing microglial pyroptosis and promoting neurogenesis

Transferring healthy and functional mitochondria to the lateral ventricles confers neuroprotection in a rat model of ischemia-reperfusion injury.Autologous mitochondrial transplantation is also beneficial in pediatric patients with cardiac ischemia-reperfusion injury.Thus,transplantation of functional exogenous mitochondria may be a promising therapeutic approach for ischemic disease.To explore the neuroprotective effect of mitochondria transplantation and determine the underlying mechanism in ischemic stroke,in this study we established a photo-thrombosis-induced mouse model of focal ischemia and administered freshly isolated mitochondria via the tail vein or to the injury site(in situ).Animal behavior tests,immunofluorescence staining,2,3,5-triphenyltetrazolium chloride(TTC)staining,mRNA-seq,and western blotting were used to assess mouse anxiety and memory,cortical infarct area,pyroptosis,and neurogenesis,respectively.Using bioinformatics analysis,western blotting,co-immunoprecipitation,and mass spectroscopy,we identified S100 calcium binding protein A9(S100A9)as a potential regulator of mitochondrial function and determined its possible interacting proteins.Interactions between exogenous and endogenous mitochondria,as well as the effect of exogenous mitochondria on recipient microglia,were assessed in vitro.Our data showed that:(1)mitochondrial transplantation markedly reduced mortality and improved emotional and cognitive function,as well as reducing infarct area,inhibiting pyroptosis,and promoting cortical neurogenesis;(2)microglial expression of S100A9 was markedly increased by ischemic injury and regulated mitochondrial function;(3)in vitro,exogenous mitochondria enhanced mitochondrial function,reduced redox stress,and regulated microglial polarization and pyroptosis by fusing with endogenous mitochondria;and(4)S100A9 promoted internalization of exogenous mitochondria by the microglia,thereby amplifying their pro-proliferation and anti-inflammatory effects.Taken together,our findings show that mitochondrial transplantation protects against the deleterious effects of ischemic stroke by suppressing pyroptosis and promoting neurogenesis,and that S100A9 plays a vital role in promoting internalization of exogenous mitochondria.

Elaidic acid leads to mitochondrial dysfunction via mitochondria-associated membranes triggers disruption of mitochondrial calcium fluxes

Elaidic acid(EA)stimulation can lead to endoplasmic reticulum stress(ERS),accompanied by a large release of Ca^(2+),and ultimately the activation of NLRP3 inflammasome in Kupffer cells(KCs).Mitochondrial instability or dysfunction may be the key stimulating factors to activate NLRP3 inflammasome,and sustained Ca^(2+)transfer can result in mitochondrial dysfunction.We focused on KCs to explore the damage to mitochondria by EA.After EA stimulation,cells produced an oxidative stress(OS)response with a significant increase in ROS release.Immunoprecipitation experiments and the addition of inhibitors revealed that the increase in the level of intracellular Ca^(2+)led to Ca^(2+)accumulation in the mitochondrial matrix via mitochondria-associated membranes(MAMs).This was accompanied by a significant release of m ROS,loss of MMP and ATP,and a significant increase in mitochondrial permeability transition pore opening,ultimately leading to mitochondrial instability.These findings confirmed the mechanism that EA induced mitochondrial Ca^(2+)imbalance in KCs via MAM,ultimately leading to mitochondrial dysfunction.Meanwhile,EA induced OS and the decrease of MMP and ATP in rat liver,and significant lesions were found in liver mitochondria.Swelling of the inner mitochondrial cristae and mitochondrial vacuolization occurred,with a marked increase in lipid droplets.

Erlotinib combination with a mitochondria-targeted ubiquinone effectively suppresses pancreatic cancer cell survival

BACKGROUND Pancreatic cancer is a leading cause of cancer-related deaths.Increased activity of the epidermal growth factor receptor(EGFR)is often observed in pancreatic cancer,and the small molecule EGFR inhibitor erlotinib has been approved for pancreatic cancer therapy by the food and drug administration.Nevertheless,erlotinib alone is ineffective and should be combined with other drugs to improve therapeutic outcomes.We previously showed that certain receptor tyrosine kinase inhibitors can increase mitochondrial membrane potential(Δψm),facilitate tumor cell uptake ofΔψm-sensitive agents,disrupt mitochondrial homeostasis,and subsequently trigger tumor cell death.Erlotinib has not been tested for this effect.AIM To determine whether erlotinib can elevateΔψm and increase tumor cell uptake ofΔψm-sensitive agents,subsequently triggering tumor cell death.METHODSΔψm-sensitive fluorescent dye was used to determine how erlotinib affectsΔψm in pancreatic adenocarcinoma(PDAC)cell lines.The viability of conventional and patient-derived primary PDAC cell lines in 2D-and 3D cultures was measured after treating cells sequentially with erlotinib and mitochondria-targeted ubiquinone(MitoQ),aΔψm-sensitive MitoQ.The synergy between erlotinib and MitoQ was then analyzed using SynergyFinder 2.0.The preclinical efficacy of the twodrug combination was determined using immune-compromised nude mice bearing PDAC cell line xenografts.RESULTS Erlotinib elevatedΔψm in PDAC cells,facilitating tumor cell uptake and mitochondrial enrichment ofΔψm-sensitive agents.MitoQ triggered caspase-dependent apoptosis in PDAC cells in culture if used at high doses,while erlotinib pretreatment potentiated low doses of MitoQ.SynergyFinder suggested that these drugs synergistically induced tumor cell lethality.Consistent with in vitro data,erlotinib and MitoQ combination suppressed human PDAC cell line xenografts in mice more effectively than single treatments of each agent.CONCLUSION Our findings suggest that a combination of erlotinib and MitoQ has the potential to suppress pancreatic tumor cell viability effectively.

Vitamin A regulates mitochondrial biogenesis and function through p38 MAPK‑PGC‑1α signaling pathway and alters the muscle fiber composition of sheep

Background Vitamin A(VA)and its metabolite,retinoic acid(RA),are of great interest for their wide range of physiological functions.However,the regulatory contribution of VA to mitochondrial and muscle fiber composition in sheep has not been reported.Method Lambs were injected with 0(control)or 7,500 IU VA palmitate into the biceps femoris muscle on d 2 after birth.At the age of 3 and 32 weeks,longissimus dorsi(LD)muscle samples were obtained to explore the effect of VA on myofiber type composition.In vitro,we investigated the effects of RA on myofiber type composition and intrinsic mechanisms.Results The proportion of type I myofiber was greatly increased in VA-treated sheep in LD muscle at harvest.VA greatly promoted mitochondrial biogenesis and function in LD muscle of sheep.Further exploration revealed that VA elevated PGC-1αmRNA and protein contents,and enhanced the level of p38 MAPK phosphorylation in LD muscle of sheep.In addition,the number of type I myofibers with RA treatment was significantly increased,and type IIx myofibers was significantly decreased in primary myoblasts.Consistent with in vivo experiment,RA significantly improved mitochondrial biogenesis and function in primary myoblasts of sheep.We then used si-PGC-1αto inhibit PGC-1αexpression and found that si-PGC-1αsignificantly abrogated RA-induced the formation of type I myofibers,mitochondrial biogenesis,MitoTracker staining intensity,UQCRC1 and ATP5A1 expression,SDH activity,and enhanced the level of type IIx muscle fibers.These data suggested that RA improved mitochondrial biogenesis and function by promoting PGC-1αexpression,and increased type I myofibers.In order to prove that the effect of RA on the level of PGC-1αis caused by p38 MAPK signaling,we inhibited the p38 MAPK signaling using a p38 MAPK inhibitor,which significantly reduced RA-induced PGC-1αand MyHC I levels.Conclusion VA promoted PGC-1αexpression through the p38 MAPK signaling pathway,improved mitochondrial biogenesis,and altered the composition of muscle fiber type.

TPOL triggers apoptosis with mitochondrial injury through activating a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signaling

AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation,before treatment with various inhibitors,N-acetyl-Lcysteine(NAC),pifithrin-αand Z-DVED-FMK.Cell viability was measured by CCK-8 assay.Annexin V/propidium iodide staining was used to count the number of apoptotic cells.DCFH-DA staining was used to detect reactive oxygen species(ROS)levels,and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry.The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot.RESULTS:TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner(P<0.05),with a decrease in Bcl-2 and increases in Bax and cytochrome C(Cyto C),followed by up-regulation of activated caspase-9 and caspase-3,and the cleavage of PARP(P<0.05).The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK(P<0.01).TPOL also led to a rapid increase in ROS,a reduction in mitochondrial membrane potential,and the release of Cyto C(P<0.01),all of which could be reversed by the ROS scavenger NAC.Moreover,the TPOL-caused alterations in p21,p27,Rb,and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0.05).The TPOL-induced changes in Bax,Bcl-2,cleaved caspase-9,activated caspase-3,and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α(P<0.05).CONCLUSION:TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis.

Pharmacological Investigation on the Qi-Invigorating Action of Schisandrin B: Effects on Mitochondrial ATP Generation in Multiple Tissues and Innate/Adaptive Immunity in Mice

Schisandrae Fructus, containing schisandrin B (Sch B) as its main active component, is recognized in traditional Chinese medicine (TCM) for its Qi-invigorating properties in the five visceral organs. Our laboratory has shown that the Qi-invigorating action of Chinese tonifying herbs is linked to increased mitochondrial ATP generation and an enhancement in mitochondrial glutathione redox status. To explore whether Sch B can exert Qi-invigorating actions across various tissues, we investigated the effects of Sch B treatment on mitochondrial ATP generation and glutathione redox status in multiple mouse tissues ex vivo. In line with TCM theory, which posits that Zheng Qi generation relies on the Qi function of the visceral organs, we also examined Sch B’s impact on natural killer cell activity and antigen-induced splenocyte proliferation, both serving as indirect measures of Zheng Qi. Our findings revealed that Sch B treatment consistently enhanced mitochondrial ATP generation and improved mitochondrial glutathione redox status in mouse tissues. This boost in mitochondrial function was associated with stimulated innate and adaptive immune responses, marked by increased natural killer cell activity and antigen-induced T/B cell proliferation, potentially through the increased generation of Zheng Qi.

Don´t give up on mitochondria as a target for the treatment of diabetes and its complications

In this editorial,we discuss an article by Wang et al,focusing on the role of mitochondria in peripheral insulin resistance and insulin secretion.Despite numerous in vitro and pre-clinical studies supporting the involvement of mitochondrial dysfunction and oxidative stress in the pathogenesis of diabetes and its complications,efforts to target mitochondria for glycemic control in diabetes using mitochondria-targeted antioxidants have produced inconsistent results.The intricate functionality of mitochondria is summarized to underscore the challenges it poses as a therapeutic target.While mitochondria-targeted antioxidants have demonstrated improvement in mitochondrial function and oxidative stress in pre-clinical diabetes models,the results regarding glycemic control have been mixed,and no studies have evaluated their hypoglycemic effects in diabetic patients.Nonetheless,pre-clinical trials have shown promising outcomes in ameliorating diabetes-related complications.Here,we review some reasons why mitochondria-targeted antioxidants may not function effectively in the context of mitochondrial dysfunction.We also highlight several alternative approaches under development that may enhance the targeting of mitochondria for diabetes treatment.

Crosstalk between degradation and bioenergetics: how autophagy and endolysosomal processes regulate energy production

Cells undergo metabolic reprogramming to adapt to changes in nutrient availability, cellular activity, and transitions in cell states. The balance between glycolysis and mitochondrial respiration is crucial for energy production, and metabolic reprogramming stipulates a shift in such balance to optimize both bioenergetic efficiency and anabolic requirements. Failure in switching bioenergetic dependence can lead to maladaptation and pathogenesis. While cellular degradation is known to recycle precursor molecules for anabolism, its potential role in regulating energy production remains less explored. The bioenergetic switch between glycolysis and mitochondrial respiration involves transcription factors and organelle homeostasis, which are both regulated by the cellular degradation pathways. A growing body of studies has demonstrated that both stem cells and differentiated cells exhibit bioenergetic switch upon perturbations of autophagic activity or endolysosomal processes. Here, we highlighted the current understanding of the interplay between degradation processes, specifically autophagy and endolysosomes, transcription factors, endolysosomal signaling, and mitochondrial homeostasis in shaping cellular bioenergetics. This review aims to summarize the relationship between degradation processes and bioenergetics, providing a foundation for future research to unveil deeper mechanistic insights into bioenergetic regulation.

Pyrroloquinoline quinone:a potential neuroprotective compound for neurodegenerative diseases targeting metabolism

Pyrroloquinoline quinone is a quinone described as a cofactor for many bacterial dehydrogenases and is reported to exert an effect on metabolism in mammalian cells/tissues.Pyrroloquinoline quinone is present in the diet being available in foodstuffs,conferring the potential of this compound to be supplemented by dietary administration.Pyrroloquinoline quinone’s nutritional role in mammalian health is supported by the extensive deficits in reproduction,growth,and immunity resulting from the dietary absence of pyrroloquinoline quinone,and as such,pyrroloquinoline quinone has been considered as a“new vitamin.”Although the classification of pyrroloquinoline quinone as a vitamin needs to be properly established,the wide range of benefits for health provided has been reported in many studies.In this respect,pyrroloquinoline quinone seems to be particularly involved in regulating cell signaling pathways that promote metabolic and mitochondrial processes in many experimental contexts,thus dictating the rationale to consider pyrroloquinoline quinone as a vital compound for mammalian life.Through the regulation of different metabolic mechanisms,pyrroloquinoline quinone may improve clinical deficits where dysfunctional metabolism and mitochondrial activity contribute to induce cell damage and death.Pyrroloquinoline quinone has been demonstrated to have neuroprotective properties in different experimental models of neurodegeneration,although the link between pyrroloquinoline quinone-promoted metabolism and improved neuronal viability in some of such contexts is still to be fully elucidated.Here,we review the general properties of pyrroloquinoline quinone and its capacity to modulate metabolic and mitochondrial mechanisms in physiological contexts.In addition,we analyze the neuroprotective properties of pyrroloquinoline quinone in different neurodegenerative conditions and consider future perspectives for pyrroloquinoline quinone’s potential in health and disease.

Protective Effect of Pueraria lobota Extracts on Mitochondria Damage of Liver

[Objective] This study aimed to investigate the protective effect of Pueraria lobota extracts on mitochondria damage of liver. [Method] The liver mitochondria injury was induced by Vc-Fe2＋ , and the influences of Pueraria lobota extracts on mitochondria ATPase activity, mitochondria swelling and protein carbonyl content were measured. In addition, the lipid peroxidation in liver mitochondria was induced by H2O2-Fe2＋ to analyze the influence of Pueraria lobota extracts on MDA content. Futhermore, NBT method was used to evaluate the inhibitory function of Pueraria lobota extracts on the superoxide anion. [Result] The results showed that Pueraria lobota extracts could significantly inhibit mitochondria oxidative damage,prevent mitochondria swelling and ATPase activity reduction,decrease protein carbonyl level,and effectively scavenge superoxide anion produced by mitochondria, indicating that Pueraria lobota extracts can protect rat liver mitochondria from oxidative damage. [Conclusion] This study provided theoretical basis for investigating the pharmacological functions of Pueraria lobota.

Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA Library was constructed with the heat shocked tomato ( Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in newer and the cold inducible character of mitochondria shsp gene in leaf were discussed.

A Novel Conservation Structure Found in Vertebrate Mitochondrial tRNA￣(phe) Gene

The nucleotide scquence of tRNAphe gene of Carp mitochondria was determined. Sequence comparisons made among Whale,Human,Xenopus laevis, Bovine, Mouse,Chicken and Carp revealed a novel conservative structure in the D. stem (dihydrouridine stem),which is known to vary in other vertebrate mitochondrial and eytoplasmic tRNA genes.This conservative structure contains 13 bp. When the first 7 bp of the conserviative structure were compared with the A domain recognized by RNA Pol III, we noticed partial homology between these two kinds of sequences among different species. In view of the tRNAphe gene's close position to the D loop,it is reasonable to expect extraordinary functions in this novel conservation structure.

平胃胶囊对恶变后GES-1细胞氧化应激的抑制作用及机制研究

目的:观察平胃胶囊对亚硝酸胺类化合物N-甲基-N’-硝基-N-亚硝基胍(N-methyl-N’-nitro-N-nitrosoguanidine,MNNG)诱导的胃癌前病变(precancerous lesions of gastric cancer,PLGC)细胞模型的影响,并初步探讨其作用机制。方法:制备空白血清和平胃胶囊含药血清备用;MNNG诱导人胃黏膜上皮细胞系GES-1制备PLGC细胞模型,采用倒置显微镜观察细胞形态,免疫荧光法检测增殖细胞相关抗原Ki67的表达水平,进行模型评价。CCK-8法筛选含药血清最佳干预浓度及时间;采用荧光探针DCFH-DA检测细胞内活性氧(reactive oxygen species,ROS)含量;ELISA检测丙二醛(malondialdehyde,MDA)含量;采用相关试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性;采用新型荧光探针JC-10检测细胞线粒体膜电位的变化;采用实时荧光定量PCR检测Ki67和黑色素瘤分化相关基因7(melanoma differentiation-associated gene-7,MDA-7)的mRNA表达水平;采用Western blot法测定Ki67、白细胞介素6(interleukin-6,IL-6)和MDA-7的蛋白表达水平。结果:与正常组相比,模型组和空白血清组ROS和MDA含量显著升高(P<0.01),SOD和GSH-Px的活性显著降低(P<0.01),线粒体膜电位显著下降(P<0.01),Ki67和IL-6蛋白表达显著升高(P<0.01),MDA-7蛋白表达显著下降(P<0.01);与空白血清组相比,模型组ROS和MDA含量,SOD和GSH-Px活性,Ki67和MDA-7 mRNA表达水平,Ki67、IL-6和MDA-7蛋白表达水平,以及线粒体膜电位均无显著差异(P>0.05);与空白血清组相比,平胃胶囊含药血清组中ROS和MDA含量显著降低(P<0.01),SOD和GSH-Px活性显著上升(P<0.05),线粒体膜电位显著升高(P<0.01),Ki67和IL-6蛋白表达水平显著下降(P<0.01),MDA-7的mRNA及蛋白表达水平显著升高(P<0.01)。结论:平胃胶囊可显著减轻MNNG诱导的胃黏膜上皮细胞氧化应激损伤和炎症反应,调控促癌基因和抑癌基因的表达,从而发挥防治PLGC的作用。

Changes of Reactive Oxygen Species and Related Enzymes in Mitochondrial Respiration During Storage of Harvested Peach Fruits

Peach fruits [Prumus persica （L.） Batsch, cv. Yuhuasanhao] were used as materials to investigate the changes of reactive oxygen species （ROS） and related enzymes in mitochondria respiration during storage and then their influence on senescence of harvested Peach fruits was studied. The results showed that low temperature （5℃） strongly inhibited the reduction of firmness and the increase in respiration rate. During storage at ambient temperature （20℃）, ROS had a cumulative process while malondialdehye （MDA） content continued to increase in associated with enhanced membrane lipid peroxidation. Lipoxygenase （LOX） activity was strongly inhibited under the low temperature condition. The activities of succinic dehydrogenase （SDH）, cytochrome C oxidase （CCO）, and Ca^2＋-ATPase declined to a certain extent at ambient temperature, while they showed higher activities at low temperature, which may be related to lower membrane lipid peroxidation at low temperature. Higher Ca^2＋ content at ambient temperature may be responsible for impairment of mitochondrial function, thus, leading to fruit senescence. The results showed that under low temperature condition, the low accumulation of ROS and the low level of membrane lipid peroxidation could maintain the function of mitochondria that would help to delay the senescence of peach fruits. These suggested a close relationship existed between ROS metabolism and mitochondrial respiration. It can be inferred that the low temperature helps to delay senescence of peach fruits via suppression of ROS and related enzymes, maintain better homeostasis of Ca^2＋ in mitochondria and thus better mitochondrial functions.

Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells

AIM To investigate the antitumor activity of α-hederin in hepatocellular carcinoma(HCC) cells and its underlying mechanisms in vitro and in vivo.METHODS SMMC-7721, Hep G-2 and Huh-7 HCC cells were cultured in vitro and treated with α-hederin(0, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, 30 μmol/L, 35 μmol/L, 40 μmol/L, 45 μmol/L, 50 μmol/L, 55 μmol/L, or 60 μmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721cells were treated with 0, 5 μmol/L, 10 μmol/L, or 20 μmol/L α-hederin for 24 h with or without DL-buthionineS,R-sulfoximine(2 mmol/L) or N-acetylcysteine(5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione(GSH) and adenosine triphosphate(ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species(ROS) levels were determined by measuring the oxidative conversion of 2',7'-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin in vivo was evaluated in a xenograft tumor model.RESULTS The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin(5 μmol/L), mid-dose α-hederin(10 μmol/L) and highdose α-hederin(20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively(P < 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth in vivo. CONCLUSION The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.

Effects of Ropivacaine and Bupivacaine on Rabbit Myocardial Energetic Metabolism and Mitochondria Oxidation

To compare the cardiotoxicity induced by ropivacaine and bupivacaine and to investigate the mechanism of cardiotoxicity, 24 mature New Zealand rabbits were divided randomly into control group (group C), ropivacaine group (group R) and bupivacaine group (group B). Hearts were drawn out rapidly from the anesthetized animals and cardiac perfu-sion was performed immediately. Ropivacaine 500 ng/ml (group R) or bupivacaine 500 ng/ml (group B) was added to the perfusion solution. Ventricular myocardial ATP, ADP and AMP were measured with high performance liquid chro-matogram. The ability of myocardial mitochondria oxidation to pyruvate or palmitoylcarnitine was detected with Clark electrode. Our results showed that myocardial ATP and ADP decreased significantly (P<0. 05) in group R and most significantly (P<0. 01) in group B as compared with group C. Myocardial ATP and ADP decreased most significantly (P<0. 01) in group B as compared with group R. The changes of myocardial AMP revealed significant difference among three groups. The changes of pyruvate oxidation exibited no significant difference among the three groups. Palmitoylcarnitine oxidation decreased markedly (P<0. 05) in group R and most significantly (P<0. 01) in group B as compared with group C. The present study indicated that the inhibition of lipid substrate oxidation may be responsible for the cardiotoxicity induced by bupivacaine and ropivacaine. The cardiotoxicity induced by ropivacaine is far more less than bupivacaine.

Changes of Reactive Oxygen Species and Related Enzymes in Mitochondria Respiratory Metabolism During the Ripening of Peach Fruit

The fruits of peach cultivar Yuhua 3 were used as materials to investigate the changes of active oxygen and related enzymes in mitochondria respiratory metabolism during ripening of peach fruit, involving their influence on the proceeding of peach fruit senescence. The results showed that the large decrease in firmness occurred between maturity II and IV. The decrease in firmness coincided with an increase in respiratory intensity. Obvious peaks of respiratory intensity lagging to the rapid change of fruit firmness could be shown during peach ripening. Reactive oxygen species （ROS） had a cumulative process and positively correlated with respiratory intensity. During peach ripening, the content of Ca^2＋ increased, the activities of succinic dehydrogenase （SDH）, cytochrome C oxidase （CCO）, H＋-ATPase, and Ca^2＋-ATPase decreased varying in different degree at the later step of ripening. These suggested a close relationship existed between ROS metabolism and mitochondrial respiration, namely, both ROS metabolism and mitochondrial respiration probably played important roles in ripening and senescing of peach fruit.