Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy:Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits

Objective:Cymbopogon citratus(DC.)Stapf is a medicinal and edible herb that is widely used for the treatment of gastric,nervous and hypertensive disorders.In this study,we investigated the cardioprotective effects and mechanisms of the essential oil,the main active ingredient of Cymbopogon citratus,on isoproterenol(ISO)-induced cardiomyocyte hypertrophy.Methods:The compositions of Cymbopogon citratus essential oil(CCEO)were determined by gas chromatography-mass spectrometry.Cardiomyocytes were pretreated with 16.9µg/L CCEO for 1 h followed by 10µmol/L ISO for 24 h.Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated.Subsequently,transcriptome sequencing(RNA-seq)and target verification were used to further explore the underlying mechanism.Results:Our results showed that the CCEO mainly included citronellal(45.66%),geraniol(23.32%),and citronellol(10.37%).CCEO inhibited ISO-induced increases in cell surface area and protein content,as well as the upregulation of fetal gene expression.Moreover,CCEO inhibited ISO-induced NLRP3 inflammasome expression,as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3,ASC,CASP1,GSDMD,and IL-1β,as well as reduced protein levels of NLRP3,ASC,pro-caspase-1,caspase-1(p20),GSDMD-FL,GSDMD-N,and pro-IL-1β.The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes.Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1,Sdhd,mt-Cytb,Uqcrq,and mt-Atp6 but had no obvious effects on mt-Col expression.Conclusion:CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Combined Oxidative Phosphorylation Deficiency-20-Exome as a Diagnostic Implement

Mitochondrial disorders are phenotypically varied, with serious clinical repercussions. Among them, there is the deficiency of combined oxidative phosphorylation of type 20, which occurs due to a defect in the VARS2 gene. This article presents a case of a 2-year-old female with progressive myoclonic epilepsy and psychomotor regression, with refractoriness to multiple anticonvulsants. The diagnosis was only made after the examination was carried out. Therefore, this article highlights the aspects of this rare disease and the importance of the exome for the diagnosis of rare conditions.

Control mechanisms in mitochondrial oxidative phosphorylation

Distribution and activity of mitochondda are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5＇- triphosphate production is regulated by many control mechanism-firstly by oxygen, substrate level, adenosine-5＇-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by ＂second control mechanisms,＂ such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, aUosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5＇-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria similarly, decline of antioxidative enzyme activities （e.g. in the elderly） enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

Effects of Chinese herbal monomers on oxidative phosphorylation and membrane potential in cerebral mitochondria isolated from hypoxia-exposed rats in vitro

Mitochondrial dysfunction is the key pathogenic mechanism of cerebral injury induced by high-altitude hypoxia. Some Chinese herbal monomers may exert anti-hypoxic effects through enhancing the efficiency of oxidative phosphorylation, in this study, effects of 10 kinds of Chinese herbal monomers on mitochondrial respiration and membrane potential of cerebral mitochondria isolated from hypoxia-exposed rats in vitro were investigated to screen anti-hypoxic drugs. Rats were exposed to a low-pressure environment of 405.35 mm Hg （54.04 kPa） for 3 days to establish high-altitude hypoxic models. Cerebral mitochondria were isolated and treated with different concentrations of Chinese herbal monomers （sinomenine, silymarin, glycyrrhizic acid, baicalin, quercetin, ginkgolide B, saffron, pipedne, ginsenoside Rgl and oxymatrine） for 5 minutes in vitro. Mitochondrial oxygen consumption and membrane potential were measured using a Clark oxygen electrode and the rhodamine 123 fluorescence analysis method, respectively. Hypoxic exposure significantly decreased the state 3 respiratory rate, respiratory control rate and mitochondrial membrane potential, and significantly increased the state 4 respiratory rate. Treatment with saffron ginsenoside Rgl and oxymatrine increased the respiratory control rate in cerebral mitochondria isolated from hypoxia-exposed rats in dose-dependent manners in vitro, while ginsenoside Rgl, piperine and oxymatrine significantly increased the mitochondrial membrane potential in cerebral mitochondria from hypoxia-exposed rats. The Chinese herbal monomers saffron, ginsenoside Rgl piperine and oxymatrine could thus improve cerebral mitochondrial disorders in oxidative phosphorylation induced by hypobaric hypoxia exposure in vitro.

Lack of transcriptional coordination between mitochondrial and nuclear oxidative phosphorylation genes in the presence of two divergent mitochondrial genomes

In most eukaryotes,oxidative phosphorylation(OXPHOS)is the main energy production process and it involves both mitochondrial and nuclear genomes.The close interaction between the two genomes is critical for the coordinated function of the OXPHOS process.Some bivalves show doubly uniparental inheritance(DUI)of mitochondria,where two highly divergent mitochondrial genomes,one inherited through eggs(F-type)and the other through sperm(M-type),coexist in the same individual.However,it remains a puzzle how nuclear OXPHOS genes coordinate with two divergent mitochondrial genomes in DUI species.In this study,we compared transcription,polymorphism,and synonymous codon usage in the mitochondrial and nuclear OXPHOS genes of the DUI species Ruditapes philippinarum using sex-and tissue-specific transcriptomes.Mitochondrial and nuclear OXPHOS genes showed different transcription profiles.Strong co-transcription signal was observed within mitochondrial(separate for F-and M-type)and within nuclear OXPHOS genes but the signal was weak or absent between mitochondrial and nuclear OXPHOS genes,suggesting that the coordination between mitochondrial and nuclear OXPHOS subunits is not achieved transcriptionally.McDonald-Kreitman and frequency-spectrum based tests indicated that M-type OXPHOS genes deviated significantly from neutrality,and that F-type and M-type OXPHOS genes undergo different selection patterns.Codon usage analysis revealed that mutation bias and translational selection were the major factors affecting the codon usage bias in different OXPHOS genes,nevertheless,translational selection in mitochondrial OXPHOS genes appears to be less efficient than nuclear OXPHOS genes.Therefore,we speculate that the coordination between OXPHOS genes may involve post-transcriptional/translational regulation.

Influence of thiol stress on oxidative phosphorylation and generation of ROS in Streptomyces coelicolor

Thiols play very important role in the intracellular redox homeostasis. Imbalance in the redox status leads to changes in the intracellular metabolism including respiration. Thiol stress, a reductive type of stress can also cause redox imbalance. When Gram-positive bacterium Strep- tomyces coelicolor was exposed to thiol stress, catalaseA was induced. Induction of catalaseA is the consequence of elevation of ROS (reactive oxygen species). The two major sources of reactive oxygen species are Fenton reaction and slippage of electrons from electron transport chain during respiration. Hence, the effect of thiol stress was checked on the rate of oxidative phosphorylation in S. coelicolor. We found correlation in the increase of oxidative phosphorylation rate and the generation of ROS, subsequently leading to induction of catalase. It was observed that thiol stress does not affect the functionality of the individual complexes of the ETC, but still there was an increase in the overall respiration, which may lead to generation of more ROS leading to induction of catalase.

Anti-esophagus cancer activity and mechanism of DN3,a novel natural diterpenoid derivative,as a dual inhibitor of glycolysis and oxidative phosphorylation

OBJECTIVE To probe into the anti-esophagus cancer activity and mechanisms of DN3,a novel natural diterpenoid derivative.METHODS The anti-tumor activity in vitro of DN3 was evaluated by MTT,and by using human esophageal carcinoma cells xenografted into athymic mice model in vivo.The specific mechanisms of DN3,as a dual inhibitor of glycolysis and oxidative phos.phorylation(OXPHOS) were explored through cell and molecular biology techniques.For instance,the manner of cancer cell death induced by DN3 was characterized by hoechst33342,FITC-Annexin V/PI staining and flow cytometric analysis,then these changes of glucose consumption,glucose uptake and lactate production in glycolysis,as well as oxygen consumption rate(OCR) and ATP content in OXPHOS caused by DN3 were performed separately through related kits and SeahorseBioscience XF24 Extra.cellular Flux Analyzer.Furthermore,in order to obtain a clear understanding of the inhibition of DN3 to glycolysis and OXPHOS,these regulatory factors were investigated by Western blot,such as PI3K/AKT,c-Myc and p53 of glycolysis,Bax and HK2 of mitochondrial function.RESULTS DN3 inhibited the growth of esophagus cancer cell EC9706,EC109 and EC1 cells in a dose and time dependent manner,but showed no significant effects on human esophageal epithelial cells(HEECs).DN3 caused significant G2/M arrest of esophagus cancer cell lines and induced apoptosis of these cell lines,which indicated DN3 inhibited the growth of esophagus cancer cell through blocking cell cycle and inducing apoptosis in a dose and time-dependent manner.Importantly,8 μM DN3 decreased the extracellular acidification rate(ECAR) by 45% in EC109,which indicated glycolysis was inhibited by DN3.Mean.while,DN3 decreased the oxygen consumption rate(OCR) and the OCR linked to intracellular ATP production in EC109 cells,but that was not obvious in HEECs,so which indicated that DN3 could selec.tively block OXPHOS of cancer cells.In addition,the accumulation of reactive oxygen species(ROS)and the drop of mitochondrial membrane potential(MMP) were also observed in EC109 incubated by DN3,which suggested mitochondrial biological function was disturbed.Furthermore,the expression of PI3K/AKT,c-Myc and HK2 related to glycolysis were down-regulated by DN3,but the p53 and Bax were up-regulated in esophageal carcinoma cells.The changes of these enzymes accounted for the decreased glycolysisand OXPHOS in esophageal carcinoma cells treated by DN3.CONCLUSION The new compound DN3 has a strong anti-esophageal carcinoma activity,and it is tolerable that DN3 is seen as a dual inhibitor of glycolysis and oxidative phosphorylation.

Simvastatin Increases the Activity of Endothelial Nitric Oxide Synthase via Enhancing Phosphorylation

3-hydroxy-3-methylgulutaryl-coenzyme A （HMG-CoA） reductase inhibitors or statins are a kind of lipid-lowering agents and have been used for the prevention and treatment of Cardiovascular diseases. Recent studies suggested that statins, besides lowering cholesterol, may protect vessels by enhancing the activity of endothelial nitric oxide synthase （eNOS）. In the present study, we investigated if simvastatin increases eNOS activity through its phosphorylation in 293 cells （293-eNOS） with stable expression of eNOS. The results showed that incubation of 293-eNOS cells with simvastatin （10 μm/L） for 2 h significantly increased in the activity of eNOS as shown by the conversion of L-arginine to L-citrulline （2889.70±201.51 versus 5630.18＋218.75 pmol/min . mg proteins） （P〈0.01）. Western blotting revealed that simvastatin increased phosphorylation of eNOS at 1177 （ser） and also 495 （thr） but did not affect the overall expression of eNOS or inducible NOS. Further study found that simvastatin raised phosphorylation levels of Akt and AMPK, and such effect could be antagonized by Akt inhibitor or AMPK inhibitor. These results suggest that simvastatin could stimulate,the activity of eNOS via its phosphorylation by Akt and AMPK, which provides a new mechanism, other than lipid-lowering effect, for the cardiovascular protection of statins.

NIR-enhanced Pt single atom/g-C_(3)N_(4)nanozymes as SOD/CAT mimics to rescue ATP energy crisis by regulating oxidative phosphorylation pathway for delaying osteoarthritis progression

Osteoarthritis(OA)progresses due to the excessive generation of reactive oxygen and nitrogen species(ROS/RNS)and abnormal ATP energy metabolism related to the oxidative phosphorylation pathway in the mitochondria.Highly active single-atom nanozymes(SAzymes)can help regulate the redox balance and have shown their potential in the treatment of inflammatory diseases.In this study,we innovatively utilised ligand-mediated strategies to chelate Pt^(4+)with modified g-C_(3)N_(4)byπ-πinteraction to prepare g-C_(3)N_(4)-loaded Pt single-atom(Pt SA/C_(3)N_(4))nanozymes that serve as superoxide dismutase(SOD)/catalase(CAT)mimics to scavenge ROS/RNS and regulate mitochondrial ATP production,ultimately delaying the progression of OA.Pt SA/C_(3)N_(4)exhibited a high loading of Pt single atoms(2.45 wt%),with an excellent photothermal conversion efficiency(54.71%),resulting in tunable catalytic activities under near-infrared light(NIR)irradiation.Interestingly,the Pt-N_(6) active centres in Pt SA/C_(3)N_(4)formed electron capture sites for electron holes,in which g-C_(3)N_(4)regulated the d-band centre of Pt,and the N-rich sites transferred electrons to Pt,leading to the enhanced adsorption of free radicals and thus higher SOD-and CAT-like activities compared with pure g-C_(3)N_(4)and g-C_(3)N_(4)-loaded Pt nanoparticles(Pt NPs/C_(3)N_(4)).Based on the use of H_(2)O_(2)-induced chondrocytes to simulate ROS-injured cartilage in vitro and an OA joint model in vivo,the results showed that Pt SA/C_(3)N_(4)could reduce oxidative stress-induced damage,protect mitochondrial function,inhibit inflammation progression,and rebuild the OA microenvironment,thereby delaying the progression of OA.In particular,under NIR light irradiation,Pt SA/C_(3)N_(4)could help reverse the oxidative stress-induced joint cartilage damage,bringing it closer to the state of the normal cartilage.Mechanistically,Pt SA/C_(3)N_(4)regulated the expression of mitochondrial respiratory chain complexes,mainly NDUFV2 of complex 1 and MT-ATP6 of ATP synthase,to reduce ROS/RNS and promote ATP production.This study provides novel insights into the design of artificial nanozymes for treating oxidative stress-induced inflammatory diseases.

The FAcilitates Chromatin Transcription complex regulates the ratio of glycolysis to oxidative phosphorylation in neural stem cells

Defects in the FAcilitates Chromatin Transcription(FACT)complex,a histone chaperone composed of SSRP1 and SUPT16H,are implicated in intellectual disability.Here,we reveal that the FACT complex promotes glycolysis and sustains the correct cell fate of neural stem cells/neuroblasts in the Drosophila 3rd instar larval central brain.We show that the FACT complex binds to the promoter region of the estrogen-related receptor(ERR)gene and positively regulates ERR expression.ERR is known to act as an aerobic glycolytic switch by upregulating the enzymes required for glycolysis.Dysfunction of the FACT complex leads to the downregulation of ERR transcription,resulting in a decreased ratio of glycolysis to oxidative phosphorylation(G/O)in neuroblasts.Consequently,neuroblasts exhibit smaller cell sizes,lower proliferation potential,and altered cell fates.Overexpression of ERR or suppression of mitochondrial oxidative phosphorylation in neuroblasts increases the relative G/O ratio and rescues defective phenotypes caused by dysfunction of the FACT complex.Thus,the G/O ratio,mediated by the FACT complex,plays a crucial role in neuroblast cell fate maintenance.Our study may shed light on the mechanism by which mutations in the FACT complex lead to intellectual disability in humans.

Single-cell transcriptomic analysis reveals transcript enrichment in oxidative phosphorylation,fluid sheer stress,and inflammatory pathways in obesity-related glomerulopathy

Obesity-related glomerulopathy(ORG)is an independent risk factor for chronic kid-ney disease and even progression to end-stage renal disease.Efforts have been undertaken to elucidate the mechanisms underlying the development of ORG and substantial advances have been made in the treatment of ORG,but relatively little is known about cell-specific changes in gene expression.To define the transcriptomic landscape at single-cell resolution,we analyzed kidney samples from four patients with ORG and three obese control subjects without kidney disease using single-cell RNA sequencing.We report for the first time that immune cells,including T cells and B cells,are decreased in ORG patients.Further analysis indicated that SPP1 was significantly up-regulated in T cells and B cells.This gene is related to inflammation and cell proliferation.Analysis of differential gene expression in glomerular cells(endothelial cells,mesangial cells,and podocytes)showed that these cell types were mainly enriched in genes related to oxidative phosphorylation,cell adhesion,thermogenesis,and inflammatory pathways(PI3K-Akt signaling,MAPK signaling).Furthermore,we found that the podocytes of ORG patients were enriched in genes related to the fluid shear stress pathway.Moreover,an evaluation of cell-cell communications revealed that there were interactions between glomerular parietal epithelial cells and other cells in ORG patients,with major interactions between parietal epithelial cells and podocytes.Altogether,our identification of molecular events,cell types,and differentially expressed genes may facilitate the development of new preventive or therapeutic approaches for ORG.

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Effects of Osmoconditioning on Mitochondrial Respiration and Phosphorylation in Soybean Seeds

Cotyledon mitochondrion respiration and oxidative phosphorylation activity were studied in two groups of soybean seeds. One group was primed with polyethylene glycol (PEG) for different periods of time, and the other was unprimed (control), and both were then exposed to imbibition at low temperatures before their germination. The results indicated that when L-Malate (L-Mal) and á-Ketoglutarate (α-Kg) were used as substrates, the ADP-stimulated mitochondria respiration rates of control seeds were mark- edly higher than state Ⅲ respiration rates of primed seeds. However, the osmoconditioning pretreatment significantly enhanced the oxidative phosphorylation activity of cotyledon mitochondrion in 12 h. The oxidative phosphorylation activity of the mitochondrion of primed seeds was normal and the ADP/O value was consistent with the theoretical one. When reduced nicotinamide adenine dinu- cleotide (NADH) was used as the substrate, the mitochondria of control seeds still had oxidative phosphorylation activity, while ADP/O value was obviously lower than that of mitochondria of primed seeds. When Succinate (Succ) was used as the substrate, the oxidative phosphorylation activity of the primed seeds was normal after priming for 24 h. When different substrates were used, the emerging order of the oxidative phosphorylation activity of the primed seeds was NADH, α-Kg, Succ and in the last place L-Mal. The mechanism of soybean imbibitionl chilling injury and protective effect of PEG priming were discussed.

橙皮苷通过氧化磷酸化途径缓解高脂饲喂诱导的小鼠肝氧化应激

旨在研究橙皮苷(Hesperidin, HDN)对高脂饲喂诱导的小鼠肝氧化应激损伤的保护作用及作用机制。将18只雄性C57BL/6(体重20~23 g)小鼠,随机分为对照(control)组、高脂饮食(HFD)组、高脂饮食+橙皮苷(HFD+HDN)组(300 mg·kg^(-1)),每组6只。control组小鼠饲喂常规饲料(脂肪含量10%、碳水化合物含量70%、蛋白质含量20%);HFD组小鼠饲喂高脂饲料(脂肪含量60%、碳水化合物含量20%、蛋白质含量20%);HFD+HDN组在饲喂高脂饲料的同时每天灌胃给药HDN 300 mg·kg^(-1)。16周后腹腔注射3%戊巴比妥钠(60 mg·kg^(-1))麻醉小鼠后进行眼球采血,采血完毕后对小鼠进行脱颈处死并解剖取肝组织;使用试剂盒检测血液中肝功能指标丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的活性;使用试剂盒检测肝组织中氧化应激指标丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)以及总抗氧化能力(T-AOC)的水平;采集肝组织进行转录组学测序,筛选出HFD组和HFD+HDN组的差异表达基因集进行KEGG通路富集分析,以P<0.05作为显著性富集的阈值,据此筛选出HDN干预后影响最显著的一条信号通路;从筛选出的信号通路上挑选显著变化的基因并采取qRT-PCR和蛋白免疫印迹的方法验证转录组学结果;检测mtDNA相对含量和线粒体外膜蛋白(TOMM20)相对表达量;检测肝组织ATP含量。结果显示:与HFD组相比,HDN干预改善了高脂饲喂引起的肝损伤,显著降低了小鼠血液中ALT以及AST活性(P<0.01);显著降低小鼠肝MDA含量(P<0.01);显著升高T-AOC、T-SOD以及GSH-Px水平(P<0.05),KEGG富集分析,筛选出氧化磷酸化(OXPHOS)途径是最显著上调的信号通路(P<0.000 1);与HFD组相比,HDN干预后肝组织Cox8b、Cox6a2、Gm10231、mt-Atp8、mt-Nd4l、Gm11237、Ndufb8、Ndufb10的mRNA相对表达量显著上调(P<0.05),Atp6v0d2、Cox6c2的mRNA的相对表达量显著下调(P<0.05)。Cox6a2、Ndufb8、Ndufb10的蛋白表达量显著升高(P<0.05),Atp6v0d2d蛋白表达量表达量显著降低(P<0.001),与转录组学结果一致;与HFD组相比,HDN干预后TOMM20相对表达量、mtDNA相对含量、ATP含量显著升高(P<0.05)。结果提示,HDN通过调节OXPHOS途径降低高脂饲喂诱导的小鼠肝氧化应激。

内皮型一氧化氮合酶在运动预适应改善心肌缺血-再灌注损伤中的作用

背景:运动是防治各种心血管疾病并保护心脏免受缺血-再灌注损伤的有效策略,其作用机制有待深入研究。目的:观察有氧运动预适应对心肌缺血-再灌注损伤的影响,并探讨内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)激活(包括偶联和磷酸化)在其间的作用。方法:取80只成年Wistar大鼠,采用随机数字表法分为安静组(n=40)和运动组(n=40),运动组进行8周有氧运动,安静组在鼠笼内安静饲养。8周后进行3项实验:①实验1:末次训练后,检测大鼠心功能、心脏NO代谢物含量及心脏eNOS、磷酸化eNOS-S1177、eNOS二聚体、eNOS单体的蛋白表达量;②实验2:将大鼠分为安静对照组、运动对照组、安静+eNOS抑制剂组、运动+eNOS抑制剂组,均进行体外心肌缺血-再灌注损伤实验,安静+eNOS抑制剂组、运动+eNOS抑制剂组再灌注前10 min持续灌注eNOS抑制剂,再灌注3 h后检测心功能与心肌梗死面积;③实验3:将大鼠分为安静对照组、运动对照组、安静+eNOS偶联剂组和运动+eNOS偶联剂组,均进行体外心肌缺血-再灌注损伤实验,安静+eNOS偶联剂组和运动+eNOS偶联剂组再灌注前10 min持续灌注eNOS偶联剂,再灌注3 h后检测心肌梗死面积、心脏NO代谢物含量及心脏eNOS、磷酸化eNOS-S1177、eNOS二聚体、eNOS单体和3-硝基酪氨酸的蛋白表达量(其中,磷酸化eNOS-S1177/eNOS比值反映eNOS磷酸化/去磷酸化水平,eNOS二聚体/单体比值反映eNOS偶联/解偶联水平)。结果与结论:①实验1:与安静组比较,运动组大鼠心输出量、左心室射血分数升高(P<0.05),亚硝酸盐和S-亚硝基硫醇含量升高(P<0.05),磷酸化eNOS-S1177、eNOS蛋白表达和磷酸化eNOS-S1177/eNOS比值上调(P<0.05),eNOS二聚体蛋白表达和eNOS二聚体/单体比值升高(P<0.05);②实验2:与安静对照组比较,运动对照组左心室发展压升高(P<0.05),心肌梗死面积下降(P<0.05);与运动对照组比较,运动+eNOS抑制剂组左心室发展压降低(P<0.05),心肌梗死面积增加(P<0.05);③实验3:与安静对照组比较,运动对照组左心室发展压升高(P<0.05),心肌梗死面积下降(P<0.05),磷酸化eNOS-S1177/eNOS比值下降(P<0.05),eNOS二聚体/单体比值下降(P<0.05),S-亚硝基硫醇含量增加(P<0.05),3-硝基酪氨酸蛋白表达量下调(P<0.05);与运动对照组比较,运动+eNOS偶联剂组左心室发展压降低(P<0.05),心肌梗死面积增加(P<0.05),磷酸化eNOS-S1177/eNOS比值升高(P<0.05),eNOS二聚体/单体比值升高(P<0.05),3-硝基酪氨酸蛋白表达升高(P<0.05);④结果表明:有氧运动预适应可诱导心脏保护效应,其机制与心脏缺血-再灌注期间eNOS解偶联以及去磷酸化进而抑制NO过度产生并降低硝基-氧化应激有关。

线粒体病分子诊断技术进展

线粒体病是一类重要的遗传代谢病,其发病可覆盖全年龄段,尤其是儿童线粒体病致死致残率极高。随着生物化学及分子与细胞生物学技术的发展,线粒体病的实验室诊断经历了快速的发展,相关诊断路径和策略也从高度有创的实验室检测逐渐过渡到以无创筛查为主。但是,线粒体病实验室诊断仍然存在单一检测策略的阳性诊断率不足、实验室漏检与待排查比例居高不下的困扰,因此新的线粒体病检测技术被开发并用于协助疾病的诊断。该文从基因、酶生物化学与代谢生物学3个层面对当前线粒体病实验室诊断的进展进行综述,旨在为特定场景下线粒体病实验室诊断策略选择提供参考,也为后续检测技术的研发提供一些建议。

穿心莲内酯对胰岛素抵抗大鼠的影响及可能作用机制分析

目的 探讨穿心莲内酯对胰岛素抵抗大鼠的影响及可能作用机制。方法 获得SPF级健康成年Wistar大鼠48只,随机选择36只大鼠经高脂高糖饲料喂养建立实验性Wistar大鼠IR模型,按照随机数字分配法分为空白对照模型组(HFD组)、穿心莲内酯高剂量组(High组)和穿心莲内酯低剂量组(Low组),选择12只大鼠接受普通饲料喂养作为正常对照组(NC组),High组大鼠予以经腹腔注射穿心莲内酯200 mg/(kg·d),Low组大鼠予以穿心莲内酯腹腔注射100 mg/(kg·d),HFD组以及NC组仅予以等量生理盐水腹腔注射,连续14 d。应用葡萄糖氧化酶法对空腹血糖值(FBG)水平进行监测,应用ELISA法检测空腹胰岛素(Fasting insulin, FINS),计算稳态模型评估胰岛素抵抗指数(homeostasis model assessment-insulin resistance, HOMA-IR);油红O染色制片检测肝脏的病理变化;应用联苯三酚自氧化法对超氧化物歧化酶(superoxide dismutase, SOD)活力,应用硫代巴比妥酸法对丙二醛(malondialdehyde, MDA)水平进行监测;免疫组化法检测大鼠肝脏组织内核糖体蛋白S6激酶(Ribosomal protein S6 kinase, S6K1)及p-胰岛素受体底物-1(p-insulin receptor substrate-1,p-IRS-1)(Tyr632)蛋白表达水平,Westernblotting法检测肝脏组织内S6K1及p-IRS-1(Tyr632)表达水平。应用SPSS 23.0软件包进行分析。结果 相比NC组比较,HFD组、High组、Low组大鼠FBG、FINS、HOMA-IR水平明显升高(P<0.05);与HFD组相比,其余3组FBG、FINS、HOMA-IR水平较低(P<0.05);与穿心莲内酯Low组相比,High组FBG、FINS、HOMA-IR水平较低(P<0.05)。相比NC组,HFD组光密度平均值增加(P<0.05);相比HFD组,穿心莲内酯High组与Low组脂滴面积/肌纤维总面积、光密度平均值均下降(P<0.05);与穿心莲内酯Low组相比,High组脂滴面积/肌纤维总面积、光密度平均值较低(P<0.05)。与NC组相比,HFD组SOD活性与MDA含量增加(P<0.05);相比HFD组,穿心莲内酯High组与Low组SOD活性与MDA含量均下降(P<0.05);与穿心莲内酯Low组相比,High组SOD活性与MDA含量较低(P<0.05)。与NC组相比,HFD组FAT/CD36蛋白相对表达量增加,CPT-1蛋白相对表达量下降(P<0.05);相比HFD组,穿心莲内酯High组与Low组FAT/CD36蛋白相对表达量下降,CPT-1蛋白相对表达量增加(P<0.05);与穿心莲内酯Low组相比,High组FAT/CD36蛋白相对表达量较低,CPT-1蛋白相对表达量较高(P<0.05)。与NC组相比,HFD组FAT/CD36mRNA相对表达量增加,CPT-1mRNA相对表达量降低(P<0.05);相比HFD组,穿心莲内酯High组与Low组FAT/CD36mRNA相对表达量下降,CPT-1mRNA相对表达量增加(P<0.05);与穿心莲内酯Low组相比,High组FAT/CD36mRNA相对表达量较低,CPT-1mRNA相对表达量较高(P<0.05)。结论 穿心莲内酯能够改善胰岛素抵抗大鼠胰岛素抵抗状态,显著改善IR大鼠的IR状态,其治疗机制与改善氧化应激状态,与下调S6K1的表达及提升IRS-1酪氨酸磷酸化水平具有密切相关性。

与肌少症发病相关的线粒体自噬靶点基因筛选及其在骨骼肌组织中表达观察

目的基于GEO数据库数据筛选与肌少症发病相关的线粒体自噬靶点基因,并观察其在肌少症患者骨骼肌组织中的表达变化。方法从GEO数据库检索肌少症的基因图谱数据,筛选肌少症发病的差异表达基因。从GeneCard数据库中检索并收集线粒体自噬相关基因。使用“VennDiagram”包将肌少症发病的差异表达基因与线粒体自噬相关基因取交集,得到与肌少症发病相关的线粒体自噬差异表达基因。运用基因本体论(GO)和京都基因与基因百科全书(KEGG)通路富集分析与肌少症发病相关的线粒体自噬差异表达基因的生物学功能,通过Cytoscape软件筛选与肌少症发病相关的线粒体自噬靶点基因,观察GSE136344基因表达图谱中肌少症、健康对照者骨骼肌与肌少症发病相关的线粒体自噬靶点基因表达情况。结果得到与肌少症发病相关的线粒体自噬差异表达基因99个。与肌少症发病相关的线粒体自噬差异表达基因主要涉及神经变性途径-多种疾病信号通路、帕金森疾病信号通路、朊毒体病信号通路等;主要调控能量代谢、细胞呼吸、氧化磷酸化调节等生物学过程,主要定位于线粒体内膜、线粒体内部的大分子蛋白质复合物等,参与调节跨膜转运活性等分子功能。与肌少症发病相关的线粒体自噬靶点基因有线粒体内膜蛋白基因(IMMT)、动态蛋白1样蛋白基因(DNM1L)及ATP合酶F1亚基α基因(ATP5A1)等;与正常骨骼肌组织相比,肌少症患者骨骼肌组织中IMMT、DNM1L表达低(P均<0.05)。结论与肌少症发病相关的线粒体自噬靶点基因为IMMT、DNM1L。与肌少症发病相关的线粒体自噬靶点基因可通过影响神经变性途径-多种疾病信号通路、帕金森疾病信号通路及朊毒体病信号通路等,参与调控能量代谢、细胞呼吸、氧化磷酸化调节等生物学过程,参与肌少症的发病。肌少症患者骨骼肌组织中IMMT、DNM1L低表达。

宰后贮藏期间氧化磷酸化调控滩羊肉色泽稳定性的机制

为阐明滩羊肉贮藏期间氧化磷酸化(oxidative phosphorylation,OXPHOS)调控色泽稳定性的机制,以6月龄滩羊背最长肌为研究对象,测定其4℃贮藏0、4、8 d时内源抗氧化物酶体系活性与活性氧(reactive oxygen species,ROS)的含量;采用基于同位素标记相对和绝对定量(isobaric tags for relative andabsolutequantitation,iTRAQ)技术的蛋白质组学研究不同贮藏时间滩羊肉中蛋白质表达情况。结果表明:贮藏0~8 d,内源抗氧化物酶系活性整体呈下降趋势(P<0.05);ROS含量随贮藏时间的延长显著上升(P<0.05);采用iTRAQ技术分析鉴定出8个差异蛋白质为影响滩羊肉色泽稳定性的核心蛋白质,分别为ATP合酶亚基α(ATP synthase F1 subunit alpha,ATP5A1)、ATP合酶亚基O(ATP synthase peripheral stalk subunit OSCP,ATP5O)、ATP合酶亚基D(ATP synthase peripheral stalk subunit D,ATP5H)、细胞色素c氧化酶亚基(cytochrome c oxidase subunit,COX)2、COX5A、琥珀酸脱氢酶复合铁硫亚基B(succinate dehydrogenase complex iron sulfur subunit B,SDHB)、ADP/ATP转位酶(ADP/ATP translocase 1,SLC25A4)和细胞色素c(cytochrome c,CYCS),这些蛋白质主要通过能量代谢过程和氧化还原过程影响滩羊肉色泽稳定性。以上结果表明宰后贮藏期间滩羊肉中抗氧化体系的失效导致ROS含量不断积累,造成肌细胞线粒体完整性受到破坏,促使OXPHOS通路上ATP5A1、ATP5O、ATP5H、COX2、COX5A、SDHB、SLC25A4和CYCS等复合物亚基表达紊乱,引起线粒体复合物结构和功能发生改变,使OXPHOS进程受到阻碍,最终影响滩羊肉色泽稳定性。

宰后贮藏期间滩羊肉线粒体氧化磷酸化与色泽稳定性的关系

为阐明滩羊肉贮藏期间氧化磷酸化与色泽稳定性的关系,以6月龄滩羊背最长肌(longissimus dorsi)为研究对象,测定其4℃贮藏0、1、2、3、4、6、8 d时色泽稳定性与氧化磷酸化各指标的变化。主要研究结果如下:贮藏0~8 d,L^(*)、a^(*)、b^(*)值均呈先上升后下降趋势(P<0.05);R_(630)/R_(580)先上升后下降(P<0.05);高铁肌红蛋白(metmyoglobin,MetMb)相对含量呈下降后上升趋势(P<0.05);氧合肌红蛋白相对含量先上升后下降(P<0.05);脱氧肌红蛋白相对含量变化整体呈下降趋势(P<0.05);线粒体膜通透性显著增大(P<0.05);线粒体膜电位显著降低(P<0.05);线粒体复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性显著下降(P<0.05),复合物V活性显著升高(P<0.05);活性氧水平显著上升(P<0.05);超氧化物歧化酶、谷胱甘肽过氧化物酶活性显著下降(P<0.05);过氧化氢酶活性呈先上升后下降趋势(P<0.05);ATP、ADP、AMP含量显著下降(P<0.05)。各肌红蛋白衍生态相对含量、肉色指标与氧化磷酸化各指标互有显著相关性。以上结果表明,宰后贮藏期间滩羊肉中内源抗氧化酶系的失效导致肌细胞内产生过多的活性氧,致使线粒体完整性受到破坏,对线粒体氧化磷酸化相关酶活性产生影响,进而影响肌细胞中高铁肌红蛋白还原酶系统的活力,致使Met Mb无法被还原,最终导致滩羊肉色泽稳定性变差。