ABSTRACT
Abstract Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).
Resumo Bemisia tabaci é um complexo de espécies que causa danos a uma ampla gama de hospedeiros vegetais por meio de alimentação séria. Ele transmite vírus de plantas de diferentes grupos para importantes safras agrícolas. Algumas safras comerciais importantes do Paquistão são cana-de-açúcar, arroz, tabaco e óleo de semente. Apresenta alta variabilidade genética e é diferenciado em raças ou biótipos. Os biótipos são: biótipo Q, biótipo B, biótipo B2, biótipo M, biótipo L, biótipo A, biótipo H, biótipo C, biótipo K, biótipo N, biótipo R, biótipo E, biótipo P, biótipo J, biótipo S, biótipo AN . Embora o relatório atual baseado no estudo bayesiano das sequências de DNA do gene 1 da oxidase do citocromo mitocondrial (CO1) tenha classificado as diferentes populações de moscas-brancas em doze grupos genéticos, que são Mediterrâneo, África Subsaariana com folha de prata, Oceano Índico, Ásia II, Ásia I, Austrália, Novo Mundo, Itália, China, África Subsaariana sem folha prateada, Batata-doce Mediterrâneo / Ásia Menor / África e Uganda. Os begomovírus são o maior grupo de vírus transmitidos por B. tabaci e causam as principais doenças de culturas, como a doença do cacho do tomate e da pimenta-malagueta, doença do mosaico da mandioca, doença do mosaico amarelo de leguminosas e doença do enrolamento da folha do algodão. O principal objetivo do presente estudo é inculpar conhecimento sobre a diversidade genética da mosca-branca em campos de algodão em todo o Paquistão por meio da análise da sequência parcial de DNA do gene mitocondrial Citocromo Oxidase I (mtCO1).
ABSTRACT
Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).
Bemisia tabaci é um complexo de espécies que causa danos a uma ampla gama de hospedeiros vegetais por meio de alimentação séria. Ele transmite vírus de plantas de diferentes grupos para importantes safras agrícolas. Algumas safras comerciais importantes do Paquistão são cana-de-açúcar, arroz, tabaco e óleo de semente. Apresenta alta variabilidade genética e é diferenciado em raças ou biótipos. Os biótipos são: biótipo Q, biótipo B, biótipo B2, biótipo M, biótipo L, biótipo A, biótipo H, biótipo C, biótipo K, biótipo N, biótipo R, biótipo E, biótipo P, biótipo J, biótipo S, biótipo AN . Embora o relatório atual baseado no estudo bayesiano das sequências de DNA do gene 1 da oxidase do citocromo mitocondrial (CO1) tenha classificado as diferentes populações de moscas-brancas em doze grupos genéticos, que são Mediterrâneo, África Subsaariana com folha de prata, Oceano Índico, Ásia II, Ásia I, Austrália, Novo Mundo, Itália, China, África Subsaariana sem folha prateada, Batata-doce Mediterrâneo / Ásia Menor / África e Uganda. Os begomovírus são o maior grupo de vírus transmitidos por B. tabaci e causam as principais doenças de culturas, como a doença do cacho do tomate e da pimenta-malagueta, doença do mosaico da mandioca, doença do mosaico amarelo de leguminosas e doença do enrolamento da folha do algodão. O principal objetivo do presente estudo é inculpar conhecimento sobre a diversidade genética da mosca-branca em campos de algodão em todo o Paquistão por meio da análise da sequência parcial de DNA do gene mitocondrial Citocromo Oxidase I (mtCO1).
Subject(s)
Genetic Variation , Genes, Mitochondrial , Begomovirus , Agricultural PestsABSTRACT
Background Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. Results We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. Conclusion Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.
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Nonalcoholic fatty liver disease （NAFLD） has gradually become the main reason affecting human liver health， and many factors are involved in the development and progression of NAFLD. Mitochondria， as the “energy factory” of cells， plays an important role in maintaining normal physiological functions. Studies have shown that hepatic mitochondrial dysfunction promotes the development and progression of NAFLD. This article briefly introduces the latest research advances in the basic characteristics and physiological function of liver mitochondria and reviews new research findings in the association of mitochondrial dysfunction with obesity， simple fatty liver disease， and nonalcoholic steatohepatitis， in order to provide new ideas for the research on targeted mitochondrial therapy for NAFLD.
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ObjectiveTo investigate the possible mechanism of Guben Fangxiao Beverage （固本防哮饮） for the prevention and treatment of chronic airway inflammation during asthma remission. MethodsThirty-six female Balb/c mice were randomly divided into normal group， model group， low-， medium-， and high-dose of Guben Fangxiao Beverage group and montelukast sodium group， with 6 mice in each group. Except for the normal group， ovalbumin and respiratory syncytial virus were used in other groups to establish a mouse model of bronchial asthma in remission stage. After molding， the low-， medium-， and high-dose groups of Guben Fangxiao Beverage were respectively given 12， 24， and 36 g/（kg·d）， the montelukast sodium group was given montelukast sodium granule 2.6 mg/（kg·d）， and the mice in the normal group and model group were given 20 ml of double-distilled water， all by gavage， once a day for 28 days. The levels of interleukin 4 （IL-4） and interleukin 5 （IL-5） in the lung tissue of mice were detected； HE staining was used to observe the pathology of the lung tissue and to score the inflammation； DHE staining was used to observe the level of reactive oxygen species （ROS） in the lung tissue， and the activities of mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， Ⅳ， and Ⅴ in the lung tissue were detected； the levels of serum superoxide dismutase （SOD）， catalase （CAT）， malondialdehyde （MDA） and adenosine triphosphate （ATP） were detected； the protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase （p-AMPK）， nuclear factor erythroid 2-related factor 2 （Nrf2）， haem oxygenase 1 （HO-1） and cAMP responsive element binding protein （CREB） in the lung tissues of the model group were detected by Western blot. ResultsCompared with the normal group， the histopathological results of the lungs of mice in the model group showed an increase in inflammatory cells around the airways and an increase in inflammatory score； DHE staining showed an increase in the level of ROS， and an increase in the levels of IL-4 and IL-5 in the lung tissues； the levels of serum SOD， CAT， and ATP were reduced， and the level of MDA was elevated； the activities of the mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， Ⅳ， and Ⅴ of the lung tissues were reduced， and the activities of p-AMPK， Nrf2， CREB protein expression decreased （P＜0.05）. Compared with the model group， the lung tissue inflammatory cells and inflammation scores of mice in each Guben Fangxiao Beverage dose group and montelukast sodium group were reduced； the levels of ROS， IL-4 and IL-5 in the lung tissue were reduced； the levels of CAT and ATP in the serum increased， and the content of MDA was reduced； and the activities of mitochondrial respiratory chain complexes Ⅰ and Ⅱ， as well as the expression of CREB protein， were elevated in the lung tissue （P＜0.05）. Compared with the high-dose group， the MDA level of the medium-dose Guben Fangxiao Beverage group decreased （P＜0.05）. The activity of mitochondrial respiratory chain complex V in the medium-dose group was higher than that in the montelukast sodium group， and the activity of mitochondrial respiratory chain complex Ⅳ in the medium- and high-dose groups was higher than that in the low-dose group （P＜0.05）. ConclusionGuben Fangxiao Beverage can inhibit oxidative stress and improve mitochondrial function to relieve chronic airway inflammation in bronchial asthma model mice during asthma remission， and its mechanism may be related to the activation of AMPK/Nrf2/HO-1 signaling pathway.
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ObjectiveTo study the effect and mechanism of Yixintai on mitochondrial fission proteins in the rat model of chronic heart failure. MethodTen of 60 SD rats were randomly selected as the sham operation group， and the remaining 50 rats were subjected to ligation of the left anterior descending coronary artery for the modeling of heart failure post myocardial infarction. The successfully modeled rats were randomized into model， low-， medium-， and high-dose （1.4， 2.8， and 5.6 g·kg-1， respectively） Yixintai， and trimetazidine （10 mg·kg-1） groups. The rats were administrated with corresponding doses of drugs by gavage， and the rats in the model group and sham operation group were given an equal volume of normal saline by gavage for 28 consecutive days. Enzyme-linked immunosorbent assay （ELISA） was then employed to measure the levels of amino-terminal pro-B-type natriuretic peptide （NT-pro BNP）， B-type natriuretic peptide （BNP）， and adenosine triphosphate （ATP） in the serum. Color Doppler ultrasound imaging was conducted to examine the cardiac function indicators. Hematoxylin-eosin staining and Masson staining were conducted to observe the pathological changes in the heart， and Image J was used to calculate collagen volume fraction （CVF）. Transmission electron microscopy was employed to observe the ultrastructural changes of myocardial cells. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to measure the apoptosis rate of myocardial cells. Western blot was employed to determine the protein levels of mitochondrial fission protein 1 （Fis1） and mitochondrial fission factor （Mff） in the outer mitochondrial membrane of the myocardial tissue. ResultCompared with the sham operation group， the model group showed elevated levels of NT-pro BNP and BNP in the serum， decreased ATP content， left ventricular ejection fraction （LVEF）， and left ventricular fraction shortening （LVFS）， increased left ventricular end-diastolic diameter （LVIDd） and left ventricular end-systolic diameter （LVIDs）， disarrangement of myocardial cells， inflammatory cell infiltration， increased collagen fibers and CVF， damaged myocardium and mitochondria， and increased apoptosis rate of myocardial cells， and up-regulated expression of Fis1 and Mff in the cardiac tissue （P<0.01）. Compared with the model group， different doses of Yixintai and trimetazidine lowered the serum levels of NT-pro BNP and BNP （P<0.05）， increased the ATP content （P<0.05）， increased LVEF and LVFS （P<0.01）， decreased LVIDd and LVIDs （P<0.01）. Moreover， the drugs alleviated the myocardial inflammatory damage and fibrosis， reduced CVF （P<0.01）， repaired the myocardial mitochondrial structure， and decreased the apoptosis rate of myocardial cells （P<0.01）. Medium- and high-dose Yixintai and trimetazidine down-regulated the expression of Fis1 and Mff in the myocardial tissue （P<0.05）. ConclusionYixintai can improve mitochondrial structure， reduce myocardial cell apoptosis， and improve cardiac function by inhibiting the expression of Fis1 and Mff in the myocardial tissue.
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Objective To investigate the role and mechanism of spliced X-box binding protein 1 (XBP1s) in the senescence of primary renal tubular epithelial cells induced by hypoxia/reoxygenation (H/R). Methods Primary renal tubular epithelial cells were divided into the normal control group (NC group), H/R group, empty adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), empty adenovirus+H/R treatment group (Ad-shNC+H/R group) and targeted silencing XBP1s adenovirus+H/R treatment group (Ad-shXBP1s +H/R group), respectively. The expression levels of XBP1s in the NC, H/R, Ad-shNC and Ad-shXBP1s groups were measured. The number of cells stained with β-galactosidase, the expression levels of cell aging markers including p53, p21 and γH2AX, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined in the Ad-shNC, Ad-shNC+H/R and Ad-shXBP1s+H/R groups. Chromatin immunoprecipitation was employed to verify Sirtuin 3 (Sirt3) of XBP1s transcription regulation, and the expression levels of Sirt3 and downstream SOD2 after down-regulation of XBP1s were detected. Mitochondrial reactive oxygen species (mtROS) were detected by flow cytometry. Results Compared with the NC group, the expression level of XBP1s was up-regulated in the H/R group. Compared with the Ad-shNC group, the expression level of XBP1s was down-regulated in the Ad-shXBP1s group (both P<0.001). Compared with the Ad-shNC group, the number of cells stained with β-galactosidase was increased, the expression levels of p53, p21 and γH2AX were up-regulated, the levels of ROS, MDA and mtROS were increased, the SOD activity was decreased, the expression level of Sirt3 was down-regulated, and the ratio of Ac-SOD2/SOD2 was increased in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the number of cells stained with β-galactosidase was decreased, the expression levels of p53, p21 and γH2AX were down-regulated, the levels of ROS, MDA and mtROS were decreased, the SOD activity was increased, the expression level of Sirt3 was up-regulated and the ratio of Ac-SOD2/SOD2 was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Conclusions Down-regulation of XBP1s may ameliorate the senescence of primary renal tubular epithelial cells induced by H/R, which probably plays a role through the Sirt3/SOD2/mtROS signaling pathway.
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Ginseng Radix et Rhizoma（GRR） has the function of replenishing vital energy and can lighten the body and prolong the life when taken for a long time， which is suitable for the development of anti-aging products， so this paper intends to sort out the progress of anti-aging research on GRR. After combing， the results of modern studies have shown that a variety of components in GRR have anti-aging effect， which can prolong the lifespan of aging animal models， as well as delay the aging of various systems. The anti-aging mechanisms mainly include anti-cellular senescence， anti-oxidative stress， inhibiting telomere shortening， maintaining mitochondrial homeostasis and so on. The anti-aging ingredients of GRR involved in the researches mainly include ginsenoside Rg1 and ginsenoside Rb1， in addition， ginsenoside Rg3， ginsenoside Rd， ginsenoside Rg2， ginsenoside Re， ginsenoside Rb2， oligosaccharides of GRR， polysaccharides of GRR， water extract of GRR， total saponins of Panax ginseng stems and leaves are also included. Therefore， under current background of population aging， the in-depth development of GRR and its transformation into anti-aging products are of great significance for delaying senility and improving the health conditions of aging population.
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ObjectiveTo investigate the protective effect of the extract of Liuwei Dihuangwan （LW） on mitochondrial damage in the Alzheimer's disease （AD） model of Caenorhabditis elegans （C. elegans）. MethodC. elegans transfected with human β-amyloid protein （Aβ） 1-42 gene was used as an AD model. The rats were divided into blank group， model group， metformin group （50 mmol·L-1）， and low， medium， and high dose （1.04， 2.08， 4.16 g·kg-1） LW groups. Behavioral methods were used to observe the sensitivity of 5-hydroxytryptamine （5-HT） in nematodes. Western blot was used to detect the expression of Aβ in nematodes. Total ATP content in nematodes was detected by the adenine nucleoside triphosphate （ATP） kit， and mitochondrial membrane potential was detected by the JC-1 method. In addition， the mRNA expression of Aβ expression gene （Amy-1）， superoxide dismutase-1 （SOD-1）， mitochondrial transcription factor A homologous gene-5 （HMG-5）， mitochondrial power-associated protein 1 （DRP1）， and mitochondrial mitoprotein 1 （FIS1） was detected by real-time fluorescence quantitative polymerase chain reaction （RT-PCR）. ResultThe extract of LW could reduce the hypersensitivity of the AD model of nematodes to exogenous 5-HT （P<0.05） and delay the AD-like pathological characteristics of hypersensitivity to exogenous 5-HT caused by toxicity from overexpression of Aβ in neurons of the AD model of nematodes. Compared with the blank group， in the model group， the mRNA expression of Aβ protein and Amy-1 increased （P<0.01）， and the mRNA expression of SOD-1 and HMG-5 decreased （P<0.01）. The mRNA expression of DRP1 and FIS1 increased （P<0.01）， and the level of mitochondrial membrane potential decreased （P<0.05）. The content of ATP decreased （P<0.01）. Compared with the model group， in the positive medicine group and medium and high dose LW groups， the mRNA expression of Aβ protein and Amy-1 decreased （P<0.05，P<0.01）， and the mRNA expression of SOD-1 and HMG-5 increased （P<0.01）. The mRNA expression of DRP1 decreased （P<0.05，P<0.01）， and that of FIS1 decreased （P<0.01）. The level of mitochondrial membrane potential increased （P<0.01）， and the content of ATP increased （P<0.05，P<0.01）. ConclusionThe extract of LW may enhance the antioxidant ability of mitochondria， protect mitochondrial DNA， reduce the fragmentation of mitochondrial division， repair the damaged mitochondria， adjust the mitochondrial membrane potential， restore the level of neuronal ATP， and reduce the neuronal damage caused by Aβ deposition.
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ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg-1） and Captopril group （0.002 57 g·kg-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.
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Diabetic peripheral neuropathy（DPN） is a neurodegenerative disease of diabetes mellitus involving peripheral nervous system damage， which is characterized by axonal degenerative necrosis， Schwann cell apoptosis and demyelination of nerve myelin sheath as the main pathological features， this disease is highly prevalent and is a major cause of disability in diabetic patients. Currently， the pathogenesis of DPN may be related to oxidative stress， inflammatory response， metabolic abnormality， and microcirculation disorder. The treatment of DPN in modern medicine mainly starts from controlling blood glucose， nourishing nerves and improving microcirculation， which can only alleviate the clinical symptoms of patients， and it is difficult to fundamentally improve the pathological damage of peripheral nerves. Mitochondrial quality control refers to the physiological mechanisms that can maintain the morphology and functional homeostasis of mitochondria， including mitochondrial biogenesis， mitochondrial dynamics， mitochondrial oxidative stress and mitochondrial autophagy， and abnormal changes of which may cause damage to peripheral nerves. After reviewing the literature， it was found that traditional Chinese medicine（TCM） can improve the low level of mitochondrial biogenesis in DPN， maintain the balance of mitochondrial dynamics， inhibit mitochondrial oxidative stress and mitochondrial autophagy， and delay apoptosis of Schwann cells and neural axon damage， which has obvious effects on the treatment of DPN. With the deepening of research， mitochondrial quality control may become one of the potential targets for the research of new anti-DPN drugs， therefore， this paper summarized the research progress of TCM in treating DPN based on four aspects of mitochondrial quality control， with the aim of providing a theoretical research basis for the discovery of new drugs.
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ObjectiveTo explore the mechanism of Buzhong Yiqitang-containing serum in alleviating the cisplatin resistance in human non-small cell lung cancer （A549/DDP） cells via regulating the nuclear factor E2-related factor 2 （Nrf2）/reactive oxygen species （ROS） signaling pathway. MethodThe serum containing Buzhong Yiqitang was prepared and A549/DDP cells were cultured and randomly grouped： blank （10% blank serum）， cisplatin （10% blank serum+20 mg·L-1 cisplatin）， Buzhong Yiqitang （10% Buzhong Yiqitang-containing serum+20 mg·L-1 cisplatin）， ML385 （10% blank serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， Buzhong Yiqitang+ML385 （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， tertiary butylhydroquinone （TBHQ） （10% blank serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）， and Buzhong Yiqitang+TBHQ （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）. The median inhibitory concentration （IC50） of cisplatin in each group was determined by the cell counting kit-8 （CCK-8） method and the resistance index （RI） was calculated. The apoptosis rate was detected by flow cytometry. The ROS content of each group was determined with the DCFH-DA fluorescence probe. Western blot was employed to determine the protein levels of Nrf2， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， cytochrome C （Cyt C）， and B-cell lymphoma-2 （Bcl-2）. ResultCompared with those in the cisplatin group， the IC50 and RI of A549/DDP cells to cisplatin in Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups decreased （P˂0.05）. Compared with the blank group， the cisplatin， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups showed increased apoptosis rate of A549/DDP cells （P˂0.05）. Compared with the blank group， cisplatin promoted the expression of Nrf2 （P˂0.05）. Compared with the cisplatin group， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 inhibited the expression of Nrf2 （P<0.05）， elevated the ROS level （P˂0.05）， up-regulated the protein levels of cleaved Caspase-3 and Cyt C， and down-regulated the protein level of Bcl-2 （P<0.05）， which were the most significant in the Buzhong Yiqitang+ML385 group. Compared with the cisplatin group， the TBHQ group showed increased IC50 and RI of cisplatin （P<0.05）， decreased apoptosis rate of A549/DDP cells （P<0.05）， up-regulated protein levels of Nrf2 and Bcl-2 （P<0.05）， lowered level of ROS （P˂0.05）， and down-regulated protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. Compared with the TBHQ group， Buzhong Yiqitang+TBHQ decreased the IC50 and RI of cisplatin in A549/DDP cells （P<0.05）， increased the apoptosis rate （P<0.05）， down-regulated the protein levels of Nrf2 and Bcl-2 （P<0.05）， increased ROS （P˂0.05）， and up-regulated the protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. ConclusionBuzhong Yiqitang induced apoptosis by inhibiting Nrf2/ROS pathway to alleviate cisplatin resistance in A549/DDP cells.
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Mitochondrial dynamics are a contraversal issue in hepatocellular carcinoma. The present study tries to illustrate the role of mitochondrial dynamics proteins (mitofusin-2 (Mfn2) and YME1L) in hepatocarcinogenesis. Five groups were used: the control group and three HCC groups (after 8, 16, and 24 weeks from DENA induction). The last group was treated with Sorafenib (SP) (10 mg/kg), via oral gavage for 4 weeks after cancer induction. This study revealed that Mfn-2 was downregulated and YME1l was overexpressed in different HCC groups. This dysregulation of mitochondrial dynamics proteins was associated with high hepatic levels of cyclin D1, MMP-9, and MDA and overexpression of ki67 as well as decreasing the hepatic expression of tissue inhibitor of matrix metalloproteinase-3 (Timp-3) and Bax. To confirm the possible role of Mfn2 and YME1L in HCC, we assessed the effect of sorafenib on these parameters and its related HCC characteristics. Sorafenib corrected the level of Mfn2 and YME1L and decreased tumor cell proliferation as well. We also elucidated that mitochondrial dynamics proteins (Mfn2 and YME1L) could be a good therapeutic target for HCC.
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Objective:To investigate the difference in the radiation sensitivity of hematopoietic stem and progenitor cells (HSPCs) derived from fetal liver and bone marrow.Methods:HSPCs from fetal liver of 14.5 d embryo or bone marrow of 8 week-old mice were isolated to receive a single dose of 5 or 10 Gy irradiation in vitro using a 60Co irradiator. Twelve hours later, the cell apoptosis, mitochondrial reactive oxygen species (ROS) level, colony formation ability and DNA damage in HSPCs were detected. Freshly isolated HSPCs were injected into lethally irradiated CD45.1 + C57BL/6J mice (4.5 Gy+ 5 Gy with an interval of 30 min) Chimerism rate, lineage constitution, and cell cycle were analyzed 12 weeks after transplantation. Results:Compared with bone marrow HSPCs after irradiation, the percentage of apoptosis in fetal liver HSPCs was significantly higher ( t=16.21, 12.27, P<0.05), the level of ROS was dramatically elevated ( t=68.72, 18.89, P<0.05). At 10 Gy, fetal liver HSPCs could not form colonies at all ( t=12.41, 15.67, 9.46, P<0.05). γ-H2AX immunofluorescence staining showed that the DNA damage of fetal liver HSPCs was more severe after irradiation, and the number of Foci formed was significantly higher than that of bone marrow HSPCs ( t=2.27, 2.03, P< 0.05), which indicated that fetal liver HSPCs were more sensitive to radiation. The chimerism rate of transplanted fetal liver HSPCs was lower than that of bone marrow cells ( t=5.84, P<0.05) with a higher proportion of myeloid lineage, suggesting that fetal liver HSPCs had lower in vivo reconstitution capacity than bone marrow HSPCs and were more prone to myeloid differentiation. The cell cycle of bone marrow HSPCs from transplanted chimeric mice was examined, and the proportion of S-phase was significantly higher in the fetal liver group than that in the bone marrow group ( t=2.89, P<0.05). Mitochondrial stress results showed that fetal liver HSPCs had higher basal respiratory capacity ( t=39.19, P<0.05), proton leakage ( t=6.64, P<0.05), ATP production ( t=9.33, P<0.05), and coupling efficiency ( t=7.10, P<0.05) than bone marrow c-Kit + cells, while respiratory reserve capacity ( t=5.53, P< 0.05) was lower than that of bone marrow c-Kit + cells. Conclusions:HSPCs derived from fetal liver display higher radiosensitivty compared with bone marrow HSPCs, laying the foundation for an in-depth illustration of the effects of radiation on hematopoietic stem cells at different developmental stages.
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Objective:To explore the protective effect of AGK2, a selective inhibitor of sirtuin 2 (SIRT2), on the mitochondria of L02 hepatocytes induced by thioacetamide (TAA) and its related mechanism.Methods:Human-derived hepatocyte line L02 cells were cultured in vitro. Different concentrations of SIRT2 inhibitor AGK2 were used as intervention drugs. Cell counting kit-8 (CCK8) was used to detect the effects of different concentrations of AGK2 on the activity of L02 cells, and the appropriate concentration was selected as the AGK2 intervention group. The normal group was not given any drug intervention. The model group was given 90 mmol/L TAA for modeling. Low, medium and high dose AGK2 groups were added with 1, 2 and 4 μmol/L AGK2, respectively 2 h before modeling. CCK8 was used to detect cell activity in each group. Morphological changes of cells were observed under inverted light microscope. The relative protein expression levels of isocitrate dehydrogenase (IDH1), malate dehydrogenase (MDH1), SIRT2 and fission protein 1 homologue (FIS1) were detected by Western blot. The expression of SIRT2 in cells of each group was observed by confocal laser scanning microscope. The mitochondrial membrane potential of cells in each group was observed under a fluorescence microscope. Results:When AGK2 concentration was 1, 2 and 4 μmol/L, the survival rate of cells were 98.05%, 95.76% and 91.65%, respectively, with no statistical significance compared with normal group (all P>0.05). When AGK2 concentration was 8, 16, 32, 64, 128 μmol/L, the cell survival rate was significantly decreased compared with normal group (all P<0.05). Compared with the model group, the L02 cells in low, medium and high AGK2 groups had better activity and adherence, and the floating cells were significantly reduced. The higher the concentration of AGK2, the better the cell activity and adherence, and the less floating cells. Compared with the model group, the red fluorescence of L02 cells in AGK2 group was enhanced, while the green fluorescence was weakened. The higher the AGK2 concentration was, the stronger the red fluorescence was, and the weaker the green fluorescence was. Compared with the model group, the fluorescence of SIRT2 in L02 cells of low, medium and high AGK2 groups was weakened, and the higher the concentration of AGK2, the weaker the fluorescence of SIRT2. The protein expressions of IDH1 and MDH1 in L02 cells of low, medium and high AGK2 groups were significantly higher than those of model group (all P<0.05), and were positively correlated with the concentration of AGK2 ( r=0.818, P<0.05; r=0.960, P<0.05); the protein expressions of SIRT2 and FIS1 were significantly lower than those of the model group (all P<0.05), and were negatively correlated with the concentration of AGK2 ( r=-0.992, P<0.05; r=-0.998, P<0.05). Conclusions:AGK2 can reduce the mitochondrial membrane potential stimulated by TAA in L02 cells, increase the protein expression of IDH1 and MDH1, and inhibit the protein expression of SIRT2 and FIS1 in L02 cells in a dose-dependent manner.
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OBJECTIVE Mitochondria plays a crucial role in cellular homeostasis by regulating various pro-cesses,including calcium signaling and mitophagy.This study aimed to explore the involvement of prohibitin 2(PHB2),an inner mitochondrial membrane protein,in the modulation of mitochondrial calcium dynamics and mitoph-agy.METHODS HEK293T cells were used as the experi-mental cells and were divided into control,PHB2 knock-down,and PHB2 overexpression groups.To evaluate mitochondrial calcium dynamics,Rhod-2 AM and Mito-Tracker Green fluorescence dyesrhod-2 staining and laser confocal microscopy were employed to visualize mito-chondrial calcium imaging.Additionally,Green-5N was utilized to measure the rate of mitochondrial calcium uptake.The mitochondrial membrane potential was assessed using JC-10 staining and laser confocal micros-copy,while cellular ATP levels were determined using ATP assay kits.Furthermore,mitochondrial autophagy was induced by treatment with CCCP,and the expression lev-els of TOM20,LC3,and PARKIN,key mitophagy-related proteins,were analyzed using Western blotting.RESULTS The results demonstrated that compared to the control group,the overexpression of PHB2 increased mitochon-drial calcium concentration,mitochondrial calcium uptake rate,ATP level and expression levels of LC3 and PAR-KIN,but decreased mitochondrial membrane potential and TOM20 expression.In contrast,PHB2 knockdown reduced mitochondrial calcium concentration,ATP level and expression levels of LC3 and PARKIN,but elevated mitochondrial membrane potential,and TOM20 expres-sion.CONCLUSION This study provides evidence that PHB2 plays a vital role in regulating mitochondrial calci-um dynamics,which in turn influences mitochondrial func-tion and modulates mitochondrial autophagy.These find-ings contribute to our understanding of the molecular mechanisms underlying the interplay between PHB2,mitochondrial calcium signaling,and mitophagy.
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OBJECTIVE The abnormal amyloid-β(Aβ)and oxidative stress assiociated with the progression of Alzheimer disease(AD).Quercetin has been reported to possess antioxidant and anti-inflammatory properties in neurodegenerative disorders.In this present study,we designed to characterize the mechanisms by which quer-cetin exerts neuroprotective effects in murine neuroblas-toma N2a cells stably expressing human Swedish mutant amyloid precursor protein(N2a/APP).METHODS N2a/APP cells were treated with quercetin at concentrations of 10,20 and 50 μ mol·L-1 for 24 h.Cell viability was examined with CCK-8 assays.The protein levels of ERK1/2 and Akt were detected by Western blotting.Intra-cellular reactive oxygen species(ROS)was detected by a fluorescent probe 2,7-dichlorofluorescein diacetate(DCFH-DA).The mitochondrial membrane potential(Δψ m)in N2a/APP cells was detected by using JC-1 staining method.Immunofluorescence was used to detect the generation of 8-hydroxy-2′-deoxyguanosine(8-OHdG)and 4-hydroxynonenal(4-HNE).RESULTS Quercetin attenuated the enhancement of p-ERK1/2,reductions of p-Akt,and decreased levels of APP expression.More-over,quercetin alleviated loss of mitochondria membrane potential(MMP)since it attenuates these oxidative stress,as reflected in the levels of ROS,4-HNE and 8-OHdG,was elevated in N2a/APP cells and these effects were again ameliorated by quercetin.CONCLUSION Neuroprotection by quercetin in N2a/APP cells involves normalizing the impaired mitochondrial function and reducing oxidative stress via inactivation of the ERK1/2 and activation of the Akt pathways.
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Objective:To explore the protective effects and mechanisms of L-carnitine (LCAR) on cognitive dysfunction in chronic cerebral hypoperfusion rats.Methods:Totally 90 SD male rats (SPF class) aged 3-4 months were divided into four groups according to random number talbe: sham operated control group (SHAM group, n=15), sham operated with L-carnitine treatment group (LCAR group, n=25), 2-vessel occlusion group (2VO group, n=25), and 2-vessel occlusion with L-carnitine treatment group (2VO+ LCAR group, n=25). The chronic cerebral hypoperfusion model was established by bilateral common carotid artery ligation, and the carotid arteries from SHAM group and LCAR group were only separated without ligation.L-carnitine was administered intraperitoneally (300 mg·kg -1·d -1) for 30 days after surgery in the LCAR and 2VO+ LCAR groups.After 30 days of L-carnitine intervention, Morris water maze was performed to test the spatial cognitive function of the rats, the ATP level of hippocampal tissue was detected by chemiluminescence, the mitochondrial structure and synaptic structure of hippocampal neurons were observed by transmission electron microscopy, the degree of mitochondrial damage was scored, the vesicle density was counted and measured, the level of N-methyl-D-aspartate receptor subunit 2A or 2B(NR2A/B) and postsynaptic density 95(PSD95) in hippocampal tissue were detected by Western blot.The expression and distribution levels of transcription factor cAMP response element-binding protein(CREB) in brain tissues were observed by immunofluorescence.SPSS 16.0 software was used for statistical analysis.The escape latency data of repeated learning training in Morris water maze was conducted by repetitive measurement ANOVA, while other data were adopted by one-way ANOVA, and Dunnett's t test was used for further pairwise comparison. Results:(1)Morris water maze results showed that the time and group interaction of escape latency was not significant among the 4 groups of rats ( F=1.4, P>0.05), but the time main effect and group main effect were significant( F=21.6, 15.2, both P<0.05). Morris water maze results showed that platform position learning from 3rd to 7th day, the escape latencies in 2VO group were longer than those in SHAM group and 2VO+ LCAR group (all P<0.05). The results of short-term memory showed that the escape latency in 2VO group was longer than those in SHAM group and 2VO+ LCAR group (all P<0.05). Meanwhile, the retention time and crossing times in the platform area of 2VO group were less than those in SHAM group and 2VO+ LCAR group (all P<0.05). (2) The absolute and relative levels of ATP in hippocampus showed that the difference among the 4 groups were statistically significant ( F=14.6, 13.2, both P<0.05). ATP level of hippocampus in 2VO group was lower than those in SHAM group and 2VO+ LCAR group (both P<0.05). Electron microscopic observation of mitochondrial morphology showed that the Flameng score of mitochondrial damage in the hippocampus of rats in 2VO group (2.82±0.17) was higher than those in SHAM group (0.25±0.07) and 2VO+ LCAR group (1.76±0.09) (both P<0.05). (3) The density of synaptic vesicles in the hippocampus of rats in 2VO group ((289.09±22.41)/μm 2)was lower than those in SHAM group ((497.49±28.89)/μm 2)and 2VO+ LCAR group ((401.23±45.09)/μm 2) (both P<0.01). Western blot results showed that the relative levels of synaptic proteins NR2A/B, PSD95 and CREB in 2VO group were lower than those in SHAM group and 2VO+ LCAR group (all P<0.05). Immunofluorescence results showed that the relative level of CREB expression in hippocampal subregions and cortex in 2VO group was lower than those in SHAM group and 2VO+ LCAR group (both P<0.01). Conclusion:L-carnitine can improve spatial learning and memory dysfunction in rats with chronic cerebral hypoperfusion, which are related with promoting ATP production and protecting mitochondrial morphology, and promoting synaptic vesicle synthesis and synaptic protein expression.
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Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease(PD).However,the mechanisms underlying this association remain unclear.In the present study,we found that high glucose(HG)levels in the cerebrospinal fluid(CSF)of diabetic rats might enhance the effect of a subthreshold dose of the neurotoxin 6-hydroxydopamine(6-OHDA)on the development of motor disorders,and the damage to the nigrostriatal dopaminergic neuronal pathway.In vitro,HG promoted the 6-OHDA-induced apoptosis in PC12 cells differentiated to neurons with nerve growth factor(NGF)(NGF-PC12).Metabolomics showed that HG promoted hyperglycolysis in neurons and impaired tricarboxylic acid cycle(TCA cycle)activity,which was closely related to abnormal mito-chondrial fusion,thus resulting in mitochondrial loss.Interestingly,HG-induced upregulation of pyruvate kinase M2(PKM2)combined with 6-OHDA exposure not only mediated glycolysis but also promoted abnormal mitochondrial fusion by upregulating the expression of MFN2 in NGF-PC12 cells.In addition,we found that PKM2 knockdown rescued the abnormal mitochondrial fusion and cell apoptosis induced by HG+6-OHDA.Furthermore,we found that shikonin(SK),an inhibitor of PKM2,restored the mito-chondrial number,promoted TCA cycle activity,reversed hyperglycolysis,enhanced the tolerance of cultured neurons to 6-OHDA,and reduced the risk of PD in diabetic rats.Overall,our results indicate that diabetes promotes hyperglycolysis and abnormal mitochondrial fusion in neurons through the upre-gulation of PKM2,leading to an increase in the vulnerability of dopaminergic neurons to 6-OHDA.Thus,the inhibition of PKM2 and restoration of mitochondrial metabolic homeostasis/pathways may prevent the occurrence and development of diabetic PD.
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@#Abstract： Objective To investigate the clinical characteristics and pathogenic genetic mutation of a case with encephalopathy due to defective mitochondrial and peroxisomal fission-1 (EMPF1). Methods The clinical data and genetic test results of a patient with EMPF1 admitted to the Department of Pediatrics, the Affiliated Hospital of Xiangya Medical College of Central South University in August 2020 were retrospectively analyzed. Results An 8-year-old girl, her main clinical features were developmental regression, microcephaly, hypotonia, refractory epilepsy, cranial MRI suggesting brain atrophy and abnormal signals in the right temporal-occipital-parietal cortex, aEEG showing slow wave discharge in the right hemisphere; Whole-exome sequencing of families suggested that the child had a heterozygous missense variant at the c.1040C>G site in the DNM1L gene and the verification results by Sanger sequencing showed that her parents had no variant in this site, which was a novel mutation in accordance with autosomal dominant inheritance; bioinformatics analysis predicted that the mutation was pathogenic. After 2 years of outpatient follow-up, the patient's condition was stable after mitochondrial cocktail therapy and antiepileptic drugs, no epileptic seizure occurred in the past year, mental state and swallowing function improved, and she could be fed orally with occasional nausea and vomiting. Conclusions The main clinical manifestations of EMPF1 are psychomotor developmental delay or regression, dystonia, limb paralysis, epilepsy and so on. According to the clinical phenotype and genetic test results, the rare disease can be diagnosed early.