Intrauterine growth restriction (IUGR) leads to adult obesity, cardiovascular disease, and non-alcoholic fatty liver disease/steatohepatitis. Animal models have shown that combined intrauterine and early postnatal calorie restriction (IPCR) ameliorates these sequelae in adult life. The mechanism by which IPCR protects against adult onset disease is not understood. Autophagy, a lysosomal degradative process, recycles cellular constituents and eliminates damaged organelles, proteins, and oxidants. In this study, we hypothesized that IPCR could regulate autophagy in the liver of male rat offspring. At birth (d1) of male IUGR rat offspring and on day 21 (p21) of life, IPCR male rat offspring had a profound decrease in hepatic autophagy in all three stages of development: initiation, elongation, and maturation. However, upon receiving a normal diet ad-lib throughout adulthood, aged IPCR rats (day 450 of life (p450)), had increased hepatic autophagy, in direct contrast to what was seen in early life. The decreased autophagy at d21 led to the accumulation of ubiquitinated proteins and lipid oxidative products, whereas the increased autophagy in late life had the opposite effect. Oxidized lipids were unchanged at d1 by IUGR treatment indicating that decreased autophagy precedes oxidative stress in early life. When cellular signaling pathways regulating autophagy were examined, the 5' adenosine monophosphate-activated protein kinase pathway (AMPK), and not endoplasmic stress pathways, was found to be altered, suggesting that autophagy is regulated through AMPK signaling pathway in IPCR rats. Taken together, this study reveals that the perinatal nutritional status establishes a nutritionally sensitive memory that enhances hepatic autophagy in late life, a process that perhaps acts as a protective mechanism to limited nutrition.
Cadmium (Cd) is a highly ubiquitous detrimental metal in the environment. It is a well-known inducer of tumorigenesis, but the mechanism is not clear. In our previous study, we found that ROS-dependent Atg4B upregulation mediated Cd-induced autophagy and autophagy played an important role in Cd-induced proliferation and invasion in A549 cells. In this study, we found that Cd induced both apoptosis and autophagy in A549 cells, and apoptosis preceded autophagy. Z-VAD-FMK repressed Cd-induced LC3 and Beclin1, indicating that apoptosis was essential for Cd-induced autophagy. 3MA destroyed the recovery of mitochondrial membrane potential and increased Cd-induced CL-CASP9 and CL-CASP3 expression, suggesting that Cd-induced autophagy prevented A549 cells from apoptosis. Further study showed that Atg4B upregulation was mediated by mitochondrial dysfunction and conversely affected mitochondrial function by decreasing Bcl-2 protein expression and its localization in mitochondria, and played an important role in Cd-induced apoptosis. Moreover, Bcl-2 was involved in Cd-induced autophagy. Co-IP assay showed that Atg4B could directly bind to Bcl-2, and consequently promote disassociation of Bcl-2-Beclin1 and released autophagic protein Beclin1 to activate autophagic pathway. Taken together, our results demonstrated that the interaction of Atg4B and Bcl-2 might play an important role in Cdinduced crosstalk between apoptosis and autophagy through disassociation of Bcl-2-Beclin1. Cd-induced autophagy is apoptosis-dependent and prevents apoptotic cell death to ensure the growth and proliferation of A549 cells.
Biomolecule (lipid and protein) oxidation products formed in plant cells exposed to photooxidative stress play a crucial role in the retrograde signaling and oxidative damage. The oxidation of biomolecules initiated by reactive oxygen species is associated with formation of organic (alkyl, peroxyl and alkoxyl) radicals. Currently, there is no selective and sensitive technique available for the detection of organic radicals in plant cells. Here, based on the analogy with animal cells, immuno-spin trapping using spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to image organic radicals in Arabidopsis leaves exposed to high light. Using antibody raised against the DMPO nitrone adduct conjugated with the fluorescein isothiocyanate, organic radicals were imaged by confocal laser scanning microscopy. Organic radicals are formed predominantly in the chloroplasts located at the periphery of the cells and distributed uniformly throughout the grana stack. Characterization of protein radicals by standard immunological techniques using anti-DMPO antibody shows protein bands with apparent molecular weights of 32 and 34 kDa assigned to D1 and D2 proteins and two protein bands below the D1/ D2 band with apparent molecular weights of 23 and 18 kDa and four protein bands above the D1/ D2 band with apparent molecular weights of 41, 43, 55 and 68 kDa. In summary, imaging of organic radicals by immuno-spin trapping represents selective and sensitive technique for the detection of organic radicals that might help to clarify mechanistic aspects on the role of organic radicals in the retrograde signaling and oxidative damage in plant cell.
Brain-derived neurotrophic factor (BDNF)/tropomyosin-related receptor kinase B (TrkB) pathway has been revealed as a novel therapeutic target for several neurological diseases. Recently, small-molecule TrkB agonist 7,8dihydroxyflavone (7,8-DHF) has received considerable attention as a novel potential candidate for the treatment of various BDNF-implicated human disorders. However, its roles in cardiac diseases are not fully understood. Here, the present study aimed to clarify the effects and mechanisms of 7,8-DHF on doxorubicin (Dox)-induced cardiotoxicity. Kunming mice and H9c2 cells were employed to investigate the functional role of 7,8-DHF both in vivo and in vitro. 7,8-DHF markedly increased cell viability and reduced cell death of Dox-treated cells. Meanwhile, 7,8-DHF significantly increased mitochondrial respiration, membrane potential, and optic atrophy 1 (OPA1) protein expression. 7,8-DHF improved cardiac function and attenuated cardiac injury in Dox mice model. Expression of AMP-activated protein kinase (AMPK) and signal transducers and activators of transcription 3 (STAT3) was restored by 7,8-DHF. Furthermore, the protective role of 7,8-DHF was abolished by ANA-12 (a specific antagonist of TrkB). In elucidating the molecular mechanism, the phosphorylation of Akt was significantly increased while extracellular regulated protein kinase (ERK) was decreased after 7,8-DHF treatment. The regulatory effects of 7,8-DHF on STAT3 and AMPK was reversed by Akt inhibitor. In summary, 7,8-DHF attenuated Dox-induced cardiotoxicity by activating Akt and increasing mitochondrial oxidative phosphorylation and thereby regulating STAT3, AMPK, and ERK signals. The present study enhanced current understanding of TrkB receptor in the cardiovascular system and provided a novel target for prevention and treatment of heart diseases.
An increasing number of studies have shown that air pollution containing particulate matter (PM) = 2.5 mu m (PM2.5) plays a significant role in the development of metabolic disorder and other chronic diseases. Inflammation and oxidative stress caused by metabolic syndrome are widely determined to be critical factors in the development of nonalcoholic fatty liver disease (NAFLD) pathogenesis. However, there is no direct evidence of this, and the underlying molecular mechanism is still not fully understood. In this study, we investigated the role of inflammation and oxidative stress caused by prolonged PM2.5 exposure in dyslipidemia-associated chronic hepatic injury, and further determined whether an increase in hepatic inflammation and oxidative stress promoted lipid accumulation in the liver, ultimately increasing the risk of NAFLD. Therefore, we studied changes in indicators of metabolic disorder and in symbolic indices of NAFLD. We confirmed increases in insulin resistance, glucose tolerance, peripheral inflammation and dysarteriotony in PM2.5-induced mice. Oxidative stress and inflammatory response in the liver caused by PM2.5 inhalation contributed to abnormal hepatic function, further promoting lipid accumulation in the liver. Moreover, we observed inhibition of oxidative stress and inflammatory response by pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) in vitro, suggesting that oxidative stress and inflammatory in liver cells aggravated by PM2.5 contributed to hepatic injury by altering normal lipid metabolism. These results indicate a new goal for preventing and treating air pollution-induced diseases: suppression of oxidative stress and inflammatory response.
Cellular memory underlies cellular identity, and thus constitutes a unifying mechanism of genetic disposition, environmental influences, and cellular adaptation. Here, we demonstrate that enduring physicochemical changes of mitochondrial networks invoked by transient stress, a phenomenon we term 'mitoengrams', underlie the transgenerational persistence of epigenetically scripted cellular behavior. Using C2C12 myogenic stem-like cells, we show that stress memory elicited by transient, low-level arsenite exposure is stored within a self-renewing subpopulation of progeny cells in a mitochondrial-dependent fashion. Importantly, we demonstrate that erasure of mitoengrams by administration of mitochondria-targeted electron scavenger was sufficient to reset key epigenetic marks of cellular memory and redirect the identity of the mitoengram-harboring progeny cells to a non-stress-like state. Together, our findings indicate that mnemonic information emanating from mitochondria support the balance between the persistence and transience of cellular memory.
The mechanism underlying the development of chronic kidney disease (CKD) after acute kidney injury (AKI) remains unclear. Maladaptive repair has been considered an important mechanism of CKD post AKI. Renal tubular cells under maladaptive repair have characteristics of premature senescence. These premature senescent cells can generate profibrotic factors that promote organ fibrosis. The purpose of this study was to investigate whether cisplatin induces premature renal senescence and the role of premature renal senescence in the progression of CKD post AKI. As oxidative stress is a major cause of senescence, we further evaluated whether antioxidant therapy could protect renal tubular cells from cisplatin-induced premature senescence and retard the progression of CKD post AKI. The molecular mechanism of this protection was also investigated. We found that cisplatin induced premature renal senescence in vitro and in vivo. In a multiple-cisplatin-treatment murine model, renal interstitial fibrosis was accompanied by premature renal senescence. N-acetylcysteine (NAC), an antioxidant, attenuated premature senescence and decreased renal fibrosis, and its effects were dependent on sirtuin1 (SIRT1) activation and p53 deacetylation. These results indicate that cisplatin can induce premature renal senescence, which is associated with the development of CKD post cisplatin-induced AKI. SIRT1 activation and p53 deacetylation might be identified as potential targets for attenuating premature renal senescence and retarding the progression of CKD post AKI.
Oxidative stress plays a pivotal and early role in the pathophysiology of Alzheimer's disease (AD). There is convincing evidence that oxidative alterations in AD and in mild cognitive impairment (MCI) patients are not limited to the brain but are extended to the blood compartment. However, the oxidative pattern in plasma is still inconclusive. Moreover, their potential association with the clinical scores MMSE (Mini-Mental State Examination) and MoCA (Montreal Cognitive Assessment) is poorly investigated. The aim of our study was to establish a pattern of blood-based redox alterations in prodromal AD and their evolution during the progression of the disease. Our results showed a reduction in the total antioxidant capacity (TAC) and an increase of the stress-response proteins apolipoprotein J (ApoJ) and Klotho in MCI subjects. For the first time, we evidenced circulating-proteasome activity. We found that the alteration of the circulating-proteasome activity is associated with the accumulation of oxidized proteins in plasma form early AD. Interestingly, the TAC, the levels of vitamin D and the activity of proteasome were positively associated to the clinical scores MMSE and MoCA. The levels of protein carbonyls and of ApoJ were negatively associated to the MMSE and MoCA scores. The levels of apolipoprotein D (ApoD) were not different between groups. Interestingly, the receiver operating characteristic (ROC) curves analysis indicated that these redox markers provide a fair classification of different groups with high accuracy. Overall, our results strengthen the notion that some specific oxidative markers could be considered as non-invasive blood-based biomarkers for an early MCI diagnosis and AD progression.
Developing anti-melanoma agents with increased activity and specificity is highly desirable due to the increasing incidence, highly metastatic malignancy, and high mortality rate of melanoma. Abnormal redox characteristics such as higher levels of tyrosinase, NAD(P) H: quinone oxidoreductase-1 (NQO1) and reactive oxygen species (ROS) observed in melanoma cells than in other cancer cells and normal cells illustrate their redox vulnerability and have opened a window for developing prooxidative anti-melanoma agents (PAAs) to target the vulnerability. However, how to design PAAs which promote selectively the ROS accumulation in melanoma cells remains a challenge. This work describes a promising redox cycle-based strategy for designing a catechol-type diphenylbutadiene as such type of PAA. This molecule is capable of constructing an efficient catalytic redox cycle with tyrosinase and NQO1 in melanoma B16F1 cells to induce selectively the ROS (mainly including hydrogen peroxide, H2O2) accumulation in the cells, resulting in highly selective suppression of melanoma B16F1 cells over tyrosinase-deficient HeLa and normal L-02 cells.
Emerging evidence supports a beneficial action of the flavan-3-ol (-)-epicatechin (EC) on insulin sensitivity and potential impact on the development/progression of type 2 diabetes (T2D). In humans, supplementation with EC-rich foods, extracts, and pure EC improves insulin sensitivity and glucose tolerance in normal weight, overweight, obese and T2D individuals. These effects of EC are also observed in rodent models of diet-induced obesity and T2D. The events involved in the development of insulin resistance and T2D are multiple and interrelated. EC has been shown to inhibit inflammation, oxidative and endoplasmic reticulum stress, to modulate mitochondrial biogenesis and function, and to regulate events in the gastrointestinal tract and the pancreas that impact glucose homeostasis. A downregulation of oxidant production, particularly through direct inhibition or suppression of NADPH oxidase expression, and of redox sensitive signals (NF-kappa B, JNK1/2) that inhibit the insulin pathway, appear to be central to the beneficial actions of EC on insulin sensitivity. Overall, EC seems to have a positive role in the regulation of glucose homeostasis, however definitive answers on its importance for the management of T2D will depend on further clinical and mechanistic studies.
Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H4BPMHC (8-((6-hydroxy-2,5,7,8-tetra-methylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an a-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H4BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H4BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons.
Alzheimer's disease (AD) is a progressive neurodegenerative disease of the brain. It cannot be cured currently, and those suffering from AD place a great burden on their caregivers and society. AD is characterized by high levels of iron ions in the brain, which catalyze radicals that damage the neurons. Knowing that the A beta 42 peptide precipitates iron by binding iron ions at amino acid residues D1, E3, H11, H13, and H14, we synthesized a 5-repeat (HAYED) sequence peptide. By treating iron-stressed SH-SY5Y cells with it and injecting it into the cerebrospinal fluid (CSF) of naturally senescence Kunming mouse, which displaying AD-similar symptoms such as learning and memory dysfunction, neuron degeneration and high level of iron in brain, we found that HAYED (5) decreased the iron and radical levels in the cell culture medium and in the CSF. Specially, the synthesized peptide prevented cell and brain damage. Furthermore, functional magnetic resonance imaging (fMRI), Morris water maze and passive avoidance tests demonstrated that the peptide ameliorated brain blood-oxygen metabolism and slowed cognitive loss in the experimental senescence mice, and clinical and blood tests showed that HAYED (5) was innoxious to the kidney, the liver and blood and offset the AD-associated inflammation and anemia.
We have previously demonstrated that acute stress decreases neuronal nitric oxide synthase (NOS) expression in the hippocampus despite increased concentrations of nitric oxide which may indicate feedback inhibition of neuronal NOS expression via inducible NOS-derived nitric oxide. Moreover, the hippocampus undergoes an initial oxidative/nitrosative insult that is rapidly followed by upregulation of protective antioxidants, including the zinc-binding metallothioneins, in order to counter this and restore redox balance following acute stress exposure. In the present study, we have utilized indicators of oxidative/nitrosative stress, members of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, antioxidant metallothioneins, and neuroinflammatory markers to observe the changes occurring in the hippocampus following short term repeated stress exposure. Male Wistar rats were subjected to control conditions or 6 h of restraint stress applied for 1, 2, or 3 days (n= 8 per group) after which the hippocampus was isolated for redox assays and relative gene expression. The hippocampus showed increased oxidative stress, transient dys-homeostasis of total zinc, and increased expression of the Nrf2 pathway members. Moreover, repeated stress increased nitrosative status, nitric oxide metabolites, and 3-nitrotyrosine, indicative of nitrosative stress in the hippocampus. However, levels of neuronal NOS decreased over all stress treatment groups, while increases were observed in inducible NOS and xanthine dehydrogenase. In addition to inducible NOS, mRNA expression of other inflammatory markers including interleukin-6 and interleukin-1 beta also increased even in the presence of increased anti-inflammatory glucocorticoids. Together, these results demonstrate that despite increases in antioxidant expression, sub-acute stress causes an inflammatory phenotype in the hippocampus by inducing oxidative/nitrosative stress, zinc dys-homeostasis, and the accumulation of nitrotyrosinated proteins which is likely driven by increased inducible NOS signaling.
Heavy ion radiotherapy has shown great promise for cancer therapy. Understanding the cellular response mechanism to heavy ion radiation is required to explore measures of overcoming devastating side effects. Here, we performed a quantitative proteomic analysis to investigate the mechanism of carbon ion irradiation on human AHH-1 lymphoblastoid cells. We identified 4602 proteins and quantified 4569 proteins showing high coverage in the mitochondria. Data are available via ProteomeXchange with identifier PXD008351. After stringent filtering, 290 proteins were found to be significantly up-regulated and 16 proteins were down-regulated. Functional analysis revealed that these up-regulated proteins were enriched in the process of DNA damage repair, mitochondrial ribosome, and particularly mitochondrial respiratory chain, accounting for approximately 50% of the accumulated proteins. Bioinformatics and functional analysis demonstrated that these up-regulated mitochondrial respiratory chain proteins enhanced ATP production and simultaneously reactive oxygen species release. More importantly, increased reactive oxygen species led to secondary organelle injury and lagged DNA double-strand breaks. Consistently, the expression of antioxidant enzymes was up-regulated for free radical scavenging. The mechanism of lagged secondary injury originated from disturbances in the mitochondrial respiratory chain. Our results provided a novel target for cell self-repair against heavy ion radiation-induced cellular damage.
Phosphatidylinositol 4-phosphate [PtdIns(4)P] plays a key role in the biogenesis of transport vesicles at the Golgi complex by recruiting coat proteins and their accessory factors. The PtdIns(4)P content of the Golgi is determined by the concerted action of PtdIns 4-kinase (PI4K) and PtdIns(4)P phosphatase enzymes. Sac1 (suppressor of actin 1) is the major PtdIns(4)P phosphatase and is localized to the Golgi and endoplasmic reticulum. The targeting of both PI4Ks and Sac1 to the Golgi membrane is extensively regulated, as is the catalytic activity of PI4Ks at the Golgi. However, regulation of the catalytic activity of Sac1 has been largely unexplored. Here we show that Sac1undergoes reversible inactivation in mammalian cells when its catalytic Cys(389) residue is oxidized by exogenous H2O2 to form an intramolecular disulfide with Cys(392). The oxidative inactivation of Sac1 results in the accumulation of PtdIns(4)P at the Golgi, with this effect also being supported by the H2O2-induced activation of p38 mitogen-activated protein kinase (MAPK), which was previously shown to promote the translocation of Sac1 from the Golgi to the endoplasmic reticulum. The increase in Golgi PtdIns(4)P due to Sac1 inactivation, however, is faster than that due to Sac1 translocation. Exposure of cells to H2O2 also increased membrane protein trafficking from the Golgi to the plasma membrane as well as protein secretion.