Reduced ATP Content Research Articles

Hyperglycemia adversely affects critical physiological events related to rat sperm capacitation

Hyperglycemia, the hallmark of diabetes mellitus (DM), is the main cause of DM-related systemic complications, including reproductive issues. Furthermore, the incidence of DM in males of reproductive ages is becoming an increasing concern, as the complexity of sperm capacitation (an essential process for fertilizing the egg) extends beyond conventional sperm parameters such as count, viability, and motility. Capacitation defects cause male infertility, and DM-related hyperglycemia may affect this process. We explore the effects of uncontrolled hyperglycemia on sperm using alloxan-induced hyperglycemic Wistar rats. In addition to assessing conventional sperm parameters, we also evaluated functional indicators, including hyperactivation (HA) with a pharmacological approach and assessed its effects with a computer-assisted sperm analysis (CASA); fluorescence indicators to monitor membrane potential (EmR, DiSC3(5)) and mitochondrial membrane potential (Ψ, JC-1); CatSper activity, using its ability to permeate Na+ ions, and ATP levels with the luciferin-luciferase reaction. We confirmed previous findings with our hyperglycemic model, which replicated the typical reduction on conventional sperm parameters. In sperm from hyperglycemic rats, we observed increased motility and HA levels after pharmacological treatment. Additionally, CatSper activity was unaffected by hyperglycemia, while EmR was hyperpolarized under non-capacitating condition. Finally, we noted a low percentage of hyperpolarized Ψ and reduced ATP content. This study highlights the significance of impact of hyperglycemia on sperm physiology and capacitation. We proposed that low ATP levels perturb energy state, signaling pathways, ion channels activity, motility, and HA. Our findings offer insight into DM-associated infertility and potential treatment strategies.

Interference with MTHFD2 induces ferroptosis in ovarian cancer cells through ERK signaling to suppress tumor malignant progression.

Ovarian cancer (OC) is a deadliest gynecological cancer with the highest mortality rate. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a crucial tumor-promoting factor, is over-expressed in several malignancies including OC. The present study aimed to explore the role and mechanisms of MTHFD2 in OC malignant progression. Thus, cell proliferation, cycling, apoptosis, migration, and invasion were evaluated by CCK-8 assay, EdU assay, flow cytometry, wound healing, transwell assay and western blotting. Additionally, glycolysis was assessed by measuring the level of glucose and lactate production, as well as the expressions of GLUT1, HK2 and PKM2. Then the expression of ferroptosis-related proteins and ERK signaling was detected using western blotting. Ferroptosis was detected through the measurement of iron level, GSH, MDA and ROS activities. The results revealed that MTHFD2 was highly expressed in OC cells. Besides, interference with MTHFD2 induced ferroptosis, promoted ROS accumulation, destroyed mitochondrial function, reduced ATP content and inhibited glycolysis in OC cells. Subsequently, we further found that interference with MTHFD2 affected mitochondrial function and glycolysis in OC cells through ERK signaling. Moreover, interference with MTHFD2 affected ferroptosis to inhibit the malignant progression of OC cells. Collectively, our present study disclosed that interference with MTHFD2 induced ferroptosis in OC to inhibit tumor malignant progression through regulating ERK signaling.

Neddylation inhibition affects early embryonic development by disrupting maternal-to-zygotic transition and mitochondrial function in mice

Post-translational modifications (PTMs) are critical for early development in mice because early cleavage-stage embryos are characterized by transcriptional inactivity. Neddylation is an important ubiquitin-like PTM that regulates multiple biophysical processes. However, the exact roles of neddylation in regulating early embryonic development remain largely unknown. In the present study, we found that inhibition of neddylation by specific inhibitor MLN4924 led to severe arrest of early embryonic development. Transcriptomic analysis showed that neddylation inhibition changed the expression of 3959 genes at the 2-cell stage. Importantly, neddylation inhibition blocked zygotic genome activation and maternal mRNA degradation, thus disrupting the maternal-to-zygotic transition. Moreover, inhibition of neddylation induced mitochondrial dysfunction including aberrant mitochondrial distribution, decreased mitochondrial membrane potential, and reduced ATP content. Further analysis showed that inhibition of neddylation resulted in the accumulation of reactive oxygen species and superoxide anion, thereby resulting in oxidative stress and severe DNA damage at the 2-cell stage. Overall, this study demonstrates that neddylation is vital for early embryonic development in mice. Our findings suggest that proper neddylation regulation is essential for the timely inter-stage transition during early embryonic development.

Vitamin E alleviates pyraclostrobin-induced toxicity in zebrafish (Danio rerio) and its potential mechanisms

Strobilurin fungicides (SFs) are commonly used in agriculture worldwide and frequently detected in aquatic environments. High toxicity of SFs to aquatic organisms has caused great concerns. To explore whether vitamin E (VE) can relieve the toxicity caused by pyraclostrobin (PY), zebrafish were exposed to PY with or without VE supplementation. When co-exposure with VE (20 μM), the 96 h-LC50 values of PY to zebrafish embryos, adult, and the 24 h-LC50 value of PY to larvae increased from 43.94, 58.36 and 38.16 μg/L to 64.72, 108.62 and 72.78 μg/L, respectively, indicating that VE significantly decreased the toxicity of PY to zebrafish at different life stages. In addition, VE alleviated the deformity symptoms (pericardial edema and brain damage), reduced speed and movement distance, and decreased heart rate caused by 40 μg/L PY in zebrafish larvae. Co-exposure of PY with VE significantly reduced PY-caused larval oxidative stress and immunotoxicity via increasing the activities of superoxide dismutase, catalase and level of glutathione, as well as reducing the malondialdehyde production and the expression levels of Nrf2, Ucp2, IL-8, IFN and CXCL-C1C. Meanwhile, the expression levels of gria4a and cacng4b genes, which were inhibited by PY, were significantly up-regulated after co-exposure of PY with VE. Moreover, co-exposure with VE significantly reversed the increased mitochondrial DNA copies and reduced ATP content caused by PY in larvae, but had no effect on the expression of cox4i1l and activity of complex III that reduced by PY, suggesting VE can partially improve PY-induced mitochondrial dysfunction. In conclusion, the potential mechanisms of VE alleviating PY-induced toxicity may be ascribed to decreasing the oxidative stress level, restoring the functions of heart and nervous system, and improving the immunity and mitochondrial function in zebrafish.

Amino Acid and Glucose Fermentation Maintain ATP Content in Mouse and Human Malignant Glioma Cells.

Energy is necessary for tumor cell viability and growth. Aerobic glucose-driven lactic acid fermentation is a common metabolic phenotype seen in most cancers including malignant gliomas. This metabolic phenotype is linked to abnormalities in mitochondrial structure and function. A luciferin-luciferase bioluminescence ATP assay was used to measure the influence of amino acids, glucose, and oxygen on ATP content and viability in mouse (VM-M3 and CT-2A) and human (U-87MG) glioma cells that differed in cell biology, genetic background, and species origin. Oxygen consumption was measured using the Resipher system. Extracellular lactate and succinate were measured as end products of the glycolysis and glutaminolysis pathways, respectively. The results showed that: (1) glutamine was a source of ATP content irrespective of oxygen. No other amino acid could replace glutamine in sustaining ATP content and viability; (2) ATP content persisted in the absence of glucose and under hypoxia, ruling out substantial contribution through either glycolysis or oxidative phosphorylation (OxPhos) under these conditions; (3) Mitochondrial complex IV inhibition showed that oxygen consumption was not an accurate measure for ATP production through OxPhos. The glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine (DON), reduced ATP content and succinate export in cells grown in glutamine. The data suggests that mitochondrial substrate level phosphorylation in the glutamine-driven glutaminolysis pathway contributes to ATP content in these glioma cells. A new model is presented highlighting the synergistic interaction between the high-throughput glycolysis and glutaminolysis pathways that drive malignant glioma growth and maintain ATP content through the aerobic fermentation of both glucose and glutamine.

The Impact of Niclosamide Exposure on the Activity of Antioxidant Enzymes and the Expression of Glucose and Lipid Metabolism Genes in Black Carp (Mylopharyngodon piceus).

Niclosamide (NIC, 2',5-dichloro-4'-nitrosalicylanilide) is a salicylanilide molluscicide, and the extensive utilization and environmental pollution associated with NIC engender a potential hazard to both human health and the wellbeing of aquatic organisms. However, the mechanism of the chronic toxicity of NIC at environmentally relevant concentrations in terms of oxidative stress, metabolic disorder, and barrier functions in black carp (Mylopharyngodon piceus) is unknown. Therefore, healthy juvenile black carp (M. piceus) (average weight: 38.2 ± 2.5 g) were exposed to NIC at an environmentally realistic concentration (0, 10, and 50 μg/L) for 28 days. The findings of this study indicate that exposure to NIC resulted in reductions in weight gain, decreased activity of antioxidant enzymes, and increased expression of the Nrf2 gene. Furthermore, the liver demonstrated a greater accumulation of NIC than that in the gut and gills, as determined with a chemical analysis. Additionally, NIC exposure led to a significant reduction in ATP content and the activity of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the gut. Meanwhile, exposure to NIC resulted in a decrease in the liver glucose (Glu) level, gut cholesterol (CHO), and glycogen (Gln) and triglyceride (TG) content in all examined tissues. Conversely, it led to an increase in tissue lactic acid (LA) and acetyl-CoA levels, as well as LDH activity. Furthermore, NIC exposure at environmentally relevant concentrations demonstrated an upregulation in the expression of genes associated with glycolysis, such as PK and GK, while concurrently downregulating the gluconeogenesis gene G6Pase. Additionally, NIC exhibited an upregulation in the expression of genes related to β-oxidation, such as CPT1 and ACOX, while downregulating genes involved in triglyceride synthesis, including SREBP1, GPAT, FAS, and ACC1. Moreover, NIC facilitated fatty acid transportation through the overexpression of FATP and Fat/cd36. These results suggest that chronic exposure to NIC is associated with oxidative stress, compromised barrier function, and metabolic disorder. Moreover, these results underscore the significance of assessing the potential consequences of NIC for black carp and aquatic environments for aquaculture.

Lycium barbarum polysaccharides attenuate oxidative stress and mitochondrial toxicity induced by mixed plasticizers in HepG2 cells through activation of Nrf2

AimsIn daily life, it is common for humans to be exposed to multiple phthalate esters (PAEs). However, there is limited research on the mechanisms and intervention of combined PAEs toxicity. This study aims to explore the cytotoxicity of combined PAEs and evaluate the potential of Lycium barbarum polysaccharides (LBP) in mitigating the aforementioned toxicity. Main methodsLBP (62.5, 125 and 250 μg/mL) were applied to intervene HepG2 cells treated with DEHP and DBP mixtures (50, 100, 200, 400 and 800 μg/mL). Western Blot and different kits were mainly performed in our study. Key findingsDEHP and DBP mixtures suppressed the expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and activated MAPK pathway by increasing ROS. Combined DEHP and DBP exposure reduced ATP content and inhibited the mitochondrial biogenesis pathway in HepG2 cells through oxidative stress, which in turn caused cytotoxicity. LBP reduced oxidative stress and cell death induced by mixed plasticizers, upregulated Nrf2 levels and mitochondrial biogenesis pathway levels and inhibited MAPK pathway activation. Notably, after treating HepG2 cells with Nrf2-specific inhibitor (ML385, 0.5 μM), we found that the activation of Nrf2 played a crucial role on LBP intervention of DEHP and DBP induced HepG2 cytotoxicity. SignificanceThis study not only enhances our understanding of the toxicological effects caused by combined PAEs exposure, but also has significant implications in devising strategies to mitigate the toxicological consequences of combined exposure to exogenous chemicals through the investigation of the role of LBP.

Protective effects of nicotinamide mononucleotide on radiation-related cardiac injury

Abstract Background Radiation therapy for cancer treatment can cause damage to the heart. There are numerous possible mechanisms related to radiation-related cardiac dysfunction, among which increased oxidative stress is an important factor. However, the detailed mechanisms remain unclear and effective treatments on radiation-related cardiac dysfunction are still lacking. Nicotinamide mononucleotide (NMN), the most direct precursor of nicotinamide adenine dinucleotide (NAD+), can effectively reduce reactive oxygen species (ROS) production and thus could be a candidate to alleviate radiation-induced damage. However, the protective effects and the underlying mechanisms of NMN on radiation-related cardiac injury remain to be elucidated. Therefore, this study was designed to identify the mechanisms of radiation-related cardiac injury and investigate the protective actions of NMN. Methods Wild-type c57 mice were divided into radiation exposure (7.2Gy radiation) and non-exposure groups. Wild-type C57BL6/J mice supplied with NMN for 12 months before radiation. Echocardiography to detect mice cardiac function. Epicardial electrical mapping technique to detect electrical conduction in the heart. Non-targeted metabolomics was used to detect cardiac different metabolites. In parallel, we observed the effect of radiation on survival and mitochondrial function (ROS production and ATP content) of H9C2 cells with or without NMN. Results In the radiation exposure group, echocardiography showed that the left ventricular ejection fraction (LVEF) and fractional shortening (FS) were reduced, whilst epicardial mapping demonstrated slowed conduction velocity and change in the direction of propagation. Non-targeted metabolomics showed the different metabolites between the radiation group and the control group were mainly in nucleotide and amino acid metabolism. Metabolic pathway enrichment analysis showed that different metabolites were implicated in the metabolism of pyrimidine, folate, arginine and proline, and tryptophan. Mice with NMN before radiation showed reduced mechanical and electrical dysfunction, with non-targeted metabolomic results showed NMN rescued the radiation-induced down-regulation of 1-(4-aminobutyl)urea, 4-aminobenzoic acid, 3-hydroxyanthranilic acid, which ultimately exerted cardioprotective effects. We also found that radiation can reduce the survival, increase ROS production, and reduce ATP content of H9C2 cells in vitro, and these harmful effects can be partially reversed by NMN. Conclusion The mechanical and electrical dysfunction caused by radiation were associated with altered nucleotide and amino acid metabolism in the myocardium, and these harmful effects could be partially mitigated by NMN supplement.

The potential mechanism of low-power water bath ultrasound to enhance the effectiveness of low-concentration chlorine dioxide in inhibiting Salmonella Typhimurium

This chapter presents a systematic study of the inhibition effect of chlorine dioxide treatment alone and in combination with ultrasound treatment of Salmonella and the physiological metabolic processes within the treated cells. The low-power ultrasound (0.03 W/mL) significantly enhanced the effectiveness (110.00 %) of low concentrations of chlorine dioxide (0.25 mg/L) in inhibiting Salmonella, which, in turn, would significantly reduce the potential environmental impact. In addition, further studies found that low-power ultrasound may enhance the structural and functional damage of chlorine dioxide on Salmonella cell membranes (significant increase in permeability of the outer and inner cell membranes) and disrupt intracellular substance metabolism (small molecule and nucleotide metabolism) and energy metabolism (significant reduction in ATP content and ATPase activity) balance to improve the bacterial inhibitory effect of chlorine dioxide. The results of the study will provide a theoretical basis and methodological guidance for the implementation of “cleaner production” in the food industry.

Inactivation action of ultrasound-assisted cinnamaldehyde on planktonic and biofilm methicillin-resistant Staphylococcus aureus and its application in beef system

There has been a growing concern for methicillin-resistant Staphylococcus aureus (MRSA) contamination in the food industry. In the present study, the highest inactivation effect (5.80 log CFU/mL) was observed in simultaneous treatment compared with sequential treatment of ultrasound (US)-assisted cinnamaldehyde (CA). The absence of sublethal bacteria indicated US-assisted CA did not induce the tolerance or adaptation to MRSA cells. The results of SEM analysis indicated that the application of US-assisted CA resulted in severe damage to the cell membrane, as demonstrated by the highest fluorescence intensity of propidium iodide dye, intracellular substance leakage, and molecular docking analysis, as well as ATPase activity. Furthermore, US-assisted CA significantly disrupted energy metabolism, as evidenced by a reduction in ATP concentration, succinate dehydrogenase, and malate dehydrogenase activities. The exacerbation of ROS-induced damage to MRSA cells was observed due to the reduction in catalase and peroxidase activities. Additionally, a significant decrease (p < 0.05) in extracellular carbohydrates and proteins within the MRSA biofilm was noted on both glass and stainless-steel surfaces exposed to US-assisted CA. Ultimately, US-assisted CA treatment resulted in a maximum reduction (p < 0.05) of MRSA cells in beef meat, while maintaining color, pH, and sensory properties during an 8-day storage period at 4 °C. These findings suggested that simultaneous US-assisted CA treatment may serve as an alternative method for ensuring microbiological safety.

Abstract P2057: The Role Of Cardiac Neurofibromin-2 In Chronic Pressure Overload

Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Rutgers University, Newark, NJ 07103.Neurofibromin 2 (NF2) is a tumor suppressor that can engage the Hippo signaling pathway and modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by acute myocardial infarction. However, the function of NF2 in the heart remains largely uncharacterized. Our current study sought to determine whether NF2 modulates heart failure due to chronic stress. We used a transverse aortic constriction (TAC) model in WT C57BL/6J mice to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. We found that NF2 is transiently upregulated in mouse myocardium in response to early phase of PO, and is downregulated during late phase PO. We generated cardiomyocyte-specific NF2 knockout (cKO) mice, which had normal cardiac morphology and function at baseline. Following TAC, NF2 cKO hearts unexpectedly showed worsened cardiac function, assessed by echocardiography and hemodynamic analysis, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation and fatty acid oxidation in NF2 cKO hearts at baseline. Additionally, we observed reduced ATP content in baseline NF2 cKO hearts compared to controls. Fractionation experiments indicated that nuclear NF2 is enriched following PO stress. RNAseq revealed the downregulation of ERRβ and ERRγ in NF2 cKO hearts, which was confirmed by qPCR. Experiments employing neonatal rat ventricular myocytes (NRMVs) confirmed that NF2 regulated expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Luciferase reporter assays demonstrated that NF2 modulates ERR function in NRVMs. Based on these findings, we propose that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.

TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation

BackgroundLung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Its role in LUAD has gained attention, but the detailed function and mechanism have not been clarified completely at present. MethodsTumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, we detected the influence of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, we comprehensively inspected the different changed proteins influenced by TIM and did global bioinformatic analysis. ResultsWe found that TIM expression was elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. We also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, we found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. We further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. ConclusionsOur study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

Mammalian Animal and Human Retinal Organ Culture as Pre-Clinical Model to Evaluate Oxidative Stress and Antioxidant Intraocular Therapeutics.

Oxidative stress (OS) is involved in the pathogenesis of retinal neurodegenerative diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR) and an important target of therapeutic treatments. New therapeutics are tested in vivo despite limits in terms of transferability and ethical concerns. Retina cultures using human tissue can deliver critical information and significantly reduce the number of animal experiments along with increased transferability. We cultured up to 32 retina samples derived from one eye, analyzed the model's quality, induced OS, and tested the efficiency of antioxidative therapeutics. Bovine, porcine, rat, and human retinae were cultured in different experimental settings for 3-14 d. OS was induced by a high amount of glucose or hydrogen peroxide (H2O2) and treated with scutellarin, pigment epithelium-derived factor (PEDF), and/or granulocyte macrophage colony-stimulating factor (GM-CSF). The tissue morphology, cell viability, inflammation, and glutathione level were determined. The retina samples showed only moderate necrosis (23.83 ± 5.05 increased to 27.00 ± 1.66 AU PI-staining over 14 d) after 14 days in culture. OS was successfully induced (reduced ATP content of 288.3 ± 59.9 vs. 435.7 ± 166.8 nM ATP in the controls) and the antioxidants reduced OS-induced apoptosis (from 124.20 ± 51.09 to 60.80 ± 319.66 cells/image after the scutellarin treatment). Enhanced mammalian animal and human retina cultures enable reliable, highly transferable research on OS-triggered age-related diseases and pre-clinical testing during drug development.

Homeostasis of flavonoids and triterpenoids most likely modulates starch metabolism for pollen tube penetration in rice.

In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male-specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α-amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α-amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding.

Potentilla anserina polysaccharide alleviates cadmium-induced oxidative stress and apoptosis of H9c2 cells by regulating the MG53-mediated RISK pathway.

Oxidative stress plays a crucial role in cadmium (Cd)-induced myocardial injury. Mitsugumin 53 (MG53) and its mediated reperfusion injury salvage kinase (RISK) pathway have been demonstrated to be closely related to myocardial oxidative damage. Potentilla anserina L. polysaccharide (PAP) is a polysaccharide with antioxidant capacity, which exerts protective effect on Cd-induced damage. However, it remains unknown whether PAP can prevent and treat Cd-induced cardiomyocyte damages. The present study was desgined to explore the effect of PAP on Cd-induced damage in H9c2 cells based on MG53 and the mediated RISK pathway. For in vitro evaluation, cell viability and apoptosis rate were analyzed by CCK-8 assay and flow cytometry, respectively. Furthermore, oxidative stress was assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and using superoxide dismutase (SOD), catalase (CAT), and glutathione/oxidized glutathione (GSH/GSSG) kits. The mitochondrial function was measured by JC-10 staining and ATP detection assay. Western blot was performed to detect the expression of proteins related to MG53, the RISK pathway, and apoptosis. The results indicated that Cd increased the levels of reactive oxygen species (ROS) in H9c2 cells. Cd decreased the activities of SOD and CAT and the ratio of GSH/GSSG, resulting in decreases in cell viability and increases in apoptosis. Interestingly, PAP reversed Cd-induced oxidative stress and cell apoptosis. Meanwhile, Cd reduced the expression of MG53 in H9c2 cells and inhibited the RISK pathway, which was mediated by decreasing the ratio of p-AktSer473/Akt, p-GSK3βSer9/GSK3β and p-ERK1/2/ERK1/2. In addition, Cd impaired mitochondrial function, which involved a reduction in ATP content and mitochondrial membrane potential (MMP), and an increase in the ratio of Bax/Bcl-2, cytoplasmic cytochrome c/mitochondrial cytochrome c, and Cleaved-Caspase 3/Pro-Caspase 3. Importantly, PAP alleviated Cd-induced MG53 reduction, activated the RISK pathway, and reduced mitochondrial damage. Interestingly, knockdown of MG53 or inhibition of the RISK pathway attenuated the protective effect of PAP in Cd-induced H9c2 cells. In sum, PAP reduces Cd-induced damage in H9c2 cells, which is mediated by increasing MG53 expression and activating the RISK pathway.

Upregulation of TLR4-Dependent ATP Production Is Critical for Glaesserella parasuis LPS-Mediated Inflammation.

Glaesserella parasuis (G. parasuis), an important pathogenic bacterium, cause Glässer's disease, and has resulted in tremendous economic losses to the global swine industry. G. parasuis infection causes typical acute systemic inflammation. However, the molecular details of how the host modulates the acute inflammatory response induced by G. parasuis are largely unknown. In this study, we found that G. parasuis LZ and LPS both enhanced the mortality of PAM cells, and at the same time, the level of ATP was enhanced. LPS treatment significantly increased the expressions of IL-1β, P2X7R, NLRP3, NF-κB, p-NF-κB, and GSDMD, leading to pyroptosis. Furthermore, these proteins' expression was enhanced following extracellular ATP further stimulation. When reduced the production of P2X7R, NF-κB-NLRP3-GSDMS inflammasome signaling pathway was inhibited, and the mortality of cells was reduced. MCC950 treatment repressed the formation of inflammasome and reduced mortality. Further exploration found that the knockdown of TLR4 significantly reduced ATP content and cell mortality, and inhibited the expression of p-NF-κB and NLRP3. These findings suggested upregulation of TLR4-dependent ATP production is critical for G. parasuis LPS-mediated inflammation, provided new insights into the molecular pathways underlying the inflammatory response induced by G. parasuis, and offered a fresh perspective on therapeutic strategies.

Heme oxygenase-1 prevents non-alcoholic steatohepatitis through modulating mitochondrial quality control.

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD) and lacks effective treatment options. Heme oxygenase-1 (HO-1) is a critical defense against oxidative stress and inflammation in the liver injury. This study aims to investigate the protective role and underlying mechanisms of HO-1 in NASH pathogenesis. The hepatocyte-specific HO-1 knockout (HO-1HEPKO ) mice on a C57BL/6J background (HO-1fl/fl /Alb-Cre) were generated and fed a high-fat/western-style diet (HFD) or methionine-choline-deficient diet (MCD). Changes in mitochondrial ultrastructure were observed by transmission electron microscopy and confocal microscopy. A mitochondrial PCR array was used to identify the crucial genes associated with mitochondrial dysfunction. Hepatocyte-specific HO-1HEPKO mice developed steatohepatitis with severe steatosis, ballooning, and necroinflammation. Dysregulated hepatic expression of mitochondria-related proteins, including DRP1, Tomm20, MFN1 and MFN2 were detected in NASH animals. Ultrastructural mitochondrial damage was observed in HO-1HEPKO mice. Mitochondrial dysfunction was recapitulated in HO-1-knockdown cells in vitro, as evidenced by decreased membrane potential, reduced ATP content, and mtDNA damage. Conversely, HO-1 overexpression restored these changes in vitro. Mechanistically, HO-1 deficiency reduced the inhibitory effect on Tomm20, leading to mitochondrial dysfunction, and thereby causing steatohepatitis. HO-1 attenuates diet-induced steatohepatitis by preventing mitochondrial dysfunction, indicating that HO-1 may constitute a potential therapeutic target for NASH.

The effects of gut microbiota colonizing on the porcine hypothalamus revealed by whole transcriptome analysis.

The roles of the microbe-gut-brain axis in metabolic homeostasis, development, and health are well-known. The hypothalamus integrates the higher nerve center system and functions to regulate energy balance, feeding, biological rhythms and mood. However, how the hypothalamus is affected by gut microbes in mammals is unclear. This study demonstrated differences in hypothalamic gene expression between the germ-free (GF) pigs and pigs colonized with gut microbiota (CG) by whole-transcriptome analysis. A total of 938 mRNAs, 385 lncRNAs and 42 miRNAs were identified to be differentially expressed between the two groups of pigs. An mRNA-miRNA-lncRNA competing endogenous RNA network was constructed, and miR-22-3p, miR-24-3p, miR-136-3p, miR-143-3p, and miR-545-3p located in the net hub. Gene function and pathway enrichment analysis showed the altered mRNAs were mainly related to developmental regulation, mitochondrial function, the nervous system, cell signaling and neurodegenerative diseases. Notably, the remarkable upregulation of multiple genes in oxidative phosphorylation enhanced the GF pigs’ hypothalamic energy expenditure. Additionally, the reduction in ATP content and the increase in carnitine palmitoyl transterase-1 (CPT1) protein level also confirmed this fact. Furthermore, the hypothalamic cell apoptosis rate in the CG piglets was significantly higher than that in the GF piglets. This may be due to the elevated concentrations of pro-inflammatory factors produced by gut bacteria. The obtained results collectively suggest that the colonization of gut microbes has a significant impact on hypothalamic function and health.

Osmoregulatory strategies of estuarine fish Scatophagus argus in response to environmental salinity changes

BackgroundScatophagus argus, an estuarine inhabitant, can rapidly adapt to different salinity environments. However, the knowledge of the molecular mechanisms underlying its strong salinity tolerance remains unclear. The gill, as the main osmoregulatory organ, plays a vital role in the salinity adaptation of the fish, and thus relative studies are constructive to reveal unique osmoregulatory mechanisms in S. argus.ResultsIn the present study, iTRAQ coupled with nanoLC-MS/MS techniques were employed to explore branchial osmoregulatory mechanisms in S. argus acclimated to different salinities. Among 1,604 identified proteins, 796 differentially expressed proteins (DEPs) were detected. To further assess osmoregulatory strategies in the gills under different salinities, DEPs related to osmoregulatory (22), non-directional (18), hypo- (52), and hypersaline (40) stress responses were selected. Functional annotation analysis of these selected DEPs indicated that the cellular ion regulation (e.g. Na+-K+-ATPase [NKA] and Na+-K+-2Cl− cotransporter 1 [NKCC1]) and ATP synthesis were deeply involved in the osmoregulatory process. As an osmoregulatory protein, NKCC1 expression was inhibited under hyposaline stress but showed the opposite trend in hypersaline conditions. The expression levels of NKA α1 and β1 were only increased under hypersaline challenge. However, hyposaline treatments could enhance branchial NKA activity, which was inhibited under hypersaline environments, and correspondingly, reduced ATP content was observed in gill tissues exposed to hyposaline conditions, while its contents were increased in hypersaline groups. In vitro experiments indicated that Na+, K+, and Cl− ions were pumped out of branchial cells under hypoosmotic stress, whereas they were absorbed into cells under hyperosmotic conditions. Based on our results, we speculated that NKCC1-mediated Na+ influx was inhibited, and proper Na+ efflux was maintained by improving NKA activity under hyposaline stress, promoting the rapid adaptation of branchial cells to the hyposaline condition. Meanwhile, branchial cells prevented excessive loss of ions by increasing NKA internalization and reducing ATP synthesis. In contrast, excess ions in cells exposed to the hyperosmotic medium were excreted with sufficient energy supply, and reduced NKA activity and enhanced NKCC1-mediated Na+ influx were considered a compensatory regulation.ConclusionsS. argus exhibited divergent osmoregulatory strategies in the gills when encountering hypoosmotic and hyperosmotic stresses, facilitating effective adaptabilities to a wide range of environmental salinity fluctuation.

Defects in the Proteome and Metabolome in Human Hypertrophic Cardiomyopathy.

Defects in energetics are thought to be central to the pathophysiology of hypertrophic cardiomyopathy (HCM); yet, the determinants of ATP availability are not known. The purpose of this study is to ascertain the nature and extent of metabolic reprogramming in human HCM, and its potential impact on contractile function. We conducted proteomic and targeted, quantitative metabolomic analyses on heart tissue from patients with HCM and from nonfailing control human hearts. In the proteomic analysis, the greatest differences observed in HCM samples compared with controls were increased abundances of extracellular matrix and intermediate filament proteins and decreased abundances of muscle creatine kinase and mitochondrial proteins involved in fatty acid oxidation. These differences in protein abundance were coupled with marked reductions in acyl carnitines, byproducts of fatty acid oxidation, in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, branched chain amino acids, and their breakdown products, were all significantly increased in HCM hearts. ATP content, phosphocreatine, nicotinamide adenine dinucleotide and its phosphate derivatives, NADP and NADPH, and acetyl CoA were also severely reduced in HCM compared with control hearts. Functional assays performed on human skinned myocardial fibers demonstrated that the magnitude of observed reduction in ATP content in the HCM samples would be expected to decrease the rate of cross-bridge detachment. Moreover, left atrial size, an indicator of diastolic compliance, was inversely correlated with ATP content in hearts from patients with HCM. HCM hearts display profound deficits in nucleotide availability with markedly reduced capacity for fatty acid oxidation and increases in ketone bodies and branched chain amino acids. These results have important therapeutic implications for the future design of metabolic modulators to treat HCM.