Mitochondrial Transmembrane Potential Research Articles

Long-term exposure to PM2.5 leads to mitochondrial damage and differential expression of associated circRNA in rat hepatocytes

Fine particulate matter (PM2.5) is one of the four major causes of mortality globally. The objective of this study was to investigate the mechanism underlying liver injury following exposure to PM2.5 and the involvement of circRNA in its regulation. A PM2.5 respiratory tract exposure model was established in SPF SD male rats with a dose of 20 mg/kg, and liver tissue of rats in control group and PM2.5-exposed groups rats were detected. The results of ICP-MS showed that Mn, Cu and Ni were enriched in the liver. HE staining showed significant pathological changes in liver tissues of PM2.5-exposed group, transmission electron microscopy showed significant changes in mitochondrial structure of liver cells, and further mitochondrial function detection showed that the PM2.5 exposure resulted in an increase in cell reactive oxygen species content and a decrease in mitochondrial transmembrane potential, while the expression of SOD1 and HO-1 antioxidant oxidase genes was upregulated. Through high-throughput sequencing of circRNAs, we observed a significant down-regulation of 10 and an up-regulation of 17 circRNAs in the PM2.5-exposed groups. The functional enrichment and pathway analyses indicated that the differentially expressed circRNAs by PM2.5 exposure were primarily associated with processes related to protein ubiquitination, zinc ion binding, peroxisome function, and mitochondrial regulation. These findings suggest that the mechanism underlying liver injury induced by PM2.5-exposure may be associated with mitochondrial impairment resulting from the presence of heavy metal constituents. Therefore, this study provides a novel theoretical foundation for investigating the molecular mechanisms underlying liver injury induced by PM2.5 exposure.

SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a ‘highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19.

Drug-Induced Fatty Liver Disease (DIFLD): A Comprehensive Analysis of Clinical, Biochemical, and Histopathological Data for Mechanisms Identification and Consistency with Current Adverse Outcome Pathways

Drug induced fatty liver disease (DIFLD) is a form of drug-induced liver injury (DILI), which can also be included in the more general metabolic dysfunction-associated steatotic liver disease (MASLD), which specifically refers to the accumulation of fat in the liver unrelated to alcohol intake. A bi-directional relationship between DILI and MASLD is likely to exist: while certain drugs can cause MASLD by acting as pro-steatogenic factors, MASLD may make hepatocytes more vulnerable to drugs. Having a pre-existing MASLD significantly heightens the likelihood of experiencing DILI from certain medications. Thus, the prevalence of steatosis within DILI may be biased by pre-existing MASLD, and it can be concluded that the genuine true incidence of DIFLD in the general population remains unknown. In certain individuals, drug-induced steatosis is often accompanied by concomitant injury mechanisms such as oxidative stress, cell death, and inflammation, which leads to the development of drug-induced steatohepatitis (DISH). DISH is much more severe from the clinical point of view, has worse prognosis and outcome, and resembles MASH (metabolic-associated steatohepatitis), as it is associated with inflammation and sometimes with fibrosis. A literature review of clinical case reports allowed us to examine and evaluate the clinical features of DIFLD and their association with specific drugs, enabling us to propose a classification of DIFLD drugs based on clinical outcomes and pathological severity: Group 1, drugs with low intrinsic toxicity (e.g., ibuprofen, naproxen, acetaminophen, irinotecan, methotrexate, and tamoxifen), but expected to promote/aggravate steatosis in patients with pre-existing MASLD; Group 2, drugs associated with steatosis and only occasionally with steatohepatitis (e.g., amiodarone, valproic acid, and tetracycline); and Group 3, drugs with a great tendency to transit to steatohepatitis and further to fibrosis. Different mechanisms may be in play when identifying drug mode of action: (1) inhibition of mitochondrial fatty acid β-oxidation; (2) inhibition of fatty acid transport across mitochondrial membranes; (3) increased de novo lipid synthesis; (4) reduction in lipid export by the inhibition of microsomal triglyceride transfer protein; (5) induction of mitochondrial permeability transition pore opening; (6) dissipation of the mitochondrial transmembrane potential; (7) impairment of the mitochondrial respiratory chain/oxidative phosphorylation; (8) mitochondrial DNA damage, degradation and depletion; and (9) nuclear receptors (NRs)/transcriptomic alterations. Currently, the majority of, if not all, adverse outcome pathways (AOPs) for steatosis in AOP-Wiki highlight the interaction with NRs or transcription factors as the key molecular initiating event (MIE). This perspective suggests that chemical-induced steatosis typically results from the interplay between a chemical and a NR or transcription factors, implying that this interaction represents the primary and pivotal MIE. However, upon conducting this exhaustive literature review, it became evident that the current AOPs tend to overly emphasize this interaction as the sole MIE. Some studies indeed support the involvement of NRs in steatosis, but others demonstrate that such NR interactions alone do not necessarily lead to steatosis. This view, ignoring other mitochondrial-related injury mechanisms, falls short in encapsulating the intricate biological mechanisms involved in chemically induced liver steatosis, necessitating their consideration as part of the AOP’s map road as well.

Decoding dynamic interactions between EGFR-TKD and DAC through computational and experimental approaches: A novel breakthrough in lung melanoma treatment.

In the quest for effective lung cancer treatments, the potential of 3,6-diaminoacridine-9-carbonitrile (DAC) has emerged as a game changer. While DAC's efficacy against glioblastoma is well documented, its role in combating lung cancer has remained largely untapped. This study focuses on CTX-1, exploring its interaction with the pivotal EGFR-TKD protein, a crucial target in lung cancer therapeutics. A meticulous molecular docking analysis revealed that CTX-1 exhibits a noteworthy binding affinity of -7.9 kcal/mol, challenging Erlotinib, a conventional lung cancer medication, which displayed a binding affinity of -7.3 kcal/mol. For a deeper understanding of CTX-1's molecular mechanics, this study employed rigorous 100-ns molecular dynamics simulations, demonstrating CTX-1's remarkable stability in comparison with erlotinib. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method further corroborated these results, with CTX-1 showing a free binding energy of -105.976 ± 1.916 kJ/mol. The true prowess of CTX-1 was tested against diverse lung cancer cell lines, including A549, Hop-62 and H-1299. CTX-1 not only significantly outperformed erlotinib in anticancer activity but also exhibited a spectrum of therapeutic effects. It effectively diminished cancer cell viability, induced DNA damage, halted cell cycle progression, generated reactive oxygen species (ROS), impaired mitochondrial transmembrane potential, instigated apoptosis and successfully inhibited EGFR-TKD. This study not only underscores the potential of CTX-1 a formidable contender in lung cancer treatment but also marks a paradigm shift in oncological therapeutics, offering new horizons in the fight against this formidable disease.

Drug-induced impairment of mitochondrial fatty acid oxidation and steatosis: assessment of causal relationship with 45 pharmaceuticals.

Drug-induced liver injury (DILI) represents a major issue for pharmaceutical companies, being a potential cause of black-box warnings on marketed pharmaceuticals, or drug withdrawal from the market. Lipid accumulation in the liver also referred to as steatosis, may be secondary to impaired mitochondrial fatty acid oxidation (mtFAO). However, an overall causal relationship between drug-induced mtFAO inhibition and the occurrence of steatosis in patients has not yet been established with a high number of pharmaceuticals. Hence, 32 steatogenic and 13 non-steatogenic drugs were tested for their ability to inhibit mtFAO in isolated mouse liver mitochondria. To this end, mitochondrial respiration was measured with palmitoyl-L-carnitine, palmitoyl-CoA + L-carnitine, or octanoyl-L-carnitine. This mtFAO tri-parametric assay was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88%. To get further information regarding the mechanism of drug-induced mtFAO impairment, mitochondrial respiration was also measured with malate/glutamate or succinate. Drugs such as diclofenac, methotrexate and troglitazone could inhibit mtFAO secondary to an impairment of the mitochondrial respiratory chain, while dexamethasone, olanzapine and zidovudine appeared to impair mtFAO directly. Mitochondrial swelling, transmembrane potential and production of reactive oxygen species were also assessed for all compounds. Only the steatogenic drugs amiodarone, ketoconazole, lovastatin and toremifene altered all these 3 mitochondrial parameters. In conclusion, our tri-parametric mtFAO assay could be useful in predicting the occurrence of steatosis in patients. The combination of this assay with other mitochondrial parameters could also help to better understand the mechanism of drug-induced mtFAO inhibition.

A potent and selective activator of large-conductance Ca2+-activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential.

Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.

Potential mitigating impact of a dipeptidyl peptidase-IV inhibitor, vildagliptin, on oxazolone-induced ulcerative colitis: Targeting the role of PI3K/AKT/mTOR and AMPK/Nrf2 signaling pathways

Growing evidence suggests that phosphoinositide 3-kinase (PI3K) and adenosine monophosphate-activated protein kinase (AMPK) signaling cascades are critical in ulcerative colitis (UC) pathophysiology by influencing gut mucosal inflammation. Recently, the coloprotective properties of dipeptidyl peptidase-IV (DPP-IV) inhibitors have emerged. Thus, this study assessed for the first time the potential mitigating impact of a DPP-IV inhibitor, vildagliptin (Vilda), on oxazolone (OXZ)-induced colitis in rats, targeting the role of PI3K/AKT/mTOR and AMPK/Nrf2 pathways. Thirty-two adult Albino rats were divided into four groups: control, Vilda (10 mg/kg/day orally), OXZ (300 µL of 5 % OXZ in 50 % aqueous ethanol solution introduced once into the colon via catheter), and Vilda+OXZ. Inflammatory cytokines (interleukin 13, tumor necrosis factor-α, interleukin 10), oxidative/endoplasmic reticulum stress markers (myeloperoxidase, reduced glutathione, catalase, CHOP), mitochondrial reactive oxygen species, adenosine triphosphate levels, and mitochondrial transmembrane potential were estimated. p-AMPK, p-AKT, beclin-1, and SQSTM1 levels were immunoassayed. Nrf2, PI3K, and mTOR expression levels were quantified using the real-time polymerase chain reaction. Furthermore, p-NF-ĸBp65 and LC3II immunoreactivity were evaluated. Vilda administration effectively ameliorated OXZ-induced colitis, as evidenced by the reduced Disease Activity Index, macroscopic colon damage score, colon weight/length ratio, ulcer index, and histopathological and electron microscopic changes in the colon tissues. Vilda treatment also counteracted OXZ-triggered inflammation, oxidative/endoplasmic reticulum stress, mitochondrial dysfunction, and enhanced autophagy in the colon. Vilda substantially suppressed PI3K/AKT/mTOR and activated the AMPK/Nrf2 pathway. Vilda has potent coloprotective and anti-ulcerogenic properties, primarily attributed to its antiinflammatory, antioxidant, and modulatory impact on mitochondrial dysfunction and autophagy activity. These effects were mostly mediated by suppressing PI3K/AKT/mTOR and activating AMPK/Nrf2 signaling cascades, suggesting a potential role of Vilda in UC therapy.

γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells.

The excessive activation of glutamate in the brain is a factor in the development of vascular dementia. γ-Oryzanol is a natural compound that has been shown to enhance brain function, but more research is needed to determine its potential as a treatment for vascular dementia. This study investigated if γ-oryzanol can delay or improve glutamate neurotoxicity in an in vitro model of differentiated HT-22 cells and explored its neuroprotective mechanisms. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h then given γ-oryzanol at appropriate concentrations or memantine (10 µmol/L) for another 24 h. Glutamate produced reactive oxygen species and depleted glutathione in the cells, which reduced their viability. Mitochondrial dysfunction was also observed, including the inhibition of mitochondrial respiratory chain complex I activity, the collapse of mitochondrial transmembrane potential, and the reduction of intracellular ATP levels in the HT-22 cells. Calcium influx triggered by glutamate subsequently activated type II calcium/calmodulin-dependent protein kinase (CaMKII) in the HT-22 cells. The activation of CaMKII-ASK1-JNK MAP kinase cascade, decreased Bcl-2/Bax ratio, and increased Apaf-1-dependent caspase-9 activation were also observed due to glutamate induction, which were associated with increased DNA fragmentation. These events were attenuated when the cells were treated with γ-oryzanol (0.4 mmol/L) or the N-methyl-D-aspartate receptor antagonist memantine. The results suggest that γ-oryzanol has potent neuroprotective properties against glutamate excitotoxicity in differentiated HT-22 cells. Therefore, γ-oryzanol could be a promising candidate for the development of therapies for glutamate excitotoxicity-associated neurodegenerative diseases, including vascular dementia.

Lactobacillus fermentum WC2020 increased the longevity of Caenorhabditis elegans via JNK-mediated antioxidant pathway.

Lactobacillus fermentum can exert antiaging effects, but their roles are strain-specific, and little is known about the molecular mechanisms in some strains. This study investigated the antiaging effects of L. fermentum WC2020 (WC2020) isolated from Chinese fermented pickles and the underlying mechanism of the action in Caenorhabditis elegans. WC2020 enhanced the mean lifespan of L1-stage and L4-stage worms by 22.67% and 12.42%, respectively, compared with Escherichia coli OP50 (OP50), a standard food source for C. elegans. WC2020-induced longevity was accompanied by an increase in body length and mitochondrial transmembrane potential and a reduction in lipid accumulation and the production of reactive oxygen species and malondialdehyde. Moreover, WC2020 increased the production of glutathione, superoxide dismutases, and catalases and altered the transcripts of many phenotype-related genes. Furthermore, WC2020-fed jnk-1 rather than akt-2 or pmk-1 loss-of-function mutants showed similar lifespans to OP50-fed worms. Correspondingly, WC2020 significantly upregulated the expression of jnk-1 rather than genes involved in insulin-like, p38 MAPK, bate-catenin, or TGF-beta pathway. Moreover, the increase in body length, mitochondrial transmembrane potential, and antioxidant capability and the decrease in lipid accumulation induced by WC2020 were not observed in jnk-1 mutants. Additionally, WC2020 increased the expression of daf-16 and the proportion of daf-16::GFP in the nucleus, and increased lifespan disappeared in WC2020-fed daf-16 loss-of-function mutants. In conclusion, WC2020 activated the JNK/DAF-16 pathway to improve mitochondria function, reduce oxidative stress, and then extend the longevity of nematodes, suggesting WC2020 could be a potential probiotic targeting JNK-mediated antioxidant pathway for antiaging in food supplements and bioprocessing. PRACTICAL APPLICATION: Aging has a profound impact on the global economy and human health and could be delayed by specific diets and nutrient resources. This study demonstrated that Lactobacillus fermentum WC2020 could be a potential probiotic strain used in food to promote longevity and health via the JNK-mediated antioxidant pathway.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

In vitro immunotoxicity effects of carbendazim were inhibited by n-acetylcysteine in microglial BV-2 cells

Carbendazim (CBZ) is a benzimidazole fungicide widely used worldwide in industrial, agricultural, and veterinary practices. Although, CBZ was found in all brain tissues causing serious neurotoxicity, its impact on brain immune cells remain scarcely understood. Our study investigated the in vitro effects of CBZ on activated microglial BV-2 cells. Lipopolysaccharide (LPS)-stimulated BV-2 cells were exposed to increasing concentrations of CBZ and cytokine release was measured by ELISA, and Cytometric Bead Array (CBA) assays. Mitochondrial superoxide anion (O2·-) generation was evaluated by Dihydroethidium (DHE) and nitric oxide (NO) was assessed by Griess reagent. Lipid peroxidation was evaluated by measuring the malonaldehyde (MDA) levels. The transmembrane mitochondrial potential (ΔΨm) was detected by cytometry analysis with dihexyloxacarbocyanine iodide (DiOC6(3)) assay. CBZ concentration-dependently increased IL-1β, IL-6, TNF-α and MCP-1 by LPS-activated BV-2 cells. CBZ significantly promoted oxidative stress by increasing NO, O2·- generation, and MDA levels. In contrast, CBZ significantly decreased ΔΨm. Pre-treatment of BV-2 cells with N-acetylcysteine (NAC) reversed all the above mentioned immunotoxic parameters, suggesting a potential protective role of NAC against CBZ-induced immunotoxicity via its antioxidant and anti-inflammatory effects on activated BV-2 cells. Therefore, microglial proinflammatory over-activation by CBZ may be a potential mechanism by which CBZ could induce neurotoxicity and neurodegenerative disorders.

Intestinal protective effects of essential oil from Chimonanthus salicifolius linked to Mfn2-tethered mitochondria-endoplasmic reticulum connection

Mitochondrial fusion protein 2 (Mfn2), a molecular tether for forming Mitochondria-associated membranes (MAMs), were dynamic platforms implicated in mitochondria-endoplasmic reticulum crosstalk. Here, 41 compounds were identified in Chimonanthus salicifolius essential oil (CSEO) by GC–MS, and its antioxidant capacities were demonstrated by free radical scavenging assays. Subsequently, cell viability results showed that treatment of intestinal epithelium-derived cell line (IEC-6 cells) with CSEO plus H2O2, considerably reduced oxidative damage, enhanced cell viability and antioxidant enzyme activities, when compared with those of treatment with H2O2 alone. Meanwhile, CSEO was demonstrated to reduce H2O2-caused apoptosis in parallel with enhancement of mitochondrial transmembrane potential, reduction of reactive oxygen species and calcium accumulation. Moreover, H2O2 significantly suppressed Mfn2 expression suggesting that intestinal impairment was involved in MAMs dysfunction. However, combination of H2O2 and CSEO significantly up-regulated Mfn2 protein. Together, CSEO exerted beneficial effects against intestinal injury by maintaining mitochondria-endoplasmic reticulum crosstalk through regulation of Mfn2.

Type 2 Diabetes: Platelets and Long-Term Metabolic Control as Estimated from Glycosylated Haemoglobin (HbA1c).

In type 2 diabetes, platelets are likely affected by impaired long-term glycaemic control, but such pathophysiological links are poorly understood. This study thus compares platelet reactivity (i.e. agonist-evoked platelet reactions) in vitro with glycosylated haemoglobin (HbA1c), a measure commonly used for monitoring long-term metabolic control of type 2 diabetes. Elders with type 2 diabetes (n = 35) were divided according to HbA1c into groups (HbA1c-low and high) consisting of 17 and 18 subjects, respectively. For estimating mitochondria disintegration, a flow cytometer determined mitochondrial transmembrane potentials after whole blood agonist stimulation. The activating agents used were α-thrombin (10 μM) and collagen (0.15 μg/mL). The same apparatus analysed the fibrinogen receptor activity, lysosomal exocytosis (surface lysosomal-associated membrane protein 1), and platelet procoagulant characteristics (membrane-attached annexin V) after stimulation. In type 2 diabetes, after in vitro agonist stimulation, platelet mitochondria injury was higher in the HbA1c-high group. The fibrinogen receptor, lysosomal secretion, and the creation of procoagulant platelets proved to be uninfluenced by HbA1c.

Telomerase inhibitors induce mitochondrial oxidation and DNA damage-dependent cell death rescued by Bcl-2/Bcl-xL

BackgroundReactivation of telomerase is a hallmark of cancer and the majority of cancers over-express telomerase. Telomerase-dependent telomere length maintenance confers immortality to cancer cells. However, telomere length-independent cell survival functions of telomerase also play a critical role in tumorigenesis. Multiple telomerase inhibitors have been developed as therapeutics and include anti-sense oligonucleotides, telomerase RNA component targeting agents, chemical inhibitors of telomerase, small molecule inhibitors of hTERT, and telomerase vaccine. In general, telomerase inhibitors affect cell proliferation and survival of cells depending on the telomere length reduction, culminating in replicative senescence or cell death by crisis. However, most telomerase inhibitors kill cancer cells prior to significant reduction in telomere length, suggesting telomere length independent role of telomerase in early telomere dysfunction-dependent cell death. MethodsIn this study, we explored the mechanism of cell death induced by three prominent telomerase inhibitors utilizing a series of genetically encoded sensor cells including redox and DNA damage sensor cells. ResultsWe report that telomerase inhibitors induce early cell cycle inhibition, followed by redox alterations at cytosol and mitochondria. Massive mitochondrial oxidation and DNA damage induce classical cell death involving mitochondrial transmembrane potential loss and mitochondrial permeabilization. Real-time imaging of the progression of mitochondrial oxidation revealed that treated cells undergo a biphasic mitochondrial redox alteration during telomerase inhibition, emphasizing the potential role of telomerase in the redox regulation at mitochondria. Additionally, silencing of hTERT confirmed its predominant role in maintaining mitochondrial redox homeostasis. Interestingly, the study also demonstrated that anti-apoptotic Bcl-2 family proteins still confer protection against cell death induced by telomerase inhibitors. ConclusionThe study demonstrates that redox alterations and DNA damage contribute to early cell death by telomerase inhibitors and anti-apoptotic Bcl-2 family proteins confer protection from cell death by their ability to safeguard mitochondria from oxidation damage.

Liver X receptor activation mitigates oxysterol-induced dysfunction in fetoplacental endothelial cells

Maintaining the homeostasis of the placental vasculature is of paramount importance for ensuring normal fetal growth and development. Any disruption in this balance can lead to perinatal morbidity. Several studies have uncovered an association between high levels of oxidized cholesterol (oxysterols), and complications during pregnancy, including gestational diabetes mellitus (GDM) and preeclampsia (PE). These complications often coincide with disturbances in placental vascular function. Here, we investigate the role of two oxysterols (7-ketocholesterol, 7β-hydroxycholesterol) in (dys)function of primary fetoplacental endothelial cells (fpEC). Our findings reveal that oxysterols exert a disruptive influence on fpEC function by elevating the production of reactive oxygen species (ROS) and interfering with mitochondrial transmembrane potential, leading to its depolarization. Moreover, oxysterol-treated fpEC exhibited alterations in intracellular calcium (Ca2+) levels, resulting in the reorganization of cell junctions and a corresponding increase in membrane stiffness and vascular permeability. Additionally, we observed an enhanced adhesion of THP-1 monocytes to fpEC following oxysterol treatment. We explored the influence of activating the Liver X Receptor (LXR) with the synthetic agonist T0901317 (TO) on oxysterol-induced endothelial dysfunction in fpEC. Our results demonstrate that LXR activation effectively reversed oxysterol-induced ROS generation, monocyte adhesion, and cell junction permeability in fpEC. Although the effects on mitochondrial depolarization and calcium mobilization did not reach statistical significance, a strong trend towards stabilization of calcium mobilization was evident in LXR-activated cells. Taken together, our results suggest that high levels of systemic oxysterols link to placental vascular dysfunction and LXR agonists may alleviate their impact on fetoplacental vasculature.

Exosomes from porcine serum as endogenous additive maintain function of boar sperm during liquid preservation at 17 °C in vitro

The supplementation of sperm culture media with serum is quite common, and improves both sperm survival and motility. However, the link between serum and sperm remains poorly understood. The present study is the first investigation of the effects on sperm quality and function of endogenous porcine serum exosomes in medium used for culturing boar sperm. Scanning electron microscopy (SEM) confirmed that serum-derived exosomes from both castrated boars (cbsExos) and sows (ssExos) exhibited typical nanostructural morphology and expressed CD63, CD9, and Alix, as shown by Western blotting. At 17 °C, the progressive motility and membrane integrity of sperm were significantly increased after incubation of fresh boar semen for 7 days with cbsExos-4 (8 × 1010 particles/mL) or ssExos-16 (32 × 1010 particles/mL). Moreover, cbsExos-4 and ssExos-16 were found to be effective sperm additives, improving mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) content, total antioxidant activity (T-AOC), superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity while reducing reactive oxygen species (ROS) levels, and malondialdehyde (MDA) content following preservation at 17 °C after a 5-day incubation. Both fluorescence and SEM showed that the serum exosomes bound directly to the sperm membrane, suggesting an interaction that could influence sperm-zona pellucida binding. Overall, this study provides new insights into the potential benefits of adding cbsExos and ssExos to enhance the quality of boar sperm during ambient temperature preservation, which may lead to advancements in sperm preservation strategies.

Quercetin Promotes the Repair of Mitochondrial Function in H9c2 Cells Through the miR-92a-3p/Mfn1 Axis.

Cardiocerebrovascular disease is a severe threat to human health. Quercetin has a wide range of pharmacological effects such as antitumor and antioxidant. In this study, we aimed to determine how quercetin regulates mitochondrial function in H9c2 cells. An H9c2 cell oxygen glucose deprivation/reoxygenation (OGD/R) model was constructed. The expression of miR-92a-3p and mitofusin 1 (Mfn1) mRNA in the cells was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Changes in the mitochondrial membrane potential of cells were examined by JC-1 staining. ATP production in the cells was detected using a biochemical assay. Mitochondrial morphological changes were observed using transmission electron microscopy. Detection of miR-92a-3p binding to Mfn1 was done using dual luciferase. Western blotting was used to detect the protein expression of Mfn1 in the cells. miR-92a-3p is essential in regulating cell viability, apoptosis, and tumor cell metastasis. OGD/R induced miR-92a-3p expression, decreased mitochondrial membrane potential and mitochondrial ATP production, and increased mitochondrial damage. Mitochondria are the most critical site for ATP production. Continued opening of the mitochondrial permeability transition pore results in an abnormal mitochondrial transmembrane potential. Both quercetin and inhibition of miR-29a-3p were able to downregulate miR-29a-3p levels, increase cell viability, mitochondrial membrane potential, and ATP levels, and improve mitochondrial damage morphology. Furthermore, we found that downregulation of miR-29a-3p upregulated the protein expression of Mfn1 in cells. Additionally, miR-92a-3p was found to bind to Mfn1 in a luciferase assay. miR- 29a-3p overexpression significantly inhibited the protein expression level of Mfn1. Quercetin treatment partially reversed the effects of miR-29a-3p overexpression in H9c2 cells. Quercetin promoted the recovery of mitochondrial damage in H9c2 cells through the miR-92a-3p/Mfn1 axis.

Polyphenol Mediated Suppression of Hepatocellular Carcinoma (HepG2) Cell Proliferation by Clerodendrum infortunatum L. Root.

Clerodendrum infortunatum L. has long been used in traditional medicine in Sri Lanka for tumours, cancer, and certain skin diseases. The present study aimed to assess the anticancer properties of the aqueous extract of C. infortunatum L. root (AECIR) through the activation of the apoptotic pathway on hepatocellular carcinoma (HepG2) and thus give it a scientific validation. Further, the contribution of polyphenols in antioxidant activity and cell cytotoxicity was investigated. Powdered plant material was boiled with water (100°C) to obtained AECIR. The DPPH assay was used to determine the antioxidant potential. The activity of AECIR on HepG2 and normal rat fibroblast (CC1) cell growth was determined using MTT assay. The morphological changes related to apoptotic pathway was examined by Ethidium Bromide/Acridine Orange (EB/AO), Rhodamine 123 (Rh123) and DNA fragmentation assay. The AECIR demonstrated antioxidant potential with an EC50 of 350.2 ± 1.5 ug/mL for DPPH assay. The HO•, H2O2 and •NO free radical scavenging activity was observed with EC50 of 19.7 ± 2.3, 11.7 ± 0.1 and 273.1 ± 0.9 ug/mL, respectively. The antiproliferative effect of AECIR on HepG2 cells was observed in a time and dose dependent manner with an EC50 of 239.1 ± 1.3 μg/mL while CC1 cells showed a nontoxic effect with an EC50 1062.7 ± 3.4 μg/mL after 24hrs treatment. A significant decrease in antioxidant activity (p<0.001) and 90% HepG2 cell viability was observed with polyphenol removed AECIR compared to the polyphenol present AECIR. The EB/AO uptake, depletion of mitochondrial transmembrane potential, and DNA fragmentation assay results revealed that the apoptosis was induced by AECIR. The obtained result of the present study demonstrates that the antioxidant potential and antiproliferative activity of AECIR is attributed to the presence of polyphenols. Furthermore, the findings provide the scientific base for anti-cancer potential of AECIR.

Antitumoral Potential of Artepillin C, a Compound Derived from Brazilian Propolis, against Breast Cancer Cell Lines.

Breast cancer is the most commonly diagnosed cancer among women worldwide with limited treatment options. Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) is one of the main constituents of Brazilian propolis presenting different activities, including antitumoral effects against various types of cancer. We evaluated the antitumoral potential and mechanisms of action of artepillin C against two distinct human breast cancer cell lines, MCF-7 and MDA-MB-231, to explore a new therapeutic candidate. Cell viability was assessed by MTT assay and the long-term cytotoxicity was performed by clonogenic assay. The morphological changes were observed by light microscopy, analysis of cell death pathway by Annexin V FITC/propidium iodide (PI), lactate dehydrogenase (LDH) by colorimetry, DNA fragmentation by agarose gel and senescence by β-galactosidase. Detection of total reactive oxygen species (ROS) by fluorescence microscopy and determination of mitochondrial transmembrane potential by flow cytometry were also performed. Artepillin C presented a strong and dose-time-dependent cytotoxic effect on MCF-7 and MDA-MB-231 cell lines, with cytotoxicity more evident in MCF-7. In both cancer cell lines, the clonogenic potential was significantly reduced and the morphology of the cells was changed. The treatment also induced death by necrosis and late apoptosis in MCF-7 and MDA-MB-231 and induced cell senescence in MCF-7. Also, artepillin C increased total ROS in both cancer cells and decreased mitochondrial membrane potential in MDA-MB-231 cells. Artepillin C presented antitumoral potential in two human breast cancer cell lines, MCF-7, and MDA-MB-231, suggesting a new promising option for the treatment and/or chemopreventive strategy for breast cancer.

Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.