Mitochondrial Membrane Potential Research Articles

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

Vismodegib Potentiates Marine Antimicrobial Peptide Tilapia Piscidin 4-Induced Cytotoxicity in Human Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is a common cancer with several accepted treatments, such as chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, and immune checkpoint inhibitors. Nevertheless, NSCLC cells often become insensitive to these treatments, and therapeutic resistance is a major reason NSCLC still has a high mortality rate. The induction of therapeutic resistance in NSCLC often involves hedgehog, and suppression of hedgehog can increase NSCLC cell sensitivity to several conventional therapies. In our previous work, we demonstrated that the marine antimicrobial peptide tilapia piscidin 4 (TP4) exhibits potent anti-NSCLC activity in both EGFR-WT and EGFR-mutant NSCLC cells. Here, we sought to further explore whether hedgehog might influence the sensitivity of NSCLC cells to TP4. Our results showed that hedgehog was activated by TP4 in both WT and EGFR-mutant NSCLC cells and that pharmacological inhibition of hedgehog by vismodegib, a Food and Drug Administration-approved hedgehog inhibitor, potentiated TP4-induced cytotoxicity. Mechanistically, vismodegib acted by enhancing TP4-mediated increases in mitochondrial membrane potential and intracellular reactive oxygen species (ROS). MitoTempo, a specific mitochondrial ROS scavenger, abolished vismodegib/TP4 cytotoxicity. The combination of vismodegib with TP4 also reduced the levels of the antioxidant proteins catalase and superoxide dismutase, and it diminished the levels of chemoresistance-related proteins, Bcl-2 and p21. Thus, we conclude that hedgehog regulates the cytotoxic sensitivity of NSCLC cells to TP4 by protecting against mitochondrial dysfunction and suppressing oxidative stress. These findings suggest that combined treatment of vismodegib and TP4 may be a promising therapeutic strategy for NSCLC.

The impact of HBx protein on mitochondrial dynamics and associated signaling pathways strongly depends on the hepatitis B virus genotype.

Chronic hepatitis B virus (HBV) infections are strongly associated with liver cirrhosis, inflammation, and hepatocellular carcinoma. In this context, the viral HBx protein is considered as a major factor influencing HBV-associated pathogenesis through deregulation of multiple cellular signaling pathways and is therefore a potential target for prognostic and therapeutic applications. However, HBV-associated pathogenesis differs significantly between genotypes, with the relevant factors and in particular the contribution of the genetic diversity of HBx being largely unknown. To address this question, we studied the specific genotype-dependent impact of HBx on cellular signaling pathways, focusing in particular on morphological and functional parameters of mitochondria. To exclusively investigate the impact of HBx of different genotypes on integrity and function of mitochondria in the absence of additional viral factors, we overexpressed HBx in Huh7 or HepG2 cells. Key signaling pathways were profiled by kinome analysis and correlated with expression levels of mitochondrial and pathogenic markers. Conclusively, HBx of genotypes A and G caused strong disruption of mitochondrial morphology alongside an induction of PTEN-induced putative kinase 1/Parkin-mediated mitophagy. These effects were only moderately dysregulated by genotypes B and E, whereas genotypes C and D exhibit an intermediate effect in this regard. Accordingly, changes in mitochondrial membrane potential and elevated reactive oxygen species production were associated with the HBx-mediated dysfunction among different genotypes. Also, genotype-related differences in mitophagy induction were identified and indicated that HBx-mediated changes in the mitochondria morphology and function strongly depend on the genotype. This indicates a relevant role of HBx in the process of genotype-dependent liver pathogenesis of HBV infections and reveals underlying mechanisms.IMPORTANCEThe hepatitis B virus is the main cause of chronic liver disease worldwide and differs in terms of pathogenesis and clinical outcome among the different genotypes. Furthermore, the viral HBx protein is a known factor in the progression of liver injury by inducing aberrant mitochondrial structures and functions. Consequently, the selective removal of dysfunctional mitochondria is essential to maintain overall cellular homeostasis and cell survival. Consistent with the intergenotypic difference of HBV, our data reveal significant differences regarding the impact of HBx of different genotypes on mitochondrial dynamic and function and thereby on radical oxygen stress levels within the cell. We subsequently observed that the induction of mitophagy differs significantly across the heterogenetic HBx proteins. Therefore, this study provides evidence that HBx-mediated changes in the mitochondria dynamics and functionality strongly depend on the genotype of HBx. This highlights an important contribution of HBx in the process of genotype-dependent liver pathogenesis.

Artesunate promotes cervical cancer cell apoptosis by regulating Bcl2 family molecules and reducing the mitochondrial membrane potential

Artesunate promotes cervical cancer cell apoptosis by regulating Bcl2 family molecules and reducing the mitochondrial membrane potential

Evaluation of cytotoxic effect of siphonochilone from African ginger: an in vitro analysis.

Plants provide a wide array of compounds that can be explored for potential anticancer properties. Siphonochilone, a furanoterpene that represents one of the main components of the African plant Siphonochilus aethiopicus, shows numerous health benefits. However, to date, its antiproliferative properties have not been tested. The aim of this study was to analyze the cytotoxic effects of siphonochilone on a panel of cancer cell lines and its underlying mechanism of action. Our results demonstrated that siphonochilone exhibited significant cytotoxic effects on pancreatic, breast, lung, colon, and liver cancer cell lines showing a IC50 ranging from 22 to 124 μM at 72 h of treatment and highlighting its cytotoxic effect against MCF7 and PANC1 breast and pancreas cancer cell lines (22.03 and 39.03 μM, respectively). Cell death in these tumor lines was mediated by apoptosis by the mitochondrial pathway, as evidenced by siphonochilone-induced depolarization of the mitochondrial membrane potential. In addition, siphonochilone treatment involves the generation of reactive oxygen species that may contribute to apoptosis induction. In this work, we described for the first time the cytotoxic properties of siphonochilone and provided data about the molecular processes of cell death. Although future studies will be necessary, our results support the interest in this molecule in relation to their clinical application in cancer, and especially in breast and pancreatic cancer.

Cholesterol nanoarchaeosomes for alendronate targeted delivery as an anti-endothelial dysfunction agent

Sodium alendronate (ALN) is a very hydrosoluble and poorly permeable molecule used as an antiresorptive agent and with vascular anticalcifying capacity. Loaded into targeted nanovesicles, its anti-inflammatory activity may be amplified towards extra-osseous and noncalcified target cells, such as severely irritated vascular endothelium. Here cytotoxicity, mitochondrial membrane potential, ATP content, and membrane fluidity of human endothelial venous cells (HUVECs) were determined after endocytosis of ALN-loaded nanoarchaeosomes (nanoARC-Chol(ALN), made of polar lipids from Halorubrum tebenquichense: cholesterol 7:3 w/w, 166 ± 5 nm, 0.16 ± 0.02 PDI, −40.8 ± 5.4 mV potential, 84.7 ± 21 µg/mg ALN/total lipids, TL). The effect of nanoARC-Chol(ALN) was further assessed on severely inflamed HUVECs. To that aim, HUVECs were grown on a porous barrier on top of a basal compartment seeded either with macrophages or human foam cells. One lighter and one more pronounced inflammatory context was modelled by adding lipopolysaccharide (LPS) to the apical or the apical and basal compartments. The endocytosis of nanoARC-Chol(ALN), was observed to partly reduce the endothelial-mesenchymal transition of HUVECs. Besides, while 10 mg/mL dexamethasone, 7.6 mM free ALN and ALN-loaded liposomes failed, 50 μg/mL TL + 2.5 μg/mL ALN (i.e., nanoARC-Chol(ALN)) reduced the IL-6 and IL-8 levels by, respectively, 75% and 65% in the mild and by, respectively, 60% and 40% in the pronounced inflammation model. This is the first report showing that the endocytosis of nanoARC-Chol(ALN) by HUVECs magnifies the anti-inflammatory activity of ALN even under conditions of intense irritation, not only surpassing that of free ALN but also that of dexamethasone.

Grifolin Induces Cell Death of Human Lung Cancer A549 Cell Line via Inhibiting KRAS-mediated Multiple Signaling Pathways.

In the current work, grifolin was obtained from the twigs and leaves of Daphne genkwa for the first time and displayed significant growth inhibition against human lung carcinoma A549 cells. Subsequent in vitro antitumor evaluation revealed that grifolin could induce remarkable cell apoptosis and G0/G1 phase arrest, as well as block cell migration and invasion. In addition, grifolin also disrupted cellular energy metabolism by inducing reactive oxygen species, reducing adenosine triphosphate and mitochondrial membrane potential, and damaging DNA synthesis. Further RNA-seq analysis demonstrated that treatment of grifolin on A549 cells led to gene enrichment in MAPK, PI3K/Akt and NF-kB signaling pathways, all of which were inhibited by grifolin according to immunoblotting experiments. Further mechanistical studies disclosed that the expression of a key upstream protein KRAS was also blocked, and the cell death triggered by grifolin could be rescued by a RAS activator ML-099. Moreover, pretreatment of ML-099 on A549 cells could reverse the grifolin-induced downregulation of key proteins in the three aforementioned pathways. These findings indicate that grifolin could induce cell death in A549 cell line by inhibiting KRAS-mediated multiple signaling pathways.

Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models

BackgroundRetinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR).MethodsAn in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted.ResultsAPI treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission.ConclusionAPI protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

Pirh2 modulates the mitochondrial function and cytochrome c-mediated neuronal death during Alzheimer’s disease

Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key pathological contributor to Alzheimer’s disease (AD), therefore, the role of Pirh2 was investigated in streptozotocin and oligomer Aβ1–42 induced rodent experimental model of AD. Pirh2 protein abundance increased during AD conditions, and transient silencing of Pirh2 inhibited the disease-specific pathological markers like level of p-Tau, βamyloid, acetylcholinesterase activity, and neuronal death. Biochemically, Pirh2 silencing significantly attenuated the oxidative stress, depleted mitochondrial membrane potential, cytochrome c translocation from mitochondria to cytosol, and depleted mitochondrial complex-I activity, and ATP level. Pirh2 silencing also inhibited the altered level of VDAC1, hsp75, hexokinase1, t-Bid, caspase-9, and altered level of apoptotic proteins (Bcl-2, Bax). MALDI-TOF/TOF, co-immunoprecipitation, and UbcH13-linked ubiquitylation assay confirmed the interaction of Pirh2 with cytochrome c and the role of Pirh2 in ubiquitylation of cytochrome c, along with Pirh2-dependent altered proteasome activity. Additionally, Pirh2 silencing further inhibited the translocation of mitochondrion-specific endonuclease G and apoptosis-inducing factors to the nucleus and DNA damage. In conclusion, findings suggested the significant implication of Pirh2 in disease pathogenesis, particularly through impaired mitochondrial function, including biochemical alterations, translocation of cytochrome c, endonuclease G and apoptosis-inducing factor, DNA damage, and neuronal apoptosis.

Ophiobolin A Covalently Targets Mitochondrial Complex IV Leading to Metabolic Collapse in Cancer Cells.

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anticancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of the OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets: lysine-72 of cytochrome c oxidase subunit 5A (COX5A) and cysteine-53 of mitochondrial hypoxia induced gene 1 domain family member 2A (HIGD2A). These two subunit proteins are part of complex IV (cytochrome C oxidase) within the electron transport chain and contributed significantly to the antiproliferative activity of OPA. OPA activated mitochondrial respiration in a COX5A- and HIGD2A-dependent manner, leading to an initial spike in mitochondrial ATP and heightened mitochondrial oxidative stress. OPA compromised mitochondrial membrane potential, ultimately leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anticancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

RUNX3 alleviates mitochondrial dysfunction and tubular damage by inhibiting TLR4/NF-κB signalling pathway in diabetic kidney disease.

The impaired function of tubular mitochondria is critical in diabetic kidney disease (DKD) progression. RUNX3 is down-regulated in DKD models. We intend to explore the effects of RUNX3 on mitochondrial dysfunction and renal tubule injury in DKD and related mechanisms. The development of diabetes models involved injecting mice with streptozotocin while treating HK-2 cells with high glucose (HG). By using immunohistochemical techniques, the renal localizations of RUNX3 were identified. Levels of adenosine triphosphate (ATP), mitochondrial membrane potential, and biochemical index were detected by appropriate kits. Reactive oxygen species (ROS) generation was assessed with dihydroethidium and MitoSOX Red staining. Apoptosis was assessed by flow cytometry and TUNEL. RUNX3 ubiquitination was measured. RUNX3 was mainly present in renal tubules. Overexpressing RUNX3 increased Mfn2, Mfn1, ATP levels, and mitochondrial membrane potential, reduced Drp1 and ROS levels and cell apoptosis, as well as Cyt-C release into the cytoplasm. RUNX3 overexpression displayed a reduction in urinary albumin to creatinine ratio, Hemoglobin A1c, serum creatinine, and blood urea nitrogen. Overexpressing TLR4 attenuated the inhibitory effect of RUNX3 overexpression on mitochondrial dysfunction and cell apoptosis. HG promoted RUNX3 ubiquitination and SMURF2 expression. RUNX3 knockdown cancelled the inhibitory effect of SMURF2 on mitochondrial dysfunction and cell apoptosis. SMURF2 interference inhibits RUNX3 ubiquitination and TLR4/NF-κB signalling pathway, thereby alleviating renal tubule injury.

AQP8 Modulates Mitochondrial H2O2 Transport to Influence Glioma Proliferation

Background Aquaporin-8 (AQP8) is involved in impacting glioma proliferation and can effect tumour growth by regulating Intracellular reactive oxygen species (ROS) signalling levels. In addition to transporting H2O2, AQP8 has been shown to affect ROS signaling, but evidence is lacking in gliomas. In this study, we aimed to investigate how AQP8 affects ROS signaling in gliomas. Materials and methods We constructed A172 and U251 cell lines with AQP8 knockdown and AQP8 rescue by CRISPR/Cas9 technology and overexpression of lentiviral vectors. We used CCK-8 and flow cytometry to test cell proliferation and cycle, immunofluorescence and Mito-Tracker CMXRos to observe the distribution of AQP8 expression in glioma cells, Amplex and DHE to study mitochondria release of H2O2, mitochondrial membrane potential (MMP) and NAD+/NADH ratio to assess mitochondrial function and protein blotting to detect p53 and p21 expression. Result We found that AQP8 co-localised with mitochondria and that knockdown of AQP8 inhibited the release of H2O2 from mitochondria and led to increased levels of ROS in mitochondria, thereby impairing mitochondrial function. We also discovered that AQP8 knockdown resulted in suppression of cell proliferation and was blocked at the G0/G1 phase with increased expression of mitochondrial ROS signalling-related p53/p21. Conclusions This finding provides further evidence for mechanistic studies of AQP8 as a prospective target for the treatment of gliomas.

A mandibular advancement device attenuates the abnormal morphology and function of mitochondria from the genioglossus in obstructive sleep apnea-hypopnea syndrome rabbits.

Obstructive sleep apnea hypopnea syndrome (OSAHS) is a serious and potentially life-threatening disease. Mandibular advancement device (MAD) has the characteristics of non-invasive, comfortable, portable and low-cost, making it the preferred treatment for mild-to-moderate OSAHS. Our previous studies found that abnormal contractility and fibre type distribution of the genioglossus could be caused by OSAHS. However, whether the mitochondria participate in these tissue changes is unclear. The effect of MAD treatment on the mitochondria of the genioglossus in OSAHS is also uncertain. To examine the morphology and function of mitochondria from the genioglossus in a rabbit model of obstructive sleep apnea-hypopnea syndrome (OSAHS), as well as these factors after insertion of a mandibular advancement device (MAD). Thirty male New Zealand white rabbits were randomised into three groups: control, OSAHS and MAD, with 10 rabbits in each group. Animals in Group OSAHS and Group MAD were induced to develop OSAHS by injection of gel into the submucosal muscular layer of the soft palate. The rabbits in Group MAD were fitted with a MAD. The animals in the control group were not treated. Further, polysomnography (PSG) and cone-beam computed tomography (CBCT) scan were used to measure MAD effectiveness. CBCT of the upper airway and PSG suggested that MAD was effective. Rabbits in the three groups were induced to sleep for 4-6 h per day for eight consecutive weeks. The genioglossus was harvested and detected by optical microscopy and transmission electron microscopy. The mitochondrial membrane potential was determined by laser confocal microscopy and flow cytometry. Mitochondrial complex I and IV activities were detected by mitochondrial complex assay kits. OSAHS-like symptoms were induced successfully in Group OSAHS and rescued by MAD treatment. The relative values of the mitochondrial membrane potential, mitochondrial complex I activity and complex IV activity were significantly lower in Group OSAHS than in the control group; however, there was no significant difference between Group MAD and the control group. The OSAHS-induced injury and the dysfunctional mitochondria of the genioglossus muscle were reduced by MAD treatment. Damaged mitochondrial structure and function were induced by OSAHS and could be attenuated by MAD treatment.

Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures

Y-27632, as a cytoskeleton protector, is commonly used for low-temperature preservation of cells. Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 °C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 µM Y-27632 significantly increased plasma membrane integrity (p< 0.05), and acrosome integrity (p < 0.05) and sperm motility (p < 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p < 0.01), increased total antioxidant capacity (T-AOC) (p < 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p < 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p < 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 μM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.

Efficacy and Safety of Asparagusic Acid against Echinococcus multilocularis In Vitro and in a Murine Infection Model

Alveolar echinococcosis (AE) stands as a perilous zoonotic affliction caused by the larvae of Echinococcus multilocularis. There is an imperative need to explore novel therapeutic agents or lead compounds for the treatment of AE. Asparagusic acid, characterized by its low toxicity and possessing antimicrobial, antioxidant, and anti-parasitic attributes, emerges as a promising candidate. The aim of this study was to investigate the in vivo and in vitro efficacy of asparagusic acid against E. multilocularis. Morphological observations, scanning electron microscopy, ROS assays, mitochondrial membrane potential assays, and Western blot were used to evaluate the in vitro effects of asparagusic acid on protoscoleces. The effects of asparagusic acid on vesicles were assessed via PGI release, γ-GGT release, and transmission electron microscopy observations. CellTiter-Glo assays, Caspase3 activity assays, flow cytometry, and Western blot were used for an evaluation of the effect of asparaginic acid on the proliferation and apoptosis of germinal cells. The in vivo efficacy of asparagusic acid was evaluated in a murine AE model. Asparagusic acid exhibited a pronounced killing effect on the protoscoleces post-treatment. Following an intervention with asparagusic acid, there was an increase in ROS levels and a decline in mitochondrial membrane potential in the protoscolex. Moreover, asparagusic acid treatment resulted in the upregulation of PGI and γ-GGT release in metacestode vesicles, concomitant with the inhibition of germinal cell viability. Furthermore, asparagusic acid led to an enhanced relative expression of Caspase3 in the culture supernatant of both the protoscoleces and germinal cells, accompanied by an increase in the proportion of apoptotic germinal cells. Notably, asparagusic acid induced an augmentation in Bax and Caspase3 protein expression while reducing Bcl2 protein expression in both the protoscoleces and germinal cells. In vitro cytotoxicity assessments demonstrated the low toxicity of asparagusic acid towards normal human hepatocytes and HFF cells. Additionally, in vivo experiments revealed that asparagusic acid administration at doses of 10 mg/kg and 40 mg/kg significantly reduced metacestode wet weight. A histopathological analysis displayed the disruption of the germinal layer structure within lesions post-asparagusic acid treatment, alongside the preservation of laminated layer structures. Transmission electron microscopy further revealed mitochondrial swelling and heightened cell necrosis subsequent to the asparagusic acid treatment. Furthermore, asparagusic acid promoted Caspase3 and Bax protein expression while decreasing Bcl2 protein expression in perilesional tissues. Subsequently, it inhibited the expression of Ki67, MMP2, and MMP9 proteins in the perilesional tissues and curbed the activation of the PI3K/Akt signaling pathway within the lesion-host microenvironmental tissues. Asparagusic acid demonstrated a pronounced killing effect on E. multilocularis, suggesting its potential as a promising therapeutic agent for the management of AE.

Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods.

For sperm cryopreservation, the conventional method, which requires glycerol, has been used for a long time. In addition, the permeable cryoprotectant-free vitrification method has been continuously studied. Although the differences of cryopreservation effects between the two methods have being studied, differences in microRNA (miRNA) profiles between them remain unclear. In this study, we investigated the differences in miRNA expression profiles among conventional freezing sperm, droplet vitrification freezing sperm and fresh human sperm. We also analyzed the differences between these methods in terms of differentially expressed miRNAs (DEmiRs) related to early embryonic development and paternal epigenetics. Our results showed no significant differences between the cryopreservation methods in terms of sperm motility ratio, plasma membrane integrity, DNA integrity, mitochondrial membrane potential, acrosome integrity, and ultrastructural damage. However, sperm miRNA-sequencing showed differences between the two methods in terms of the numbers of DEmiRs (28 and 19 with vitrification using a nonpermeable cryoprotectant and the conventional method, respectively) in postthaw and fresh sperm specimens. DEmiRs related to early embryonic development and paternal epigenetics mainly included common DEmiRs between the groups. Our results showed that the differences between conventional freezing and droplet vitrification were minimal in terms of miRNA expression related to embryonic development and epigenetics. Changes in sperm miRNA expression due to freezing are not always detrimental to embryonic development. This study compared differences in miRNA expression profiles before and after cryopreservation between cryopreservation by conventional and vitrification methods. It offers a new perspective to evaluate various methods of sperm cryopreservation.

Reactive oxygen species-responsive supramolecular deucravacitinib self-assembly polymer micelles alleviate psoriatic skin inflammation by reducing mitochondrial oxidative stress

IntroductionThe new topical formula is urgent needed to meet clinical needs for majority mild patients with psoriasis. Deucravacitinib exerts outstanding anti-psoriatic capacity as an oral TYK2 inhibitor; however, single therapy is insufficient to target the complicated psoriatic skin, including excessive reactive oxygen species (ROS) and persistent inflammation. To address this need, engineered smart nano-therapeutics hold potential for the topical delivery of deucravacitinib.Methodshydrophobic Deucravacitinib was loaded into polyethylene glycol block-polypropylene sulphide (PEG-b-PPS) for transdermal delivery in the treatment of psoriasis. The oxidative stress model of HaCaT psoriasis was established by TNF-α and IL-17A in vitro. JC-1 assay, DCFH-DA staining and mtDNA copy number were utilized to assess mitochondrial function. 0.75% Carbopol®934 was incorporated into SPMs to produce hydrogels and Rhb was labeled to monitor penetration by Immunofluorescence. In vivo, we established IMQ-induced psoriatic model to evaluate therapeutic effect of Car@Deu@PEPS.ResultsDeu@PEPS exerted anti-psoriatic effects by restoring mitochondrial DNA copy number and mitochondrial membrane potential in HaCaT. In vivo, Car@Deu@PEPS supramolecular micelle hydrogels had longer retention time in the dermis in the IMQ-induced ROS microenvironment. Topical application of Car@Deu@PEPS significantly restored the normal epidermal architecture of psoriatic skin with abrogation of splenomegaly in the IMQ-induced psoriatic dermatitis model. Car@Deu@PEPS inhibited STAT3 signaling cascade with a corresponding decrease in the levels of the differentiation and proliferative markers Keratin 17 and Cyclin D1, respectively. Meanwhile, Car@Deu@PEPS alleviated IMQ-induced ROS generation and subsequent NLRP3 inflammasome-mediated pyroptosis.ConclusionDeu@PEPS exerts prominent anti-inflammatory and anti-oxidative effects, which may offers a more patient-acceptable therapy with fewer adverse effects compared with oral deucravacitinib.

2-(4-Methylthiazol-5-yl) Ethyl Nitrate Hydrochloride Ameliorates Cognitive Impairment via Modulation of Oxidative Stress and Nuclear Factor Kappa B (NF-κB) Signaling Pathway in Chronic Cerebral Hypoperfusion-Associated Spontaneously Hypertensive Rats

Hypertension reduces the bioavailability of vascular nitric oxide (NO) and contributes to the onset of vascular dementia (VaD). A loss of NO bioavailability increases inflammation and oxidative stress. 2-(4-Methylthiazol-5-yl) ethyl nitrate hydrochloride (W1302) is a novel nitric oxide donor (NOD) which is undergoing phase I clinical trials in China for the treatment of VaD. In this study, we investigated the protective effects of W1302 in VaD rats induced by the permanent occlusion of a bilateral common carotid arteries model related to spontaneous hypertension (SHR-2VO), and we further explored the underlying mechanisms. Nimodipine was used as a positive control. Our results showed that W1302 treatment for 4 weeks (10 mg/Kg/day) exhibited stronger improvement in the spatial learning and memory deficits in SHR-2VO rats compared with nimodipine with slightly lower systolic blood pressure (SBP). Meanwhile, W1302 treatment significantly increased NO and cGMP production, restored mitochondrial membrane potential and attenuated oxidative stress as evidenced by increasing ATP production and reducing malondialdehyde (MDA) levels in the brain. Furthermore, W1302 treatment markedly inhibited the iNOS activity and decreased TNF-α expression via inhibiting the nuclear factor kappa B (NF-κB) signaling pathway. Nimodipine treatment also restored these aberrant changes, but its ATP production was weaker than that of W1302, and there was no significant effect on NO release. Taken together, W1302 exhibited beneficial effects on complications in VaD with hypertension, which is involved in suppressing oxidative damage, and the inflammatory reaction might be mediated by an increase in NO release. Therefore, W1302 has therapeutic potential for the treatment of VaD caused by chronic cerebral hypoperfusion-associated spontaneous hypertension.

A novel pathogenic mitochondrial DNA variant m.4344T>C in tRNAGln causes developmental delay.

Mitochondrial diseases are a group of genetic diseases caused by mutations in mitochondrial DNA and nuclear DNA. However, the genetic spectrum of this disease is not yet complete. In this study, we identified a novel variant m.4344T>C in mitochondrial tRNAGln from a patient with developmental delay. The mutant loads of m.4344T>C were 95% and 89% in the patient's blood and oral epithelial cells, respectively. Multialignment analysis showed high evolutionary conservation of this nucleotide. TrRosettaRNA predicted that m.4344T>C variant would introduce an additional hydrogen bond and alter the conformation of the T-loop. The transmitochondrial cybrid-based study demonstrated that m.4344T>C variant impaired the steady-state level of mitochondrial tRNAGln and decreased the contents of mitochondrial OXPHOS complexes I, III, and IV, resulting in defective mitochondrial respiration, elevated mitochondrial ROS production, reduced mitochondrial membrane potential and decreased mitochondrial ATP levels. Altogether, this is the first report in patient carrying the m.4344T>C variant. Our data uncover the pathogenesis of the m.4344T>C variant and expand the genetic mutation spectrum of mitochondrial diseases, thus contributing to the clinical diagnosis of mitochondrial tRNAGln gene variants-associated mitochondrial diseases.