Membrane Potential Levels Research Articles

Життєздатність ацинарних клітин підшлункової залози та рівень мембранного потенціалу мітохондрій за дії жовчевої кислоти TLC-S

To investigate the mechanism of action of bile acids, TLC-S, the viability of pancreatic acinar cells, and the level of mitochondrial membrane potential were studied using various oxidation substrates. The experiments were conducted on male Wistar rats weighing 250–300 g. A suspension of isolated pancreatic acinar cells was obtained using collagenase (type IV, 0.2 mg/ml). The trypan blue exclusion test assessed cell viability after isolation and the amount was >90 %. The survival of acinar cells after prolonged incubation in different environments was determined by fluorescent dyes propidium iodide (0.5 mg/ml) and Hoechst 33258 (5 mg/ml) dyes. Mitochondrial membrane potential was recorded by adding the dye TMRM (50 nM). Cells were photographed using an inverted microscope Olympus IX73 with a digital camera DP-74. Cell counting and fluorescence intensity of TMRM were analyzed using ImageJ software. It was found that the presence of TLC-S in the medium at a concentration of 0.5 mmol/L, when used as an oxidation substrate for glucose (10 mmol/L), pyruvate (2 mmol/L), or alanine (2 mmol/L), did not significantly affect the proportion of live cells after 2 and 4 hours of incubation. However, when TLC-S was added to the medium at a concentration of 2 mmol/L, the proportion of live cells significantly decreased. This decrease was least pronounced during pyruvate oxidation. Under the action of TLC-S (after 25 min) in the presence of glucose alone, the mitochondrial membrane potential decreased by 9.8 % (P<0.05, n=3), in the presence of alanine (in the presence of glucose) – by 23.5 % (P<0.05, n=4). When pyruvate was added to the medium (in the presence of glucose), the mitochondrial membrane potential likely did not change significantly. These results suggest the presence of several different mechanisms of TLC-S’s negative effects on pancreatic acinar cells: at low concentrations (or at early stages), it disrupts alanine transamination reactions, leading to the disruption of mitochondrial membrane potential generation, while at high concentrations, it affects the integrity of plasma and/or intracellular membranes.

Oocyte quality is impaired in a hyperandrogenic PCOS mouse model by increased Foxo1 expression

One of the most important characteristics of patients with polycystic ovary syndrome (PCOS) is excess androgen, which has adverse effects on pregnancy outcomes and increases the risk of offspring developing metabolic disorders. Foxo1 has been shown to play an important role in PCOS, but whether it has an affect on oocyte’s quality in PCOS remains unclear. The current research investigated the effect of excess androgen exposure on mouse oocyte quality, as well as the possible molecular mechanism. Timelapse incubator was used to culture oocytes in vitro and evaluate the maturation process. The level of reactive oxygen species (ROS) and mitochondrial membrane potential were detected by laser confocal microscope. Immunofluorescence staining assays were performed to examine the expression of Foxo1 and γ-H2AX. Relative mRNA level of Foxo1 and Caspase3 were examined by RT-qPCR. Results showed Germinal vesicle breakdown and maturation rates of oocytes from hyperandrogenic PCOS mice were significantly decreased in vitro, while in vitro maturation showed a marked delay from the germinal vesicle breakdown to metaphase II stage in PCOS group. Expression levels of reactive oxygen species, Foxo1, Caspase3, and γ-H2AX were significantly increased, whereas mitochondrial membrane potential was significantly decreased in oocytes from PCOS mice. These results indicate that excess androgen exposure induced oxidative stress, which further induced high expression of Foxo1, resulting in more DNA damage and apoptosis in oocytes. The current findings provide new knowledge for exploring the mechanism of decreased oocyte quality in hyperandrogenic PCOS.

Silencing USP19 alleviates cigarette smoke extract-induced mitochondrial dysfunction in BEAS-2B cells by targeting FUNDC1.

Chronic obstructive pulmonary disease (COPD) is commonly caused by smoking. FUN14 domain-containing protein 1 (FUNDC1) plays a fundamental role in mitochondrial autophagy and apoptosis in cigarette smoke extract (CSE)-treated BEAS-2B cells. The present study investigated the mechanism of action of FUNDC1 in mitochondrial dysfunction and apoptosis in CSE-treated BEAS-2B cells. The interaction between ubiquitin-specific peptidase 19 (USP19) and FUNDC1 was analyzed using co-immunoprecipitation. Effects of USP19 knockdown and/or FUNDC1 overexpression on the survival, apoptosis, mitochondrial membrane potential, and oxygen consumption rate (OCR) of BEAS-2B cells treated with 15% CSE were determined. In BEAS-2B cells, CSE inhibited cell survival, promoted apoptosis, increased the expression of USP19 and FUNDC1, increased the ratio of LC3 II to LC3 I (LC3 II/I), and decreased mitochondrial membrane potential and TOM20 levels. In CSE-treated BEAS-2B cells, USP19 knockdown reduced FUNDC1 and LC3 II/I, increased the levels of TOM20, improved cell survival, mitochondrial membrane potential, and OCR, and inhibited apoptosis. USP19 deubiquitinates FUNDC1. FUNDC1 overexpression inhibited the effect of USP19 knockdown in CSE-treated BEAS-2B cells. Overall, decreasing USP19 expression alleviates CSE-induced mitochondrial dysfunction in BEAS-2B cells by downregulating FUNDC1, providing novel insights into the molecular mechanism of FUNDC1 regulation in COPD.

Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1.

Tanshinone IIA (TSN) extracted from danshen (Salviamiltiorrhiza) could protect cardiomyocytes against myocardial ischemia/reperfusion injury (IRI), however the underlying molecular mechanisms of action remain unclear. The aim of the present study was to identify the protective effects of TSN and its mechanisms of action through invitro studies. An anoxia/reoxygenation (A/R) injury model was established using H9c2 cells to simulate myocardial IRI invitro. Before A/R, H9c2 cardiomyoblasts were pretreated with 8 µM TSN or 10µM ferrostatin‑1 (Fer‑1) or erastin. The cell counting kit 8 (CCK‑8) and lactate dehydrogenase (LDH) assay kit were used to detect the cell viability and cytotoxicity. The levels of total iron, glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde (MDA), ferrous iron, caspase‑3 activity, and reactive oxygen species (ROS) were assessed using commercial kit. The levels of mitochondrial membrane potential (MMP), lipid ROS, cell apoptosis, and mitochondrial permeability transition pore (mPTP) opening were detected by flow cytometry. Transmission electron microscopy (TEM) was used to observed the mitochondrial damage. Protein levels were detected by western blot analysis. The interaction between TSN and voltage‑dependent anion channel 1 (VDAC1) was evaluated by molecular docking simulation. The results showed that pretreatment with TSN and Fer‑1 significantly decreased cell viability, glutathione peroxidase4 (GPX4) protein and GSH expression and GSH/GSSG ratio and inhibited upregulation of LDH activity, prostaglandin endoperoxide synthase 2 and VDAC1 protein expression, ROS levels, mitochondrial injury and GSSG induced by A/R. TSN also effectively inhibited the damaging effects of erastin treatment. Additionally, TSN increased MMP and Bcl‑2/Bax ratio, while decreasing levels of apoptotic cells, activating Caspase‑3 and closing the mPTP. These effects were blocked by VDAC1 overexpression and the results of molecular docking simulation studies revealed a direct interaction between TSN and VDAC1. In conclusion, TSN pretreatment effectively attenuated H9c2 cardiomyocyte damage in an A/R injury model and VDAC1‑mediated ferroptosis and apoptosis served a vital role in the protective effects of TSN.

Regulatory role of phospholipase A2 inhibitor in oxidative stress and inflammation induced by an experimental mouse migraine model

Migraine is a complex neurological problem whose primary symptom is headache and is common in the human population. It is well known that neuroinflammation plays a vital role in the pathogenesis of migraine, with adverse effects on the nervous system, including headache disorders such as migraine. The infusion of the nitric oxide donor glyceryl trinitrate (GTN) is often used in experimental models of migraine because it is the best-known model of migraine provocation. N-(p-amyl cinnamoyl) anthranilic acid (ACA) has been shown to inhibit both TRPM2 and phospholipase A2 (PLA2). Recent research has explored potential interventions to mitigate GTN-induced neurotoxicity. One such candidate is ACA, a compound with anti-inflammatory and antioxidant properties.
 Thirty-six C57BL/6j black mice were divided into the control groups of ACA, GTN, and ACA+GTN. Mice in the ACA were treated intraperitoneally with ACA (25 mg/kg) for three days. Mice in the GTN were treated intraperitoneally with a single dose of GTN (10 mg/kg) for migraine induction. After the experimental stages were completed, the mice in all groups were sacrificed, and brain tissue and erythrocyte samples were taken from the mice.
 The levels of inflammatory cytokines (TNF α, IL 1β, and IL 6), apoptosis, intracellular ROS, lipid peroxidation, caspase 3-9, and mitochondrial membrane potential increased in the GTN group. However, their levels were decreased in the ACA+GTN group by the injection of ACA. The treatment of ACA regulated the GTN treatment-induced decreases of glutathione levels, glutathione peroxidase activation, and cell viability in the brain and erythrocytes.
 In conclusion, GTN plays a role in neurotoxicity caused by increased apoptosis and ROS. We observed that ACA modulated the brain and erythrocyte oxidant, antioxidant parameters, and apoptotic processes. The neuro-protective role of ACA treatment may be explained by its modulating activity against increased apoptosis and oxidative stress.

Size-controllable food-grade nanoparticles based on sea cucumber polypeptide with good anti-oxidative capacity to prolong lifespan in tumor-bearing mice

Liver cancer, a malignancy with a rising global incidence, poses a significant challenge in achieving effective treatment outcomes. As food-derived nutrient, sea cucumber peptide (SCP) has shown promising anticancer effects. Therefore, we explored the nanodelivery systems to encapsulate SCP to enhance its stability in the gastrointestinal tract and improve absorption within the tumor microenvironment. This study aimed to develop size-controllable multifunctional nanoparticles using SCP, procyanidins (PCs), and vanillin through molecular assembly via a one-pot Mannich condensation approach. These food-grade nanoparticles demonstrated water solubility and exhibited a spherical structure with sizes ranging from 441 to 1360 nm, depending on the concentration of the reactants. In vitro cell experiments demonstrated that SCP nanoparticles modified with PCs effectively reduced the generation of reactive oxygen species from H2O2 and acrylamide while maintaining normal levels of mitochondrial membrane potential. Furthermore, in vivo nutrition intervention studies conducted on tumor-bearing mice revealed that mice treated with SCP nanoparticles exhibited a survival rate of 40 %, which was significantly higher than the 0 % and 20 % survival rates observed in the control and SCP-treated groups, respectively. These findings suggest that SCP nanoparticles, possessing antioxidative properties and controllable sizes, hold potential for precision nutrition in the field of cancer treatment.

Multilevel synchronization of human β-cells networks.

β-cells within the endocrine pancreas are fundamental for glucose, lipid and protein homeostasis. Gap junctions between cells constitute the primary coupling mechanism through which cells synchronize their electrical and metabolic activities. This evidence is still only partially investigated through models and numerical simulations. In this contribution, we explore the effect of combined electrical and metabolic coupling in β-cell clusters using a detailed biophysical model. We add heterogeneity and stochasticity to realistically reproduce β-cell dynamics and study networks mimicking arrangements of β-cells within human pancreatic islets. Model simulations are performed over different couplings and heterogeneities, analyzing emerging synchronization at the membrane potential, calcium, and metabolites levels. To describe network synchronization, we use the formalism of multiplex networks and investigate functional network properties and multiplex synchronization motifs over the structural, electrical, and metabolic layers. Our results show that metabolic coupling can support slow wave propagation in human islets, that combined electrical and metabolic synchronization is realized in small aggregates, and that metabolic long-range correlation is more pronounced with respect to the electrical one.

Qibai Pingfei capsule induces apoptosis of pulmonary artery smooth muscle cells in hypoxic rats by regulating PI3K/AKT/mitochondrial signaling pathway

Purpose: To investigate the effect of Qibai Pingfei capsules (QBPF) medicated serum on the apoptosis of rat pulmonary artery smooth muscle cells (PASMC) in hypoxic rats, and to determine the relationship between that effect and PI3K/Akt/mitochondrial apoptosis pathway.Methods: Rat PASMCs were isolated, cultured, and the optimal hypoxic time and concentration of QBPF were determined by CCK-8 method. Hypoxic rats were treated with QBPF, QBPF + LY294002, or QBPF + SC79. Apoptosis and mitochondrial membrane potential were assessed using Annexin VFITC/ PI, Hoechst 33258, and Rho123 staining. The protein expression levels of AKT, P-AKT, and apoptosis-related proteins were evaluated via western blot.Results: CCK-8 studies showed that the optimal hypoxic time was 24 h, while the optimal concentration of QBPF was 20 %. Annexin V-FITC/PI double staining and Hoechst 33258 assay revealed that QBPF significantly promoted the apoptosis of PASMCs in hypoxic rats (p < 0.05). Rho123 test results showed that QBPF inhibited mitochondrial membrane potential level in hypoxic rats' PASMCs, which was enhanced by PI3K inhibitor LY29002 and inhibited by AKT agonist SC79 (p < 0.05). Western blot showed that QBPF reduced the protein level of P-AKT and Bcl-2, and raised the protein levels of Bad, Bax, CytC, casepase-9 and casepase-3, which was enhanced by LY29002 and blocked by SC79 (p < 0.05). No major changes in AKT protein expression were seen between the groups.Conclusion: In hypoxic rats, QBPF blocks PI3K/AKT signaling pathway and regulates the activation of downstream Bcl-2 family members, thus activating mitochondrial apoptosis pathway and triggering PASMC death.

Suberoylanilide hydroxamic acid upregulates reticulophagy receptor expression and promotes cell death in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a common clinical condition with a poor prognosis and few effective treatment options. Potent anticancer agents for treating HCC must be identified. Epigenetics plays an essential role in HCC tumorigenesis. Suberoylanilide hydroxamic acid (SAHA), the most common histone deacetylase inhibitor agent, triggers many forms of cell death in HCC. However, the underlying mechanism of action remains unclear. Family with sequence similarity 134 member B (FAM134B)-induced reticulophagy, a selective autophagic pathway, participates in the decision of cell fate and exhibits anticancer activity. This study focused on the relationship between FAM134B-induced reticulophagy and SAHA-mediated cell death. To elucidate potential roles and underlying molecular mechanisms of reticulophagy in SAHA-induced HCC cell death. The viability, apoptosis, cell cycle, migration, and invasion of SAHA-treated Huh7 and MHCC97L cells were measured. Proteins related to the reticulophagy pathway, mitochondria-endoplasmic reticulum (ER) contact sites, intrinsic mitochondrial apoptosis, and histone acetylation were quantified using western blotting. ER and lysosome colocalization, and mitochondrial Ca2+ levels were characterized via confocal microscopy. The level of cell death was evaluated through Hoechst 33342 staining and propidium iodide colocalization. Chromatin immunoprecipitation was used to verify histone H4 lysine-16 acetylation in the FAM134B promoter region. After SAHA treatment, the proliferation of Huh7 and MHCC97L cells was significantly inhibited, and the migration and invasion abilities were greatly blocked in vitro. This promoted apoptosis and caused G1 phase cells to increase in a concentration-dependent manner. Following treatment with SAHA, ER-phagy was activated, thereby triggering autophagy-mediated cell death of HCC cells in vitro. Western blotting and chromatin immunoprecipitation assays confirmed that SAHA regulated FAM134B expression by enhancing the histone H4 lysine-16 acetylation in the FAM134B promoter region. Further, SAHA disturbed the Ca2+ homeostasis and upregulated the level of autocrine motility factor receptor and proteins related to mitochondria-endoplasmic reticulum contact sites in HCC cells. Additionally, SAHA decreased the mitochondrial membrane potential levels, thereby accelerating the activation of the reticulophagy-mediated mitochondrial apoptosis pathway and promoting HCC cell death in vitro. SAHA stimulates FAM134B-mediated ER-phagy to synergistically enhance the mitochondrial apoptotic pathway, thereby enhancing HCC cell death.

Neuroprotective Effect of Zishen Huoxue Decoction treatment on Vascular Dementia by activating PINK1/Parkin mediated Mitophagy in the Hippocampal CA1 Region

Ethnopharmacological relevanceZishen Huoxue Decoction (ZSHXD) is a Traditional Chinese Medicine (TCM) prescription for the treatment of vascular dementia (VD). Although the clinical effects of ZSHXD have been demonstrated, the molecular mechanisms underlying the neuroprotective effects of ZSHXD remain unclear. Aim of the studyTo explore whether the neuroprotective effect of Zishen Huoxue Decoction (ZSHXD) treatment is associated with the PINK1/Parkin pathway-mediated mitophagy in hippocampal CA1 region of 2-VO model rats. Materials and methodsSeventy-two male SD rats were randomly divided into the sham group, model group, Donepezil (0.45 mg/kg) group, ZSHXD low dose group (8.9 g/kg), ZSHXD medium dose group (17.8 g/kg), and ZSHXD high dose group (35.6 g/kg). Two-vessel occlusion (2-VO) rat model is established to evaluate the therapeutic effect of ZSHXD pretreatment. Hematoxylin-eosin (HE) staining is conducted to detect the morphological changes of neurons and the number of normal neurons in the hippocampal CA1 region. Then, the mitochondrial function and structure were reflected by the mitochondrial membrane potential (MMP) levels and transmission electron microscopy (TEM). Meanwhile, the expression of mitophagy related proteins mediated by PINK1/Parkin was detected by western blot (WB). After that, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured by Elisa. At last, the apoptosis-related proteins Caspase-3、Bax、Bcl-2 were measured by WB. ResultsThe results depict that ZSHXD has dose-dependently improved the cognitive function in 2-VO model rats. It has also been showed that ZSHXD can alleviate neuron damage, rescue the mitochondrial structural injury and dysfunction in hippocampal CA1 region. Besides, ZSHXD has increased the activity of SOD and decreased the activity of MDA. In addition, ZSHXD can inhibit apoptosis with Caspase-3, Bax decreasing and Bcl-2 increasing. Specially, the protection of ZSHXD showed in 2-VO model rats is along with the upregulation of PINK1, Parkin and LC3-Ⅱ/Ⅰ, and downregulation of p62 in the hippocampal CA1 region. ConclusionsThis study reveals that ZSHXD protects the 2-VO model rats from ischemic injury by activating the PINK1/Parkin-mediated mitophagy in the hippocampal CA1 region.

Quality of fresh and cryopreserved bovine sperm is reduced by BPA and BPF exposure.

Bisphenol A (BPA) is an endocrine disrupting compound, used as the key monomer of polycarbonate plastics and epoxy resins. BPA has been detected in both humans and farm animals and has been correlated with decreased sperm counts and motility. BPS and BPF are structural analogs of BPA and are increasingly being used in manufacturing as BPA substitutes. In this study we aim to assess the direct outcomes of BPA, BPS and BPF exposure on bovine sperm parameters in vitro to elucidate how they affect sperm quality and fertilization potential, and to assess whether BPS and/or BPF are less harmful than BPA. Sperm from three or more bulls was obtained from either fresh samples or cryopreserved straws and exposed to 0.05 mg/mL of BPA, BPS and BPF in vitro. After 4h incubation, motility, capacitation, apoptosis/necrosis, and mitochondrial membrane potential levels were measured by CASA or computational flow cytometry. Results showed that BPA exposure significantly reduced both fresh and cryopreserved sperm motility, capacitation, viability and mitochondrial membrane potential levels. Furthermore, BPF significantly decreased motility, capacitation and mitochondrial membrane potential in cryopreserved sperm only. BPS did not have any significant effects on any of the parameters measured. Our results suggest that BPA is the most harmful to sperm, while BPF is toxic under certain conditions, and BPS seems to be the least detrimental. Overall, this study provides an understanding of how the ubiquitous environmental chemicals, bisphenols, may impact male fertility even after ejaculation.

Aralia saponin A isolated from Achyranthes bidentata Bl. ameliorates LPS/D-GalN induced acute liver injury via SPHK1/S1P/S1PR1 pathway in vivo and in vitro

ObjectiveAcute liver injury (ALI) refers to a disease in which the liver is affected by factors such as chemical substances, alcohol, and virus infection in a short time, resulting in damage to liver cells. Achyranthes bidentata Bl. with the hepatoprotective activity has attracted great attention. In this study, a pentacyclic triterpenoid (Aralia saponin A, AsA) was isolated from roots of Achyranthes bidentata Bl. and its anti-ALI activity, as well as the mechanisms, were investigated for the first time. MethodsAsA (10 or 20 mg/kg, i.g.) was administered over a period of 1 weeks, following which liver injury was induced by LPS (10 µg/kg)/D-GalN (700 mg/kg). H&E staining of liver, Aspartate amino transferase (AST), Alanine transaminase (ALT) and cytokines was employed to investigate ALI relevant features. The mitochondrial morphology and levels of mitochondrial membrane potential (MMP), oxidative stress balance, apoptosis, average fluorescence intensity of 2-DG, natural killer (NK) cells in liver tissues were determined to assess the oxidative stress damage and inflammatory injury. Transcriptomics and metabonomics analysis were employed to clarify the mechanisms. Additionally, the mRNA and protein expression levels of Sphingosine 1-phosphate (S1P), Sphingosine kinase-1 (SPKH1), Sphingosine 1 phosphate receptor 1 (S1PR1), Sphingosine 1 phosphate receptor 3 (S1PR3), TNF receptor associated factor 2 (TRAF-2), Phospho-NF- kappaB p65 (p-P65), NF- kappaB p65 (P65), Proto-oncogene ras (Ras), Ras-related C3 botulinum toxin substrate (Rac), Phospholipase C (PLC), Interleukin 6 (IL-6), Tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), Vascular cell adhesion molecule 1 (Vcam1), CC chemokine ligand-2 (Ccl2) were analyzed. The mediating role of SPHK1 in the observed effects caused by AsA was assessed by investigatin SPHK1 transfection silencing/overexpression against AsA in AML12 cells induced by LPS/D-GalN. ResultsAsA can ameliorate liver function, inflammation, mitochondrial structure and oxidative stress in the ALI model. Meanwhile, AsA led to downregulated expression of proteins associated with sphingolipid signaling pathway. Silencing of SPHK1 led to enhanced protective effects of AsA, while over-expression of SPHK1 led to degraded protective effects of AsA in LPS/D-GalN-induced AML12 cells, suggesting that ALI is regulated by active molecules of AsA by means of SPHK1 mediation. ConclusionsAsA can ameliorate LPS/D-GalN-induced ALI by inhibiting inflammation and oxidative stress via the SPHK1/S1P/S1PR1 pathway. In this way, a molecular justification is provided for AsA application in ALI treatment.

Codium fragile Suppressed Chronic PM2.5-Exposed Pulmonary Dysfunction via TLR/TGF-β Pathway in BALB/c Mice.

This study investigated the ameliorating effect of the aqueous extract of Codium fragile on PM2.5-induced pulmonary dysfunction. The major compounds of Codium fragile were identified as palmitic acid, stearic acid, and oleamide using GC/MS2 and hexadecanamide, oleamide, and 13-docosenamide using UPLC-Q-TOF/MSE. Codium fragile improved pulmonary antioxidant system deficit by regulating SOD activities and reducing GSH levels and MDA contents. It suppressed pulmonary mitochondrial dysfunction by regulating ROS contents and mitochondrial membrane potential levels. It regulated the inflammatory protein levels of TLR4, MyD88, p-JNK, p-NF-κB, iNOS, Caspase-1, TNF-α, and IL-1β. In addition, it improved the apoptotic protein expression of BCl-2, BAX, and Caspase-3 and attenuated the fibrous protein expression of TGF-β1, p-Smad-2, p-Smad-3, MMP-1, and MMP-2. In conclusion, this study suggests that Codium fragile might be a potential material for functional food or pharmaceuticals to improve lung damage by regulating oxidative stress inflammation, cytotoxicity, and fibrosis via the TLR/TGF-β1 signaling pathway.

Quality of testicular spermatozoa improves with changes in composition of culture medium

BackgroundSpermatozoa retrieved from the testis and epididymis are deprived of the beneficial effects of seminal fluid. Thus applying an artificial medium with normal seminal fluid characteristics, known as artificial seminal fluid (ASF), may provide an appropriate condition for improving some sperm parameters in azoospermia. The objective was to investigate the impact of in vitro exposure of testicular and epididymal spermatozoa to ASF on sperm quality. The study was conducted on testicular (n = 20) and epididymal (n = 20) sperm specimens obtained from azoospermic men. Each sample was divided into two equal parts: Part I) for processing and incubation with Ham’s F10 medium; Part II) for processing and incubation with ASF.ResultsAfter 2 h incubation, testicular sperm motility was significantly higher in ASF than in Ham’s F10 medium. In comparison to 0 h, mitochondrial membrane potential levels of testicular spermatozoa were significantly higher after 2 h and 24 h in ASF and after 24 h in Ham’s F10 medium. Furthermore, the data indicated significantly lower rates of epididymal spermatozoa with high MMP in both media after 24 h. There were no significant differences in the DNA fragmentation index of testicular and epididymal spermatozoa between ASF and Ham’s F10 medium at different time points.ConclusionThe results demonstrated that in vitro incubation of testicular spermatozoa improved their motility more effectively than Ham’s F10 medium in the short term (2 h), but had no effect on epididymal spermatozoa. Since the physiology of testicular spermatozoa is different from that of ejaculated spermatozoa, it seems that a special environment should be designed and used for each of them.

Dandelion (Taraxacum mongolicum) Extract Alleviated H2O2-Induced Oxidative Damage: The Underlying Mechanism Revealed by Metabolomics and Lipidomics.

Dandelion has received wide attention in food and medicine fields due to its excellent antioxidant properties. Nonetheless, the underlying mechanism of this action has not yet been fully clarified, particularly at the metabolic level. Herein, the effects of dandelion extract (DE) on H2O2-induced oxidative damage was investigated. The results indicate that the DE alleviated H2O2-induced cell damage (increased by 14.5% compared to H2O2 group), reduced the reactive oxygen species (ROS) level (decreased by 80.1% compared to H2O2 group), maintained the mitochondrial membrane potential (MMP) level, and increased antioxidant-related enzyme activities. Importantly, the metabolic response of PC12 cells indicates that H2O2 disturbed phospholipid metabolism and damaged cell membrane integrity. In addition, energy metabolism, the central nervous system, and the antioxidant-related metabolism pathway were perturbed. In contrast, DE rescued the H2O2-induced metabolic disorder and further alleviated oxidative damage. Collectively, these findings provide valuable stepping stones for a discussion of the mechanism and show the promise of DE as a suitable additive for functional food products.

Cloning and functional analysis prohibitins protein-coding gene EuPHB1 in Eucommia ulmoides Oliver

As multifunctional proteins, prohibitins(PHBs) participate in many cellular processes and play essential roles in organisms. In this study, using rapid amplification of cDNA end (RACE) technology, EuPHB1 was cloned from Eucommia ulmoides Oliver (E. ulmoides). A subcellular localization assay preliminarily located EuPHB1 in mitochondria. Then EuPHB1 was transformed into tobacco, and phenotype analyses showed that overexpression of EuPHB1 caused leaves to become chlorotic and shrivel. Furthermore, genes related to hormone and auxin signal transduction, auxin binding, and transport, such as ethylene-responsive transcription factor CRF4-like and ABC transporter B family member 11-like, were significantly inhibited in response to EuPHB1 overexpression. Its overexpression disturbs the original signal transduction pathway, thus causing the corresponding phenotypic changes in transgenic tobacco. Indeed, such overexpression caused fading of palisade tissue and an increase in the number of certain mesophyll cells. It also increased adenosine triphosphate (ATP) synthase activity, mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS) levels in cells. Our results suggest that EuPHB1 expression promotes cellular energy metabolism by accelerating the oxidative phosphorylation of the mitochondrial respiratory chain. Elevated levels of EuPHB1 in the mitochondria, which helps supply the extra energy required to support rapid rates of cell division.

Trastuzumab-induced cardiomyopathy via ferroptosis-mediated mitochondrial dysfunction.

Trastuzumab (TRZ) is a first-line chemotherapeutic agent for HER-2 (ErbB2)-positive breast cancer. Unfortunately, its clinical use is limited due to its cardiotoxicity, referred to as TRZ-induced cardiotoxicity (TIC). However, the exact molecular mechanisms underlying the development of TIC remain unclear. Iron and lipid metabolism and redox reactions participate in the development of ferroptosis. Here, we show that ferroptosis-mediated mitochondrial dysfunction is involved in TIC in vivo and in vitro. We first established TIC models with BALB/c mice or neonatal rat cardiomyocytes and confirmed cardiomyopathy with echocardiography and inhibition of cell viability with a cell counting kit-8 examination, respectively. We showed that TRZ downregulated glutathione peroxidase 4 (GPx4) and elevated lipid peroxidation by-products, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), by inactivating the ErbB2/PI3K/AKT/Nrf2 signalling pathway. Additionally, upregulated mitochondrial 4-HNE binds to voltage-dependent anion channel 1 (VDAC1), increases VDAC1 oligomerization, and subsequently induces mitochondrial dysfunction, as evidenced by mitochondrial permeability transition pore (mPTP) opening and decreased mitochondrial membrane potential (MMP) and ATP levels. Concomitantly, TRZ affected the mitochondrial levels of GSH/GSSG and iron ions and the stability of mitoGPx4. Ferroptosis inhibitors, such as ferrostatin-1 (Fer-1) or the iron chelator deferoxamine (DFO), ameliorate TRZ-induced cardiomyopathy. Overexpression of mitoGPx4 also suppressed mitochondrial lipid peroxidation and prevented TRZ-induced ferroptosis. Our study strongly suggests that targeting ferroptosis-mediated mitochondrial dysfunction is a potential cardioprotective strategy.

Sexually dimorphic extracellular vesicle responses after chronic spinal cord injury are associated with neuroinflammation and neurodegeneration in the aged brain

BackgroundMedical advances have made it increasingly possible for spinal cord injury (SCI) survivors to survive decades after the insult. But how SCI affects aging changes and aging impacts the injury process have received limited attention. Extracellular vesicles (EVs) are recognized as critical mediators of neuroinflammation after CNS injury, including at a distance from the lesion site. We have previously shown that SCI in young male mice leads to robust changes in plasma EV count and microRNA (miR) content. Here, our goal was to investigate the impact of biological sex and aging on EVs and brain after SCI.MethodsYoung adult age-matched male and female C57BL/6 mice were subjected to SCI. At 19 months post-injury, total plasma EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA). EVs miR cargo was examined using the Fireplex® assay. The transcriptional changes in the brain were assessed by a NanoString nCounter Neuropathology panel and validated by Western blot (WB) and flow cytometry (FC). A battery of behavioral tests was performed for assessment of neurological function.ResultsTranscriptomic changes showed a high number of changes between sham and those with SCI. Sex-specific changes were found in transcription networks related to disease association, activated microglia, and vesicle trafficking. FC showed higher microglia and myeloid counts in the injured tissue of SCI/Female compared to their male counterparts, along with higher microglial production of ROS in both injured site and the brain. In the latter, increased levels of TNF and mitochondrial membrane potential were seen in microglia from SCI/Female. WB and NTA revealed that EV markers are elevated in the plasma of SCI/Male. Particle concentration in the cortex increased after injury, with SCI/Female showing higher counts than SCI/Male. EVs cargo analysis revealed changes in miR content related to injury and sex. Behavioral testing confirmed impairment of cognition and depression at chronic time points after SCI in both sexes, without significant differences between males and females.ConclusionsOur study is the first to show sexually dimorphic changes in brain after very long-term SCI and supports a potential sex-dependent EV-mediated mechanism that contributes to SCI-induced brain changes.

Synthesis, Structural Investigations, DNA/BSA Interactions, Molecular Docking Studies, and Anticancer Activity of a New 1,4-Disubstituted 1,2,3-Triazole Derivative.

We report herein a new 1,2,3-triazole derivative, namely, 4-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde, which was synthesized by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The structure of the compound was analyzed using Fourier transform infrared spectroscopy (FTIR), 1H NMR, 13C NMR, UV-vis, and elemental analyses. Moreover, X-ray crystallography studies demonstrated that the compound adapted a monoclinic crystal system with the P21/c space group. The dominant interactions formed in the crystal packing were found to be hydrogen bonding and van der Waals interactions according to Hirshfeld surface (HS) analysis. The volume of the crystal voids and the percentage of free spaces in the unit cell were calculated as 152.10 Å3 and 9.80%, respectively. The evaluation of energy frameworks showed that stabilization of the compound was dominated by dispersion energy contributions. Both in vitro and in silico investigations on the DNA/bovine serum albumin (BSA) binding activity of the compound showed that the CT-DNA binding activity of the compound was mediated via intercalation and BSA binding activity was mediated via both polar and hydrophobic interactions. The anticancer activity of the compound was also tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using human cell lines including MDA-MB-231, LNCaP, Caco-2, and HEK-293. The compound exhibited more cytotoxic activity than cisplatin and etoposide on Caco-2 cancer cell lines with an IC50 value of 16.63 ± 0.27 μM after 48 h. Annexin V suggests the induction of cell death by apoptosis. Compound 3 significantly increased the loss of mitochondrial membrane potential (MMP) levels in Caco-2 cells, and the reactive oxygen species (ROS) assay proved that compound 3 could induce apoptosis by ROS generation.

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) protects hair cells from cisplatin-induced ototoxicity in vitro: possible relation to the activities of p38 MAPK signaling pathway.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) gene encodes rate-limiting enzyme in cholesterol biosynthesis, which is related to cell proliferation and mitochondrial function. The present study was designed to explore the expression of HMGCR in murine cochlear hair cells and HEI-OC1 cells and the possible mechanisms underpinning the actions of HMGCR in cisplatin-induced ototoxicity, with special attention given to p38 mitogen-activated protein kinase (MAPK) activities in vitro. The expressions of HMGCR, p-p38, cleaved caspase-3 and LC3B was measured by immunofluorescence and western blot. JC-1 staining and MitoSOX Red were used to detect mitochondria membrane potential (MMP) and reactive oxygen species (ROS) levels respectively. The apoptosis of auditory cells was assessed by TUNEL staining and flow cytometry. Protein levels of bcl2/bax and beclin1 were examined by western blot. We found that HMGCR was widely expressed in the auditory cells, of both neonatal mice and 2-month-old mice, in cytoplasm, nucleus and stereocilia. Moreover, 30μM cisplatin elicited the formation of ROS, which, in turn, led to HMGCR reduction, activating p38 kinase-related apoptosis and autophagy in auditory cells. Meanwhile, co-treatment with ROS scavenger at a concentration of 2mM, N-acetyl-L-cysteine (NAC), could alleviate the aforementioned changes. In addition, HMGCR silencing resulted in higher p38 MAPK-mediated apoptosis and autophagy under cisplatin injury. Taken together, we demonstrate that, for the first time, that HMGCR is expressed in the cochlear. Furthermore, HMGCR exerts protective benefit on auditory cells against cisplatin-mediated injury stimulated by ROS, culminating in regulation of p38 MAPK-dependent apoptosis and autophagy.