Changes In Membrane Potential Research Articles

Podophyllotoxin reduces the aggressiveness of human oral squamous cell carcinoma through myeloid cell leukemia‑1.

Podophyllotoxin (PPT), which is derived from the podophyllum plant, exhibits marked cytotoxic effects against cancer cells; however, the precise molecular mechanism underlying its activity against human oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, the mechanism by which PPT induced cytotoxicity in two OSCC cell lines, HSC3 and HSC4, was determined. The effects of PPT on cytotoxicity in HSC3 and HSC4 cells were analyzed using Annexin V/PI double staining, Sub‑G1 analysis, soft agar assays, western blotting, and quantitative PCR. The changes in the mitochondrial membrane potential were assessed using a JC‑1 assay and cytosolic and mitochondrial fractionation. A myeloid cell leukemia‑1 (Mcl‑1) overexpression cell lines were also established to study the role of Mcl‑1 on apoptosis. The results showed that PPT inhibited the growth of the two human OSCC cell lines and induced apoptosis, which was accompanied by mitochondrial membrane depolarization. Compared with the control, PPT reduced the expression of Mcl‑1 in both cell lines through a proteasome‑dependent protein degradation process. Overall, these results suggested that targeting of Mcl‑1 protein by PPT induced apoptosis, providing a foundation for further pre‑clinical and clinical study of its value in the management of OSCC.

Anticancer effect of umbelliferone on MKN-45 and MIA PaCa-2 cell lines

In this study, the anticancer activity of umbelliferone (7-hydroxycoumarin-UMB) was investigated in MKN-45 human gastric cancer and MIA PaCa-2 human pancreatic cancer cells. The cytotoxic effect of UMB on MKN-45 and MIA PaCa-2 cells was determined by WST-8 cell viability assay; the effect on colony formation and migration potential by colony forming assay and wound healing/cell migration assay. Apoptotic effect of UMB was determined by measuring the change in mitochondrial membrane potentials, reactive oxygen species levels, and Caspase-3 activities in cells. Anticancer drugs cisplatin and gemcitabine were used as positive controls in experiments, and NIH/Swiss 3 T3 mouse embryonic fibroblast cells were used as a healthy cell group.The results of this study showed that umbelliferone had a significant cytotoxic effect in MKN-45 and MIA PaCa-2 cells, especially after 72 h treatment, while its cytotoxic effect in NIH/3 T3 cells was low. Furthermore, UMB reduces significantly the potential of cells to colonize and migrate; it has been determined that it causes apoptosis by decreasing the mitochondrial membrane potential, increasing intracellular ROS levels and Caspase-3 activity. UMB was found to have more anticancer effect on MIA PaCa-2 cells compared to MKN-45 cells. This showed that UMB has a cell-selective effect.

Mesoporous Titanium Dioxide Nanoparticles-Poly(N-isopropylacrylamide) Hydrogel Prepared by Electron Beam Irradiation Inhibits the Proliferation and Migration of Oral Squamous Cell Carcinoma Cells.

An urgently needed approach for the treatment of oral squamous cell carcinoma (OSCC) is the development of novel drug delivery systems that offer targeted specificity and minimal toxic side effects. In this study, we developed an injectable and temperature-sensitive composite hydrogel by combining mesoporous titanium dioxide nanoparticles (MTNs) with Poly(N-isopropylacrylamide) (PNIPAAM) hydrogel to serve as carriers for the model drug Astragalus polysaccharide (APS) using electron beam irradiation. The characteristics of MTNs, including specific surface area and pore size distribution, were analyzed, and the characteristics of MTNs-APS@Hyaluronic acid (HA), such as microscopic morphology, molecular structure, crystal structure, and loading efficiency, were examined. Additionally, the swelling ratio, gel fraction, and microscopic morphology of the composite hydrogel were observed. The in vitro cumulative release curve was plotted to investigate the sustained release of APS in the composite hydrogels. The effects on the proliferation, migration, and mitochondrial membrane potential of CAL-27 cells were evaluated using MTT assay, scratch test, and JC-1 staining. The results indicated successful preparation of MTNs with a specific surface area of 147.059 m2/g and an average pore diameter of 3.256 nm. The composite hydrogel displayed temperature-sensitive and porous characteristics, allowing for slow release of APS. Furthermore, it effectively suppressed CAL-27 cells proliferation, migration, and induced changes in mitochondrial membrane potential. The addition of autophagy inhibitors chloroquine (CQ) and 3-methyladenine (3-MA) attenuated the migration inhibition (p < 0.05).

Optical Mapping of Cardiomyocytes in Monolayer Derived from Induced Pluripotent Stem Cells.

Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca2+ variations in excitable tissues using voltage-sensitive dyes and Ca2+ indicators, respectively. This powerful tool has rapidly become indispensable in the field of cardiac electrophysiology for studying depolarization wave propagation, estimating the conduction velocity of electrical impulses, and measuring Ca2+ dynamics in cardiac cells and tissues. In addition, mapping these electrophysiological parameters is important for understanding cardiac arrhythmia mechanisms. In this review, we delve into the fundamentals of cardiac optical mapping technology and its applications when applied to hiPSC-derived cardiomyocytes and discuss related advantages and challenges. We also provide a detailed description of the processing and analysis of optical mapping data, which is a crucial step in the study of cardiac diseases and arrhythmia mechanisms for extracting and comparing relevant electrophysiological parameters.

Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury

To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

Combined treatment of polygonatum and Scutellaria baicalensis suppresses lung cancer cell proliferation through inducing ferroptosis

ObjectiveTo investigate the effects of combined treatment of Scutellaria baicalensis (SB) and polygonatum (HH) for lung cancer and explore the underlying mechanism. PurposeThe study was designed to investigate the anti-tumor effect of HH and reveal its mechanisms. MethodsBased on the Traditional Chinese Medicine (TCM) theory and network pharmacological analysis. The inhibitory effect of HH on the proliferation of A549 and LLC lung cancer cells was predicted by CCK8 assay in a subcutaneous tumor model in vivo. Tumor tissues were collected for proteomics, qPCR and immunohistochemistry assays. A549 and LLC cell proliferation was detected by CCK8 assay after treatment with HH combined with ferroptosis inhibitors and ferroptosis inducer Erastin for 48 h respectively. The expression of Transferrin Receptor (TFRC) was detected by qPCR and Western Blot. Changes in free iron content and lipid peroxide in cytoplasm and mitochondria were observed by laser confocal microscopy. Changes in Mitochondrial Membrane Potential (MMP, Δψm) and ROS content were detected by flow cytometry. ResultsSearch of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database demonstrated that HH played a role in lung cancer treatment. CCK8 assay showed that lung cancer cell proliferation was inhibited by HH in a dose-dependent manner. The animal experiment results showed that the tumor volume in HH-treated group was significantly smaller than that in Control group in the LLC allograft model (P<0.01). The expression level of TFRC in HH-treated group was higher than that in Control group, which was verified by proteomics, qPCR assay and immunohistochemistry. Two ferroptosis specific inhibitors could block HH-induced ferroptosis (P<0.05). Compared with Control group, free iron and lipid peroxide contents were significantly increased in cytoplasm and mitochondria while MMP was decreased and the level of ROS was augmented in HH group (P<0.01). ConclusionHH increased the content of free iron and lipid peroxide in cytoplasm and mitochondria by upregulating the expression of TFRC in lung cancer cells, which further led to the accumulation of lipid peroxide, caused mitochondrial dysfunction, and finally induced ferroptosis in lung cancer cells.

Low-dose of polystyrene microplastics induce cardiotoxicity in mice and human-originated cardiac organoids

Microplastic particles (MP) are prevalent in both industrial production and the natural environment, posing a significant concern for human health. Daily diet, air inhalation, and skin contact are major routines of MP intake in human. The main injury target systems of MPs include the digestive system, respiratory system, and cardiovascular system. However, the study on MPs’ adverse effects on the heart is less than other target organs. Previous in vivo studies have demonstrated that MPs can induce heart injuries, including abnormal heart rate, apoptosis of cardiomyocytes, mitochondrial membrane potential change, and fibrin overexpression. To address animal welfare concerns and overcome inter-species variations, this study employed a human pluripotent stem cell-derived in vitro three-dimensional cardiac organoid (CO) model to investigate the adverse effects of MPs on the human heart. The distinct cavities of COs allowed for the observation of MPs’ aggregation and spatial distribution following polystyrene-MP (PS) exposure in a dynamic exposure system. After exposure to various concentrations of PS (0.025, 0.25 and 2.5 µg/mL, with the lowest concentration equivalent to human internal exposure levels), the COs exhibited increased oxidative stress, inflammatory response, apoptosis, and collagen accumulation. These findings were consistent with in vivo observations, in terms of increases in the interventricular septal thickness. The expression of hypertrophic-related genes of COs (MYH7B/ANP/BNP/COL1A1) changed noticeably and the cardiac-specific markers MYL2/MYL4/CX43 were also markedly elevated. Our findings revealed the PS could induced cardiac hypertrophy in vivo and in vitro, indicating that MP may be an under-recognized risk factor for cardiovascular system.

Acid ground nano-realgar processed product inhibits breast cancer by inducing mitophagy via the p53/BNIP3/NIX pathway.

Breast cancer is a highly prevalent malignancy with the first morbidity and the primary reason for female cancer-related deaths worldwide. Acid ground nano-realgar processed product (NRPP) could inhibit breast cancer cell proliferation and induce autophagy in our previous research; however, the underlying mechanisms are still unclear. Therefore, this research aimed to verify whether NRPP induces breast cancer mitophagy and explore the mitophagy-mediated mechanism. Primarily, rhodamine-123 assay and transmission electron microscopy were applied to detect mitochondrial membrane potential (MMP) and ultrastructural changes in the MDA-MB-435S cells, respectively. Mito-Tracker Green/Lyso-Tracker Red staining, western blot, immunofluorescence and RT-PCR were used to explore molecular mechanisms of NRPP-induced mitophagy in vitro. MDA-MB-435S breast cancer xenograft models were established to assess the activity and mechanisms of NRPP in vivo. Our results showed that NRPP decreased MMP and increased autophagosome numbers in MDA-MB-435S cells and activated mitophagy. Furthermore, mitophagy was consolidated because mitochondria and lysosomes colocalized phenomenology were observed, and the expression of LC3II/I and COXIV was upregulated. Additionally, we found the p53/BNIP3/NIX pathway was activated. Finally, NRPP inhibited tumour growth and downregulated the levels of TNF-α, IL-1β and IL-6. Necrosis, damaged mitochondria and autophagosomes were observed in xenograft tumour cells, and proteins and mRNA levels of LC3, p53, BNIP3 and NIX were increased. Overall, NRPP inhibited MDA-MB-435S cell proliferation and tumour growth by inducing mitophagy via the p53/BNIP3/NIX pathway. Thus, NRPP is a promising candidate for breast cancer treatment.

Immunometabolic actions of trabectedin and lurbinectedin on human macrophages: relevance for their anti-tumor activity.

In recent years, the central role of cell bioenergetics in regulating immune cell function and fate has been recognized, giving rise to the interest in immunometabolism, an area of research focused on the interaction between metabolic regulation and immune function. Thus, early metabolic changes associated with the polarization of macrophagesinto pro-inflammatoryorpro-resolving cells under different stimuli have been characterized. Tumor-associated macrophages are among the most abundant cells in the tumor microenvironment; however, it exists an unmet need to study the effect of chemotherapeutics on macrophage immunometabolism. Here, we use a systems biology approach that integrates transcriptomics and metabolomics to unveil the immunometabolic effects of trabectedin (TRB) and lurbinectedin (LUR), two DNA-binding agents with proven antitumor activity. Our results show that TRB and LUR activate human macrophages toward a pro-inflammatory phenotype by inducing a specific metabolic rewiring program that includes ROS production, changes in the mitochondrial inner membrane potential, increased pentose phosphate pathway, lactate release, tricarboxylic acids (TCA) cycle, serine and methylglyoxal pathways in human macrophages. Glutamine, aspartate, histidine, and proline intracellular levels are also decreased, whereas oxygen consumption is reduced. The observed immunometabolic changes explain additional antitumor activities of these compounds and open new avenues to design therapeutic interventions that specifically target the immunometabolic landscape inthe treatment of cancer.

Sialic Acid Engineered Prodrug Nanoparticles for Codelivery of Bortezomib and Selenium in Tumor Bearing Mice.

Most cancer patients rarely benefit from monodrug therapy because of both cancer complexity and tumor environment. One of the main reasons for this failure is insufficient accumulation of the optimal dose at the tumorous site. Our investigation implies a promising strategy to engineer prodrug nanoparticles (NPs) of bortezomib (BTZ) and selenium (Se) using sialic acid (SAL) as a ligand to improve breast cancer therapy. BTZ was conjugated with SAL and HPMA (N-2-hydroxypropyl methacrylamide) to prepare a prodrug conjugate; BTZ-SAL-HPMA (BSAL-HP) and then fabricated into prodrug NPs with Se (Se_BSAL-HP prodrug NPs). The self-assembly of prodrug NPs functionalized with Se showed size (204.13 ± 0.02 nm) and zeta potential (-31.0 ± 0.11 mV) in dynamic light scattering (DLS) experiments and spherical shape in TEM and SEM analysis. Good stability and low pH drug release profile were characterized by Se_BSAL-HP prodrug NPs. The tumor-selective boronate-ester-based prodrug NPs of BTZ in combination with Se endowed a synergistic effect against cancer cells. Compared to prodrug conjugate, Se_BSAL-HP prodrug NPs exhibited higher cell cytotoxicity and enhanced cellular internalization with significant changes in mitochondria membrane potential (MMP). Elevated apoptosis was observed in the (G2/M) phase of the cell cycle for Se_BSAL-HP prodrug NPs (2.7-fold) higher than BTZ. In vivo studies were performed on Sprague-Dawley rats and resulted in positive trends. The increased therapeutic activity of Se_BSAL-HP prodrug NPs inhibited primary tumor growth and showed 43.05 fold decrease in tumor volume than the control in 4T1 tumor bearing mice. The surprising and remarkable outcomes for Se_BSAL-HP prodrug NPs were probably due to the ROS triggering effect of boronate ester and selenium given together.

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

Neuroprotective effects of idebenone on hydrogen peroxide-induced oxidative damage in retinal ganglion cells-5.

To investigate the neuroprotective effect of idebenone against hydrogen peroxide (H2O2)-induced oxidative damage in retinal ganglion cells-5 (RGC-5 cells). RGC-5 cells were pre-treated with various idebenone concentrations (5, 10, and 20µM) for 12h and were then subjected to 300µM H2O2 for a further 12h. Apoptosis in RGC-5 was measured by flow cytometry. The changes of mitochondrial membrane potential (MMP) were detected by JC-1 staining. Autophagy in RGC-5 cells was observed by transmission electron microscopy. Western blots were used to measure the expression of autophagy-related protein light chain 3 (LC3), Beclin-1, and the release of Cytochrome c (Cyt-c). Flow cytometry showed that the apoptosis rates in the normal control group, H2O2 group, and idebenone groups were 6.48 ± 0.55%, 27.3 ± 0.51%, 22.8 ± 0.52%, 15.45 ± 0.81%, and 12.59 ± 0.58%, respectively (F = 559.7, P < 0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, whereas it was decreased in the idebenone groups. After incubation of RGC-5 cells with H2O2, MMP levels were significantly decreased, while idebenone could prevent the decrease in MMP levels. Compared with that in the normal control group, LC3 II/I, the expression levels of Beclin-1 and Cyt-c were increased significantly in the H2O2 group (P < 0.05). Compared with that in the H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c was significantly decreased in idebenone groups (P < 0.05). Idebenone protects RGC-5 cells against H2O2-induced oxidative damage by reducing mitochondrial damage and autophagic activity.

Abstract P3181: A Cyclophilin D-mitochondria-redox Differentiation Pathway Regulates Neonatal Heart Bioenergetics And Function

Background: During birth, demands on the heart increase when exposed to high oxidative stress. Cardiac myocyte differentiation is dependent on changes in mitochondrial function that regulate cell stress pathways to enhance differentiation. We have shown these changes are dependent on closure of the mitochondrial permeability transition pore (mPTP) controlled by the protein cyclophilin D (CypD). These studies aim to determine the effects of independently manipulating CypD, mitochondrial membrane potential, and reactive oxygen species (ROS) production on mitochondrial function, myocyte differentiation, and cardiac function in the neonate. Methods: Neonatal C57Bl6 mice were intraperitoneally injected on postnatal day (P) 1-6 of with mitoparaquat (0.025-0.05 mg/kg), Tempol (0.7 mg/kg), mitoTempo (0.7 mg/kg), 2,4-dinitrophenol (2 mg/kg), or vehicle. Hearts were harvested at P7 and mitochondrial respiration, enzymatic activity of the electron transport chain (ETC) complex (Cx) I and 2, and assembly of mitochondrial supercomplexes were measured by native gel electrophoresis. Results: Treatment with mitoparaquat to increase mitochondrial ROS caused a dose-dependent decrease of Cx I activity measured by oxygen consumption and enzyme assays. This corresponded to decrease in assembly of Cx-I containing supercomplexes. Decreasing mitochondrial ROS using mitoTempo had no effect on Cx I activity but increased assembly of Cx-III containing supercomplexes. Decreasing ROS in the cell with Tempol decreased Cx-I activity but had no effect on supercomplex assembly. Uncoupling at the mitochondrial membrane using 2,4-dinitrophenol increased electron flux from Cx I to Cx III but may decrease supercomplex assembly. Treatment of isolated mitochondria with cyclosporin A to inhibit CypD had no effects on these measures. Conclusions: Changes in ROS and mitochondrial membrane potential have selective effects on ETC assembly, activity in the neonatal period, and are not inhibited by closure of the mPTP. This data suggests a pathway where CypD regulates the mPTP to regulate the ETC, membrane potential, and ROS production to regulate myocyte differentiation; such a pathway could be targeted to increase cardiac maturation in infants with poor cardiac function.

Antiprotozoal Activity of Benzoylthiourea Derivatives against Trypanosoma cruzi: Insights into Mechanism of Action.

For decades, only two nitroheterocyclic drugs have been used as therapeutic agents for Chagas disease. However, these drugs present limited effectiveness during the chronic phase, possess unfavorable pharmacokinetic properties, and induce severe adverse effects, resulting in low treatment adherence. A previous study reported that N-(cyclohexylcarbamothioyl) benzamide (BTU-1), N-(tert-butylcarbamothioyl) benzamide (BTU-2), and (4-bromo-N-(3-nitrophenyl) carbamothioyl benzamide (BTU-3) present selective antiprotozoal activity against all developmental forms of Trypanosoma cruzi Y strain. In this study, we investigated the mechanism of action of these compounds through microscopy and biochemical analyses. Transmission electron microscopy analysis showed nuclear disorganization, changes in the plasma membrane with the appearance of blebs and extracellular arrangements, intense vacuolization, mitochondrial swelling, and formation of myelin-like structures. Biochemical results showed changes in the mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and plasma membrane fluidity. In addition, the formation of autophagic vacuoles was observed. These findings indicate that BTU-1, BTU-2, and BTU-3 induced profound morphological, ultrastructural, and biochemical alterations in epimastigote forms, triggering an autophagic-dependent cell death pathway.

Mechanism of aconitine mediated neuronal apoptosis induced by mitochondrial calcium overload caused by MCU

Aconitine is a crucial toxic component in Chinese herbal medicines such as Aconitum, Aconitum coreanum, and Aconitum soongaricum. The poisoning symptoms of the central nervous system and cardiovascular system caused by it are relatively common in China, and there are many studies on cardiovascular system diseases caused by aconitine. However, the specific mechanism of neurotoxicity induced by aconitine is still unclear. This study explored the effect and mechanism of mitochondrial calcium uniporter on mitochondrial energy metabolism disorder in aconitine poisoning hippocampal neurons. The results showed that after treatment with 400μmol/L aconitine, mitochondrial energy metabolism was abnormal in rat hippocampal neuron cells, the expression of MCU in mitochondria was up-regulated, calcium overload in mitochondria, ATP production decreased, and mitochondrial membrane potential Changes, increased expression of the apoptosis gene Cleaved-Caspase-3. After treatment with the MCU agonist spermine, mitochondrial energy metabolism was significantly abnormal, and cell apoptosis was increased considerably. However, pretreatment with calcium ion channel inhibitor Ruthenium Red (RR) effectively promoted the generation of ATP, thereby improving mitochondrial energy metabolism disorders and reducing cell apoptosis. These results suggest that aconitine induces mitochondrial energy metabolism dysfunction in hippocampal neurons, which may be related to the increased expression of MCU.

Real-time and in situ visualizing the details of mitochondrial fragmentation with a leakage-free fluorescent probe in living cells

Mitochondrial fragmentation is a crucial physiologic process that is closely associated with energy production, metabolism, and apoptosis. However, the details of morphological changes are rarely reported. To visualize them, a leakage-free fluorescent probe composed of an indole-pyridinium and a C8-alkyl chain, IP-8, was constructed. IP-8 could firmly immobilize in mitochondria by strong hydrophobic interaction between the C8-alkyl chain and phospholipid bilayer of the mitochondrial inner membrane, thus realizing the long-term staining without leakage regardless of mitochondrial membrane potential (MMP) changes. Importantly, using IP-8, three details during mitochondrial fragmentation process, from granule to donut, eventually formed cluster, were successfully visualized real-time and in situ. Therefore, IP-8 was suggested as a useful tool for visualizing and tracking of mitochondrial morphology changes.

KCa 2.2 (KCNN2): A physiologically and therapeutically important potassium channel.

One group of the K+ ion channels, the small-conductance Ca2+ -activated potassium channels (KCa 2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca2+ -activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca2+ . According to the phylogenic research done on the KCa 2.x channels family, there are three channels' subtypes: KCa 2.1, KCa 2.2, and KCa 2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The KCa 2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. KCa 2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the KCa 2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express KCa 2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The KCa 2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective KCa 2.2 modulators are promising potential therapeutics for SCAs.

VDAC1 Protein Regulation of Oxidative Damage and Mitochondrial Dysfunction-Mediated Cytotoxicity by Silica Nanoparticles in SH-SY5Y Cells.

Silica nanoparticles (SiNPs) have been widely used in industry, electronics, and pharmaceutical industries. In addition, it is also widely used in medicine, tumor treatment and diagnosis, as well as other biomedical and biotechnology fields. The opportunities for people to contact SiNPs through iatrogenic, occupational, and environmental exposures are gradually increasing. The damage and biological effects of SiNPs on the nervous system have attracted widespread attention in the field of toxicology. Central nerve cells are rich in mitochondria. It is suggested that the effects of SiNPs on mitochondrial damage of nerve cells may involve the maintenance of neuronal membrane potential, the synthesis and operation of neurotransmitters, and the transmission of nerve pulses, and so on. We established an experimental model of SH-SY5Y cells to detect the cell survival rate, apoptosis, changes of reactive oxygen species and mitochondrial membrane potential, and the expression of mitochondrial function-related enzymes and proteins, so as to reveal the possible mechanism of SiNPs on neuronal mitochondrial damage. It was found that SiNPs could cause oxidative damage to cells and mitochondria, destroy some normal functions of mitochondria, and induce apoptosis in SH-SY5Y cells. The voltage-dependent anion channel 1(VDAC1) protein inhibitor DIDS could effectively reduce intracellular oxidative stress, such as the reduction of ROS content, and could also usefully restore some functional proteins of mitochondria to normal levels. The inhibition of VDAC1 protein may play an important role in the oxidative damage and dysfunction of neuronal mitochondria induced by SiNPs.

The role of protein kinase R in placental inflammation, mtUPR and apoptosis

IntroductionPlacental inflammation is implicated in the pathophysiology of many pregnancy complications, including fetal growth restriction, preeclampsia, gestational diabetes, and choriocarcinoma. Mitochondrial dysfunction, one of the outcomes of placental inflammation, is characterized by loss of membrane potential, accumulation of oxygen radicals, mitochondrial protein folding defects, and disturbances in mitochondrial dynamics. Protein kinase R (PKR) is stimulated by double-stranded RNA and bacterial endotoxins in the presence of pathogens and is a critical immune response enzyme. PKR is also correlated with the cell death response during endoplasmic reticulum stress. In this study, we aim to investigate the effects of PKR activity stimulated by lipopolysaccharide (LPS), and double-stranded RNA analog (Poly I:C) on mitochondrial unfolded protein response (mtUPR), mitochondrial membrane potential, apoptosis, and oxidative stress in placental trophoblasts. MethodsWe applied LPS and Poly I:C to BeWo cells to induce PKR activation. In addition, cells were treated with 2-aminopurine (2-AP) to inhibit the kinase activity of PKR. Protein levels of ATP-dependent Clp protease proteolytic subunit (CLPP) and heat shock protein 60 (HSP60) were determined after treatments. Apoptotic markers were detected by real-time PCR and flow cytometry. PKR-induced reactive oxygen radicals (ROS) accumulation and mitochondrial membrane potential change were assessed by flow cytometry. ResultsIt was determined that PKR activation-induced apoptosis in BeWo cells by reducing the levels of mtUPR proteins (CLPP and HSP60) and caused a decrease in mitochondrial membrane potential. PKR inhibition was sufficient for decreases in apoptotic markers and caused a reduction in the ratio of depolarized and ROS (+) cells. DiscussionOur results showed that LPS and Poly I:C administration stimulated PKR in BeWo cells in vitro. Furthermore, PKR activation is correlated with the levels of proteins involved in mitochondrial homeostasis and apoptosis. Our findings will contribute to understanding the role of PKR activation in placental inflammation and related diseases.

Triterpene acids from Rosa roxburghii Tratt fruits exert anti-hepatocellular carcinoma activity via ROS/JNK signaling pathway-mediated cell cycle arrest and mitochondrial apoptosis

BackgroundRosa roxburghii Tratt (RRT) is a famous healthy and medicinal edible fruit in southwest China and has been shown to have some hepatoprotective properties. However, whether the active components, such as the triterpene acids from Rosa roxburghii Tratt fruits (TAR), have anti-hepatocellular carcinoma (HCC) effects and the potential molecular mechanisms are still unclear. PurposeThis study aimed to investigate the anti-HCC effects and potential action mechanisms of triterpene components in RRT fruits. MethodsThe triterpene acids in TAR were analyzed by using UPLC-Q-Exactive Orbitrap/MS, and the main components were virtual screening for targets based on pharmacophore and then performed enrichment analysis. HepG2 cells were used for in vitro experiments, including MTT assay, wound healing assay, and flow cytometry to detect cell cycle, reactive oxygen species (ROS) level, caspase-3 activity, and mitochondrial membrane potential (MMP) changes. Moreover, the western blot was used to detect mitochondrial apoptosis and ROS/ c-Jun N-terminal kinase (JNK) signaling pathway-related proteins. ResultsThe main components in TAR are pentacyclic triterpene acids (mainly euscaphic acid and roxburic acid). TAR could inhibit cell viability, cell migration ability and suppress the proliferation of HepG2 cells through G2/M cell cycle arrest. On the other hand, TAR could induce HepG2 cells apoptosis, which was achieved by causing the accumulation of ROS and activation of the JNK signaling pathway, and our research showed that this apoptosis was mediated through the mitochondrial pathway. In addition, the free radical scavenger N-acetyl cysteine (NAC) could attenuate TAR-induced ROS accumulation and JNK signaling pathway activation, which ultimately reversed mitochondrial apoptosis. ConclusionTAR could activate the ROS/JNK signaling pathway, which could inhibit the proliferation through G2/M cell cycle arrest and promote apoptosis through the mitochondrial pathway in HCC cells. This supports the anti-tumor potential in RRT fruits.