JC-1 Staining Research Articles

Qiangji decoction mitigates neuronal damage, synaptic and mitochondrial dysfunction in SAMP8 mice through the regulation of ROCK2/Drp1-mediated mitochondrial dynamics.

Qiangji Decoction (QJD), a Chinese medicine, is widely used in Traditional Chinese Medicine to treat amnesia and Alzheimer's disease (AD), showing significant anti-AD effects. However, the precise mechanisms behind these effects are not well understood and require more research. This study aims to elucidate the mechanisms by which QJD ameliorates neuronal damage, synaptic dysfunction, and mitochondrial impairment in AD through the regulation of ROCK2/Drp1-mediated mitochondrial dynamics. UPLC-Q-TOF-MS/MS was used to identify active components in QJD extract. The study used SAMP8 mice for AD modeling and SAMR1 mice as controls. Cognitive function in SAMP8 mice was assessed with the Morris Water Maze after following treatment with QJD and the mitochondrial fission inhibitor Mdivi-1. Nissl and FJB staining evaluated QJD's effect on hippocampal injury. Synaptic integrity was examined with Golgi-Cox staining, transmission electron microscopy, and immunofluorescence. Mitochondrial function in hippocampal neurons was assessed using electron microscopy, JC-1 staining, and reagent kits. Western blot analyzed expression of proteins related to mitochondrial fission (ROCK2, Drp1, Fis1, Mff) and fusion (Mfn1, Mfn2, OPA1). The analysis of QJD extract via UPLC-Q-TOF-MS/MS led to the identification of 46 active compounds. In SAMP8 mice, administration of QJD resulted in decreased escape latency, increased platform crossings, and extended duration in the target quadrant. Additionally, QJD exhibited neuroprotective effects on the hippocampus of SAMP8 mice, effectively preventing neuronal loss and damage. QJD also facilitated the extension and thickening of dendritic spines, enhanced the ultrastructure of hippocampal synapses, and upregulated synaptic function-related proteins, including PSD95 and SYN1. Furthermore, QJD ameliorated mitochondrial damage, improved mitochondrial membrane potential and ATP content, and reduced ROS expression in hippocampal neurons of SAMP8 mice. These effects were mediated through the downregulation of ROCK2, phosphorylated Drp1 (Ser616), Fis1, and Mff, as well as the upregulation of Mfn1, Mfn2, and OPA1. QJD may reduce neuronal damage, synaptic dysfunction, and mitochondrial impairment in SAMP8 mice by regulating mitochondrial dynamics through the ROCK2/Drp1 pathway.

Hederagenin ameliorates ferroptosis-induced damage by regulating PPARα/Nrf2/GPX4 signaling pathway in HT22 cells: An in vitro and in silico study.

Hederagenin (HG), derived from ivy seeds, is known to offer protection against Alzheimer's disease (AD). However, the specific molecular pathways through which it counters ferroptosis-induced neurotoxicity are not fully elucidated. This investigation seeks to delineate the processes by which HG mitigates neurotoxic effects in HT22 cells subjected to glutamate (Glu)-induced ferroptosis. HT22 cell ferroptosis was prompted by Glu exposure. Cell viability was assessed using CCK-8 and LDH assays, while Fe2+ fluorescence and assays of iron-related proteins served to gauge intracellular Fe2+ concentrations. Evaluations of mitochondrial structure and functionality employed JC-1 staining and transmission electron microscopy. Assessments of ROS, lipid peroxidation, MDA, 4-HNE, and the GSSG/GSH ratio were conducted to ascertain HG's antioxidative efficacy. The expression of proteins within the PPARα/Nrf2/GPX4 pathway was quantified via western blotting, with molecular docking (MD), and molecular dynamics simulations (MDS) used to explore protein interactions. HG diminished the cellular toxicity triggered by Glu in HT22 cells, lowered Fe2+ within cells, and rejuvenated mitochondrial morphology and performance. Concurrently, it modulated proteins critical to Fe2+ metabolism, diminished ROS and lipid peroxidation, and elevated GSH/GSSG ratios. Enhanced PPARα/Nrf2/GPX4 protein levels were corroborated by western blot results. Furthermore, molecular docking revealed favorable binding of HG to the proteins PPARα, Nrf2, and GPX4, with binding energies of -7.751, -7.535, and -7.414kcal/mol, respectively. MDS confirmed robust interactions between HG and these pivotal targets. The evidence suggests that HG effectively mitigates Glu-induced ferroptosis in HT22 cells by activating the PPARα/Nrf2/GPX4 signaling pathway. These findings endorse HG's potential as a nutritional adjunct for AD management.

Akkermansia muciniphila relieves inflammatory response in DSS-induced ulcerative colitis in mice through regulating macrophage polarization via SCFAs-SLC52A2/FFAR2 pathway.

Ulcerative colitis (UC) remains an intractable and relapsing disease featured by intestinal inflammation. The anti-UC activity of Akkermansia muciniphila (AKK), an intestinal microorganism, has been widely investigated. The current work is to explore the impacts of AKK on UC and its possible reaction mechanism. In vivo UC model was induced by dextran sulfate sodium (DSS) and phorbol-12-myristate-13-acetate (PMA)-induced THP-1-M0 and raw264.7 macrophages were treated by lipopolysaccharide (LPS). H&E staining evaluated tissue damage. Inflammatory and oxidative stress levels were assessed by relevant kits. The high-throughput analysis of fatty acids was performed by the LC/MS method. RT-qPCR and Western blot detected related gene expression. Flow cytometry measured cell apoptosis and macrophage polarization. Energy metabolism was detected by ELISA, related assay kits, JC-1 staining, and Western blot. AKK reduced the pathological damage of mice colon tissues, alleviated oxidative stress and inflammatory response, upregulated the expression of Occludin-1 and SCFAs receptors, and stimulated M1 to M2 macrophage polarization in vivo. After FFAR2 was silenced, the promoting role of AKK in the viability and M1 to M2 macrophage polarization and the inhibitory role in oxidative stress, inflammation, apoptosis, energy metabolism disorder, necroptosis, and pyroptosis were both reverted. Conclusively, AKK might mediate SCFAs-SLC52A2/FFAR2 pathways to exert protective activities against intestinal inflammatory response in UC, suggesting that AKK might represent a novel and promising candidate for UC therapy.

Emamectin Benzoate and Microplastics Led to Skeletal Muscle Atrophy in Common Carp via Induced Oxidative Stress, Mitochondrial Dysfunction, and Protein Synthesis and Degradation Imbalance.

Pesticides and plastics have brought convenience to agricultural production and daily life, but they have also led to environmental pollution through residual chemicals. Emamectin benzoate (EMB) is among the most widely used insecticides, which can cause environmental pollution and harm the health of organisms. Additionally, microplastics (MPs), a relatively new type of pollutant, not only are increasing in residual amounts within water bodies and aquatic organisms but also exacerbate pollution by adsorbing other pollutants, leading to a mixed pollution scenario. Nevertheless, the toxicity and mechanism of EMB and MPs on common carp skeletal muscle have not been elucidated. Therefore, we established exposure models for EMB and MPs, and methods such as hematoxylin and eosin staining, immunofluorescence staining, JC-1 staining, and western blotting were employed to investigate the underlying mechanisms of skeletal muscle damage. The results of in vivo and in vitro experiments indicated that exposure to EMB or MPs led to oxidative stress, which in turn caused mitochondrial fusion/fission imbalance (with decreased Mfn1, Mfn2, and OPA1 and increased DRP1), reduced mitochondrial membrane potential, decreased ATP content, reduced protein synthesis, and increased degradation, ultimately resulting in skeletal muscle atrophy. Joint exposure caused more severe damage than single exposure, and the addition of NAC can effectively alleviate skeletal muscle atrophy. In summary, exposure to EMB and/or MPs induced excessive reactive oxygen species (ROS) production, giving rise to mitochondrial dysfunction and an imbalance in skeletal muscle protein synthesis and degradation, ultimately resulting in skeletal muscle atrophy in common carp.

Mitochondria-Targeted Antioxidant (MitoQ) and Nontargeted Antioxidant (Idebenone) Mitigate Mitochondrial Dysfunction in Corneal Endothelial Cells.

To investigate the effectiveness of mitochondrial-targeted antioxidant mitoquinone (MitoQ) and nontargeted antioxidant idebenone (Idb) in alleviating mitochondrial dysfunction in corneal endothelial cells (CEnCs). In vitro experiments were conducted using immortalized normal human corneal endothelial cells (HCEnC-21T; SVN1-67F) and Fuchs endothelial corneal dystrophy (FECD) cells (SVF5-54F; SVF3-76M). Cells were pretreated with MitoQ or Idb and then exposed to menadione (MN) with simultaneous antioxidant treatment. Mitochondrial parameters were evaluated through adenosine triphosphate viability assays, JC-1 staining for mitochondrial membrane potential, and Tom-20 antibody staining for fragmentation, with analysis performed using ImageJ software. HCEnC-21T cells were additionally exposed to ultraviolet-A (25 J/cm2) to assess drug effects under physiological stress. Mitochondrial fragmentation in FECD specimens was analyzed pre- and post-treatment with the drugs. Statistical analysis was conducted using 1-/2-way analysis of variance with post-hoc Tukey test. MitoQ and Idb enhanced cell viability and mitochondrial membrane potential in both normal and FECD cells under MN-induced stress. Idb reduced MN-induced mitochondrial fragmentation by 32% more than MitoQ in HCEnC-21T cells and by 13% more in SVF5-54F cells. Under ultraviolet-A stress, Idb and MitoQ improved mitochondrial function by 31% and 25%, respectively, with MitoQ increasing mitochondrial function by 42% in FECD specimens. Differential responses in mitochondrial dysfunction across cell lines highlight disease heterogeneity. MitoQ and Idb protected CEnCs from oxidative stress and improved mitochondrial bioenergetics, suggesting that mitochondrial-targeted antioxidants could be considered for mitochondrial dysfunction in CEnCs.

20-HETE mediates Ang II-induced cardiac hypertrophy via ROS and Ca2+ signaling in H9c2 cells

In the vascular system, angiotensin II (Ang II) mediated vasoconstriction by inducing the production of 20-hydroxyeicosatetraenoic acid (20-HETE). However, the role of 20-HETE in Ang II-induced cardiac dysfunction had yet to be fully elucidated. This study investigated the effects of Ang II on CYP4A expression and 20-HETE production in H9c2 cells using RT-qPCR, Western blot, and ELISA. The role of 20-HETE in Ang II-induced cardiac hypertrophy was examined using DHE, MitoSOX, and JC-1 staining to evaluate reactive oxygen species (ROS) generation and mitochondrial membrane potential changes. The ERK/Akt and CaN/NFAT3 signaling pathways were analyzed through Western blot. Ang II was found to promote CYP4A expression and 20-HETE production in H9c2 cells via an AT1 receptor-dependent mechanism. Additionally, the upregulation of AT1 receptor expression by 20-HETE further confirms its facilitatory effect on the Ang II signaling pathway. Inhibition of 20-HETE synthesis or blockade of its receptor, G-protein-coupled receptor 75 (GPR75), significantly reversed Ang II-induced cardiac hypertrophy. This reversal was closely associated with 20-HETE-induced ROS production, oxidative stress, and activation of the Ca2+/CaN/NFAT3 signaling pathway. This study demonstrated that 20-HETE mediated Ang II-induced cardiac hypertrophy and, for the first time, highlighted the significant role of the GPR75 receptor in this process. These findings suggested that targeting 20-HETE reduction or blocking its receptor action could offer a novel therapeutic approach for cardiovascular diseases associated with Ang II.

Icariin mediates autophagy and apoptosis of hepatocellular carcinoma cells induced by the β-catenin signaling pathway through lncRNA LOXL1-AS1.

To investigate the effect of icariin (ICA) on hepatocellular carcinoma (HCC) and its autophagy/apoptosis mechanism in HCC. The anti-HCC mechanism of ICA was investigated using HCC cells treated with 20 µmol/L ICA. Cell viability and proliferation were assessed using CCK-8 and colony formation assays, respectively, while TUNEL staining evaluated anti-apoptotic effects. DHE staining quantified intracellular ROS levels, and JC-1 staining assessed mitochondrial membrane potential. The expression of LC3 was detected by immunofluorescence staining. Additionally, HepG2 cells (2.0 × 106) were implanted into the thymus of BALB/c nude mice, which received intraperitoneal injections of 40 mg/kg ICA. Western blotting was used to evaluate the expression of proteins related to apoptosis and autophagy. ICA effectively inhibited the proliferation and invasion of HCC cells, enhancing autophagy and apoptosis. Silencing of lncRNA LOXL1-AS1 reduced β-catenin expression and downregulated PI3K/AKT/mTOR pathway phosphorylation. Targeting β-catenin with siRNA augmented apoptosis in HepG2 cells through elevated levels of Bax and caspase-3/8/9 and boosted autophagy via increased expression of LC3-II, Atg5, Atg7, Atg8, and Beclin-1. ICA reversed this autophagic effect, while rapamycin enhanced ICA's efficacy. In vivo, ICA suppressed tumor growth and promoted autophagy and apoptosis in mice. Icariin induces autophagy and apoptosis in HCC cells via the β-catenin signaling pathway mediated by lncRNA LOXL1-AS1, offering a novel approach to HCC clinical management.

MiR-361-5p regulates SLC25A24 to maintain mitochondrial function and alleviate granulosa cell dysfunction in diminished ovarian reserve.

The aim of this study was to investigate the role of miR-361-5p (a tumor suppressor) in regulating granulosa cell function by targeting SLC25A24, a key mitochondrial protein, to uncover potential therapeutic targets for diminished ovarian reserve (DOR). This study included patients undergoing assisted reproductive technology treatment at our hospital. Granulosa cells were isolated from follicular fluid, and KGN cells were used for in vitro experiments. miR-361-5p and SLC25A24 expression levels were manipulated using miRNA mimics and inhibitors, and their effects on cell viability, apoptosis, and mitochondrial function were assessed. Techniques employed included qRT-PCR, Western blot analysis, ELISA, JC-1 staining, and dual-luciferase reporter assays. Key quantitative metrics included changes in mitochondrial DNA (mtDNA), ATP production, and reactive oxygen species (ROS) levels. miR-361-5p expression was significantly lower in DOR patients' granulosa cells compared to controls (P < 0.01). miR-361-5p inhibition markedly decreased KGN cells viability and increased apoptosis (P < 0.01), while miR-361-5p overexpression had the opposite effects (P < 0.01). SLC25A24 expression was inversely correlated with miR-361-5p levels, and its knockdown reversed the effects of miR-361-5p inhibition. Additionally, miR-361-5p modulation significantly affected mitochondrial function, with its overexpression reducing ROS levels and increasing ATP production (P < 0.01). miR-361-5p plays a pivotal role in maintaining mitochondrial function and reducing KGN cells dysfunction by targeting SLC25A24. These findings offer new insights into the molecular mechanisms of DOR and highlight miR-361-5p as a potential therapeutic target to enhance ovarian reserve and improve fertility outcomes.

Astragaloside IV Relieves Central Sensitization by Regulating Astrocytic ROS/NF-κB Nuclear Translocation Signaling in Chronic Migraine Male Rats.

Chronic migraine (CM) is a disabling neurological disease. Astragaloside IV (AS-IV), a natural product derived from Astragalus membranaceus, shows great potential in treating chronic pain by relieving inflammation and oxidative stress. This study aimed to investigate the effects and mechanisms of action of AS-IV on CM. An inflammatory soup comprising histamine, bradykinin, serotonin, and prostaglandin E2 was used to establish a CM rat model, while lipopolysaccharide was applied to induce an inflammatory response in primary astrocytes. Pain threshold measurements were used to evaluate nociceptive hypersensitivity, while qPCR and Western blotting were applied to detect inflammatory indicators and synaptic protein expression, and Golgi-Cox staining was applied to observe dendritic spine density, while transmission electron microscopy was used to observe synaptic ultrastructure. Mitochondrial function and oxidative stress were assessed using JC-1 staining, Mitotracker staining, reactive oxygen species (ROS) quantification, and glutathione content. AS-IV pretreatment alleviated central sensitization and ameliorated astrocyte activation and neuroinflammation. AS-IV pretreatment alleviated mitochondrial dysfunction invitro, and reduced the nuclear translocation of NF-κB and the production of IL-1β, which were reversed by ROS scavengers invitro or mitochondrial respiratory chain disruptors invivo. Our study indicates that AS-IV can inhibit neuroinflammation by alleviating astrocyte mitochondrial dysfunction to mitigate central sensitization in CM, thereby providing an experimental basis for AS-IV and A. membranaceus in CM prevention and treatment.

Kuanxiong Aerosol Attenuates Ischemic Stroke Injury via Modulation of the TRPV1 Channel.

To evaluate the therapeutic effects of Kuanxiong Aerosol (KXA) on ischemic stroke with reperfusion and elucidate the underlying pharmacological mechanisms. In vivo pharmacological effects on ischemic stroke with reperfusion was evaluated using the transient middle cerebral artery occlusion (t-MCAO) mice model. To evaluate short-term outcome, 30 mice were randomly divided into vehicle group (n=15) and KXA group (n=15). Mice in KXA and vehicle groups received 69 mg KXA and vehicle for 1 day, respectively. To evaluate long-term outcome, 35 mice were randomly divided into sham group (n=5), vehicle group (n=15), and KXA group (n=15). Mice in KXA and vehicle groups received 69 mg KXA and vehicle for 7 days, respectively. Pathological changes in the brain were observed by 2,3,5-triphenyltetrazolium chloride or Nissl stainings, and behavioral assessments, including the Modified Neurologic Severity Score, Bederson score, rotarod test, and adhesive removal test were conducted. The penetration ability of KXA and KX (KXA without propellants) through the blood-brain barrier was assessed both in vitro using a transwell model and in vivo. Furthermore, in vitro effects of KX (5, 10, and 20 µL/L) on oxygen and glucose deprivation/re-oxygenation (OGD/R)-induced injury, transient receptor potential vanilloid type 1 (TRPV1) modulation, calcium influx, and mitochondrial function were explored through Western blot, CCK-8 assay, JC-1 staining, calcium imaging, adenosine triphosphate (ATP) and antioxidant measurements. In in vivo experiments, KXA reduced brain infarct volume and neuron loss in t-MCAO mice. Behavioral assessments showed marked improvement in the neurological deficit of t-MCAO mice with KXA treatment (P<0.05 or P<0.01). Additionally, in vitro findings indicated that KX ameliorated OGD/R-induced injury through TRPV1 channel modulation. KX increased cell viability in OGD/R-treated SH-SY5Y cells and prevented OGD/R-induced calcium overload by downregulating TRPV1 expression and constraining calcium influx through TRPV1 (P<0.05 or P<0.01). Furthermore, KXA maintained the membrane potential and function of mitochondria in OGD/R-treated SH-SY5Y cells. KXA could attenuate ischemic stroke injury through TRPV1 channel modulation, indicating its potential as a promising therapeutic option for stroke in clinical practice.

Exploration of the cytotoxic and microtubule disruption potential of novel imidazo[1,5-a]pyridine-based chalcones.

In continuation of our efforts to develop new anticancer compounds, a new series of imidazo[1,5-a]pyridine-chalcone derivatives was designed, synthesized, characterized, and evaluated for its cytotoxicity against five human cancer cell lines, i.e., breast (MDA-MB-231), colon (RKO), bone (Mg-63), prostate (PC-3), and liver (HepG2) cell lines, as well as a normal cell line (HEK). Among the synthesized compounds, two exhibited promising cytotoxicity against the MDA-MB-231 cell line with IC50 values of 4.23 ± 0.25 μM and 3.26 ± 0.56 μM. We also studied apoptotic induction of the compounds using annexin V-FITC/PI staining, and ROS-mediated mitochondrial damage was elucidated using DCFDA, followed by JC-1 staining. The potential activity of the compounds was further confirmed by immuno-fluorescence and molecular docking studies, which revealed the anticancer activity of active compounds through binding and microtubule disruption.

The UCP2/PINK1/LC3b-mediated mitophagy is involved in the protection of NRG1 against myocardial ischemia/reperfusion injury.

Available evidence indicates that neuregulin-1 (NRG-1) can provide a protection against myocardial ischemia/reperfusion (I/R) injury and is involved in various cardioprotective interventions by potential regulation of mitophagy. However, the molecular mechanisms linking NRG-1 and mitophagy remain to be clarified. In this study, both an in vivo myocardial I/R injury model of rats and an in vitro hypoxia/reoxygenation (H/R) model of H9C2 cardiomyocytes were applied to determine whether NRG-1 postconditioning attenuated myocardial I/R injury through the regulation of mitophagy and to explore the underlying mechanisms. In the in vivo experiment, cardioprotective effects of NRG-1 were determined by infarct size, cardiac enzyme and histopathologic examinations. The potential downstream signaling pathways and molecular targets of NRG-1 were screened by the RNA sequencing and the Protein-Protein Interaction Networks. The expression levels of mitochondrial uncoupling protein 2 (UCP2) and mitophagy-related proteins in both the I/R myocardium and H/R cardiomyocytes were measured by immunofluorescence staining and Western blots. The activation of mitophagy was observed with transmission electron microscopy and JC-1 staining. The KEGG and GSEA analyses showed that the mitophagy-related signaling pathways were enriched in the I/R myocardium treated with NRG-1, and UCP2 exhibited a significant correlation between mitophagy and interaction with PINK1. Meanwhile, the treatment with mitophagy inhibitor Mdivi-1 significant eliminated the cardioprotective effects of NRG-1 postconditioning in vivo, and the challenge with UCP2 inhibitor genipin could also attenuate the activating effect of NRG-1 postconditioning on mitophagy. Consistently, the in vitro experiment using H9C2 cardiomyocytes showd that NRG-1 treatment significantly up-regulated the expression levels of UCP2 and mitophagy-related proteins, and activated the mitophagy, whereas the challenge with small interfering RNA-mediated UCP2 knockdown abolished the effects of NRG-1. Thus, it is conclused that NRG-1 postconditioning can produce a protection against the myocardial I/R injury by activating mitophagy through the UCP2/PINK1/LC3B signaling pathway.

Senkyunolide A attenuates cerebral ischemia-reperfusion injury by inhibiting NLRP3-mediated ferroptosis in PC12 cells.

Cerebral ischemia-reperfusion (I/R) is a serious complication in patients with ischemic stroke. Senkyunolide A (SenA) can alleviate neuronal cell damage induced by cerebral I/R; however, the exact action mechanism remains unclear. An in vitro cellular injury model was established by inducing PC-12 cells with OGD/R. The viability of SenA-treated PC-12 cells with or without OGD/R induction was detected with CCK-8 assay while the cell apoptosis was detected using TUNEL. The secretion of inflammatory cytokines, the activity of ROS, mitochondrial membrane potential and mtROS level were measured with ELISA, ROS assay kits, JC-1 staining and MitoSOX Red assay, respectively. The level of Fe2+ was detected with Fe2+ assay kits and lipid peroxidation was detected with TBARS assay. The expressions of lipid peroxides were measured using corresponding assay kits. Western blot was used to measure the expressions of NLRP3, apoptosis-, and ferroptosis-related proteins. The transfection efficiency of OV-NLRP3 was also detected using Western blot. The present study showed that SenA could attenuate viability damage, inflammatory response, oxidative stress, apoptosis and ferroptosis in OGD/R-induced PC-12 cells and it was identified that the cytoprotective effects of SenA on PC-12 cells stimulated by OGD/R may be associated with the inhibition of NLPR3. Collectively, SenA protects neuronal cells against cerebral I/R injury through the inhibition of NLRP3-mediated ferroptosis.

Icariside II relieves radiation enteritis by regulating PINK/Parkin-mediated mitophagy.

Radiation enteritis (RE) is one of the major side effects of radiotherapy. So far, there are no effective drugs for preventing the disease process. Icariside II (ICS II) is a highly efficient monomer compound extracted and purified from the classic Chinese medicinal herb Epimedium. It has anti-inflammatory, antioxidant, and immunomodulatory effects. However, the role and mechanism of ICS II on radiation enteritis are not clear. Here, we reveal the role of ICS II in radiation enteritis by using an irradiation-induced rat model and a human colorectal cancer cell (CaCo2). After intragastric administration, HE staining and Tunel staining to observe the histopathological changes in the colon, and TEM to observe the ultrastructure of mitochondria; The antioxidant indexes and mitochondrial function-related markers of colon tissues were determined; DCFH-DA fluorescent probe were used to detect the cellular ROS level, JC-1 staining was used to detect the changes in mitochondrial membrane potential, and Western Blot was used to detect related protein expression. The results showed that ICS II could reduce intestinal injury and attenuate the radiation-induced oxidative stress and inflammatory response. In addition, ICS II could effectively attenuate mitochondrial damage and activate mitochondrial autophagy in rats. Mechanistically, ICS II activates mitochondrial autophagy-related protein expression to rescue radiation-induced damage to mitophagy. We found that by inhibiting mitophagy, the therapeutic effect of ICS II can be eliminated and our data suggest that ICS II may be a new and effective drug candidate for the treatment of radiation enteritis.

EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction in lipopolysaccharide-induced tubular epithelial cells

Sepsis represents an organ dysfunction resulting from the host’s maladjusted response to infection, and can give rise to acute kidney injury (AKI), which significantly increase the morbidity and mortality of septic patients. This study strived for identifying a novel therapeutic strategy for patients with sepsis-induced AKI (SI-AKI). Rat tubular epithelial NRK-52E cells were subjected to lipopolysaccharide (LPS) exposure for induction of in-vitro SI-AKI. The expressions of E1A binding protein p300 (EP300) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in NRK-52E cells were assessed by western blot and qRT-PCR, and their interaction was explored by chromatin immunoprecipitation performed with antibody for H3K27 acetylation (H3K27ac). The effect of them on SI-AKI-associated mitochondrial dysfunction of tubular epithelial cells was investigated using transfection, MTT assay, TUNEL staining, 2′,7′-Dichlorodihydrofluorescein diacetate probe assay, Mitosox assay, and JC-1 staining. MTHFD2 and EP300 were upregulated by LPS exposure in NRK-52E cells. LPS increased the acetylation of H3 histone in the MTHFD2 promoter region, and EP300 suppressed the effect of LPS. EP300 ablation inhibited the expression of MTHFD2. MTHFD2 overexpression antagonized LPS-induced viability reduction, apoptosis promotion, reactive oxygen species overproduction, and mitochondrial membrane potential collapse of NRK-52E cells. By contrast, MTHFD2 knockdown and EP300 ablation brought about opposite consequences. Furthermore, MTHFD2 overexpress and EP300 ablation counteracted each other’s effect in LPS-exposed NRK-52E cells. EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction of tubular epithelial cells in SI-AKI.

Fumarprotocetraric acid and geraniin were identified as novel inhibitors of human respiratory syncytial virus infection in vitro.

Respiratory syncytial virus (RSV) remains a major international public health concern. However, disease treatment is limited to preventive care with monoclonal antibodies and supportive care. In this study, natural products were screened to identify novel anti-RSV inhibitors. The antiviral effect of 320 compounds on RSV in HEp-2 cells was tested using a Cytopathic effect (CPE) inhibition assay. The antiviral effect of fumarprotocetraric acid (FUM) and geraniin (GE) were confirmed by Real-time reverse transcription quantitative PCR (Real-time RT-PCR), plaque reduction test, immunofluorescence assay, and Western blot analysis. Real-time PCR was used to detect inflammatory factor expression. ATP assay and JC-1 stain were used to evaluate mitochondrial protection function. The experiment of administration time was used to determine the stages in the RSV life cycle inhibited by FUM and GE. Human metapneumovirus (HMPV) and human rhinovirus (HRV) were used to evaluate the antiviral activities of other respiratory viruses of FUM and GE. Finally, Air-liquid interface human airway epithelium (ALI-HAE) cells were used to evaluate the antiviral effect and mechanism of FUM and GE to RSV. The results showed that FUM and GE can inhibit the replication of RSV in multiple-cell models. Both compounds could dose-dependent inhibit the viral load, RSV nucleic acids level, and RSV-F protein level. Besides, FUM and GE showed good anti-inflammatory activity, mitochondrial protection, and antiviral activity to HMPV and HRV. Meanwhile, our result indicated that FUM and GE can inhibit RSV replication in ALI-HAE cells. FUM and GE were identified as new inhibitors of RSV infection. At the same time, FUM and GE have anti-inflammatory activity, mitochondrial protection function, and broad-spectrum antiviral activity. These results provide evidence that FUM and GE are potential candidates for the development of novel anti-RSV drugs.

Shikonin induces the apoptosis and pyroptosis of EGFR-T790M-mutant drug-resistant non-small cell lung cancer cells via the degradation of cyclooxygenase-2

BackgroundThe T790M mutation in the epidermal growth factor receptor (EGFR) gene is the primary cause of resistance to EGFR-tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients. Previous research demonstrated that certain traditional Chinese medicine (TCM) monomers exhibit anti-tumor effects against various malignancies. This study aims to investigate the potentials of shikonin screened from a TCM monomer library containing 1060 monomers in killing EGFR-T790M drug-resistant NSCLC cells and elucidate the underlying mechanisms.MethodsMTT method was used to screen for the TCM monomers with significant killing effects on H1975 cells carrying the EGFR-T790M mutation. The influences of the identified monomer shikonin on cell growth were determined by the colony formation assay. Annexin-V/PI staining and JC-1 staining were applied to detect the effects of shikonin on cell apoptosis. The influences of shikonin on cell membrane integrity were detected by lactate dehydrogenase (LDH) release assay. Reactive oxygen species (ROS) generation was analyzed using DCFH-DA as probe. The mechanisms of shikonin affecting the stability of cyclooxygenase-2 (COX-2) were evaluated by using specific inhibitors for protein degradation pathways. Western blotting was performed to assess the effects of the alteration of COX-2 expression or enzymatic activity on the related signal pathways as well as the apoptotic and pyroptotic markers.ResultsShikonin was identified as a potent cytotoxic compound against EGFR-T790M-mutant NSCLC cells. Shikonin induced cell apoptosis and pyroptosis by triggering the activation of the caspase cascade and cleavage of poly (ADP-ribose) polymerase and gasdermin E by elevating intracellular ROS levels. Further investigations revealed that shikonin induced the degradation of COX-2 via the proteasome pathway, thereby decreasing COX-2 protein level and enzymatic activity and subsequently inhibiting the downstream PDK1/Akt and Erk1/2 signaling pathways through the induction of ROS production. Notably, COX-2 overexpression attenuated shikonin-induced apoptosis and pyroptosis, whereas COX-2 inhibition with celecoxib enhanced the cytotoxic effects of shikonin.ConclusionsCombination treatment with shikonin and COX-2 inhibitor may be a suitable therapeutic strategy for EGFR-T790M-mutant NSCLC treatment.

Inhibition of TFAM-Mediated Mitophagy by Oroxylin A Restored Sorafenib Sensitivity Under Hypoxia Conditions in HepG2 Cells.

Background: Liver cancer treatment encounters considerable therapeutic challenges, especially because hypoxic microenvironments markedly reduce sensitivity to chemotherapeutic agents. TFAM (mitochondrial transcription factor A) plays a crucial role in maintaining mitochondrial function. Oroxylin A (OA), a flavonoid with potential therapeutic properties, demonstrated prospects in cancer treatment. However, the mechanism of the sensitizing effect of OA on cancer cells has not been elucidated. Methods: MTT assays were utilized to evaluate a hypoxia-induced resistance model. Plate colony formation assays, TEM, and JC-1 staining were used to examine the effects of siTFAM on proliferation and mitochondrial damage of HepG2 cells. Cox8-EGFP-mCherry plasmid transfection, LysoTracker and MitoTracker colocalization analysis, and WB were conducted to evaluate the influence of OA on mitophagy. The effect of OA on p53 ubiquitination levels was investigated by Co-IP and the CHX chase assay. A mouse xenograft tumor model was utilized to assess the therapeutic effect of OA on HepG2 cells in vivo. Results: OA significantly improved the inhibitory effect of sorafenib by inhibiting mitophagy on HepG2 cells in in vitro and in vivo models. Notably, the molecular docking and thermal shift assays indicated a clear binding of OA and TFAM. Further research revealed that OA suppressed p53 acetylation and promoted its degradation by downregulating TFAM expression, which ultimately inhibited mitophagy in hypoxia. Conclusions: OA has demonstrated the potential to enhance the efficacy of sorafenib treatment for liver cancer, and TFAM may be one of its targets.

Mechanism of WAVE1 regulation of lipopolysaccharide-induced mitochondrial metabolic abnormalities and inflammatory responses in macrophages

To explore the mechanism by which Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) regulates lipopolysaccharide (LPS)-induced mitochondrial metabolic abnormalities and inflammatory responses in macrophages. Macrophage cell lines with overexpressed WAVE1 (mouse BMDM and human THP1 cells) were prepared. The macrophages were treated with LPS (500 ng/mL) to simulate sepsis-induced inflammatory responses. The experiment consisted of two parts. The first part included control, LPS, vector (LPS+oe-NC), WAVE1 overexpression (LPS+oe-WAVE1) groups. The second part included LPS, LPS+oe-NC, LPS+oe-WAVE1 and exogenous high mobility group box-1 (HMGB1) intervention (LPS+oe-WAVE1+HMGB1) groups. RT-PCR was used to measure mitochondrial DNA content, and RT-qPCR was used to detect the mRNA expression levels of WAVE1, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. Western blot was performed to measure the protein expression of WAVE1, hexokinase 2, and pyruvate kinase M2. ELISA was utilized to detect the levels of TNF-α, IL-1β, IL-6, and HMGB1. JC-1 staining was used to assess mitochondrial membrane potential. Seahorse XP96 was used to evaluate oxygen consumption rate and extracellular acidification rate. MitoSOX probe was employed to measure mitochondrial reactive oxygen species levels, and 2-NBDG method was used to assess glucose uptake. Kits were used to measure pyruvate kinase activity, lactate, adenosine triphosphate (ATP), and HMGB1 levels. Compared with the control group, the LPS group showed lower levels of WAVE1 protein and mRNA expression, mitochondrial membrane potential, oxygen consumption rate, and mitochondrial DNA content (P<0.05), while TNF-α, IL-1β, IL-6 levels and mRNA expression, mitochondrial reactive oxygen species, glucose uptake, lactate, ATP, hexokinase 2, and pyruvate kinase M2 protein expression levels as well as extracellular acidification rate, pyruvate kinase activity, and HMGB1 release were significantly increased (P<0.05). Compared with the LPS+oe-NC group, the LPS+oe-WAVE1 group showed increased WAVE1 protein and mRNA expression, mitochondrial membrane potential, oxygen consumption rate, and mitochondrial DNA content (P<0.05), while TNF-α, IL-1β, IL-6 levels and mRNA expression, mitochondrial reactive oxygen species, glucose uptake, lactate, ATP, hexokinase 2, and pyruvate kinase M2 protein expressions, as well as extracellular acidification rate, pyruvate kinase activity, and HMGB1 release were decreased (P<0.05). Compared with the LPS+oe-WAVE1 group, the LPS+oe-WAVE1+HMGB1 group exhibited increased glucose uptake, lactate, ATP levels, and extracellular acidification rate (P<0.05). WAVE1 participates in the regulation of LPS-induced inflammatory responses in macrophages by modulating the release of inflammatory factors, mitochondrial metabolism, and HMGB1 release.

Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation.

Hepatocellular carcinoma (HCC), the third leading cancer mortality worldwide, shows rising incidence. The mitochondria in HCC cells are prone to damage from metabolic stress and oxidative stress, necessitating heightened mitophagy for mitochondrial homeostasis and cell survival. Thus, mitophagy inhibition is a promising HCC therapy. The traditional Chinese medicinal herb ailanthone have proved promote mitochondrial dysfunction and inhibits HCC. However, the underlying mechanism remains unclear. CCK8 assay was applied to detect the proliferation. JC-1, MitoTracker Red/Green and MitoSOX staining were applied to detect the mitochondrial homeostasis. Inflammatory factors were analysed via ELISA and WB assay. Mitochondria and cytoplasm separation, genome extraction and qPCR were used to detect mitochondrial DNA (mtDNA) leakage. Mitochondria ultrastructure was detected by transmission electron microscopy. WB and IHC experiments were applied to detect protein expression. Protein-protein interactions detected by immunoprecipitation and immunofluorescence imaging. The in vivo antitumor effect was validated by the xenograft mouse model. In this study, we demonstrated the potent anti-HCC properties of ailanthone and revealed its molecular mechanism. In vitro studies demonstrated that ailanthone effectively inhibited PINK1-PRKN mediated mitophagy and promoted BAX-BAK1 mitochondrial pores formation through PRKN inhibition. This process led to the mitochondrial mtDNA leakage into the cytoplasm, which subsequently triggered the induction of inflammatory factors. The inhibition of mitophagy and the activation of inflammatory response ultimately led to HCC proliferation inhibition. In vivo studies demonstrated that ailanthone exhibited stronger anti-HCC activity than 5-Fluorouracil (5-FU), with no significant adverse effects on animal body weight or the physiological functions of vital organs. This study highlighted the efficacy of ailanthone against HCC and elucidated its underlying molecular mechanisms, suggesting the promising therapeutic potential of ailanthone for HCC.