Bax Mitochondrial Translocation Research Articles

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

BackgroundNeutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood.MethodsUsing the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice.ResultsWe identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the “FOXO1-transferrin receptor (TFRC)” mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI.ConclusionsFOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

Inhibition of Drp1- Fis1 interaction alleviates aberrant mitochondrial fragmentation and acute kidney injury

BackgroundAcute kidney injury (AKI) is a common clinical disorder with complex etiology and poor prognosis, and currently lacks specific and effective treatment options. Mitochondrial dynamics dysfunction is a prominent feature in AKI, and modulation of mitochondrial morphology may serve as a potential therapeutic approach for AKI.MethodsWe induced ischemia–reperfusion injury (IRI) in mice (bilateral) and Bama pigs (unilateral) by occluding the renal arteries. ATP depletion and recovery (ATP-DR) was performed on proximal renal tubular cells to simulate in vitro IRI. Renal function was evaluated using creatinine and urea nitrogen levels, while renal structural damage was assessed through histopathological staining. The role of Drp1 was investigated using immunoblotting, immunohistochemistry, immunofluorescence, and immunoprecipitation techniques. Mitochondrial morphology was evaluated using confocal microscopy.ResultsRenal IRI induced significant mitochondrial fragmentation, accompanied by Dynamin-related protein 1 (Drp1) translocation to the mitochondria and Drp1 phosphorylation at Ser616 in the early stages (30 min after reperfusion), when there was no apparent structural damage to the kidney. The use of the Drp1 inhibitor P110 significantly improved kidney function and structural damage. P110 reduced Drp1 mitochondrial translocation, disrupted the interaction between Drp1 and Fis1, without affecting the binding of Drp1 to other mitochondrial receptors such as MFF and Mid51. High-dose administration had no apparent toxic side effects. Furthermore, ATP-DR induced mitochondrial fission in renal tubular cells, accompanied by a decrease in mitochondrial membrane potential and an increase in the translocation of the pro-apoptotic protein Bax. This process facilitated the release of dsDNA, triggering the activation of the cGAS-STING pathway and promoting inflammation. P110 attenuated mitochondrial fission, suppressed Bax mitochondrial translocation, prevented dsDNA release, and reduced the activation of the cGAS-STING pathway. Furthermore, these protective effects of P110 were also observed renal IRI model in the Bama pig and folic acid-induced nephropathy in mice.ConclusionsDysfunction of mitochondrial dynamics mediated by Drp1 contributes to renal IRI. The specific inhibitor of Drp1, P110, demonstrated protective effects in both in vivo and in vitro models of AKI.

Augmented mitochondrial apoptotic signaling impairs C2C12 myoblast differentiation following cellular aging through sequential passaging.

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self-renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age-related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high-passage (HP) myoblasts exhibit greater mitochondrial-mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase-9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts.

Hederagenin suppresses ovarian cancer via targeting mitochondrial fission through dynamin-related protein 1

A triterpenoid isolated from the plant Hedera helix, hederagenin was discovered to have anti-cancer, anti-inflammatory, anti-depressant and anti-fibrosis properties both in vivo and in vitro. In this study, the relationship between mitochondrial fission and hederagenin-induced apoptosis in ovarian cancer (OC) was investigated and the underlying mechanisms were deciphered. Hederagenin's cytotoxicity on OC cells was analyzed using colony formation and CCK-8 assays. The effect of hederagenin on OC cells was also verified by a mouse xenograft tumor model. Flow cytometric analysis was conducted to examine hederagenin's effects on mitochondrial membrane potential, apoptosis, and cell cycle OC cells. MitoTracker Red (CMXRos) staining was performed to observe the mitochondrial morphology. The protein levels of Bak, Bcl-2, Caspase 3, Caspase 9, Cyclin D1 and Bax were measured by Western blot. This study found that hederagenin could suppress the in vivo and in vitro SKOV3 and A2780 cell proliferation in an effective manner. Besides, hederagenin altered the mitochondrial membrane potential, induced S-phase and G0/G1-phase arrest, mitochondrial morphology changes, and apoptosis in OC cells. Additionally, our findings further demonstrated that hederagenin changed the mitochondrial morphology by suppressing dynamin-related protein 1 (Drp1), a crucial mitochondrial division factor. Moreover, Drp1 overexpression could reverse hederagenin-induced apoptosis, whereas the Drp1 knockdown had the opposite effect. Furthermore, hederagenin may trigger BAX mitochondrial translocation and apoptosis in OC cells. These results provided a novel perspective on the relationship between the modulation of mitochondrial morphology and the suppression of ovarian cancer by hederagenin.

DUSP1 protects against ischemic acute kidney injury through stabilizing mtDNA via interaction with JNK

The mechanism underlying acute kidney injury (AKI) and AKI-to-Chronic kidney disease (CKD) transition remains unclear, but mitochondrial dysfunction may be a key driving factor. Literature reports suggest that dual-specificity phosphatase 1 (DUSP1) plays a critical role in maintaining mitochondrial function and structural integrity. In this study, ischemic Acute Kidney Injury (AKI) and post-ischemic fibrosis models were established by clamping the renal pedicle with different reperfusion times. To investigate the role of DUSP1, constitutional Dusp1 knockout mice and tubular-specific Sting knockout mice were used. Mitochondrial damage was assessed through electron microscopy observation, measurements of mitochondrial membrane potential, mtDNA release, and BAX translocation. We found that Dusp1 expression was significantly upregulated in human transplant kidney tissue and mouse AKI tissue. Dusp1 gene deletion exacerbated acute ischemic injury, post-ischemic renal fibrosis, and tubular mitochondrial dysfunction in mice. Mechanistically, DUSP1 could directly bind to JNK, and DUSP1 deficiency could lead to aberrant phosphorylation of JNK and BAX mitochondria translocation. BAX translocation promoted mitochondrial DNA (mtDNA) leakage and activated the cGAS-STING pathway. Inhibition of JNK or BAX could inhibit mtDNA leakage. Furthermore, STING knockout or JNK inhibition could significantly mitigate the adverse effects of DUSP1 deficiency in ischemic AKI model. Collectively, our findings suggest that DUSP1 is a regulator for the protective response during AKI. DUSP1 protects against AKI by preventing BAX-induced mtDNA leakage and blocking excessive activation of the cGAS-STING signaling axis through JNK dephosphorylation.

Mitochondrial IRG1 traps MCL-1 to induce hepatocyte apoptosis and promote carcinogenesis

Hepatocarcinogenesis is initiated by repeated hepatocyte death and liver damage, and the underlying mechanisms mediating cell death and the subsequent carcinogenesis remain to be fully investigated. Immunoresponsive gene 1 (IRG1) and its enzymatic metabolite itaconate are known to suppress inflammation in myeloid cells, and its expression in liver parenchymal hepatocytes is currently determined. However, the potential roles of IRG1 in hepatocarcinogenesis are still unknown. Here, using the diethylnitrosamine (DEN)-induced hepatocarcinogenesis mouse model, we found that IRG1 expression in hepatocytes was markedly induced upon DEN administration. The DEN-induced IRG1 was then determined to promote the intrinsic mitochondrial apoptosis of hepatocytes and liver damage, thus enhancing the subsequent hepatocarcinogenesis. Mechanistically, the mitochondrial IRG1 could associate and trap anti-apoptotic MCL-1 to inhibit the interaction between MCL-1 and pro-apoptotic Bim, thus promoting Bim activation and downstream Bax mitochondrial translocation, and then releasing cytochrome c and initiating apoptosis. Thus, the inducible mitochondrial IRG1 promotes hepatocyte apoptosis and the following hepatocarcinogenesis, which provides mechanistic insight and a potential target for preventing liver injury and HCC.

PGAM5 exacerbates acute renal injury by initiating mitochondria-dependent apoptosis by facilitating mitochondrial cytochrome c release.

Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular cell death has not been fully elucidated. Here, we reported that phosphoglycerate mutase 5 (PGAM5) was upregulated in renal tubular epithelial cells during ischaemia/reperfusion or cisplatin-induced AKI in mice. PGAM5 knockout significantly alleviated the activation of the mitochondria-dependent apoptosis pathway and tubular apoptosis. Apoptosis inhibitors alleviated the activation of the mitochondria-dependent apoptosis pathway. Mechanistically, as a protein phosphatase, PGAM5 could dephosphorylate Bax and facilitate Bax translocation to the mitochondrial membrane. The translocation of Bax to mitochondria increased membrane permeability, decreased mitochondrial membrane potential and facilitated the release of mitochondrial cytochrome c (Cyt c) into the cytoplasm. Knockdown of Bax attenuated PGAM5 overexpression-induced Cyt c release and tubular cell apoptosis. Our results demonstrated that the increase in PGAM5-mediated Bax dephosphorylation and mitochondrial translocation was implicated in the development of AKI by initiating mitochondrial Cyt c release and activating the mitochondria-dependent apoptosis pathway. Targeting this axis might be beneficial for alleviating AKI.

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax.

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochromec (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cytcrelease, closely resembling the natural Bax function in the cellular context. This regulated cell death process wasreverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.

Ginsenoside Rg1 can restore hematopoietic function by inhibiting Bax translocation-mediated mitochondrial apoptosis in aplastic anemia

The present study investigated, the anti-apoptotic activity of Ginsenoside Rg1 (Rg1) via inhibition of Bax translocation and the subsequent recovery of hematopoietic function. Mitochondrial apoptosis in bone marrow mononuclear cells (BMNCs) was observed in aplastic anemia (AA) patients. To establish a mouse model of AA, BALB/c mice were transplanted with lymph node cells from DBA/2 donor mice via vein injection after treatment with Co60 γ-radiation. After treatment with Rg1 for 14 days, the peripheral blood and Lin–Sca-1 + c-Kit + (LSK) cell counts of the treated group were increased compared with those of the untreated model mice. In in vivo and in vitro tests of LSKs, Rg1 was found to increase mitochondrial number and the ratio of Bcl-2/Bax and to decrease damage to the mitochondrial inner and outer membranes, the mitochondrial Bax level and the protein levels of mitochondrial apoptosis-related proteins AIF and Cyt-C by decreasing the ROS level. Rg1 also improved the concentration–time curve of MAO and COX and levels of ATP, ADP and AMP in an in vitro test. In addition, high levels of Bax mitochondrial translocation could be corrected by Rg1 treatment. Levels of markers of mitochondrial apoptosis in the Rg1-treated group were significantly better than those in the AA model group, implying that Rg1 might improve hematopoietic stem cells and thereby restore hematopoietic function in AA by suppressing the mitochondrial apoptosis mediated by Bax translocation.

Genistein nanoformulation promotes selective apoptosis in oral squamous cell carcinoma through repression of 3PK-EZH2 signalling pathway

BackgroundOverexpression of polycomb protein contributes to epigenetic repression in oral squamous cell carcinoma (OSCC) ensuing in poor prognosis and aggressive phenotype. Several plant-based compounds could help prevent epigenome alteration and cancer progression, but their low bioavailability limits their therapeutic activity. HypothesisIn this study, we have synthesized genistein nanoformulation (GLNPs) and evaluated its epigenetic regulation mechanism for selective apoptosis induction in OSCC. MethodsLactalbumin was used to prepare nanoformulation of Genistein. The mechanism of epigenetic regulation and selective apoptosis by Genistein loaded nanoparticles was studied in OSCC cell line JHU011 and fibroblast cell line L929 using immunofluorescence, Western blotting and ChIP-qPCR assay. ResultsWe have found that GLNPs treatment selectively induced apoptosis in OSCC compared to the normal fibroblast cells. This selective effect in OSCC is achieved through enhanced reactive oxygen species (ROS) generation followed by Bax mitochondrial translocation and caspase 3 activation. Further, GLNPs induced withdrawal of epigenetic transcription repression through concurrent downregulation of the polycomb group proteins (PcG) Bmi 1 and EZH2 along with their successive targets, UbH2AK119 and H3K27me3, which have immense therapeutic implications in the treatment of OSCC. Last, we have established that GLNPs regulate EZH2expression through proteasomal mediated degradation and 3PK inhibition; 3PK protein was found physically linked with EZH2 protein and its promoter region (-1107 to -1002). This event indicates that 3PK might play some crucial role in EZH2 expression and epigenetic control of OSCC. Moreover, the formulation showed improved biodistribution, aqueous dispersibility and enhanced biocompatibility In-vivo. ConclusionsThese results provide evidence that GLNPs may withdraw epigenetic transcriptional repression and selectively induce apoptosis in human oral squamous cell carcinoma.

NPD1 rapidly targets mitochondria-mediated apoptosis after acute injection protecting brain against ischemic injury

Mitochondria-related cell death pathways play a major role in ischemic brain injury. Thus, mitochondrial “protective” molecules could be considered for new therapeutic regimens. We recently reported that acute administration of docosahexaenoic acid (DHA) triglyceride lipid emulsion, immediately after hypoxic-ischemic (HI) insult, markedly attenuated brain infarct size. This was associated with an early change of DHA-derived specialized pro-resolving mediator (SPM) profiles. Specifically, DHA treatment induced a 50% increase of neuroprotectin D1 (NPD1) levels in ischemic brain. Based on these findings, we questioned if direct administration of NPD1 after HI injury also affords neuroprotection, and if so, by what mechanisms.Using HI insult to mimic ischemic stroke in neonatal mice, we observed that acute intraperitoneal injection of NPD1 immediately after HI injury prevented the expansion of the ischemic core by ~40% and improved coordination and motor abilities compared to the control group. At 7 days after HI injury, NPD1 treatment decreased ipsilateral hemisphere atrophy and preserved motor functions in wire-holding and bridge-crossing tests compared to control littermates. Brain mitochondria, isolated at 4 h after reperfusion from mice treated with NPD1, showed an increase in the capacity to buffer calcium after HI injury, as result of the preservation of mitochondrial membranes. Further, NPD1 induced a reduction of mitochondrial BAX translocation and oligomerization, attenuated cytochrome C release and decreased AIF nuclear translocation. To confirm whether NPD1 acts as BAX inhibitor, we evaluated NPD1 action co-administrated with a pro-apoptotic agent, staurosporine, using mouse embryonic fibroblasts as in vitro model of apoptosis. NPD1 exposure markedly decreased mitochondria-mediated apoptosis, blocking BAX translocation from cytosol to mitochondria and subsequently reducing caspase-3 activation. Our findings provide novel evidence that the neuroprotective action of NPD1 is elicited rapidly in the first few hours after ischemic injury and is associated with both preserved mitochondrial membrane structure and reduced BAX mitochondrial translocation and activation.

Voltage-Dependent Anion Channels Influence Cytotoxicity of ME-344, a Therapeutic Isoflavone.

ME-344 is a second-generation cytotoxic isoflavone with anticancer activity promulgated through interference with mitochondrial functions. Using a click chemistry version of the drug together with affinity-enriched mass spectrometry, voltage-dependent anion channels (VDACs) 1 and 2 were identified as drug targets. To determine the importance of VDAC1 or 2 to cytotoxicity, we used lung cancer cells that were either sensitive (H460) or intrinsically resistant (H596) to the drug. In H460 cells, depletion of VDAC1 and VDAC2 by small interfering RNA impacted ME-344 effects by diminishing generation of reactive oxygen species (ROS), preventing mitochondrial membrane potential dissipation, and moderating ME-344–induced cytotoxicity and mitochondrial-mediated apoptosis. Mechanistically, VDAC1 and VDAC2 knockdown prevented ME-344–induced apoptosis by inhibiting Bax mitochondrial translocation and cytochrome c release as well as apoptosis in these H460 cells. We conclude that VDAC1 and 2, as mediators of the response to oxidative stress, have roles in modulating ROS generation, Bax translocation, and cytochrome c release during mitochondrial-mediated apoptosis caused by ME-344.SIGNIFICANCE STATEMENTDissecting preclinical drug mechanisms are of significance in development of a drug toward eventual Food and Drug Administration approval.

Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation

BackgroundOxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF). AimsTo investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation. Materials and methodsIn vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2−/− were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection. ResultsOur results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2−/− HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2−/− mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2−/− mice. ConclusionsThese investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.

Drp1 regulates mitochondrial dysfunction and dysregulated metabolism in ischemic injury via Clec16a-, BAX-, and GSH- pathways

The adaptation of mitochondrial homeostasis to ischemic injury is not fully understood. Here, we studied the role of dynamin-related protein 1 (Drp1) in this process. We found that mitochondrial morphology was altered in the early stage of ischemic injury while mitochondrial dysfunction occurred in the late stage of ischemia. Drp1 appeared to inhibit mitophagy by upregulating mito-Clec16a, which suppressed mito-Parkin recruitment and subsequently impaired the formation of autophagosomes in vascular tissues after ischemic injury. Moreover, ischemia-induced Drp1 activation enhanced apoptosis through inducing mitochondrial translocation of BAX and thereby increasing release of Cytochrome C to activate caspase-3/-9 signalling. Furthermore, Drp1 mediated metabolic disorders and inhibited the levels of mitochondrial glutathione to impair free radical scavenging, leading to further increases in ROS and the exacerbation of mitochondrial dysfunction after ischemic injury. Together, our data suggest a critical role for Drp1 in ischemic injury.

Sulforaphane improves voiding function via the preserving mitochondrial function in diabetic rats

Hyperglycemia evoked oxidative stress contributing to diabetes (DM)-induced voiding dysfunction. We explored whether antioxidant sulforaphane,a NF-E2-related nuclear factor erythroid-2 (Nrf-2) activator, may ameliorate DM-induced bladder dysfunction. DM was induced by streptozotocin andsulforaphanewas administered before DM induction.Bladder reactive oxygen species (ROS) were determined by an ultrasensitive chemiluminescence analyzer. Mitochondrial function index mitochondrial Bax and cytosolic cytochrome c, antioxidant defense Nrf-2/HO-1, endoplasmic reticulum stress marker ATF-6/CHOP, and caspase 3/PARP were evaluated by Western blot. DM increased Keap1 and reduced Nrf-2 expression, associated with increase of bladder ROS, mitochondrial Bax translocation, cytosolic cytochrome c release, ATF-6/CHOP, caspase-3/PARP in bladders which resulted in voiding dysfunction by increased intercontraction intervals and micturition duration. However,sulforaphanesignificantly increased nuclear Nrf-2/HO-1axis expression, decreased bladder ROS amount, mitochondrial Bax translocation, cytochrome c release, ATF-6/CHOP and caspase 3/PARP/apoptosis, thereby improved the voiding function by the shortened intercontraction intervals and micturition duration. We suggest that sulforaphanevia activating Nrf-2/HO-1 signaling preserved mitochondrial function and suppressed DM-induced ROS, endoplasmic reticulum stress, apoptosis and voiding dysfunction.

Emerging and established modes of cell death during acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.

Rutin hydrate inhibits apoptosis in the brains of cadmium chloride-treated rats via preserving the mitochondrial integrity and inhibiting endoplasmic reticulum stress

ABSTRACTRecent evidence has suggested that cadmium (Cd) ions-induced neurotoxicity is associated with increased oxidative stress and mitochondrial-dependent and endoplasmic reticulum (ER) stress-induced apoptosis. This study aimed to investigate if rutin hydrate (RH), a well-reported neuroprotective and an antioxidant flavonoid, can ameliorate cadmium chloride (CdCl2)-induced neurotoxicity by inhibiting the resultant ER stress. Rats were divided into 4 groups (n = 16/group) of control, control + RH (100 mg/kg), CdCl2 (5 mg/kg), and CdCl2 + RH. All treatments were administered orally for 30 days, on daily basis. Brain homogenates from CdCl2-treated rats showed increased oxidative stress and induced activation of ER stress characterized by increasing mRNA and protein levels of GRP78, ATF-6, CHOP and Xbp-1 and protein levels of p-elF2α, p-JNK1/2 and cleaved caspase-12. Also, CdCl2 significantly reduced Bcl-2, enhanced Bax translocation to the mitochondrial membrane, increased cytoplasmic levels of cytochrome-C and caspase-3, and reduced mitochondrial membrane potential (Δψm) (increased Vmax and reduced time to Vmax). In contrast, RH significantly enhanced levels GSH and activities of SOD, GSH-Px, decreased levels of MDA and inhibited mitochondrial permeability transition pore (mtPTP) in the brains of both control and CdCl2-treated rats. Interestingly, in brain homogenates of CdCl2-treated rats only, RH reduced all markers of ER stress, increased Bcl-2, reduced mitochondrial Bax translocation and improved mitochondrial coupling. It also reduced cytosolic levels of cytochrome-C, cleaved caspase-3, and cleaved caspase-12. Overall, these findings support the efficiency of RH to inhibit ER stress in brains CdCl2-treated rats which is added to its existing mechanisms of neuroprotection.Abbreviations: ATF-6: activating transcription factor-6; Bax: Bcl-associated x; BBB: blood-brain barrier; Bcl-2: B-cell lymphoma 2; BiP: immunoglobulin heavy-chain-binding protein; [Ca2+]i: intracellular free Ca2+ concentration; Cd: cadmium; CdCl2: cadmium chloride; CHOP: CCAAT/enhancer-binding protein-homologous protein; CMC: carboxymethyl cellulose; Δψm: mitochondrial membrane potential; elF2α: phospho-eukaryotic translation initiation factor 2-alpha; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERK1/2: extracellular signal-regulated kinases 1 and 2; GADD 153: growth arrest and DNA damage-inducible protein 153; GRP78, 78 kDa glucose-regulated protein; GSH: reduced glutathione; GSH: reduced glutathione; GSH-Px: glutathione peroxidase; GSSG: glutathione disulfide (oxidized glutathione); IRE-1: inositol-requiring enzyme-1; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; mTOR: Akt/mammalian target of rapamycin; mtPTP: mitochondrial permeability transition pore; ONOO−: peroxynitrite; PCR: polymerase chain reaction; PERK: protein kinase RNA-like ER kinase; p-JNK: phospho-JNK; qPCR: quantitative PCR; RCR: respiratory control ratio; RH: rutin hydrate; RHoGDI: Rho-GDP-dissociation inhibitor; ROS: reactive oxygen species; SOD: superoxide dismutase; UPR: unfolded protein response; VDAC: voltage-dependent anion channel; Vmax: maximal rate of pore opening; Xbp-1: X-box binding protein 1.

Small-molecule allosteric inhibitors of BAX.

BAX is a critical effector of the mitochondrial cell death pathway in response to a diverse range of stimuli in physiological and disease contexts. Upon binding by BH3-only proteins, cytosolic BAX undergoes conformational activation and translocation, resulting in mitochondrial outer membrane permeabilization. Efforts to rationally target BAX and develop inhibitors have been elusive, despite the clear therapeutic potential of inhibiting BAX-mediated cell death in a host of diseases. Here, we describe a class of small molecule BAX inhibitors, termed BAIs, which bind directly to a previously unrecognized pocket and allosterically inhibit BAX activation. BAI-binding around the hydrophobic helix α5 using hydrophobic and hydrogen bonding interactions stabilizes key areas of the hydrophobic core. BAIs inhibit conformational events in BAX activation that prevent BAX mitochondrial translocation and oligomerization. Our data highlight a novel paradigm for effective and selective pharmacological targeting of BAX to enable rational development of inhibitors of BAX-mediated cell death.

Farrerol Ameliorates APAP-induced Hepatotoxicity via Activation of Nrf2 and Autophagy.

Farrerol has been shown to have antioxidative potential via Nrf2 activation, which in turn is involved in the prevention of hepatotoxicity. The purpose of the current study was to explore the protective effect of farrerol against acetaminophen-induced hepatotoxicity and its underlying mechanisms. Mice were used to evaluate the hepatoprotective effect of farrerol on liver injury induced by acetaminophen in vivo. HepG2 cells were utilized to further determine the functional role and mechanisms by which Nrf2 and autophagy are involved in the hepatoprotective effect of farrerol in vitro. We found that treatment with farrerol leads to a significant reduction in acetaminophen-induced hepatotoxicity by decreasing mortality, histopathological liver changes, and ALT and AST levels. Furthermore, farrerol effectively suppressed mitochondrial dysfunction by reducing JNK phosphorylation, Bax mitochondrial translocation, AIF and cytochrome c release. Further investigations revealed that the activation of Nrf2 and the induction of autophagy via the AMPK/AKT pathway by farrerol contributed to its hepatoprotective activity in vitro. In addition, farrerol inhibited acetaminophen-induced the mortality and histopathological changes in WT mice were evidently alleviated but not abrogated in Nrf2 -/- mice, which attributed to the induction of autophagy. Together, farrerol has protective potential against acetaminophen-induced hepatotoxicity which may be associated with activation of Nrf2 and autophagy.

Macrophage migration inhibitory factor regulates mitochondrial dynamics and cell growth of human cancer cell lines through CD74–NF-κB signaling

The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.