MEMS-based two-photon microscopy with Lissajous scanning and image reconstruction under a feed-forward control strategy.

Two-photon microscopy (TPM) based on two-dimensional micro-electro-mechanical (MEMS) system mirrors shows promising applications in biomedicine and the life sciences. To improve the imaging quality and real-time performance of TPM, this paper proposes Lissajous scanning control and image reconstruction under a feed-forward control strategy, a dual-parameter alternating drive control algorithm and segmented phase synchronization mechanism, and pipe-lined fusion-mean filtering and median filtering to suppress image noise. A 10 fps frame rate (512 × 512 pixels), a 140 µm × 140 µm field of view, and a 0.62 µm lateral resolution were achieved. The imaging capability of MEMS-based Lissajous scanning TPM was verified by ex vivo and in vivo biological tissue imaging.

Food-Origin Mycotoxin-Induced Neurotoxicity: Intend to Break the Rules of Neuroglia Cells.

Mycotoxins are key risk factors in human food and animal feed. Most of food-origin mycotoxins could easily enter the organism and evoke systemic toxic effects, such as aflatoxin B1 (AFB1), ochratoxin A (OTA), T-2 toxin, deoxynivalenol (DON), zearalenone (ZEN), fumonisin B1 (FB1), and 3-nitropropionic acid (3-NPA). For the last decade, the researches have provided much evidences in vivo and in vitro that the brain is an important target organ on mycotoxin-mediated neurotoxic phenomenon and neurodegenerative diseases. As is known to all, glial cells are the best regulator and defender of neurons, and a few evaluations about the effects of mycotoxins on glial cells such as astrocytes or microglia have been conducted. The fact that mycotoxin contamination may be a key factor in neurotoxicity and glial dysfunction is exactly the reason why we reviewed the activation, oxidative stress, and mitochondrial function changes of glial cells under mycotoxin infection and summarized the mycotoxin-mediated glial cell proliferation disorders, death pathways, and inflammatory responses. The purpose of this paper is to analyze various pathways in which common food-derived mycotoxins can induce glial toxicity and provide a novel perspective for future research on the neurodegenerative diseases.

Targeting HMGB1 inhibits T-2 toxin-induced neurotoxicity via regulation of oxidative stress, neuroinflammation and neuronal apoptosis.

T-2 toxin, a food-derived mycotoxin, has been identified as a neurotoxin. Nonetheless, T-2 toxin-induced neuroinflammation has never been revealed. As an important therapeutic target for inflammatory diseases and cancers, the role of high mobility group B1 (HMGB1) in mycotoxin-mediated neurotoxicity remains a mystery. In current study, we found that PC12 cells were sensitive to trace amounts of T-2 toxin less than 12 ng/mL, distinguished by decreased cell viability and increased release of lactate dehydrogenase (LDH). Oxidative stress and mitochondrial damage were observed in PC12 cells, manifested as accumulation of oxidative stress products, up-regulation of Nrf2/HO-1 pathway and decrease of mitochondrial membrane potential (MMP), leading to mitochondria-dependent apoptosis. Meanwhile, we first discovered that tiny amounts of T-2 toxin triggered neuroinflammation directly, including raising the expression and translocation of NF-κB and promoting secretion of proinflammatory cytokines such as TNF-α, IL-6, IL-8 and IL-1ß. Most interestingly, the increased of HMGB1 was detected both inside and outside the cells. Conversely, HMGB1 siRNA reduced T-2 toxin-mediated oxidative stress, apoptosis and neuroinflammatory outbreak, accompanied by lessened caspase-3 and caspase-9, and decreased secretion of pro-inflammatory cytokines. Taken together, T-2 toxin-stimulated PC12 cells simultaneously displayed apoptosis and inflammation, whereas HMGB1 played a critical role in these neurotoxic processes.

A Comprehensive Toxicological Assessment of Fulvic Acid.

Fulvic acid (FA), a humic substance, has several nutraceutical properties, including anti-inflammation, antimicrobial, and immune regulation abilities. However, systematic safety assessment remains insufficient. In the present study, a battery of toxicological studies was conducted per internationally accepted standards to investigate the genotoxicity and repeated-dose oral toxicity of FA. Sprague-Dawley (SD) rats or ICR mice were used. Compared to the control group, there were no significant changes (all p > 0.05) in all FA treatment groups in the bacterial reverse mutation test, in vitro mammalian chromosome aberration test, in vivo sperm shape abnormality assay, and in vivo mouse micronucleus assay. The acute toxicity test showed that no mortality or toxic effect was observed following oral administration of the maximum dose of 5,000 mg/kg BW/day to mice or rats. A 60-day subchronic study was conducted at 0 (control), 200, 1,000, and 5,000 mg/kg/day. Compared to the control group, there were no significant changes (all p > 0.05) in the body weights, feed consumption, clinical signs, hematology, clinical chemistry, organ weights, or histopathology examinations. In conclusion, the no-observed-adverse-effect-level (NOAEL) of FA supplementation from the 60-day study was determined to be 5,000 mg/kg body weight/day, the highest dose tested. Our findings suggest that the oral administration of FA may have higher safety.

Olaquindox-Induced Liver Damage Involved the Crosstalk of Oxidative Stress and p53 In Vivo and In Vitro.

Olaquindox (OLA), a member of the quinoxaline-N,N-dioxide family, has been widely used as a growth-promoting feed additive and treatment for bacterial infections. The toxicity has been a major concern, and the precise molecular mechanism remains poorly understood. The present study was aimed at investigating the roles of oxidative stress and p53 in OLA-caused liver damage. In a mouse model, OLA administration could markedly cause liver injury as well as the induction of oxidative stress and activation of p53. Antioxidant N-acetylcysteine (NAC) inhibited OLA-induced oxidative stress and p53 activation in vivo. Furthermore, knockout of the p53 gene could significantly inhibit OLA-induced liver damage by inhibiting oxidative stress and the mitochondria apoptotic pathway, compared to the p53 wild-type liver tissue. The cell model in vitro further demonstrated that p53 knockout or knockdown in the HCT116 cell and L02 cell significantly inhibited cell apoptosis and increased cell viability, presented by suppressing ROS production, oxidative stress, and the Nrf2/HO-1 pathway. Moreover, loss of p53 decreased OLA-induced mitochondrial dysfunction and caspase activations, with the evidence of inhibited activation of phosphorylation- (p-) p38 and p-JNK and upregulated cell autophagy via activation of the LC3 and Beclin1 pathway in HCT116 and L02 cells. Taken together, our findings provided a support that p53 primarily played a proapoptotic role in OLA-induced liver damage against oxidative stress and mitochondrial dysfunction, which were largely dependent on suppression of the JNK/p38 pathway and upregulation of the autophagy pathway via activation of LC3 and Beclin1.

Molecular mechanism of olaquindox-induced hepatotoxicity and the hepatic protective role of curcumin.

Olaquindox (OLA) is a chemosynthetic growth promoter, which could promote the treatment of bacterial infections and improve feed energy efficiency. Hepatotoxicity is still a poor feature associated with the adverse effects of OLA. The present study aimed to investigate the molecular mechanism of OLA-induced hepatotoxicity and the protective role of curcumin in mice and HepG2 cells. The result showed that representative biomarkers involved in mitochondrial pathway, p53 pathway, mitogen-activated protein kinase (MAPK) pathway, autophagy and antioxidant pathway were activated. Furthermore, curcumin attenuated OLA-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and liver damage in mice. In addition, cell viability of HepG2 was enhanced by curcumin pretreatment at 5, 10 and 20 µM. Meanwhile, curcumin markedly ameliorated OLA-induced oxidative stress, apoptosis and mitochondrial dysfunction. Moreover, curcumin pretreatment significantly up-regulated the expressions of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1) and down-regulated the expressions of nuclear factor-kappaB (NF-kB) and p53 through reduced the nuclear translocation of NF-kB induced by OLA. In summary, our findings indicated that OLA-induced hepatotoxicity involved in mitochondrial apoptosis, autophagy, p53 pathway, Nrf2/HO-1 pathways, and curcumin regulated OLA-induced liver damage, oxidative stress and apoptosis via activation of Nrf2/HO-1 pathway and suppression of p53 and NF-kB pathway.

Baicalin inhibits the TGF-ß1/p-Smad3 pathway to suppress epithelial-mesenchymal transition-induced metastasis in breast cancer.

TGF-ß1 is an epithelial-mesenchymal transition (EMT)-inducing factor that is critical in tumor progression. However, whether the effect of TGF-ß1 on breast cancer is through the EMT pathway remains to be determined, and drug development based on this mechanism needs to be improved. Results of this study showed that TGF-ß1 dysregulation significantly correlated with the expression levels of EMT-associated markers and transcriptional factors. Exogenous expression of TGF-ß1 promoted breast cancer cell metastasis and EMT progression. In addition, direct binding of baicalin to TGF-ß1 caused its inactivation, which subsequently blocked signal transduction and inhibited breast cancer cell metastasis. In vivo experiment results further invalidated the inhibitory effect of baicalin on TGF-ß1-induced tumor metastasis. These results suggest that baicalin, an active ingredient used in traditional Chinese medicine, exhibits a potential therapeutic effect on breast cancer metastasis by regulating TGF-ß1-dependent EMT progression.

Molecular Insights of Copper Sulfate Exposure-Induced Nephrotoxicity: Involvement of Oxidative and Endoplasmic Reticulum Stress Pathways.

The precise pathogenic mechanism in Cu exposure-cause nephrotoxicity remains unclear. This study investigated the underlying molecular mechanism of copper sulfate (CuSO4)-induced nephrotoxicity. Mice were treated with CuSO4 at 50, 100, 200 mg/kg/day or co-treated with CuSO4 (200 mg/kg/day) and 4-phenylbutyric acid (4-PBA, 100 mg/kg/day) for 28 consecutive days. HEK293 cells were treated with CuSO4 (400 µM) with or without superoxide dismutase, catalase or 4-PBA for 24 h. Results showed that CuSO4 exposure can cause renal dysfunction and tubular necrosis in the kidney tissues of mice. CuSO4 exposure up-regulated the activities and mRNA expression of caspases-9 and -3 as well as the expression of glucose-regulated protein 78 (GRP78), GRP94, DNA damage-inducible gene 153 (GADD153/CHOP), caspase-12 mRNAs in the kidney tissues. Furthermore, superoxide dismutase and catalase pre-treatments partly inhibited CuSO4-induced cytotoxicity by decreasing reactive oxygen species (ROS) production, activities of caspases-9 and -3 and DNA fragmentations in HEK293 cells. 4-PBA co-treatment significantly improved CuSO4-induced cytotoxicity in HEK293 cells and inhibited CuSO4 exposure-induced renal dysfunction and pathology damage in the kidney tissues. In conclusion, our results reveal that oxidative stress and endoplasmic reticulum stress contribute to CuSO4-induced nephrotoxicity. Our study highlights that targeting endoplasmic reticulum and oxidative stress may offer an approach for Cu overload-caused nephrotoxicity.

Polymyxins-Curcumin Combination Antimicrobial Therapy: Safety Implications and Efficacy for Infection Treatment.

The emergence of antimicrobial resistance in Gram-negative bacteria poses a huge health challenge. The therapeutic use of polymyxins (i.e., colistin and polymyxin B) is commonplace due to high efficacy and limiting treatment options for multidrug-resistant Gram-negative bacterial infections. Nephrotoxicity and neurotoxicity are the major dose-limiting factors that limit the therapeutic window of polymyxins; nephrotoxicity is a complication in up to ~60% of patients. The emergence of polymyxin-resistant strains or polymyxin heteroresistance is also a limiting factor. These caveats have catalyzed the search for polymyxin combinations that synergistically kill polymyxin-susceptible and resistant organisms and/or minimize the unwanted side effects. Curcumin-an FDA-approved natural product-exerts many pharmacological activities. Recent studies showed that polymyxins-curcumin combinations showed a synergistically inhibitory effect on the growth of bacteria (e.g., Gram-positive and Gram-negative bacteria) in vitro. Moreover, curcumin co-administration ameliorated colistin-induced nephrotoxicity and neurotoxicity by inhibiting oxidative stress, mitochondrial dysfunction, inflammation and apoptosis. In this review, we summarize the current knowledge-base of polymyxins-curcumin combination therapy and discuss the underlying mechanisms. For the clinical translation of this combination to become a reality, further research is required to develop novel polymyxins-curcumin formulations with optimized pharmacokinetics and dosage regimens.

Nerve Growth Factor Confers Neuroprotection against Colistin-Induced Peripheral Neurotoxicity.

Neurotoxicity is an unwanted side effect for patients when receiving parenteral colistin therapy. The development of effective neuroprotective agents that can be coadministered during colistin therapy remains a priority area in antimicrobial chemotherapy. The present study aimed to investigate the protective effect of nerve growth factor (NGF) against colistin-induced peripheral neurotoxicity using a murine model. C57BL/6 mice were randomly divided into the following 6 groups: (i) untreated control, (ii) NGF alone (36 µg/kg/day administered intraperitoneally), (iii) colistin alone (18 mg/kg/day administered intraperitoneally), and (iv-vi) colistin (18 mg/kg/day) plus NGF (9, 18, and 36 µg/kg/day). After treatment for 7 days, neurobehavioral and electrophysiology changes, histopathological assessments of sciatic nerve damage, and oxidative stress biomarkers were examined. The mRNA expression levels of Nrf2, HO-1, Akt, Bax, and caspase-3 and -9 were assessed using quantitative RT-PCR. The results showed that, across all the groups wherein NGF was coadministered with colistin, a marked attenuation of colistin-induced sciatic nerve damage and improved sensory and motor function were observed. In comparison to the colistin only treatment group, animals that received NGF displayed upregulated Nrf2 and HO-1 mRNA expression levels and downregulated Bax and caspase-3 and -9 mRNA expression levels. In summary, our study reveals that NGF coadministration protects against colistin-induced peripheral neurotoxicity via the activation of Akt and Nrf2/HO-1 pathways and inhibition of oxidative stress. This study highlights the potential clinical application of NGF as a neuroprotective agent for coadministration during colistin therapy.

Curcumin Attenuates Colistin-Induced Peripheral Neurotoxicity in Mice.

Peripheral neurotoxicity often occurs in patients receiving parenteral polymyxin therapy (i.e., colistin methanesulfonate or polymyxin B). The present study aimed to investigate the protective effect of curcumin on colistin-induced peripheral neurotoxicity using a murine model. Female C57BL/6 mice (n = 10 in each group) were randomly divided into the following: (1) control group (saline), (2) curcumin only group (200 mg/kg/day; orally), (3) colistin only group (18 mg/kg/day; i.p.), (4) colistin (18 mg/kg/day) plus curcumin 50 mg/kg/day group, (5) colistin (18 mg/kg/day) plus curcumin 100 mg/kg/day group, (6) colistin (18 mg/kg/day) plus curcumin 200 mg/kg/day group; all mice were treated for 7 days. Orally applied curcumin was detected in the brain, cerebellum, and sciatic nerve. Co-administration of oral curcumin markedly improved colistin-induced impaired sensory and motor dysfunctions in a dose-dependent manner. Curcumin supplementation at 100 and 200 mg/kg significantly decreased lipid peroxidation and upregulated catalase (CAT) and superoxide dismutase (SOD) activities, ATP levels, and Na+/K+-ATPase activity in sciatic nerve tissue, compared to the colistin alone group. Curcumin supplementation at 200 mg/kg upregulated the levels of AKT, NGF, mTOR, Nrf2, and HO-1 mRNA and concomitantly downregulated Bax, caspases-3, and -9 mRNA; it also decreased caspase-3 and caspase-9 activity. In summary, for the first time, our study reveals that the protective effect of oral curcumin on colistin induced peripheral neurotoxicity is associated with the activation of NGF/Akt and Nrf2/HO-1 pathways and inhibition of oxidative stress. This study highlights the potential clinical application of curcumin as an oral neuroprotective agent coadministered during colistin therapy.

Identification of Plant Soot as Novel Safe Feed Additive: Evaluation of 90-Day Oral Toxicity and Prenatal Developmental Toxicity in Rats.

Plant soot, as a novel feed additive, could not only improve digestive function but also adsorb mycotoxins and inhibit bacterial infections. The subchronic toxicity and prenatal developmental effects of plant soot were studied for the first time. Our results indicated that there was no subchronic toxicity in the range of 2,000-50,000 mg/kg plant soot added in the feed, and there was no significant difference in reproductive function, embryo development, and teratogenicity between the pregnant rats exposed to 312.5, 1,250, and 5,000 mg/kg plant soot and the control group. The maximum no-observed effect level (NOEL) of supplemental dosage in feed could be set to 50,000 mg/kg, and the maximum intragastric NOEL could be set to 5,000 mg/kg, which preliminarily provided guidance on daily additive amount or clinical protocols for plant soot, as well as promoting the development and application of this harmless antibiotic substitutes.

T-2 toxin neurotoxicity: role of oxidative stress and mitochondrial dysfunction.

Mycotoxins are highly diverse secondary metabolites produced in nature by a wide variety of fungi. Mycotoxins cause animal feed and food contamination, resulting in mycotoxicosis. T-2 toxin is one of the most common and toxic trichothecene mycotoxins. For the last decade, it has garnered considerable attention due to its potent neurotoxicity. Worryingly, T-2 toxin can cross the blood-brain barrier and accumulate in the central nervous system (CNS) to cause neurotoxicity. This review covers the current knowledge base on the molecular mechanisms of T-2 toxin-induced oxidative stress and mitochondrial dysfunction in the CNS. In vitro and animal data have shown that induction of reactive oxygen species (ROS) and oxidative stress plays a critical role during T-2 toxin-induced neurotoxicity. Mitochondrial dysfunction and cascade signaling pathways including p53, MAPK, Akt/mTOR, PKA/CREB and NF-κB contribute to T-2 toxin-induced neuronal cell death. T-2 toxin exposure can also result in perturbations of mitochondrial respiratory chain complex and mitochondrial biogenesis. T-2 toxin exposure decreases the mitochondria unfolded protein response and dampens mitochondrial energy metabolism. Antioxidants such as N-acetylcysteine (NAC), activation of Nrf2/HO-1 and autophagy have been shown to provide a protective effect against these detrimental effects. Clearly, translational research and the discovery of effective treatment strategies are urgently required against this common food-borne threat to human health and livestock.

Colistin induced peripheral neurotoxicity involves mitochondrial dysfunction and oxidative stress in mice.

Polymyxin is a critical antibiotic against the infection caused by multidrug-resistant gram-negative bacteria. Neurotoxicity is one of main dose-limiting factors. The present study aimed to investigate the underlying molecular mechanism on colistin induced peripheral neurotoxicity using a mouse model. Forty mice were divided into control, colistin 1-, 3- and 7-day groups, the mice were intravenously injected with saline or colistin (sulfate) at the dose of 15 mg/kg/day for 1, 3 and 7 days, respectively. The results showed that, colistin treatment for 7 days markedly resulted in the demyelination, axonal degeneration and mitochondria swelling in the mice's sciatic tissues. Colistin treatment induces oxidative stress as well as the increases of mitochondrial permeability transition, decreases of membrane potential (ΔΨm) and activities of mitochondrial respiratory chain in the mice's sciatic nerve tissues. Furthermore, in the colistin-7 day group, adenosine-triphosphate (ATP) level Na+/K+-ATPase activity decreased to 75.2% (p < 0.01) and 80.1% (p < 0.01), respectively. Meanwhile, colistin treatment down-regulates the expression of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) mRNAs and up-regulates the expression of Bax and caspase-3 mRNAs. Our results reveal that colistin induced sciatic nerves damage involves oxidative stress, mitochondrial dysfunction and the inhibition of Akt/mTOR pathway.

Molecular Mechanisms of Neurotoxicity Induced by Polymyxins and Chemoprevention.

Neurotoxicity is one major unwanted side-effects associated with polymyxin (i.e., colistin and polymyxin B) therapy. Clinically, colistin neurotoxicity is characterized by neurological symptoms including dizziness, visual disturbances, vertigo, confusion, hallucinations, seizures, ataxia, and facial and peripheral paresthesias. Pathologically, colistin-induced neurotoxicity is characterized by cell injury and death in neuronal cell. This Review covers our current understanding of polymyxin-induced neurotoxicity, its underlying mechanisms, and the discovery of novel neuroprotective agents to limit this neurotoxicity. In recent years, an increasing body of literature supports the notion that polymyxin-induced nerve damage is largely related to oxidative stress and mitochondrial dysfunction. P53, PI3K/Akt, and MAPK pathways are also involved in colistin-induced neuronal cell death. The activation of the redox homeostasis pathways such as Nrf2/HO-1 and autophagy have also been shown to play protective roles against polymyxin-induced neurotoxicity. These pathways have been demonstrated to be upregulated by neuroprotective agents including curcumin, rapamycin and minocycline. Further research is needed toward the development of novel polymyxin formulations in combination with neuroprotective agents to ameliorate this unwanted adverse effect during polymyxins therapy in patients.

Chloroquine ameliorates carbon tetrachloride-induced acute liver injury in mice via the concomitant inhibition of inflammation and induction of apoptosis.

This is the first study to investigate the hepatoprotective effect of CQ on acute liver injury caused by carbon tetrachloride (CCl4) in a murine model and the underlying molecular mechanisms. Ninety-six mice were randomly divided into the control (n = 8), CQ (n = 8), CCl4 (n = 40), and CCl4 + CQ (n = 40) treatment groups. In the CCl4 group, mice were intraperitoneally (i.p) injected with 0.3% CCl4 (10 mL/kg, dissolved in olive oil); in the CCl4 + CQ group, mice were i.p injected with CQ at 50 mg/kg at 2, 24, and 48 h before CCl4 administration. The mice in the control and CQ groups were administered with an equal vehicle or CQ (50 mg/kg). Mice were killed at 2, 6, 12, 24, 48 h post CCl4 treatment and their livers were harvested for analysis. The results showed that CQ pre-treatment markedly inhibited CCl4-induced acute liver injury, which was evidenced by decreased serum transaminase, aspartate transaminase and lower histological scores of liver injury. CQ pretreatment downregulated the CCl4-induced hepatic tissue expression of high-mobility group box 1 (HMGB1) and the levels of serum HMGB1 as well as IL-6 and TNF-α. Furthermore, CQ pre-treatment inhibited autophagy, downregulated NF-kB expression, upregulated p53 expression, increased the ratio of Bax/Bcl-2, and increased the activation of caspase-3 in hepatic tissue. This is the first study to demonstrate that CQ ameliorates CCl4-induced acute liver injury via the inhibition of HMGB1-mediated inflammatory responses and the stimulation of pro-apoptotic pathways to modulate the apoptotic and inflammatory responses associated with progress of liver damage.

Curcumin Attenuates Colistin-Induced Neurotoxicity in N2a Cells via Anti-inflammatory Activity, Suppression of Oxidative Stress, and Apoptosis.

Neurotoxicity is an unwanted side-effect seen in patients receiving therapy with the "last-line" polymyxin antibiotics. This is the first study to show that colistin-induced neurotoxicity in neuroblastoma-2a (N2a) cells gives rise to an inflammatory response involving the IL-1ß/p-IκB-α/NF-κB pathway. Pretreatment with curcumin at 5, 10, and 20 µM for 2 h prior to colistin (200 µM) exposure for 24 h, produced an anti-inflammatory effect by significantly down-regulating the expression of the pro-inflammatory mediators cyclooxygenase-2 (COX-2), phosphorylation of the inhibitor of nuclear factor-kappa B (NF-κB) (p-IκB)-α, and concomitantly NF-κB levels. Moreover, curcumin significantly decreased intracellular reactive oxygen species (ROS) production and increased the activities of the anti-ROS enzymes superoxide dismutase, catalase, and the intracellular levels of glutathione. Curcumin pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation, and subsequent apoptosis. Overall, our findings demonstrate for the first time, a potential role for curcumin for treating polymyxin-induced neurotoxicity through the modulation of NF-κB signaling and its potent anti-oxidative and anti-apoptotic effects.

Rapamycin Confers Neuroprotection against Colistin-Induced Oxidative Stress, Mitochondria Dysfunction, and Apoptosis through the Activation of Autophagy and mTOR/Akt/CREB Signaling Pathways.

Our previous studies showed that colistin-induced neurotoxicity involves apoptosis and oxidative damage. The present study demonstrates a neuroprotective effect of rapamycin against colistin-induced neurotoxicity in vitro and in vivo. In a mouse model, colistin treatment (18 mg/kg/d; 14 days) produced marked neuronal mitochondria damage in the cerebral cortex and increased activation of caspase-9 and -3. Rapamycin cotreatment (2.5 mg/kg/d) effectively reduced this neurotoxic effect. In an in vitro mouse neuroblastoma-2a (N2a) cell culture model, rapamycin pretreatment (500 nM) reduced colistin (200 µM) induced cell death from ∼50% to 72%. Moreover, rapamycin showed a marked neuroprotective effect in the N2a cells by decreasing intracellular reactive oxygen species (ROS) production and by up-regulating the activities of the anti-ROS enzymes superoxide dismutase and catalase and recovering glutathione (GSH) levels to normal. Moreover, rapamycin pretreatment protected against colistin-induced mitochondrial dysfunction, caspase activation, and subsequent apoptosis by up-regulating autophagy and activating the Akt/CREB, NGF, and Nrf2 pathways, while inhibiting p53 signaling. Taken together, this is the first study to demonstrate that rapamycin protects against colistin-induced neurotoxicity by activating autophagy, inhibiting oxidative stress, mitochondria dysfunction, and apoptosis. Our data highlight that regulating autophagy to rescue neurons from apoptosis may become a new targeted therapy to relieve the adverse neurotoxic effects associated with colistin therapy.

DIDS inhibits overexpression BAK1-induced mitochondrial apoptosis through GSK3ß/ß-catenin signaling pathway.

Bcl-2 homologous antagonist/killer (BAK1) is a critical regulator of mitochondrial apoptosis. Although upregulation of BAK1 induces apoptosis has been established, the underlying molecular mechanism is far from clear. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an organic anion used as a blocker of anion exchangers and chloride channels, has been proved to rescue cell apoptosis both in vitro and in vivo. However, whether DIDS can inhibit BAK1-induced mitochondrial apoptosis remains undefined. Thus, this study aimed to explore whether DIDS could protect BAK1-induced apoptosis through GSK3ß/ß-catenin signaling pathway. The results showed overexpression BAK1 in 293T cells induced mitochondrial apoptosis accompanied by increasing the expression levels of cleaved caspase-9, -3, poly (ADP-ribose) polymerase (PARP) and reducing the MMP. Furthermore, overexpression BAK1 decreased the expression levels of Ser9-GSK3ß and ß-catenin. In addition, lithium chloride (LiCl), an activator of Wnt/ß-catenin signaling pathway, markedly attenuated overexpression BAK1-induced mitochondrial apoptosis by restoring the expression levels of Ser9-GSK3ß and ß-catenin. Finally, DIDS absolutely abolished overexpression BAK1-mediated mitochondrial apoptosis through recovering the expression levels of Ser9-GSK3ß and ß-catenin. Taken together, our results reveal that DIDS blocks overexpression BAK1-induced mitochondrial apoptosis through GSK3ß/ß-catenin pathway.

ROS-mediated oligomerization of VDAC2 is associated with quinocetone-induced apoptotic cell death.

Quinocetone (QCT) has been approved and widely used as an animal feed additive in China since 2003. However, investigations indicate that QCT shows potential toxicity both in vitro and in vivo. Although voltage dependent anion channel 1 (VDAC1) involved in regulating QCT-induced apoptotic cell death has been established, the role of voltage dependent anion channel 2 (VDAC2) in QCT-induced toxicity remains unclear. In this study, we showed that QCT-induced cell death was coupled to VDAC2 oligomerization. Moreover, VDAC inhibitor 4, 4'-diisothiocyano stilbene-2, 2'-disulfonic acid (DIDS) alleviated QCT-induced cell death and VDAC2 oligomerization. Meanwhile, overexpression VDAC2 aggravated QCT-induced VDAC2 oligomerization. In addition, caspase inhibitor Z-VAD-FMK and reactive oxidative species (ROS) scavenger N-acetyl-l-cysteine (NAC) apparently blocked QCT-induced cell death and VDAC2 oligomerization. Finally, overexpression N-terminal truncated VDAC2 attenuated QCT-induced VDAC2 oligomerization but had no influence on its localization to mitochondria when comparing to the full length of VDAC2. Taken together, our results reveal that ROS-mediated VDAC2 oligomerization is associated with QCT-induced apoptotic cell death. The N-terminal region of VDAC2 is required for QCT-induced VDAC2 oligomerization.