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Network oscillations at γ frequency band (30–80 Hz), generated by the interaction between inhibitory interneurons and excitatory neurons, have been proposed to be associated with higher brain functions such as learning and memory. Dopamine (DA), one of the major CNS transmitters, modulates hippocampal γ oscillations but the intracellular mechanisms involved remain elusive. In this study, we recorded kainate-induced γ oscillations in the CA3 area of rat hippocampal slices, and found that DA strongly enhanced γ power, which was largely blocked by dopamine receptor 1 (DR1) antagonist SCH23390, receptor tyrosine kinase (RTK) inhibitor UNC569 and ERK inhibitor U0126, partially blocked by D2/3R antagonist raclopride, PKA inhibitor H89 and PI3K inhibitor wortmannin, but not affected by AKT inhibitor TCBN or NMDAR antagonist D-AP5. Our results indicate that DA-mediated γ enhancement is involved in the activation of signaling pathway of DR1/2-RTK-ERK. Our data demonstrate a strong, rapid modulation of DA on hippocampal γ oscillations and provide a new insight into cellular mechanisms of DA-mediated γ oscillations.

The aim of this study was to determine the antiproliferative effect of an ethanolic leaf extract of Combretum apiculatum (CA) against human epidermoid carcinoma (A431) and human malignant melanoma (UCT-MEL1) cells, the toxic potential of CA on non-cancerous human keratinocytes (HaCat) and to evaluate the inhibitory activity against the phosphatidylinositol-3-kinase (PI3K) enzyme, which is involved in tumor survival and proliferation. The protective effect of CA on hepatocellular carcinoma (HepG2) cells, in which toxicity was induced using acetaminophen, was further determined. The CA extract was found to exhibit a 50% inhibitory concentration (IC50) of 56.40 ± 6.11 and 90.53 ± 4.94 µg/mL on A431 and UCT-MEL-1 cells respectively. Against non-cancerous HaCat cells an IC50 value of 62.20 ± 3.52 µg/mL was obtained, resulting in a selectivity index of 1.10 and 0.69 on A431 and UCT-MEL-1 cells respectively. Therefore, CA showed a higher selective antiproliferative effect towards squamous cell carcinoma cells. Combretum apiculatum was also found to have a protective effect against acetaminophen induced toxicity in HepG2 cells, with an 11% protection at 5 µg/mL. Lastly, the results from the PI3K assay revealed that CA was able to significantly inhibit the PI3K enzyme at each of the tested concentrations, with the highest inhibition noted at a concentration of 0.4 µg/mL with a relative percentage of 106.38 ± 26.36 %, which was comparable to the positive control, wortmannin. This study provides the first report of the activity of Combretum apiculatum against skin cancer cell lines and the potential inhibitory activity of this species on the PI3K enzyme.

Background Protein tyrosine phosphatase 1B (PTP1B) inhibitors are potential candidates for the treatment of peripheral insulin resistance and diabetes mellitus. Similar to peripheral action within the brain also, PTP1B activation impairs insulin signaling pathways. Activation of PTP1B in brain also accentuates neuroinflammation, oxidative stress and decreases neurotrophic factors in various brain dysfunctions including cognitive decline. Objectives The main objective of our study was to elucidate the role of alendronate, a potent PTP1B inhibitor (blood brain barrier crossing bisphosphonate) in central insulin resistance and associated memory deficits. Methodology To induce central insulin resistance, streptozotocin (3 mg/kg) intracerebroventricular (ICV) was administered in two alternate days (1st and 3rd). After 21 days, memory was assessed via using the passive avoidance and Morris water maze paradigm. At the end of behavioral studies, animals were sacrificed to assess a variety of biochemical and molecular parameters in the hippocampus and cerebral cortex region of the brain. Treatment drug alendronate (3 mg/kg/day, p.o) and standard drug donepezil (3 mg/kg/i.p.) were administered from the 3rd day of STZ administration till the end of the study. Inhibition of PTP1B activates phosphoinsotide-3 kinase (PI3 K) (down-stream regulator of insulin signaling pathway).Thus, to illuminate the mechanism of action of alendronate, PI3 K inhibitor, wortmannin was administered in presence of alendronate in one group. Results Administration of alendronate to ICV streprozotocin treated rats resulted in modulation of the insulin signaling pathway and associated behavioral, biochemical and molecular changes in central insulin resistance. However, the protective effect of alendronate was entirely vanished when it was administered in the presence of wortmannin. Conclusion Alendronate can be an important treatment strategy in central insulin signaling pathway dysfunction and associated cognitive deficits. Protective effect of alendronate is via modulation of PI3-K/Akt signaling pathway.

Background As the symbolic pathological changes of Alzheimer’s disease (AD), hyperphosphorylated tau and amyloid plaque play important roles in the progression of the disease. In AD patients, the neural activity in default mode network is abnormal at different stages of the disease, and showed a hypoconnective status. Inhibition of phosphatidylinositol-3-kinase (PI3K) activates glycogen synthase kinase 3 beta (GSK-3β) and induces tau phosphorylation. Objective We speculated that inhibiting cerebral PI3K altered the glucose metabolism in DMN. We aimed to explore the impacts of PI3K inhibition on tau phosphorylation, cerebral glucose metabolism, and synaptic plasticity. Methods We injected wortmannin, an inhibitor of PI3K, lateral ventricularly in rats to mimic the pathology of AD. Immunohistochemistry was carried out to analyze the expression of phosphorylated tau. Region-specific glucose metabolism in the brain was analyzed using 18F-FDG PET imaging. In vivo long-term potentiation (LTP) in the hippocampus was detected to assess the synaptic plasticity. Results The results show that the phosphorylated tau at T231 increased and the hippocampal LTP was suppressed 24 h after wortmannin administration. In the DMN, glucose uptake was significantly high, indicating a neural activity disturbance. Conclusion We conclude that targeting PI3K-GSK-3β pathway to mimic AD tau pathology interrupted the glucose metabolism of DMN brain regions.

Endosome trafficking has been reported to play an essential role in pollen tube polar growth and NtGNL1 (Nicotiana tabacum GNOM-LIKE 1) regulates the polar growth through endosome trafficking. However, the regulation network and detailed molecular mechanisms underlying endosome trafficking remain unclear. Here, comparative proteomic analysis was carried out to survey the overall effect of NtGNL1 on pollen tube polar growth and NtGNL1-dependent endosome trafficking. With multiple comparative systems (RNAi, Wild type, and BFA or wortmannin treatments), 481 distinct proteins were identified including 43 common DEPs (differentially expressed proteins), of which 16 significant DEPs were common among RNAi, BFA, and wortmannin treated pollen tubes, indicating their close relation to the endosome trafficking. GO annotation indicates that the vesicle trafficking of gnl1HE pollen tubes differs from that of the BFA and wortmannin treated pollen tubes in the COPII-coated vesicle budding process. KEGG pathway analysis suggests that the Pentose phosphate pathway is critical for the NtGNL1-dependent endosome trafficking. Yeast two-hybrid further confirmed that the NtGNL1-Sec7 domain interacted strongly with VPS32.2, TCTP, PIS2, and PDIL2-1, suggesting that the core functional region of NtGNL1 is the Sec7 domain. Therefore, NtGNL1 likely functions via its Sec7 binding with these proteins to affect endosome trafficking. Our results provide a clear outline of proteins involving in NtGNL1-dependent endosome trafficking and valuable clues for understanding the regulatory mechanism of NtGNL1 guided pollen tube polar growth.

Aims Apelin is the endogenous ligand of G protein-coupled receptor APJ and play an important role in the regulation of cardiovascular homeostasis. We aimed to investigate whether apelin ameliorates vascular calcification (VC) by inhibition of endoplasmic reticulum stress (ERS). Methods and results VC model in rats was induced by nicotine plus vitamin D, while calcification of vascular smooth muscle cell (VSMC) was induced by beta-glycerophosphate. Alizarin Red S staining showed dramatic calcium deposition in the aorta of rats with VC, while calcium contents and ALP activity also increased in calcified aorta. Protein levels of apelin and APJ were decreased in the calcified aorta. In rats with VC, apelin treatment significantly ameliorated aortic calcification, compliance and stimulation of ERS. The ameliorative effect of apelin on VC and ERS was also observed in calcified VSMCs. ERS stimulator (tunicamycin or DTT) blocked the beneficial effect of apelin. Apelin treatment activated the PI3K/Akt signaling, blockage of which by wortmannin or inhibitor IV prevented the ameliorative effect of apelin, while ERS inhibitor 4-PBA rescued the blockade effect of wortmannin. Akt-induced GSK inhibition prevented the phosphorylation of PERK and IRE1, and the activation of these two major ERS branches. F13A blocked the ameliorative effect of apelin on VC and ERS, which was reversed by treatment with 4-PBA or Akt activator SC79 Conclusions Apelin ameliorated VC by binding to APJ and then prevented ERS activation by stimulating Akt signaling. These results might provide new target for therapy and prevention of VC.

This research aims to study the therapeutic effect of dexmedetomidine on type 2 diabetic rats reperfusion injury model. A rat model of type 2 diabetes was induced by high-fat and high-sugar feed, and a reperfusion rat model was obtained by ligating the left anterior descending branch and reperfusion. The diabetic rat model sham operation group was used as the control group (DM-S), the rats were divided into diabetic reperfusion group (DM-IR) and dexmedetomidine treatment group (DM-D), dexmedetomidine combined with DM-DW (DM-DW), and Wortmannin treatment group (DM-W). In this study, the MB isoenzyme of creatine kinase (CK-MB) concentration was determined by ELISA competition method, superoxide dismutase (SOD) activity in rat plasma was detected by superoxide dismutase WST-1, and the content of malonaldehyde (MDA) in rat plasma was determined by thiobarbituric acid method. Besides, Western Blot was used to detect the expression changes of Akt, P-Akt, GSK-3β, and P-GSK-3β in rat myocardial tissue. The results showed that the content of CK-MB and SOD in DM-D group was significantly higher than that in DM-IR group (P

Reactive Oxygen Species (ROS) are generated as by-products of aerobic metabolism. The production of ROS increases during xenobiotic stress and under multiple pathological conditions. Although ROS are considered harmful historically, mounting evidence recently indicates a signaling function of ROS, preceding to and regulating transcriptional or post-transcriptional events, contributing to cell death or cell survival and adaptation. Among the cellular defense mechanisms activated by ROS is formation of stress granules (SGs). The stalled translational apparatus, together with mRNA, aggregates into microscopically detectable and molecularly dynamic granules. We found that with H2O2, the dose most potent for inducing SGs in HeLa cells is 400–600 μM. With 200 μM H2O2, 2 h treatment induced the highest percentage of cells containing SGs. Whether ROS signaling pathways regulate the formation of SGs was tested using pharmacological inhibitors. We probed the potential role of PI3K, MAPKs, PKC or histone deacetylation in SG formation. Using deferoxamine as a positive control, we found a lack of inhibitory effect of wortmannin, LY-294002, JNK-I, SB-202190, PD-98059, or H89 when the percentage of cells containing SGs was counted. About 35% inhibition was observed with HDAC6 inhibitor Tubastatin A, whereas general HDAC inhibitor Trichostatin A provided a complete inhibition of SG formation. Our data point to the need of investigating the role of HDACs in SG formation during oxidative stress.

Electroacupuncture (EA), a traditional Chinese replacement therapy, is widely accepted to treat ischemic stroke. Increasing evidence show that autophagy is involved in the process of cerebral ischemia injury and the Wnt/GSK3β pathway, playing an important role in protecting central nervous system. In this study, rats were treated with EA prior to focal ischemia by middle cerebral artery occlusion (MCAO). Deficit score, infarct volumes and levels of autophagy markers, such as LC3I, LC3II and p62, were assessed with either PI3K inhibitor wortmannin or a GSK-3β inhibitor LiCl. Oxygen-glucose deprivation/re-oxygenation (OGD/R) was made in the primitive neuron in vitro, and was respectively treated with autophagy inhibitors 3-MA, LiCl, GSK3β siRNA, or mTOR inhibitor rapamycin. The results indicated that EA pretreatment increased the levels of autophagy marker LC3-II and reduced the levels of p62. Meanwhile, deficit outcome was improved, and infarct volumes were reduced by EA pretreatment. Furthermore, the beneficial effects of EA pretreatment were reversed by wortmannin. LiCl and GSK3β siRNA can mimic the neuroprotective effects of EA pretreatment by downregulating autophagy, and increasing protein levels of p-mTOR, p-GSK3β and β-catenin in OGD/R neurons. However, the protective effects of GSK3β siRNA were blocked by rapamycin. These results suggest that EA pretreatment induces tolerance to cerebral ischemia by inhibiting autophagy via the Wnt pathway through the inhibition of GSK3β.

T315I mutation found in chronic myelogenous leukemia (CML) and Ph + ALL patients is the most serious one among resistance against BCR/ABL kinase inhibitors including imatinib and is only responsive to ponatinib (PNT). However, the novel strategy is required to reduce life-threatening adverse effects of PNT including ischemic cardiovascular disease. We examined the mechanism of PNT-induced cytotoxicity against a T315I(+) Ph + ALL cell line, TccY/Sr. PNT induced apoptosis (increased sub G1 cells, and cleaved caspase3 and PARP), and suppressed protein expression of MCL1, cyclin D2 and c-myc, which were reversed by a proteasome inhibitor, MG132, suggesting enhanced proteasomal degradation by PNT. Among BCL2 family inhibitors, MCL1 inhibitors (maritoclax and AZD5991) robustly induced cell death, showing the MCL1-dependent survival of TccY/Sr cells. Decreased MCL1 and c-myc expression by PNT was also observed in T315I(+) MEGA2/STIR cells. PNT suppressed PI3K activation followed by AKT inhibition and GSK3 dephosphorylation. PI3K/AKT inhibitors mimicked PNT, suggesting that PI3K/AKT signaling is important for survival of TccY/Sr cells. Moreover, GSK3 inhibitor (SB216763) reduced PNT-induced cytotoxicity and degradation of c-myc and MCL1. AZD5991 exhibited the synergistic action with PNT, anti-cancer drugs and venetoclax (BCL2 inhibitor), suggesting the utility of MCL1 inhibitor alone or in combination as a future clinical option for Ph + leukemia patients.

Selenocyanates and diselenides are potential antitumor agents. Here we report two series of selenium derivatives related to selenocyanates and diselenides containing carboxylic, amide and imide moieties. These compounds were screened for their potency and selectivity against seven tumor cell lines and two non-malignant cell lines. Results showed that MCF-7 cells were especially sensitive to the treatment, with seven compounds presenting GI50 values below 10 μM. Notably, the carboxylic selenocyanate 8b and the cyclic imide 10a also displayed high selectivity for tumor cells. Treatment of MCF-7 cells with these compounds resulted in cell cycle arrest at S phase, increased levels of pJNK and pAMPK and caspase independent cell death. Autophagy inhibitors wortmannin and chloroquine partially prevented 8b and 10a induced cell death. Consistent with autophagy, increased Beclin1 and LC3-IIB and reduced SQSTM1/p62 levels were detected. Our results point to 8b and 10a as autophagic cell death inducers. The research leading to these results has received funding from 'La Caixa' Banking Foundation. P. Garnica wishes to express his gratitude to the Asociación de Amigos de la Universidad de Navarra for the pre-doctoral fellowship. Furthermore, the authors wish to express their gratitude to the Plan de Investigación de la Universidad de Navarra, PIUNA (Ref 2014–26 ) as well as Calgary Foundation for financial support for the project.

Chondrocyte death due to apoptosis is central for osteoarthritis (OA) pathogenesis. The family with sequence similarity 3A (FAM3A) is a mitochondrial protein that plays an important role for cellular adaptation to stress and cell survival. Yet, whether FAM3A is associated with chondrocyte apoptosis and OA pathogenesis remains uncharacterized. In this study, we found that FAM3A expression was downregulated in cartilage tissue from an experimental OA mouse model. Besides, FAM3A expression was also reduced in chondrocytes treated with interleukin-1β (IL-1β), an inflammatory cytokine that promotes cartilage degradation. Moreover, we discovered that FAM3A attenuated chondrocyte apoptosis induced by IL-1β treatment in vitro, suggesting a protective effect of FAM3A against chondrocyte apoptosis. Moreover, mechanistically, FAM3A activated PI3K/Akt/mTOR pathway in IL-1β-treated chondrocytes, and blockade of PI3K/Akt/mTOR pathway with specific inhibitors, wortmannin and LY294002, diminished FAM3A effect on IL-1β-induced chondrocyte apoptosis, hence demonstrating that FAM3A attenuates IL-1β-induced chondrocyte apoptosis through activating the pro-survival PI3K/Akt/mTOR pathway. In conclusion, our study may identify FAM3A as a potential regulator of chondrocyte apoptosis involved in OA pathogenesis.

Painful peripheral neuropathy is the most dose-limiting side effect of paclitaxel (PTX), a widely used anti-cancer drug to treat solid tumours. The understanding of the mechanisms involved in this side effect is crucial to the development of new therapeutic approaches. CXCL1 chemokine and its receptor CXCR2 have been pointed as promising targets to treat chronic pain. Herein, we sought to evaluate the possible involvement of CXCL1 and CXCR2 in the pathogenesis of PTX-induced neuropathic pain in mice. PTX treatment led to increased levels of CXCL1 in both dorsal root ganglion and spinal cord samples. Systemic treatment with the anti-CXCL1 antibody (10 μg/kg, i.v.) or the selective CXCR2 antagonist (SB225002, 3 mg/kg, i.p.) had minor effect on PTX-induced mechanical hypersensitivity. On the other hand, the intrathecal (i.t.) treatment with anti-CXCL1 (1 ng/site) or SB225002 (10 μg/site) consistently inhibited the nociceptive responses of PTX-treated mice. Similar results were obtained by inhibiting the PI3Kγ enzyme a downstream pathway of CXCL1/CXCR2 signalling with either the selective AS605240 (5 μg/site, i.t.) or the non-selective wortmannin PI3K inhibitor (0.4 μg/site, i.t.). Overall, the data indicates that the up-regulation of CXCL1 is important for the development and maintenance of PTX-induced neuropathic pain in mice. Therefore, the spinal blockage of CXCL1/CXCR2 signalling might be a new innovative therapeutic approach to treat this clinical side effect of PTX.

Despite multiple antitumor activities, interferon-alpha (IFNα) therapy alone is less effective in solid tumors. Autophagy has been reported to play a key role in tumor chemoresistance. Therefore, it is meaningful to explore whether autophagy can be activated by IFNα in head and neck squamous cell carcinoma (HNSCC) and serve as a potential target to improve efficacy of IFNα therapy. In this study, we report that IFNα not only exhibits anti-proliferation activity and induces apoptosis, but also activates autophagy in HNSCC cells. Moreover, silencing autophagy-related protein 5 (ATG5) and signal transducer and activator of transcription 1 (STAT1) suppresses autophagy flux. Furthermore, IFNα and autophagy inhibitors (hydroxychloroquine and wortmannin) show clear synergistic effects on inhibiting growth and promoting apoptosis in HNSCC cells and xenograft models. Our findings indicate that IFNα-induced autophagy plays a cytoprotective role and blocking autophagy flux promotes IFNα-mediated apoptosis in HNSCC. These results suggest that the combination of IFNα and autophagy inhibitors represents a novel strategy for HNSCC treatment.

Autophagy plays a key role in the stress response of tumor cells, which contributes to cancer cell survival and resistance to chemotherapy by degrading cytoplasmic proteins to provide energy and clear the hazardous substances. Therefore, combined treatment of chemotherapeutics and autophagy inhibitors is thought to obtain a desirable antitumor effect. Nanoparticles (NPs) show potential in tumor-targeting drug delivery because of the enhanced permeability and retention (EPR) effect. However, NPs with fixed particle size cannot achieve optimal delivery effect. Herein, a strategy based on Cu (I)-catalyzed click chemistry-triggered aggregation of azide/alkyne-modified micelles was developed for the co-delivery of the chemotherapeutic drug doxorubicin (Dox) and the autophagy inhibitor wortmannin (Wtmn). In vitro experiments showed that the size of micelles increased in a time-dependent manner, which enhanced micelle accumulation in both B16F10 and 4 T1 cells. The fluorescence resonance energy transfer (FRET) experiment and biodistribution study further demonstrated that the aggregation of micelles through click cycloaddition significantly improved the accumulation of drug-loading micelles at the tumor region. Furthermore, the decreased amount of autophagosomes observed by transmission electron microscopy (TEM), the declined expression of LC3-II, and the increased level of p62 by western blotting and immunohistochemistry (IHC) confirmed the obvious inhibition of autophagy induced by Dox/Wtmn co-loaded size-adjustable micelles, which had a synergistic effect in cancer suppression. In addition, the co-loaded size-adjustable micelles showed outstanding cytotoxicity and antitumor effect. Therefore, this strategy effectively suppressed melanoma and breast cancer in mice. Statement of Significance The therapeutic effects of chemotherapy can be limited by autophagy; hence, combined use of autophagy inhibitors with chemotherapeutics achieves desirable anticancer efficacy. In the present study, we designed size-adjustable micelles by modifying the click reaction substrate azide group and the alkyne group on the surface of micelles, and subsequently, the autophagy inhibitor wortmannin and the chemotherapeutic drug doxorubicin were co-loaded. The micelles could aggregate by click reaction at the tumor site when the catalysts were intratumorally injected. The results showed that the size-adjustable micelles achieved efficient drug delivery, penetration, and retention in tumors; through the combined effect of wortmannin-mediated autophagy inhibition and doxorubicin-mediated cytotoxicity, this strategy exerted significant anticancer effect in melanoma and breast cancer treatment.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein–lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro. We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

alpha-Melanocyte-stimulating hormone (-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that -MSH suppresses the transient outward A-type K+ current (I-A) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to -MSH concentration-dependently decreased I-A. This -MSH-induced I-A decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated I-A response. Blocking G(i/o)-protein signaling with pertussis toxin or by dialysis of TG neurons with the G-blocking synthetic peptide QEHA abolished the -MSH-mediated decrease in I-A. Further, -MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38 abrogated the -MSH-induced I-A response. Additionally, -MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by I-A blockade. Taken together, our findings suggest that -MSH suppresses I-A by activating MC4R, which is coupled sequentially to the G complex of the G(i/o)-protein and downstream class I PI3K-dependent p38 signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.

To comprehensively understand the endocytosis of Sapelovirus A (PSV) entry into PK-15 cells, we studied PSV infection in the context of cell perturbations through drug inhibition, siRNA silencing and overexpression of dominant negative (DN) mutants. We showed here that PSV infection of PK-15 cells was unaffected by pretreated with chlorpromazine, EIPA, knockdown of the clathrin heavy chain or overexpression of Eps15 DN mutant. Conversely, PSV infection was sensitive to NH4Cl, chloroquine, dynasore, nystatin, MβCD and wortmannin with reduced PSV VP1 expression levels and virus titer. Additionally, PSV invasion leaded to rapid actin rearrangement and disruption of the cellular actin network enhanced PSV infection. After internalization the virus was transported to late endosomes and/or cycling endosomes that requires the participation of Rab7 and Rab11. Our findings demonstrate that PSV uses caveolae-dependent endocytosis as the predominant entry portal into PK-15 cells which requires low pH, dynamin, Rab7 and Rab11.

Balancing microglia M1/M2 polarization has been shown as a prospective therapeutic strategy for Parkinson's disease (PD). Various vital signaling pathways are likely to govern the microglial phenotype. The implication of 5HT1A receptors in neurodegenerative disorders has raised interest in exploring the repositioning of flibanserin (Flib), a 5HT1A agonist, as an effective neuroprotective agent for PD. Therefore, this study was designed to assess the ability of Flib to modulate microglia phenotype switching from M1 to M2 via PI3K/AKT downstream targets in a rotenone model of PD. Rats received rotenone (1.5 mg/kg) every other day and were concurrently treated with Flib (40 mg/kg/day) with or without wortmannin (15 μg/kg/day), a PI3K inhibitor, for 21 days. Flib improved the motor perturbations induced by rotenone, as confirmed by the reversion of histopathological damage and tyrosine hydroxylase immunohistochemical alterations in both the striata and substantia nigra. The molecular signaling of Flib was elaborated by inducing striatal AKT phosphorylation and the expression of its substantial target, KLF4. Flib induced STAT6 phosphorylation to promote M2 polarization as demonstrated by the increased CD163

Dibutyl phthalate (DBP) is an endocrine disruptor that has been widely used in various products of human use. DBP exposure has been associated with reproductive and cardiovascular diseases and metabolic disorders. Although dysfunction of the vascular endothelium is responsible for many cardiovascular and metabolic diseases, little is known about the effects of DBP on human endothelium. In this study, we investigated the effect of three concentrations of DBP (10−6, 10−5, and 10−4 M) on angiogenesis in human endothelial cell (EC) line EA.hy926 after acute exposure. Tube formation assay was used to investigate in vitro angiogenesis, whereas qRT-PCR was employed to measure mRNA expression. The effect of DBP on extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt), and endothelial nitric oxide (NO) synthase (eNOS) activation was examined using Western blotting, whereas the Griess method was used to assess NO production. Results show that the 24-h-long exposure to 10−4 M DBP increased endothelial tube formation, which was prevented by addition of U0126 (ERK1/2 inhibitor), wortmannin (PI3K-Akt inhibitor), and L-NAME (NOS inhibitor). Short exposure to 10−4 M DBP (from 15 to 120 min) phosphorylated ERK1/2, Akt, and eNOS in different time points and increased NO production after 24 and 48 h of exposure. Application of nuclear estrogen receptor (ER) and G protein-coupled ER (GPER) inhibitors ICI 182,780 and G-15, respectively, abolished the DBP-mediated ERK1/2, Akt, and eNOS phosphorylation and increase in NO production. In this study, we report for the first time that DBP exerts a pro-angiogenic effect on human vascular ECs and describe the molecular mechanism involving ER- and GPER-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways.

Schistosomiasis is a disease of global importance caused by parasitic flatworms, schistosomes, which cause pathogenicity through eggs laid by the female worm inside the host's blood vessels. Maintenance of cellular homeostasis is crucial for parasites, as for other organisms, and is quite likely important for schistosome reproduction and vitality. We hypothesize a role for autophagy in these processes, an evolutionarily conserved and essential cellular degradation pathway. Here, for the first known time, we shed light on the autophagy machinery and its involvement in pairing-dependent processes, vitality and reproduction of Schistosoma mansoni. We identified autophagy genes by in silico analyses and determined the influence of in vitro culture on the transcriptional expression in male and female worms using quantitative real-time PCR. Among the identified autophagy genes were Beclin, Ambra1, Vps34, DRAM, DAP1, and LC3B, of which some showed a sex-dependent expression. Specifically, the death-associated protein DAP1 was significantly more highly expressed in females compared with males, while for the damage-regulated autophagy modulator DRAM it was the opposite. Furthermore, in-vitro culture significantly changed the transcript expression level of DAP1 in female worms. Next, worms were treated with an autophagy inducer (rapamycin) or inhibitors (bafilomycin A1, wortmannin and spautin-1) to evaluate effects on autophagy protein expression, worm vitality, and reproduction. The conversion of the key autophagy protein LC3B, a marker for autophagic activity, was increased by rapamycin and blocked by bafilomycin. All inhibitors affected worm fitness, egg production, and negatively affected the morphology of gonads and intestine. In summary, autophagy genes in S. mansoni show an interesting sex-dependent expression pattern and manipulation of autophagy in S. mansoni by inhibitors induced detrimental effects, which encourages subsequent studies to identify antischistosomal targets within the autophagy machinery.

Therapeutic failure and drug resistance are common sequelae to insulin resistance associated with type 2 diabetes mellitus (T2DM). Consequently, there is an unmet need of alternative strategies to overcome insulin resistance associated complications.To demonstrate whether Kutkin (KT), iridoid glycoside enriched fraction of Picrorhiza kurroa extract (PKE) has potential to increase the insulin sensitivity vis à vis glucose uptake in differentiated adipocytes.Molecular interaction of KT phytoconstituents, picroside-I (P-I)picroside- II (P-II) with peroxisome proliferator-activated receptor gamma (PPARγ), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were analyzed in silico. Cellular viability and adipogenesis were determined by following 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium bromide (MTT) assay and Oil Red-O staining. Further, ELISA kit based triglycerides and diacylglycerol-O-Acyltransferase-1 (DGAT1) were assessed in differentiated adipocytes. ELISA based determination were performed to check the levels of adiponectin and tumor necrosis factor alpha (TNF-α). However, Flow cytometry and immunofluorescence based assays were employed to measure the glucose uptake and glucose transporter 4 (glut4) expression in differentiated adipocytes, respectively. Further to explore the targeted signaling axis, mRNA expression levels of PPARγ, CCAAT/enhancer binding protein α (CEBPα), and glut4 were determined using qRT-PCR and insulin receptor substrate-1 (IRS-1), Insulin receptor substrate-2 (IRS-2), PI3K/Akt, AS160, glut4 followed by protein validation using immunoblotting in differentiated adipocytes.In silico analysis revealed the binding affinities of major constituents of KT (P-IP-II) with PPARγ/PI3K/Akt. The enhanced intracellular accumulation of triglycerides with concomitant activation of PPARγ and C/EBPα in KT treated differentiated adipocytes indicates augmentation of adipogenesis in a concentration-dependent manner. Additionally, at cellular level, KT upregulated the expression of DAGT1, and decreases fatty acid synthase (FAS), and lipoprotein lipase (LPL), further affirmed improvement in lipid milieu. It was also observed that KT upregulated the levels of adiponectin and reduced TNFα expression, thus improving the secretory functions of adipocytes along with enhanced insulin sensitivity. Furthermore, KT significantly promoted insulin mediated glucose uptake by increasing glut4 translocation to the membrane via PI3/Akt signaling cascade. The results were further validated using PI3K specific inhibitor, wortmannin and findings revealed that KT treatment significantly enhanced the expression and activation of p-PI3K/PI3K and p-Akt/Akt even in case of treatment with PI3K inhibitor wortmannin alone and co-treatment with KT in differentiated adipocytes and affirmed that KT as activator of PI3K/Akt axis in the presence of inhibitor as well.Collectively, KT fraction of PKE showed anti-diabetic effects by enhancing glucose uptake in differentiated adipocytes via activation of PI3K/Akt signaling cascade. Therefore, KT may be used as a promising novel natural therapeutic agent for managing T2DMand to the best of our knowledge, this is the first report, showing the efficacy and potential molecular mechanism of KT in enhancing insulin sensitivity and glucose uptake in differentiated adipocytes.

Aims The impaired angiogenesis is the major cause of diabetic delayed wound healing. The molecular insight remains unknown. Previous study has shown that high glucose (HG) activates Na+/H+ exchanger 1 (NHE1) and induces intracellular alkalinization, resulting in endothelial dysfunction. The aim of this study is to investigate whether activation of NHE1 in endothelial cells by HG damages the angiogenesis in vitro and in vivo. Methods and results We used western blot to detect the phosphorylations of both Akt and Girdin, and pH-sensitive BCECF fluorescence to assay NHE1 activity and pHi value, respectively. The angiogenesis was evaluated by measuring the number of tube formation in vitro, and blood perfusion by laser doppler and neovascularization by staining CD31 in vivo. Our results indicated that induction of intracellular acidosis (IA) increased p-Akt and p-Girdin in human umbilical vein endothelial cells (HUVEC). HG activated NHE1 and increased pHi value in a time-dependent manner, associated with the decreased phosphorylations of both Akt and Gridin, while inhibition of NHE1 by amiloride abolished the HG-induced reductions of p-Akt and p-Girdin. However, silence of Akt by siRNA transfection or pharmacological inhibitors (wortmannin and LY294002) bypassed IA-induced Girdin phosphorylation. Overexpression of constitutively active Akt abolished HG-reduced Girdin phosphorylation. In addition, upregulation of Akt or inhibition of NHE1 remarkably attenuated HG-impaired tube formation in HUVEC. In vivo study revealed that amiloride dramatically rescued hyperglycemia-delayed blood perfusion and neovascularization by augmenting ischemia-induced angiogenesis. Conclusion IA promotes ischemia-induced angiogenesis via Akt-dependent Girdin phosphorylation in diabetic mice.

Background and Aim Caloric restriction (CR) improves insulin sensitivity and is one of the dietetic strategies most commonly used to enlarge life and to prevent aging-induced cardiovascular alterations. The aim of this study was to analyze the possible beneficial effects of caloric restriction (CR) preventing the aging-induced insulin resistance in the heart of male Wistar rats. Methods and results Three experimental groups were used: 3 months old rats (3m), 24 months old rats (24m) and 24 months old rats subjected to 20% CR during their three last months of life (24m-CR). After sacrifice hearts were mounted in a perfusion system (Langendorff) and heart function in basal conditions and in response to accumulative doses of insulin (10−9-10−7 M), in the presence or absence of Wortmannin (10−6 M), was recorded. CR did not attenuate the aging-induced decrease in coronary artery vasodilation in response to insulin administration, but it prevented the aging-induced downregulation of cardiac contractility (dp/dt) through activation of the PI3K/Akt intracellular pathway. Insulin stimulated in a greater extent the PI3K/Akt pathway vs the activation of the MAPK pathway and increased the protein expression of IR, GLUT-4 and eNOS in the hearts of 3m and 24m-CR rats, but not in the hearts of 24m rats. Furthermore, CR prevented the aging induced increase in endothelin-1 protein expression in myocardial tissue. Conclusion In conclusion CR partially improves cardiac insulin sensitivity and prevents the aging induced decrease in myocardial contractility in response to insulin administration through activation of PI3K/Akt pathway.

Wortmannin, a known inhibitor of phosphoinositide 3-kinases (PI3Ks), their low selectivity and high toxicity is still problematic and less is known about their effects on PI3Ks in cellular systems. Hence, we have synthesized a series of multifunctional wortmannin probes with the ability to self-activate, by installing a clickable handle at C11 site, and secondary amine and cancer-targeting moiety at C20 site, respectively. MTT assay first confirmed that self-activating probes have better inhibition potency and biotin enhanced their cancer cell uptake. Further experiments showed most of probes can target PI3K/Akt/mTOR pathway with prolonged turn-over time. Protein profiling and pull-down results were observed that the derivatives not only labelled four PI3Ks with selectivity, but also engaged in covalent interactions with numerous cellular proteins which could be the major reason of their high toxicity.

Volatile anesthetics, including isoflurane, have been reported to have negative effects on cognitive dysfunction characterized by cognitive deficits following anesthesia. The aim of the current study was to investigate the effects involved with disintegrin and metallopeptidase domain 2 (ADAM2) silencing on isoflurane-induced cognitive dysfunction via the P13 K/Akt signaling pathway in immature rats. One week old healthy Sprague-Dawley (SD) rats were recruited and administered isoflurane anesthesia. The rats were then subjected to shADAM2 or wortmannin (PI3K/Akt signaling pathway inhibitor) to identify the effects of ADAM2 and the PI3K/Akt signaling pathway on the cognitive function of rats. Morris water maze and passive-avoidance tests were performed to examine the cognitive function of the rats. TUNEL staining was conducted to detect neuronal apoptosis in the hippocampal CA1 region. The obtained experimental results demonstrated that isoflurane anesthesia led to increased escape latency, reaction time, number of errors and TUNEL-positive neurons, along with a decreased latency time. In response to treatment with shADAM2, escape latency, reaction time, number of errors and TUNEL-positive cells were all noted to have decreased, in addition to elevated latency time, while contrasting trends were observed in regard to treatment with wortmannin. Taken together, the key findings of the present study revealed that shADAM2 activated the PI3K/Akt signaling pathway, resulting in elevated expressions of PI3K and Akt. Our study ultimately identified that ADAM2 silencing alleviates isoflurane-induced cognitive dysfunction by activating the P13 K/Akt signaling pathway in immature rats.

The effect of insulin priming on Il-10 expression, regulation of inflammatory cytokines and participation of intra-cellular signalling events, primarily ERK1/2 and PI3K/Akt, has been investigated in high glucose (HG) and/or lipopolysaccharide (LPS)-induced murine macrophages. Our results demonstrate that congruent with sharp increase in ERK1/2 and CREB phosphorylation, insulin stimulation in vitro promotes significant increase in Il-10 expression in mouse peritoneal macrophage and RAW 264.7 cells, both positive for anti-IRβ. Pharmacological inhibition of MEK/MAPK, but not PI3K/Akt cascade, abrogates CREB phosphorylation and Il-10 synthesis indicating functional relevance of insulin action. Conversely, priming with PI3K inhibitor wortmannin prevents insulin attenuation of HG- and/or LPS-induced p38 MAPK and NF-κB activation, Tnf-α, Il-1β expression as well as NO production. Congruent with reduced Il-10 expression, MEK inhibition abrogates insulin action allowing significant increase in Tlr4 expression and LPS response indicating insulin-induced Il-10 might have pivotal influence in regulation of chronic as well as acute inflammatory response.

Antioxidant therapy is considered as promising strategy for treating oxidative stress-induced cardiovascular disease. Bis (β-elemene-13-yl) glutarate (BEG) is a novel β-elemene derivative. Herein, we examined the antioxidant activity of BEG on human umbilical vein endothelial cells (HUVECs) after injury with hydrogen peroxide (H2O2) and investigated the mechanism involved. HUVECs were divided into the following groups: control group (untreated cells); treated groups (cells treated with 0.1, 1, 10 μmol/L of BEG); positive control group (cells treated with 0.1 mM Vitamin E); model group (cells treated with 0.5 mM H2O2 alone). Cells were pre-incubated with or without BEG for 24 h and then incubated for a further 2 h with 0.5 mM H2O2. Our results showed that BEG significantly reduced H2O2 induced loss in endothelial cell viability, reactive oxygen species (ROS) production, reduced lactate dehydrogenase (LDH) release, and malonyldialdehyde (MDA) level in a concentration-dependent manner. Also, BEG increased the cellular the superoxide dismutase (SOD) activity. Moreover, we found that H2O2 decreased Akt and eNOS phosphorylation, which perhaps, indirectly reduced nitric oxide (NO) production. These effects induced by H2O2, however, were reduced by pre-treatment with BEG. BEG effects were inhibited by a PI3K inhibitor (wortmannin) and eNOS inhibitor (L-NAME). In conclusion, the present study demonstrated that BEG has antioxidant activity. Furthermore, BEG reduced H2O2-induced endothelial cells injury by the involvement of antioxidation and PI3K/Akt/eNOS/NO signaling pathways.

BRS-3 has an important role in glucose homeostasis. Its expression was reduced in skeletal muscle from obese and/or diabetic patients, and BRS-3 KO-mice developed obesity. In this work, focused on rat/human adipose tissue, BRS-3 gene-expression was lower than normal-levels in hyperlipidemic, type-2-diabetic (T2D), and type-1-diabetic rats and also in obese (OB) and T2D patients. Moreover, BRS-3 protein levels were decreased in diabetic rat and in obese and diabetic human fat pieces; but neither mutation nor even polymorphism in the BRS-3-gene was found in OB or T2D patients. Interestingly, in rat and human adipocytes, without metabolic alterations, [D-Tyr6,β-Ala11,Phe13,Nle14]bombesin6-14 -BRS-3-agonist-, as insulin, enhanced BRS-3 gene/protein expression, increased, PKB, p70s6K, MAPKs and p90RSK1 phosphorylation-levels, and induced a concentration-related stimulation of glucose transport, GLUT-4 membrane translocation and lipogenesis, exclusively mediated by BRS-3, and abolished by wortmannin, PD98059 or rapamacyn. These results confirm that BRS-3 and/or its agonist are a potential therapeutic tool for obesity/diabetes.

Apoptosis is a cellular mechanism contributing to cardiac surgery using cardiopulmonary bypass (CPB)-induced lung injury. The ubiquitous PI3K/Akt pathway regulates proliferation, apoptosis and differentiation by controlling a broad range of target proteins including NF-κB and GSK-3β. The roles of the PI3K/Akt/NF-κB and PI3K/Akt/GSK-3β pathways in CPB-related lung injury are unclear.Seventy-two male Sprague-Dawley rats were assigned into sham, CPB, Wortmannin (Wtn) and insulin-like growth factor-I (IGF-I) groups (n = 18, each). Six subjects per group were evaluated at each of three time points: Prior to CPB (T1); opening of the left hilus pulmonis (T2); and 90 min after CPB (T3). Arterial blood specimens were obtained at each time point to test respiratory and oxygenation indices. Left lung tissues were processed for HE and TUNEL staining. Western blot was employed to evaluate protein levels and activities of Akt, phospho-Akt (p-Akt), GSK-3β, phospho-GSK-3β (p-GSK-3β) and nuclear NF-κB.Lung ischemia/reperfusion and CPB caused notable lung injury, as evidenced by lung functional decline and pathological deterioration, accompanied by increases in apoptosis and expression levels of p-Akt, p-GSK-3β and nuclear NF-κB in lungs (all P0.05 vs. Sham). At T3, Wtn-treated CPB subjects showed worsened lung function and pathological lung structures, as well as apoptosis in lungs (all P0.05 vs. CPB); additionally, Wtn inhibited Akt phosphorylation and slightly, but significantly increased expression of nuclear NF-κB (both P0.001 vs. CPB). Conversely, treatment of subjects with IGF-I increased Akt phosphorylation (P0.001 vs. CPB), inhibited expression of nuclear NF-κB (P = 0.008 vs. CPB), improved lung function and tissue morphology (both P0.05 vs. CPB), and reduced apoptosis in lungs (P0.001 vs. CPB). Neither Wtn nor IGF-I did alter GSK-3β phosphorylation levels (P = 0.836 and P = 0.669 vs. CPB, respectively).The PI3K/Akt/NF-κB pathway played a role in CPB-related lung injury, possibly through mediating apoptosis in lungs. GSK-3β, a signaling effector that also participated in CPB-induced apoptosis in lungs, but was not regulated by the PI3K/Akt pathway in this context.

Progesterone effects on vascular smooth muscle cell (VSMC) relaxation and the mechanism were investigated in cultured human umbilical vein VSMCs. Membrane progesterone receptors mPRα, mPRβ, and mPRγ were highly expressed in VSMCs, whereas nuclear progesterone receptor (nPR) had low expression. Progesterone (20 nM) and 02–0 (mPR-selective agonist), but not R5020 (nPR agonist), induced muscle relaxation in both a VSMC collagen gel disk contraction assay and an endothelium-denuded human umbilical artery ring tension assay. Progesterone and 02–0 increased ERK and Akt phosphorylation and decreased cAMP levels. These effects were blocked by preincubation with pertussis toxin. Progestin–induced muscle relaxation was blocked by pretreatment with mPRα, but not nPR, siRNAs, and by co-treatment with 8-Br-cAMP, AZD6244 (MAP kinase inhibitor), and wortmannin (PI3K inhibitor). Progestins reduced myosin light chain phosphorylation which was blocked with AZD6244 and wortmannin. These results demonstrate progesterone directly relaxes human VSMCs through mPRα/Gi and MAP kinase/ERK-, Akt/PI3K-, and cAMP-dependent pathways.

Neonatal injury-induced exaggeration of pain hypersensitivity after adult trauma is a significant clinical challenge. However, the underlying mechanisms remain poorly understood. Growing evidence shows that spinal Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) contributes to chronic pain in adult rodents. Here we demonstrated that the phosphorylation and expression of SHP2 in synaptosomal fraction of the spinal dorsal horn are elevated in adult rats subjected to neonatal and adult incisions (nIN-IN), and the upregulation of SHP2 is highly correlated with pain hypersensitivity. Intrathecal blockade of SHP2 phosphorylation using a SHP2 protein tyrosine phosphatase inhibitor NSC-87877, or knockdown of SHP2 by intrathecal delivery of small interfering RNA (siRNA), ameliorates mechanical allodynia and heat hyperalgesia in nIN-IN rats. Moreover, the expression of phosphatidylinositol 3-kinase (PI3K) in the spinal dorsal horn is significantly increased in nIN-IN rats. Intrathecal application of PI3K inhibitor, LY294002 or wortmannin, alleviates pain hypersensitivity in nIN-IN rats. Additionally, intrathecal administration of NSC-87877 or SHP2 siRNA attenuates the upregulation of PI3K. Finally, no alternation of SHP2 phosphorylation in the dorsal root ganglion and dorsal root of nIN-IN rats as well as PI3K expression in the dorsal root of nIN-IN rats intrathecally treated with NSC-87877 or SHP2 siRNA is observed. These results suggest that the phosphorylation and expression of SHP2 in the spinal dorsal horn play vital roles in neonatal incision-induced exaggeration of adult incisional pain via PI3K. Thus, SHP2 and PI3K may serve as potential therapeutic targets for exaggerated incisional pain induced by neonatal and adult injuries.

Milk fat globule membrane (MFGM) protein is known to have several health benefits, including an anti-sarcopenia effect; however, its mechanism is unclear. The aim of this study was to investigate the potential mechanism of action of the MFGM protein. The MFGM protein was extracted and separated into 4 fractions, and Fraction 2 (57% of total MFGM) demonstrated the greatest effect on C2C12 cell proliferation. Milk fat globule-EGF factor 8 (MFG-E8) accounted for 82.35% of the MFGM protein. The effects of whole Fraction 2 (100μg/mL, 200μg/mL and 300μg/mL) on cell proliferation and morphology were measured. Using qRT-PCR or a Western blot assay, several regulatory factors, e.g., PI3K P85α, p-pI3K p85α (Tyr 508), Akt, p-Akt (Ser 473), mTOR and p-mTOR (Ser 2448), were measured in cells incubated with 200μg/mL of Fraction 2 with or without wortmannin. The results demonstrated that Fraction 2 induced C2C12 cell proliferation in a dose-dependent manner, upregulated the mRNA expression of mTOR and p70S6K, and activated PI3K, Akt, mTOR and P70S6K phosphorylation; however, Fraction 2 inhibited FOXO3a and 4E-BP. The results demonstrate that the MFGM protein, predominantly MFG-E8, promotes cell proliferation through the PI3K/Akt/mTOR signaling pathway. This study elucidated the molecular mechanism of the MFGM protein, primarily MFG-E8, in promoting C2C12 cell proliferation via the PI3K/Akt/mTOR/P70S6K signal pathway.

Most ovarian cancer patients respond well to initial platinum-based chemotherapy. However, within a year, many patients experience disease recurrence with a platinum resistant phenotype that responds poorly to second line chemotherapies. As a result, new strategies to address platinum resistant ovarian cancer (PROC) are needed. Herein, we report that NP co-delivery of cisplatin (CP) and wortmannin (Wtmn), a DNA repair inhibitor, synergistically enhances chemoradiotherapy (CRT) and reverses CP resistance in PROC. We encapsulated this regimen in FDA approved poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) NPs to reduce systemic side effects, enhance cellular CP uptake, improve Wtmn stability, and increase therapeutic efficacy. Treatment of platinum-sensitive ovarian cancer (PSOC) and PROC murine models with these dual-drug loaded NPs (DNPs) significantly reduced tumor burden versus treatment with combinations of free drugs or single-drug loaded NPs (SNPs). These results support further investigation of this NP-based, synergistic drug regimen as a means to combat PROC in the clinic.

Membrane deterioration is an inherent aspect of the advancement in senescence and loss in fruit quality during storage. Postharvest technologies used for extending shelf life and quality are targeted to reduce membrane damage through downregulating or blocking ethylene action. In this study, mature green tomato fruit were treated with inhibitors of ethylene receptor (ETR), phosphatidylinositol 3-kinase (PI3K) and phospholipase D (PLD), all recognized to be targets of regulation of fruit ripening. The inhibitors used included 1-methylcyclopropene (1-MCP, an ethylene receptor blocker), wortmannin (an inhibitor of PI3K), and hexanal (a PLD inhibitor). Fruit were treated at optimal levels of the inhibitors and were stored at 21 °C for 10 days. Color development was strongly delayed in wortmannin treated tomatoes just as in 1-MCP treated fruit; while, changes in respiration, firmness and ethylene evolution were very similar to that of control fruit. Hexanal delayed the initiation of these changes; while 1-MCP and wortmannin blocked the ripening process. Changes in expression levels of key genes involved in ethylene signalling, phosphoinositide metabolism, and lycopene synthesis that occurred in response to inhibitors, suggested potential roles for PI3K and PLD in ethylene signalling. Furthermore, fruit treated with all the three inhibitors showed a marked reduction in PLD transcript levels; suggesting that, regulation of PLD gene expression is a common critical regulatory point that regulates ripening. Lowered PLD levels may reduce membrane lipid catabolism and the generation of phosphatidic acid (PA), an intermediate in ethylene signalling regulation through downstream components.

Prions are protein-based infectious agents that autocatalytically convert the cellular prion protein PrPC to its pathological isoform PrPSc. Subsequent aggregation and accumulation of PrPSc in nervous tissues causes several invariably fatal neurodegenerative diseases in humans and animals. Prions can infect recipient cells when packaged into endosome-derived nanoparticles called exosomes, which are present in biological fluids such as blood, urine, and saliva. Autophagy is a basic cellular degradation and recycling machinery that also affects exosomal processing, but whether autophagy controls release of prions in exosomes is unclear. Our work investigated the effect of autophagy modulation on exosomal release of prions and how this interplay affects cellular prion infection. Exosomes isolated from cultured murine central neuronal cells (CAD5) and peripheral neuronal cells (N2a) contained prions as shown by immunoblotting for PrPSc, prion-conversion activity, and cell culture infection. We observed that autophagy stimulation with the mTOR inhibitor rapamycin strongly inhibited exosomal prion release. In contrast, inhibition of autophagy by wortmannin or CRISPR/Cas9-mediated knockout of the autophagy protein Atg5 (autophagy-related 5) greatly increased the release of exosomes and exosome-associated prions. We also show that a difference in exosomal prion release between CAD5 and N2a cells is related to differences at the level of basal autophagy. Taken together, our results indicate that autophagy modulation can control lateral transfer of prions by interfering with their exosomal release. We describe a novel role of autophagy in the prion life cycle, an understanding that may provide useful targets for containing prion diseases.

As the incretin hormone, Glucose-dependent insulinotropic peptide (GIP) plays an important role in lipid metabolism, glucose uptake, and bone metabolism in mammals. To evaluate the roles of GIP in the teleost, the gip gene was isolated from the foregut of grass carp by RT-PCR. The ORF of grass carp gip was 327 bp and encodes 108 amino acids. And the mature peptide of grass carp was 31 amino acids. The tissue distribution results showed that gip mRNA was highly expressed in the gut, and was also at high levels in the spleen and brain region. In the oral glucose tolerance test (OGTT), the gip expression was significantly increased in foregut by glucose treatment for 1 and 3 h. The primary hepatocytes and intraperitoneal injection studies showed that the glut2, g6pase, pepck, acc1, fas, hsl, lpl expression were increased and the gk and cpt1 expression were decreased by GIP treatment. And the expression of pi3k, akt and pka were also increased by GIP treatment. In the primary hepatocytes, the effects of GIP on g6pase, glut2, lpl and cpt1 expression were relieved by Wortmannin (PI3K inhibitor), and the effects of GIP on pepck, gk and hsl expression were alleviated by H89•2HCl (PKA inhibitor). And glucose and triglyceride (TG) contents are promoted in primary hepatocytes by GIP treatment. In the intraperitoneal injection experiment, the serum glucose, total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C) contents were reduced, and serum TG and high-density lipoprotein cholesterol (HDL-C) contents were elevated after GIP injection. Our studies for the first time unveil the potential role of GIP as a regulatory factor of glucose and lipid metabolism in teleost.

As a new member of the Insulin-like growth factors (IGFs) family of proteins, Igf3 has been found to be involved in fish reproduction. To evaluate the functions of Igf3 in grass carp, the igf3 gene was isolated from the gonads via RT-PCR (reverse transcription polymerase chain reaction). The ORF of grass carp igf3 was found to be 606 bp long, encoding 201 amino acids. The grass carp Igf3 precursor contains five domains, termed B-C-A-D-E. Tissues distribution analysis showed that igf3 was widely expressed in multiple tissues, and is highly expressed in the gonads, cerebellum and hypothalamus of grass carp. The oral glucose tolerance test (OGTT) showed that igf3 expression levels increased in the hypothalamus, hepatopancreas and muscle after glucose treatment. The fasting/refeeding showed that igf3 expression in hepatopancreas and muscle were markedly inhibited in the fasting group and were significantly increased in refeeding group. A recombinant grass carp Igf3 protein was obtained using a prokaryotic expression system. The function of this recombinant Igf3 were assessed in vitro and in vivo experiments. The expression of igf1, igf2b, and ghr was significantly increased, and igf2a expression was markedly decreased by Igf3 treatment in vitro and in vivo. The expression of igf3 and igf1r was significantly increased by Igf3 treatment in vitro. The expression of igfbp1 decreased and the expression of igfbp2, igfbp3, akt and pi3k increased after Igf3 treatment in vivo and in vitro. The expression of igfbp5a and igfbp5b were also increased by Igf3 treatment in vitro. The effects of Igf3 treatment on the expression of igf1, igf2, and ghr were relieved after treatment with the PI3K inhibitor wortmannin in grass carp primary hepatocytes. To our knowledge, our study was the first to reveal that Igf3 regulates the expression of growth-related genes in teleosts.

Rationale The migration of osteoblastic cells to bone formation surface is an essential step for bone development and growth. However, whether the migration capacity of osteoblastic cells is compromised during osteoporosis occurrence and how it contributes to bone formation reduction remain unexplored so far. In this work, we found, as a positive regulator of cell migration, microtubule actin crosslinking factor 1 (MACF1) enhanced osteoblastic cells migration. We also examined whether MACF1 could facilitate osteoblastic cells' migration to bone formation surface to promote bone formation through another cytoskeleton protein, microtubule associated protein 1 (MAP1B). Methods Preosteoblast cell line MC3T3-E1 with different MACF1 level was used for in vitro and in vivo cell migration assay; Primary cortical bone derived mesenchymal stem cells (C-MSCs) from bone tissue of MACF1 conditional knock out (cKO) mice was used for in vitro cell migration assay. Cell migration ability in vitro was evaluated by wound healing assay and transwell assay and in vivo by bone marrow cavity injection. Small interfering RNA (siRNA) was used for knocking down Map1b in MC3T3-E1 cell. Lithium chloride (LiCl) and Wortmannin (Wort) were used for inhibiting/activating GSK3β pathway activity. Luciferase report assay was performed for detection of transcriptional activity of TCF7 for Map1b; Chromatin immunoprecipitation (ChIP) was engaged for the binding of TCF7 to Map1b promoter region. Results We found MACF1 enhanced MC3T3-E1 cell and C-MSCs migration in vitro through promoting microtubule (MT) stability and dynamics, and increased the injected MC3T3-E1 cell number on bone formation surface, which indicated a promoted bone formation. We further authenticated that MAP1B had a similar function to MACF1 and was regulated by MACF1 in osteogenic cell, and silencing map1b repressed MC3T3-E1 cell migration in vitro. Mechanistically, by adopting MC3T3-E1 cell with different MACF1 level or treated with LiCl/Wort, we discovered that MACF1 decreased the levels of 1265 threonine phosphorylated MAP1B (p[T1265] MAP1B) through inhibiting GSK3β activity. Additionally, total MAP1B mRNA expression level was upregulated by MACF1 through strengthening the binding of TCF7 to the map1b promoter sequence. Conclusion Our study uncovered a novel role of MACF1 in bone formation and MAP1B regulation, which suggested that MACF1 could be a potential therapeutic target for osteoporosis.

Background Bao Yuan Capsule (BYC) is a patented Chinese medicinal formula for health promotion but its application for ischemic stroke remains unknown. In this study, we proposed the hypothesis that BYC could promote neurogenesis and neurological functional recovery through promoting mitochondrial function and activating PI3K/Akt signaling pathway. Methods We firstly performed chemical identification studies by using QIT-TOF-MS technology. Then, we investigated the effects of BYC (1 g/kg, 2 g/kg, 4 g/kg per day) on improving the recovery of the neurological functions in transient middle cerebral artery occlusion (MCAO) ischemic mice. Results We tentatively characterized 36 compounds from the BYC extractions. At dosage of 4 g/kg, BYC effectively improved locomotor ability, attenuated anxiety-like behaviors, and enhanced the exploring behaviors, learning and memory capability in the transient MCAO ischemic mice. BYC treatment promoted neural stem cell differentiations in the subventricular zone (SVZ) and subgranular zone (SGZ) of the MCAO mice. BYC also up-regulated the expression of Aconitase 2 (ACO2), Succinate dehydrogenase complex, subunit A (SDHA), phosphorylation of AMP-activated protein kinase (p-AMPK), protein kinase B (p-Akt) and glycogen synthase kinase 3β (p-GSK3β) in the hippocampus of the MCAO mice. BYC (200 µg/ml) significantly improved the mitochondrial functions in cultured mouse multipotent neural stem like C17.2 cells. BYC treatment also promoted neuronal differentiations in the C17.2 cells under oxygen-glucose deprivation (OGD) condition. The neurogenetic effects were abolished by co-treatments of ATP synthesis inhibitor oligomycin and PI3K/Akt inhibitor wortmannin. Moreover, Akt phosphorylation was dramatically reduced by oligomycin. Conclusion BYC could promote neurogenesis and neurological functional recovery in post ischemic brains by regulating the mitochondrial functions and Akt signaling pathway.

Both renin–angiotensin systems and insulin participate in kidney-involved blood pressure regulation. Activation of angiotensin II type 2 receptor (AT 2 R) decreases sodium reabsorption in renal proximal tubule (RPT) cells, whereas insulin produces the opposite effect. We presume that AT 2 R has an inhibitory effect on insulin receptor expression in RPT cells, which may affect renal sodium transport and therefore be of physiological or pathological significance. Our present study found that activation of AT 2 R inhibited insulin receptor expression in a concentration and time-dependent manner in RPT cells from Wistar-Kyoto (WKY) rats. In the presence of a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19–31, 10 −6 mol/L) or a phosphatidylinositol 3 kinase inhibitor (wortmannin, 10 −6 mol/L), the inhibitory effect of AT 2 R on insulin receptor was blocked, indicating that both PKC and phosphatidylinositol 3 kinase were involved in the signaling pathway. There was a linkage between AT 2 R and insulin receptor which was determined by both laser confocal microscopy and coimmunoprecipitation. However, the effect of AT 2 R activation on insulin receptor expression was different in RPT cells from spontaneously hypertensive rats (SHRs). Being contrary to the effect in WKY RPT cells, AT 2 R stimulation increased insulin receptor in SHR RPT cells. Insulin (10 −7 mol/L, 15 minutes) enhanced Na + -K + -ATPase activity in both WKY and SHR RPT cells. Pretreatment with CGP42112 decreased the stimulatory effect of insulin on Na + -K + -ATPase activity in WKY RPT cells, whereas pretreatment with CGP42112 increased it in SHR RPT cells. It is suggested that activation of AT 2 R inhibits insulin receptor expression and function in RPT cells. The lost inhibitory effect of AT 2 R on insulin receptor expression may contribute to the pathophysiology of hypertension.

Hydrogen sulfide (H2S) serves as a neuromodulator and regulator of neuroinflammation. It is reported to be therapeutic for Parkinson's disease (PD) animal and cellular models. However, whether it affects α-synuclein accumulation in dopaminergic cells, the key pathological feature in PD, is poorly understood. In this study we reported that exogenous H2S donors NaHS and GYY4137 (GYY) enhanced the autophagy activity, as indicated by the increases of autophagy marker LC3-II expression and LC3 dots formation even during lysosome inhibition in dopaminergic cell lines and HEK293 cells. The enhancement of H2S donors on autophagic flux was mediated by adenosine 5′-monophosphate-activated protein kinase (AMPK)-dependent mammalian target of rapamycin (mTOR) inhibition, as H2S donors activated AMPK but reduced the mTOR activity and H2S donors-induced LC3-II increase was diminished by mTOR activator. Moreover, point mutation of Cys302 into alanine (C302A) in AMPKα2 subunit abolished the AMPK activation and mTOR inhibition, as well as autophagic flux increase elicited by NaHS. Interestingly, NaHS triggered AMPK S-sulfuration, which was not observed in AMPK C302A-transfected cells. Further, NaHS was able to attenuate α-synuclein accumulation in a cellular model induced by dopamine oxidized metabolite 3, 4-dihydroxyphenylacetaldehyde (DOPAL), and this effect was interfered by autophagy inhibitor wortmannin and also eliminated in AMPK Cys302A-transfected cells. In sum, the findings identified a role of Cys302 S-sulfuration in AMPK activation induced by exogenous H2S and demonstrated that H2S donors could enhance the autophagic flux via AMPK-mTOR signaling and thus reduce α-synuclein accumulation in vitro.

Background Studies of insulin-like growth factor 1 (IGF-1) as a novel therapy for the treatment of cardiovascular diseases have proven promising. However, elevated IGF-1 levels have also been associated with poor patient outcomes in heart failure with reduced ejection fraction. IGF-1 therapy has additionally been shown to not be beneficial in the percutaneous coronary intervention setting. Although IGF-1 activation of the PI3K/Akt and ERK1/2 pathways have been demonstrated as cardioprotective, other cellular mechanisms have not been fully investigated. Methods Neonatal rat cardiac myocytes (NCMs) and fibroblasts (NCFs) were isolated from 1 to 2-day old pups using enzymatic digestion. NCMs and NCFs were pre-treated with IGF binding protein 6, inhibitors for the PI3K/Akt Wortmannin, ERK1/2 U0126, Rho Associated Protein Kinase (ROCK) GSK576371, Apoptosis Signal-regulating Kinase-1 (ASK-1) G2261818A, and p38MAPK RWJ67657 pathways before stimulation with IGF-1 for 62 and 50 h, respectively. Cardiac myocyte hypertrophy and fibroblast collagen synthesis were determined by 3H-leucine and 3H-proline incorporation, respectively. Results IGF-1 dose-dependently stimulated NCM hypertrophy and NCF collagen synthesis. Treatment with IGFBP6 and the kinase inhibitors, Wortmannin, U0126, GSK576371, G2261818A and RWJ67657 significantly inhibited IGF-1 stimulated NCM hypertrophy and NCF collagen synthesis. Conclusion This study is the first to demonstrate that IGF-1 treatment in NCMs and NCFs activates the ROCK, ASK-1 and p38MAPK pathways. Future research may be guided by consideration of the PI3K/Akt and ERK1/2 pathways potentially increasing collagen synthesis, and the utilisation of a biased agonist to reduce activation of the ROCK, ASK-1 and p38MAPK pathways to maximise cardioprotective benefit whilst mitigating risks.

Diabetes mellitus affects ion channel physiology. We have previously reported that acute application of insulin suppresses the KCNQ1/KCNE1 currents that play an important role in terminating ventricular action potential. In this study, we investigated the effect of long-term insulin treatment on KCNQ1/KCNE1 currents using the Xenopus oocyte expression system. Insulin treatment with a duration longer than 6 h had an opposite effect to acute insulin application, that is, it augmented the KCNQ1/KCNE1 currents. Inhibitors of PI3K, wortmannin and LY294002, and a MEK inhibitor, U0126, abolished the potentiating effect of long-term insulin treatment. The long-term treatment with insulin had no effect on KCNQ1 currents indicating an essential role of KCNE1 in the insulin effect, which is similar to the acute insulin effect. Cycloheximide, an inhibitor of protein synthesis, and brefeldin A, an inhibitor of protein transport from endoplasmic reticulum, suppressed the long-term insulin effect. Western blotting analysis combined with these pharmacological data suggest that long-term insulin treatment augments KCNQ1/KCNE1 currents by increasing KCNE1 protein expression.

Status epilepticus (SE, a prolonged seizure activity) is a high risk factor of developing vasogenic edema, which leads to secondary complications following SE. In the present study, we investigated whether transient receptor potential canonical channel-3 (TRPC3) may link vascular endothelial growth factor (VEGF) pathway to NFκB/ETB receptor axis in the rat piriform cortex during vasogenic edema formation. Following SE, TRPC3 and ETB receptor independently activated phosphatidylinositol 3 kinase (PI3K)/AKT/eNOS signaling pathway. SN50 (a NFκB inhibitor) attenuated the up-regulations of eNOS, TRPC3 and ETB receptor expressions following SE, accompanied by reductions in PI3K/AKT phosphorylations. Inhibition of SE-induced VEGF over-expression by leptomycin B also abrogated PI3K and AKT phosphorylations, but not TRPC3 expression. Wortmannin (a PI3K inhibitor) and 3CAI (an AKT inhibitor) effectively inhibited up-regulation of eNOS expressions and vasogenic edema lesion following SE. These findings indicate that PI3K/AKT may be common down-stream molecules for TRPC3- and ETB receptor signaling pathways during vasogenic edema formation. In addition, the present data demonstrate for the first time that TRPC3 may integrate VEGF- and NFκB-mediated vasogenic edema formation following SE. Thus, we suggest that PI3K/AKT signaling pathway may be one of considerable therapeutic targets for vasogenic edema.

Background and aims Saffron is an antioxidant herbal derivative; however, its efficacy as a nutritional cardioprotective agent has not been fully elucidated. We investigated the cardioprotective properties of a standardized saffron aqueous extract (SFE) against ischemia/reperfusion (I/R) injury in Wild-Type (WT) and ApoE (−/−) mice and the underlying molecular mechanisms. Methods and results WT and ApoE (−/−) mice were subjected to 30 min I and 2 h R, with the following per os interventions for 4 weeks: 1) WT Control Group, receiving Water for Injection (WFI); 2) WT Crocus Group, receiving SFE at a dose of 60 mg/kg/day; 3) WT Crocus + Wort group, receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus 15 min before R; 4) ApoE (−/−) Control Group, receiving WFI; 5) ApoE (−/−) Crocus Group, receiving SFE at a dose of 60 mg/kg/day and 6) ApoE (−/−) Crocus + Wort: receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus, 15 min before R. Ischemic area/area at risk (I/R%) ratio was measured. Blood samples and ischemic myocardial tissue were collected at the 10th min of reperfusion for assessment of troponin I, malondialdehyde (MDA), nitrotyrosine (NT), p-eNOS, eNOS, p-Akt, Akt, p-p42/p-p44, p-GSK3β, GSK3β, IL-6, Nrf2, HO-1 and MnSOD expression. The effect of SFE on Nrf2 expression was also evaluated in vitro . SFE reduced infarct size in WT (16.15 ± 3.7% vs 41.57 ± 2.48%, ***p (−/−) mice (16.14 ± 1.47% vs 45.57 ± 1.73%, ***p (−/−) mice). Mice receiving SFE showed increased levels of eNOS, p-Akt, p-ERK1/2, p-44/p-42 and p-GSK3β-Ser9 and reduced expression of IL-6 and iNOS; furthermore, SFE reduced the levels of MDA and NT. SFE induced Nrf2 expression and its downstream targets, HO-1 and MnSOD in the myocardium of the treated animals, and induced Nrf2 expression in vitro in a dose-dependent manner. Conclusions SFE limits myocardial infarction in Wild-Type and ApoE (−/−) mice in a multifaceted manner including activation of Akt/eNOS/ERK1/2/GSK3-β and through Nrf2 pathway, bestowing antioxidant protection against I/R.

Chitosan has been investigated for its protective effect on nucleus pulposus (NP) cells against intervertebral disc degeneration(IDD), but its high molecular weight prohibits its clinical application. The low molecular derivatives, chitosan oligosaccharides (COS) are easier to absorb; however, the protective effect of COS against IDD remains unclear. In this study, we investigated the effects of COS on NP degeneration. NP cells derived from rats were treated with H2O2 to induce an IDD-like transition. Then, COS was used to pre-treat cells before administering H2O2 and changes occurring in the cells were observed. As shown by the result of a cell counting kit-8(CCK-8) assay, COS protected the viability of the cells. The induced apoptosis rate fell when cells were pre-treated with COS, revealed by annexin-V FITC/PI double staining analysis and Hoechst 33342 staining. COS administration also protected ECM synthesis and prevented its degradation, as shown by western blotting(WB) and polymerase chain reaction(PCR). We analyzed the activity of the PI3K/Akt pathway in H2O2 treated NP cells by WB and the result showed that COS could enhance activity of the pathway. To investigate the relationship between the PI3K/Akt pathway and the protective effects of COS on NP cells, the PI3K/Akt pathway was inhibited by wortmannin, and we subsequently found that this abolished the protective effects. These results support the hypothesis that COS exerts its protective effect on NP cells against H2O2-induced apoptosis via the PI3K/Akt pathway.

The present study investigates the role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes, NADPH oxidase and NO synthase, in the 1-methyl-3-octylimidazolium tetrafluoroborate ([omim][BF 4 ])-mediated toxic mode of action in mussel hemocytes. Specifically, cell viability (using the neutral red uptake assay) was primarily tested in hemocytes treated with different concentrations of [omim][BF 4 ] (0.1–10 mg L −1 ) and thereafter [omim][BF 4 ]-mediated oxidative (in terms of superoxide anions/O 2 - and nitric oxide/NO generation, as well as the enhancement of lipid peroxidation by-products, in terms of malondialdehyde/MDA) and genotoxic (in terms of DNA damage) effects were determined in hemocytes treated with 1 mg L −1 [omim][BF 4 ]. Moreover, in order to investigate, even indirectly and non-entirely specific, the role of PI3-kinase, NADPH oxidase and NO synthase, the [omim][BF 4 ]-mediated effects were also investigated in hemocytes pre-incubated with wortmannin (50 nM), diphenyleneiodonium chloride (DPI 10 μM) and N G -nitro- l -arginine methyl ester ( l -NAME 10 μM), respectively. The results showed that [omim][BF 4 ] ability to enhance O 2 - , NO, MDA and DNA damage, via its interaction with cellular membranes, was significantly attenuated in the presence of each inhibitor in almost all cases. The current findings revealed for the first time that certain signaling molecules, such as PI3-kinase, as well as respiratory burst enzymes activation, such as NADPH oxidase and NO synthase, could merely attribute to the [omim][BF 4 ]-mediated mode of action, thus enriching our knowledge for the molecular mechanisms of ILs toxicity.

The protection exerted by neutrophils against invading microbes is partially mediated via the generation of neutrophil extracellular traps (NETs). In sterile conditions NETs are damaging species, enriched in autoantigens and endowed with the ability to damage the vessel wall and bystander tissues, to promote thrombogenesis, and to impair wound healing. To identify and reposition agents that can be used to modulate the formation of NETs is a priority in the research agenda. Low molecular weight heparins (LMWH) are currently used, mostly on an empirical basis, in conditions in which NETs play a critical role, such as pregnancy complications associated to autoimmune disease. Here we report that LMWHs induce a profound change in the ability of human neutrophils to generate NETs and to mobilize the content of the primary granules in response to unrelated inflammatory stimuli, such as IL-8, PMA and HMGB1. Autophagy consistently accompanies NET generation in our system and autophagy inhibitors, 3-MA and wortmannin, prevent NET generation. Pretreatment with LMWH in vitro critically jeopardizes neutrophil ability to activate autophagy, a mechanism that might contribute to neutrophil unresponsiveness. Finally, we verified that treatment of healthy volunteers with a single prophylactic dose of parnaparin abrogated the ability of neutrophils to activate autophagy and to generate NETs. Together, these results support the contention that neutrophils, and NET generation in particular, might represent a preferential target of the anti-inflammatory action of LMWH.

Several nicotinic acetylcholine receptor (nAChR) subtypes are expressed in mitochondria to regulate the internal pathway of apoptosis in ion channel-independent manner. However, the mechanisms of nAChR activation in mitochondria and targeting to mitochondria are still unknown. Nicotine has been shown to favor nAChR pentamer assembly, folding, and maturation on the way of biosynthesis. The idea of the present work was to determine whether nicotine affects the content, glycosylation, and function of mitochondrial nAChRs. Experiments were performed in isolated liver mitochondria from mice, that either consumed or not nicotine with the drinking water (200 μL/L) for 7 days. Mitochondria detergent lysates were studied by sandwich or lectin ELISA for the presence and carbohydrate composition of different nAChR subunits. Intact mitochondria were examined by flow cytometry for the binding of fluorescently labeled α-cobratoxin and were tested in functional assay of cytochrome c release under the effect of either Ca 2+ or wortmannin in the presence or absence of nAChR-selective ligands, including PNU-282987 (1 nM), dihydro-β-erythroidine (DhβE, 1 μM), PNU-120596 (0.3, 3, or 10 μM) and desformylflustrabromine hydrochloride (dFBr, 0.001, 0.3, or 1 μM). It was found that nicotine consumption increased the ratio of mitochondrial vs non-mitochondrial nAChRs in the liver, enhanced fucosylation of mitochondrial nAChRs, but prevented the binding of α-cobratoxin and the cytochrome c release-attenuating effects of nAChR-specific agonists, antagonists, or positive allosteric modulators. It is concluded that nicotine consumption in vivo favors nAChR glycosylation and trafficking to mitochondria but makes them less susceptible to the effects of specific ligands.