The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Autophagy modulation changes mechano-chemical sensitivity of T24 bladder cancer cells.

Bladder cancer cells possess unique adaptive capabilities: shaped by their environment, cells face a complex chemical mixture of metabolites and xenobiotics accompanied by physiological mechanical cues. These responses might translate into resistance to chemotherapeutical regimens and can largely rely on autophagy. Considering molecules capable of rewiring tumor plasticity, compounds of natural origin promise to offer valuable options. Fungal derived metabolites, such as bafilomycin and wortmannin are widely acknowledged as autophagy inhibitors. Here, their potential to tune bladder cancer cells´ adaptability to chemical and physical stimuli was assessed. Additionally, dietary occurring mycotoxins were also investigated, namely deoxynivalenol (DON, 0.1-10 µM) and fusaric acid (FA, 0.1-1 mM). Endowing a Janus' face behavior, DON and FA are on the one side described as toxins with detrimental health effects. Concomitantly, they are also explored experimentally for selective pharmacological applications including anticancer activities. In non-cytotoxic concentrations, bafilomycin (BAFI, 1-10 nM) and wortmannin (WORT, 1 µM) modified cell morphology and reduced cancer cell migration. Application of shear stress and inhibition of mechano-gated PIEZO channels reduced cellular sensitivity to BAFI treatment (1 nM). Similarly, for FA (0.5 mM) PIEZO1 expression and inhibition largely aligned with the modulatory potential on cancer cells motility. Additionally, this study highlighted that the activity profile of compounds with similar cytotoxic potential (e.g. co-incubation DON with BAFI or FA with WORT) can diverge substantially in the regulation of cell mechanotransduction. Considering the interdependence between tumor progression and response to mechanical cues, these data promise to provide a novel viewpoint for the study of chemoresistance and associated pathways.

Nicorandil attenuates cisplatin-induced acute kidney injury in rats via activation of PI3K/AKT/mTOR signaling cascade and inhibition of autophagy.

Cisplatin is a highly effective antitumor agent, but its clinical use is limited due to critical adverse reactions including acute kidney injury (AKI). Nicorandil is an approved antianginal agent decreasing ischemia by potassium channel opening. The aim of this study was to investigate the nephroprotective effects of nicorandil and the possible role of activating PI3K/AKT/mTOR pathway in ameliorating cisplatin-induced AKI. Forty male Wistar rats were randomly allocated in 4 groups (n = 10). Group I: rats received the vehicle and served as control. Group II: rats received a single dose of cisplatin (7 mg/kg, i.p) on the 10th day of the experiment and served as AKI group. Group III: rats received cisplatin as in group II and nicorandil (3 mg/kg/day, p.o) for 14 days. Group IV: rats received cisplatin and nicorandil as in group III as well as wortmannin (15 µg/kg, i.v) for 14 days. Nicorandil exhibited obvious nephroprotective effects via the activation of PI3K/AKT/mTOR pathway. Moreover, nicorandil succeed to reduce the expression of the autophagy markers beclin-1 and LC-3II/I. In parallel, nicorandil showed anti-inflammatory and antiapoptotic effects via inhibition of NF-κB inflammatory pathway and depression of Bax/Bcl-2 ratio. Wortmannin, the PI3K inhibitor, was used to demonstrate the proposed pathway. Our study showed the nephroprotective effects of nicorandil in cisplatin-induced AKI in rats via activation of PI3K/AKT/mTOR signaling cascade, inhibition of autophagy, anti-inflammatory, anti-apoptotic, anti-oxidant activities. Thus, nicorandil could represent a promising renoprotective agent in cancer patients treated with cisplatin.

Sequestration of translation initiation factors in p62 condensates.

Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.

Endothelial Progenitor-Cell-Derived Exosomes Induced by Astragaloside IV Accelerate Type I Diabetic-wound Healing via the PI3K/AKT/mTOR Pathway in Rats.

OBJECTIVE: We explore the effects of endothelial progenitor cell (EPC)-derived exosomes (EPCexos) and of astragaloside IV (ASIV)-stimulated EPCexos (ASIV-EPCexos) on type I diabetic-wound healing, and determine the basic molecular mechanisms of action. METHODS: EPCs were exposed to different concentrations of ASIV to generate ASIV-EPCexos. A chronic-wound healing model involving streptozotocin-stimulated diabetic rats was established. These rats were treated with EPCexos, ASIV-EPCexos, rapamycin, and wortmannin. Wound healing was evaluated by direct photographic observation, hematoxylin and eosin staining, and Masson's trichrome staining. RESULTS: ASIV treatment increased the abilities of EPCs (e.g., proliferation), as well as exosome secretion. EPCexo showed a "cup holder" like structure. Treatment with ASIV-EPCexos increased the wound-healing rate, collagen-deposition area, bromodeoxyuridine uptake, VEGF expression, and the number of CD31- and αSMA- positive cells, whereas decreased epidermal thickness and CD45 expression. The expression of the PI3K/AKT/mTOR pathway increased, whereas the expression of inflammatory factor decreased. However, rapamycin and wortmannin reversed these changes. CONCLUSIONS: ASIV-EPCexos may accelerate type I diabetic-wound healing via the PI3K/AKT/mTOR pathway. This study may lay the foundation for new clinical treatment options for patients with type I diabetic wounds.

Autophagic activation in porcine oocytes is independent of meiotic progression.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

Mineralocorticoid receptor agonist aldosterone rescues hippocampal neural stem cell proliferation defects and improves postoperative cognitive function in aged mice.

OBJECTIVES: Hippocampal neurogenesis is closely related to learning and memory, and hippocampal neurogenesis disorders are involved in the development of many neurodegenerative diseases. Mineralocorticoid receptor (MR) plays a vital role in regulating stress response, neuroendocrine and cognitive functions, and is involved in regulating the integrity and stability of neural networks. However, the potential role of MR in the pathogenesis of postoperative cognitive dysfunction (POCD) is unclear. Therefore, this study evaluated the effect and mechanism of MR activation on postoperative hippocampal neurogenesis and cognitive function in aged mice. METHODS: 18-month-old male Kunming mice were randomly divided into Control group (C group), Surgery group (S group), Surgery+ Aldosterone group (S+Aldo group), Surgery + Wortmannin group (S+Wort group), Surgery + Aldosterone + Wortmannin group (S+Aldo+Wort group). Laparotomy was used to establish an animal model of postoperative cognitive dysfunction. After surgery, mice were intraperitoneally injected with aldosterone (100 ug/kg,150 ug/kg,200 ug/kg) and / or wortmannin (1 mg/kg); One day before the sacrifice, mice were injected intraperitoneally with BrdU (100 mg / kg / time, 3 times in total). Mice were subjected to Morris water maze and field tests at 1, 3, 7, and 14 days after surgery. Immunofluorescence was used to detect the number of BrdU +, Nestin +, BrdU/Nestin + positive cells in the hippocampal dentate gyrus of mice at 1, 3, 7 and 14 days after surgery. Western-blot was used to detect PI3K/Akt/GSK-3ß signaling pathway related proteins Akt, p-Akt, GSK-3ß, P-GSK-3ß expression. RESULTS: Stress impairs the performance of aged mice in water maze and open field tests, reduces the number of BrdU/Nestin+ cells in the hippocampal dentate gyrus, and inhibits the phosphorylation of Akt and GSK-3ß proteins in the hippocampus. Aldosterone treatment promotes P-Akt, P-GSK-3ß protein expression and hippocampal neural stem cell proliferation, and improves postoperative cognitive dysfunction. However, wortmannin treatment significantly reversed these effects of aldosterone. CONCLUSIONS: The mineralocorticoid receptor agonist aldosterone promotes the proliferation of hippocampal neural stem cells and improves cognitive dysfunction in aged mice after surgery, and the mechanism may be related to activation of PI3K/Akt/GSK-3ß signaling.

Quantitative and temporal measurement of dynamic autophagy rates.

Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process. Established methods to quantify flux use steady-state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady-state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 min, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannin's inhibition of autophagy, which is further accelerated by rapamycin. Furthermore, we applied this approach to study the effect of serum and glutamine starvation on autophagy. Serum starvation led to a rapid and transient increase in all the rates. Glutamine starvation led to a decrease in the rates on a longer timescale. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.

Limonin, a Component of Immature Citrus Fruits, Activates Anagen Signaling in Dermal Papilla Cells.

Hair loss remains a significant problem that is difficult to treat; therefore, there is a need to identify safe natural materials that can help patients with hair loss. We evaluated the hair anagen activation effects of limonin, which is abundant in immature citrus fruits. Limonin increased the proliferation of rat dermal papilla cells (rDPC) by changing the levels of cyclin D1 and p27, and increasing the number of BrdU-positive cells. Limonin increased autophagy by decreasing phosphorylated mammalian target of rapamycin levels and increasing the phospho-Raptor, ATG7 and LC3B. Limonin also activated the Wnt/ß-catenin pathway by increasing phospho-ß-catenin levels. XAV939, a Wnt/ß-catenin inhibitor, inhibited these limonin-induced changes, including induced autophagy, BrdU-positive cells, and cell proliferation. Limonin increased the phosphorylated AKT levels in both two-dimensional cultured rDPC and three-dimensional spheroids. Treatment with the PI3K inhibitor wortmannin inhibited limonin-induced proliferation, and disrupted other limonin-mediated changes, including decreased p27, increased BrdU-positive cells, induced autophagy, and increased ATG7 and LC3B levels. Wortmannin also inhibited limonin-induced cyclin D1 and LC3 expression in spheroids. Collectively, these results indicate that limonin can enhance anagen signaling by activating autophagy via targeting the Wnt/ß-catenin and/or PI3K/AKT pathways in rDPC, highlighting a candidate nutrient for hair loss treatment.

Ang II acutely stimulates Na,K-pump in cells from proximal tubules by increasing its phosphorylation at S938 via a PI3K/AKT pathway.

Angiotensin II (Ang II)-dependent stimulation of the AT1 receptor in proximal tubules increases sodium reabsorption and blood pressure. Reabsorption is driven by the Na,K-pump that is acutely stimulated by Ang II, which requires phosphorylation of serine-938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II-dependent phosphorylation of S938. We tested the hypothesis that Ang II-dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT-dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co-expressing the AT1 receptor and either the wild-type (α-1.wild-type) or an alanine substituted (α-1.S938A) form of rat kidney Na,K-pump. A 5-min exposure to 10 pM Ang II significantly activated Na,K-pump activity (56%) measured as short-circuit current across polarized α-1.wild-type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II-dependent activation. Ang II did not stimulate Na,K-pump activity in α-1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α-1.wild-type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H-89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H-89. These data support our hypothesis and show that Ang II-dependent phosphorylation at S938 stimulates Na,K-pump activity and transcellular sodium transport.

Crosstalk between PI3K/AKT/KLF4 signaling and microglia M1/M2 polarization as a novel mechanistic approach towards flibanserin repositioning in parkinson's disease.

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++ microglial count with striatal arginase activity. In parallel, it markedly inhibited M1 activation as evidenced by the reduction in CD86++ microglia count with striatal proinflammatory mediators, IL-1ß and iNOS. The pre-administration of wortmannin mostly negated Flib's neuroprotective effects. In conclusion, Flib AKT/ KLF4-dependently amended M1/M2 microglial imbalance to exert a promising neuroprotective effect, highlighting its potential as a revolutionary candidate for conquering PD.

Dibutyl phthalate promotes angiogenesis in EA.hy926 cells through estrogen receptor-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways.

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.

Inhibition of NLRP1-Dependent Pyroptosis Prevents Glycogen Synthase Kinase-3ß Overactivation-Induced Hyperphosphorylated Tau in Rats.

Our previous study indicated that inhibition of NLRP1-dependent pyroptosis could decrease intracerebroventricular (ICV) injection of a protein kinase A (PKA) agonist- or streptozotocin (STZ)-induced hyperphosphorylated tau. In this study, we used a glycogen synthase kinase-3ß (GSK-3ß) overactivation rat model to reconfirm our previous results. ICV injection of wortmannin (WT, a PI3K inhibitor) and GF-109203X (GFX, a PKC inhibitor) was used to induce overactivation of GSK-3ß in rats. We injected NLRP1 siRNA together with WT/GFX to evaluate the effect of the inhibition of NLRP1-dependent neuronal pyroptosis on hyperphosphorylated tau. Our results indicated that ICV injection of NLRP1 siRNA prevented ICV-WT/GFX-induced neuronal death, further improving the spatial memory of the rats in the Morris water maze test. ICV injection of NLRP1 siRNA downregulated the expression of ASC, caspase-1, and GSDMD and the contents of IL-1ß and IL-18 in rat brains. ICV injection of NLRP1 siRNA also decreased hyperphosphorylated tau and the activity of GSK-3ß. Thus, these results support our previous study that NLRP1-dependent pyroptosis could enhance hyperphosphorylation of tau protein.

VEGF-A promotes the motility of human melanoma cells through the VEGFR1-PI3K/Akt signaling pathway.

Vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1 and R2) play important roles in the progression of malignant melanoma through tumor angiogenesis. However, it is not clear whether the VEGF-A/VEGFR1 signaling pathway is involved in the proliferation and migration of melanoma cells. Thus, the effect of VEGF-A on cell migration was investigated in human melanoma cell lines. Of several splicing variants of VEGF-A, VEGF165 is the most abundant and responsible for VEGF-A biological potency. VEGF165 facilitated the migration of melanoma cells in both a chemotactic and chemokinetic manner, but cell proliferation was not affected by VEGF165. VEGF165 also induced the phosphorylation of Akt. In addition, VEGF165-induced cell migration was inhibited significantly by VEGFR1/2 or a VEGFR1-neutralizing antibody. Furthermore, the downregulation of VEGFR1 via the transfection of VEGFR1-targeting antisense oligonucleotides suppressed VEGF165-induced cell migration. Moreover, wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI3K) in the PI3K/Akt pathway, suppressed VEGF165-induced Akt phosphorylation and VEGF165-induced cell migration. These findings suggest that the motility of melanoma cells is regulated by signals mediated through the PI3K/Akt kinase pathway with the activation of VEGFR1 tyrosine kinase by VEGF165. Thus, the downregulation of signaling via VEGF-A/VEGFR1 might be an effective therapeutic approach that could prevent the progression of malignant melanoma.

Signaling Cascade Mediating the Effect of FTY720P on the Na+/K+ ATPase in LLC-PK1.

BACKGROUND/AIMS: In renal ischemia, the Na+/K+ ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions. Sphingosine-1-phosphate (S1P) was shown to play a protective role against this ischemic injury. Whether the sphingolipid targets the Na+/K+ ATPase is a possibility that has not been explored before. This work aims at investigating the effect of S1P on renal Na+/K+ ATPase using its analogue FTY720P and LLC-PK1 cells. METHODS: The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme while its protein expression was studied by western blot analysis. RESULTS: FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and a dose of 80 nM. The protein expression was also increased. The stimulation of the Na+/K+ ATPase disappeared completely in presence of JTE-013, a specific blocker of S1PR2, as well as in presence of Y-27632, a Rho kinase inhibitor, BAPTA-AM, a Ca2+ chelator, wortmannin, a PI3K inhibitor, carboxy-PTIO, a scavenger for nitric oxide (NO), and KT 5823, a PKG inhibitor. CYM 5520, a S1PR2 agonist mimicked the effect of FTY720P. FTY720P increased the expression of p-Akt, a direct effector of PI3K, however, this increase disappeared when Rho kinase was inhibited, revealing that Rho kinase acts upstream PI3K. Glyco-SNAP-1, a NO donor, activated the pump in both presence and absence of wortmannin, indicating that PI3K is upstream NO. Interestingly, glyco-SNAP-1 and 8-bromo-cGMP, a PKG activator, exerted no effect on the Na+/K+ ATPase in absence of free Ca2+ revealing that the NO mediated effect is calcium-dependent. The involvement of calcium was further confirmed by the translocation of NFAT to the nucleus. The presence of verapamil or extracellular EGTA abolished the stimulatory effect of FTY720P, indicating that the source of calcium is extracellular. CONCLUSION: The results suggest that FTY720P activates sequentially S1PR2, Rho kinase, PI3K, leading to NO release and PKG stimulation. The latter phosphorylates calcium channels in the cell membrane, leading to calcium influx, and translocation of the ATPase units to the membrane.

Sevoflurane Postconditioning Inhibits Pulmonary Apoptosis via PI3K/AKT in Dog Cardiopulmonary Bypass Model.

AIMS: The study aimed to investigate the protective effects and regulatory mechanism of sevoflurane postconditioning (SPC) in pulmonary apoptosis induced by cardiopulmonary bypass (CPB). METHODS: Twenty-four healthy dogs were divided into a control (C group), ischemia/reperfusion (I/R group), sevoflurane postconditioning (S group), and wortmannin group (S+W group). At 10 min after the establishment of CPB, the left pulmonary artery was blocked. When the pulmonary artery was reopened, 2% sevoflurane was administered. Wortmannin was delivered 10 min before the pulmonary artery was open. Before thoracotomy was implemented (T1), when the artery was reopened (T2) and 2 h after CPB (T3), blood and the inferior lobe of the left lung were isolated and subjected to gas analysis, pathological examination, western blot, and TUNEL staining. RESULTS: No obvious changes were observed in the C group throughout the experiment. The conditions of all treated groups progressively deteriorated, and no difference could be found except in the number of apoptotic cells of T3 between the S+W and I/R groups. At T2, the treated groups showed similar conditions. At T3, the lung function and structure of the S group were improved in I/R and S+W groups. The S group showed the highest p-Akt expression, the lowest cleaved-caspase 3 expression, and apoptotic cell percentage. CONCLUSIONS: Ischemia-reperfusion of the lung during CPB reduces lung function and injures the pulmonary structure via inducing lung apoptosis. Sevoflurane postconditioning preserves lung function and structure by alleviating apoptosis via activation of PI3K/Akt.

PI3K/Akt pathway mediates the positive inotropic effects of insulin in Langendorff-perfused rat hearts.

Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs-Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.

Kutkin, iridoid glycosides enriched fraction of Picrorrhiza kurroa promotes insulin sensitivity and enhances glucose uptake by activating PI3K/Akt signaling in 3T3-L1 adipocytes.

BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: In silico analysis revealed the binding affinities of major constituents of KT (P-I& P-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. CONCLUSION: 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.

Targeting TROY-mediated P85a/AKT/TBX3 signaling attenuates tumor stemness and elevates treatment response in hepatocellular carcinoma.

BACKGROUND: Previous in vitro hepatocyte differentiation model showed that TROY was specifically expressed in liver progenitor cells and a small proportion of hepatocellular carcinoma cells, suggesting that TROY may participate in hepatocellular carcinoma (HCC) stemness regulation. Here, we aim to investigate the role and mechanism of TROY in HCC pathogenesis. METHOD: Bioinformatics analysis of the TCGA dataset has been used to identify the function and mechanism of TROY. Spheroid, apoptosis, and ALDH assay were performed to evaluate the stemness functions. Validation of the downstream pathway was based on Western blot, co-immunoprecipitation, and double immunofluorescence. RESULTS: HCC tissue microarray study found that a high frequency of TROY-positive cells was detected in 53/130 (40.8%) of HCC cases, which was significantly associated with poor prognosis and tumor metastasis. Functional studies revealed that TROY could promote self-renewal, drug resistance, tumorigenicity, and metastasis of HCC cells. Mechanism study found that TROY could interact with PI3K subunit p85α, inducing its polyubiquitylation and degradation. The degradation of p85α subsequently activate PI3K/AKT/TBX3 signaling and upregulated pluripotent genes expression including SOX2, NANOG, and OCT4, and promoted EMT in HCC cells. Interestingly, immune cell infiltration analysis found that upregulation of TROY in HCC tissues was induced by TGF-ß1 secreted from CAFs. PI3K inhibitor wortmannin could effectively impair tumor stemness to sorafenib. CONCLUSION: We demonstrated that TROY is an HCC CSC marker and plays an important role in HCC stemness regulation. Targeting TROY-positive CSCs with PI3K inhibitor wortmannin combined with chemo- or targeted drugs might be a novel therapeutic strategy for HCC patients.

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels.

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of -70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near -3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 µM), Gd3+ (10 µM), SKF-96365 (50 µM) and 2-aminoethoxydiphenyl borane (2-APB; 100 µM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-ß-cyclodextrin (MßCD; 1-6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 µM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MßCD (3 mg/ml), wortmannin (20 µM) and La3+ (10 µM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 µM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 µM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.