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Traditional Chinese medicine has growing importance in the treatment of ischemia stroke due to its abundance and low drug resistance. In this study, we aim to investigate the therapeutic potential of daucosterol palmitate against ischemia stroke, as well as its neuro-protective mechanism. The dose-response effects of daucosterol palmitate in the protection from brain damage were evaluated in a cerebral ischemia/reperfusion (I/R) rat model. The correlation of neuro-protective effects of daucosterol palmitate with apoptosis inhibition was examined and the possible signaling targets were identified. Our findings revealed that daucosterol palmitate treatment after 2 h' ischemia significantly lowered brain damage, and neuronal cell apoptosis caused by I/R injury in a dose-response mode (20, 40 and 80 mg/kg). Western blot analysis indicated that daucosterol palmitate could reverse the effects of I/R injury on protein expression of PI3K and mTOR, and phosphorylation of Akt. Contrarily, inactivation of PI3K using wortmannin dramatically antagonized the effect of daucosterol palmitate for I/R injury. With these findings, it supports the application potential of daucosterol palmitate in the treatment of ischemia stroke. Besides, the PI3K/Akt/mTOR pathway might be potential cellular targets for daucosterol palmitate.

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.

Background/aims Ischemic stroke is the main cause of nerve damage and brain dysfunction, accompanied by strong brain cell apoptosis. This study aimed to investigate the effect of kaempferol-3-O-rhamnoside (K-3-rh) on cerebral ischemia-reperfusion (I/R) injury. Methods and materials A rat model of cerebral I/R injury was established. The effects of K-3-rh on cerebral infarction size, brain water content and neurological deficits in rats were evaluated. Apoptosis of ischemic brain cells after mouse I/R was observed by TUNEL staining and flow cytometry. Western blot and qRT-PCR were used to detect the effect of K-3-rh on the expression of apoptosis-related proteins. Results K-3-rh can improve the neurological deficit score, reduce the infarct volume and brain water content, and inhibit cell apoptosis. In addition, K-3-rh significantly downregulated the expression of Bax and p53 and upregulated the expression of Bcl-2, and the phosphorylation level of Akt. Blockade of PI3K activity by the PI3K inhibitor wortmannin not only reversed the effects of K-3-rh on infarct volume and brain water content but also reversed the expression level of p-Akt. Conclusion K-3-rh had obvious neuroprotective effects on brain I/R injury and neuronal apoptosis, and its mechanism may be related to activation of PI3K/Akt signaling pathway.

Thioredoxin (Trx) is a hydrogen acceptor of ribonucleotide reductase and a regulator of some enzymes and receptors. It has been previously shown that significantly elevated levels of Trx expression are associated with the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but it is not clear how Trx regulates the effects of hydrogen peroxide (H2O2) on myogenic differentiation of BMSCs. Here, we report that rat BMSCs treated with a high dose (150 µm) of H2O2 exhibited a significant reduction in viability, cell cycling, and superoxide dismutase and glutathione peroxidase levels, and an increase in reactive oxygen species and malondialdehyde levels, which was accompanied by reductions in protein kinase B activation and forkhead Box O1, myogenic differentiation 1 and myogenin expression during myogenic differentiation. Furthermore, treatment with recombinant human Trx significantly mitigated the effects of H2O2 on the myogenic differentiation of BMSCs, and this was abrogated by cotreatment with wortmannin [a specific phosphatidylinositol 3‐kinase inhibitor]. In summary, our results suggest that treatment with recombinant human Trx mitigates H2O2‐induced oxidative stress and may promote myogenic differentiation of rat BMSCs by enhancing phosphatidylinositol 3‐kinase/protein kinase B/forkhead Box O1 signaling., Hydrogen peroxide can cause significant peroxidative damage to and inhibit the proliferation and myogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In this study, we found that recombinant human Trx significantly mitigated hydrogen peroxide‐induced oxidative stress and may promote myogenic differentiation of rat BMSCs by enhancing phosphatidylinositol 3‐kinase/protein kinase B/forkhead Box O1 signaling, which may provide an experimental basis for the BMSC‐based treatment of sarcopenia.

Cardiomyocyte autophagy plays an important role in myocardial ischemia-reperfusion injury (MIRI). P300/CBP-associated factor (PCAF) was involved in the regulation of autophagy. However, the role of PCAF in MIRI is currently unknown. This study was to investigate whether downregulation of PCAF attenuate MIRI. The results showed that the expression of PCAF was significantly increased in MIRI in vivo and in vitro. Downregulation of PCAF not only inhibited autophagy and damage of H9c2 cells induced by hypoxia-reoxygenation, but also reduced autophagy and myocardial infarct size during myocardial ischemia-reperfusion in rats. In addition, downregulation of PCAF promoted activation of PI3K/Akt/mTOR signaling pathway in cardiomyocytes during hypoxia-reoxygenation. Wortmannin, a PI3K/Akt inhibitor, could abrogate the effects of downregulation of PCAF on cardiomyocytes autophagy. These results demonstrated that downregulation of PCAF alleviated MIRI by inhibiting cardiomyocyte autophagy through PI3K/Akt/mTOR signaling pathway. Thus, PCAF may be a potential target for prevention and treatment of MIRI.

A decrease in oxygen concentration is a hallmark of inflammatory reactions resulting from infection or homeostasis disorders. Mast cells interact with extracellular matrix and other cells by adhesion receptors. We investigated the effect of hypoxia on integrin-mediated mast cell adhesion to fibronectin. We found that it was mediated by the α5/β1 receptor and that hypoxia significantly upregulated this process. Hypoxia-mediated increases in mast cell adhesion occurred without increased surface expression of integrins, suggesting regulation by inside-out integrin signaling. Hypoxia also mediated an increase in phosphorylation of Akt, and PI3’kinase inhibitors abolished hypoxia-mediated mast cell adhesion. Hypoxia upregulates the function of integrin receptors by PI3’ kinase-dependent signaling. This process might be important for the location of mast cells at inflammatory sites

The PI3K‐AKT signalling pathway is a crucial part of the signal transduction network and is involved in many important physiological processes and the stress response to environmental alteration. In this study, gill and kidney transcriptome data demonstrated that the PI3K‐AKT signalling pathway plays an important role in the stress response of the turbot Scophthalmus maximus when exposed to low salinity. Low‐salinity stress led to down‐regulation of gene expression, protein content and phosphorylation of AKT1 at Thr308 of the PI3K‐AKT signalling pathway. However, the abundance and phosphorylation of AKT1 protein at Ser473 and Thr308 were detected only in the gill and not in the kidney. Inhibition of the PI3K‐AKT signalling pathway mediated by wortmannin showed that wortmannin remarkably inhibited PI3K gene expression and protein synthesis as well as phosphorylation of AKT1 at Thr308 with or without low‐salinity stress. In the context of pathway inhibition, suppressed expression levels of ion channel genes demonstrated that the PI3K‐AKT signalling pathway acts on osmoregulation by positively regulating ion transport.

By being at the interface of the exocytic and endocytic pathways, the plant trans-Golgi network (TGN) is a multitasking and highly diversified organelle. Despite governing vital cellular processes, the TGN remains one of the most uncharacterized organelle of plant cells. In this review, we highlight recent studies that have contributed new insights and to the generation of markers needed to answer several important questions on the plant TGN. Several drugs specifically affecting proteins critical for the TGN functions have been extremely useful for the identification of mutants of the TGN in the pursuit to understand how the morphology and the function of this organelle are controlled. In addition to these chemical tools, we review emerging microscopy techniques that help visualize the TGN at an unpreceded resolution and appreciate the heterogeneity and dynamics of this organelle in plant cells.

Glioblastoma remains the most malignant of all primary adult brain tumours with poor patient survival and limited treatment options. This study adopts a drug repurposing approach by investigating the anti-cancer activity of a derivative of the antipsychotic drug phenothiazine (DS00329) in malignant U251 and U87 glioblastoma cells. Results from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays showed that DS00329 inhibited short-term glioblastoma cell viability and long-term survival while sparing non-cancerous cells. Western blot analysis with an antibody to γH2AX showed that DS00329 induced DNA damage and flow cytometry and western blotting confirmed that it triggered a G1 cell cycle arrest which correlated with decreased levels in Cyclin A, Cyclin B, Cyclin D1 and cyclin dependent kinase 2 and an increase in levels of the cyclin dependent kinase inhibitor p21. DS00329 treated glioblastoma cells exhibited morphological and molecular markers typical of apoptotic cells such as membrane blebbing and cell shrinkage and an increase in levels of cleaved PARP. Flow cytometry with annexin V-FITC/propidium iodide staining confirmed that DS00329 induced apoptotic cell death in glioblastoma cells. We also show that DS00329 treatment of glioblastoma cells led to an increase in the autophagosome marker LC3-II and autophagy inhibition studies using bafilomycin A1 and wortmannin, showed that DS00329-induced-autophagy was a pro-death mechanism. Furthermore, DS00329 treatment of glioblastoma cells inhibited the phosphatidylinositol 3'-kinase/Akt cell survival pathway. Our findings suggest that DS00329 may be an effective treatment for glioblastoma and provide a rationale for further exploration and validation of the use of phenothiazines and their derivatives in the treatment of glioblastoma.

Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM., Journal of Radiation Research, 61(2), pp.171-176; 2020

Objective: Xanthotoxin (XAT) is a linear furanocoumarin mainly extracted from the plants Ammi majus L. XAT has been reported the apoptosis of tumor cells, anti-convulsant, neuroprotective effect, antioxidative activity, and vasorelaxant effects. This study aimed to investigate the vascular protective effects and underlying molecular mechanisms of XAT. Methods: XAT’s activity was studied in rat thoracic aortas, isolated with aortic rings, and human umbilical vein endothelial cells (HUVECs). Results: XAT induced endothelium-dependent vasodilation in a concentration-dependent manner in the isolated rat thoracic aortas. Removal of endothelium or pretreatment of aortic rings with L-NAME, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, and wortmannin significantly inhibited XAT-induced relaxation. In addition, treatment with thapsigargin, 2-aminoethyl diphenylborinate, Gd3+, and 4-aminopyridine markedly attenuated the XAT-induced vasorelaxation. XAT increased nitric oxide production and Akt- endothelial NOS (eNOS) phosphorylation in HUVECs. Moreover, XAT attenuated the expression of TNF-α-induced cell adhesion molecules such as intercellular adhesion molecule, vascular cell adhesion molecule-1, and E-selectin. However, this effect was attenuated by the eNOS inhibitors L-NAME and asymmetric dimethylarginine. Conclusions: This study suggests that XAT induces vasorelaxation through the Akt-eNOS-cGMP pathway by activating the KV channel and inhibiting the L-type Ca2+ channel. Furthermore, XAT exerts an inhibitory effect on vascular inflammation, which is correlated with the observed vascular protective effects.

Background Mulberry (Morus alba L.) leaf tea benefits the control of diabetes in Asian nations. This study was aim to investigate if the flavonoids, which extracts from mulberry leaves, could regulate the metabolism of glycolipid, and to investigate if flavonoids could regulate IRS1/PI3K/AKT pathway signal to affect the expression of FAS and membrane transfer capacity GLUT4 in 3T3-L1 adipocytes. Results Results revealed that flavonoids decreased the levels of free fatty acid and increased the glucose consumption and the levels of adiponectin and leptin in a dose-dependent manner, and remarkably increased the protein expression levels of p-IRS1, p-PI3K, p-Akt, total GLUT4, and membrane GLUT4, and decreased the protein expression levels of PTEN and FAS in 3T3-L1 adipocytes IR model. On the other hand, wortmannin (2 nM), a selective and irreversible PI3K inhibitor, significantly decreased the glucose consumption and the adiponectin and leptin levels, and increased the free fatty acid level in flavonoids treated 3T3-L1 adipocytes IR model. Furthermore, wortmannin (2 nM) partly eliminated the activation of PI3K/AKT signaling, the suppression of FAS, and the up-regulated membrane transfer capacity of GLUT4 in flavonoids treated 3T3-L1 adipocytes IR model. Conclusion In conclusion, our results illustrated that mulberry leaf extracts flavonoids alleviated the glycolipid metabolic abnormalities in 3T3-L1 adipocytes IR model, and the effect was associated with the activation of IRS1/PI3K/AKT pathway, the suppression of FAS, and the up-regulation of membrane transfer capacity of GLUT4.

Lactobacillus salivarius AR809 is a newly discovered probiotic strain from a healthy human pharynx and has potential ability to adhere to the pharyngeal epithelium and inhibit Staphylococcus aureus (S. aureus)-induced inflammatory response. Pharyngeal spray administration of AR809 exhibited protective effects in a S. aureus-induced mouse model of pharyngitis. The inhibitory effect and underlying molecular mechanism of AR809 on S. aureus-stimulated pharyngitis were further investigated. AR809 significantly increased phagocytosis and bactericidal activity, reduced the production of inflammatory mediators (intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), nitric oxide (NO), inducible NOS (iNOS)) and the expression of inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)), and induced macrophages to adopt the M2 phenotype. AR809 also attenuated S. aureus-induced phosphorylations of protein kinase B (Akt) and rapamycin (mTOR), and elevated the autophagic protein (light chain 3 from II (LC3-II) and Beclin-1) level. Furthermore, AR809 inhibited nuclear transcription factor kappa-B (NF-κB) activation by suppressing the nuclear translocation of NF-κB p65. Likewise, 740Y-P (a PI3K activator) decreased the anti-inflammatory effect of AR809 against S. aureus-induced inflammatory response, while AR809 treatments with wortmannin (a PI3K inhibitor) markedly reversed this inflammatory response. AR809 prevents S. aureus-induced pharyngeal inflammatory response, possibly by regulating TLR/PI3K/Akt/mTOR signalling pathway-related autophagy and TLR/PI3K/Akt/IκB/NF-κB pathway activity, and therefore has potential for use in preventing pharyngitis and other inflammatory diseases.

Guavanoic acid functionalized gold nanoparticles exhibit anti-diabetic potential by improving insulin dependent glucose uptake in L6 rat skeletal muscle cells. The mode of action of the gold nanoparticles was established from the glucose uptake assay in the presence and absence of genistein and wortmannin. The anti-diabetic efficacy of guavanoic acid functionalized gold nanoparticles was put forth by in vitro assays like for PTP 1B, α-amylase and α-glucosidase enzyme activities. Studies on cytotoxicity revealed 50% inhibition of cells at 265 ± 0.01 μg mL(–1). In the LDH enzyme release assay on differentiated L6 myoblasts treated with different concentrations (1–100 μg mL(–1)) of guavanoic acid functionalized gold nanoparticles, a viability of 75% at 100 μg mL(–1) was observed.

This study examined the possible upstream cellular signaling pathway associated with nitric oxide (NO)-mediated inhibition of phenylephrine-induced contraction in isolated rat aortae in response to mild hypothermia, with a particular focus on endothelial Rho-kinase. We examined the effects of mild hypothermia (33°C), wortmannin, Nω-nitro-L-arginine methyl ester (L-NAME), Y-27632, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and methylene blue, alone and combined, on phenylephrine-induced contraction in isolated rat aortae. Finally, we examined the effects of mild hypothermia, wortmannin, Y-27632 and L-NAME, alone and combined, on endothelial nitric oxide synthase (eNOS) and endothelial Rho-kinase membrane translocation induced by phenylephrine. Mild hypothermia attenuated phenylephrine-induced contraction only in endothelium-intact aortae. L-NAME, wortmannin, ODQ and methylene blue increased phenylephrine-induced contraction of endothelium-intact aortae pretreated at 33°C. Wortmannin did not significantly alter the L-NAME-induced enhancement of phenylephrine-induced maximal contraction of endothelium-intact aortae pretreated at 33°C. Wortmannin abolished the ability of Y-27632 to magnify the hypothermic inhibition of maximal phenylephrine-induced contraction. Wortmannin and L-NAME inhibited the enhancing effect of mild hypothermia on phenylephrine-induced eNOS phosphorylation. Y-27632 and L-NAME attenuated the enhancing effect of hypothermia on phenylephrine-induced endothelial Rho-kinase membrane translocation. The results suggest that hypothermia-induced, NO-dependent inhibition of phenylephrine-induced contraction is mediated by phosphoinositide 3-kinase and inhibited by endothelial Rho-kinase activation.

BackgroundPrevious 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.MethodBioinformatics 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.ResultsHCC 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-β1 secreted from CAFs. PI3K inhibitor wortmannin could effectively impair tumor stemness to sorafenib.ConclusionWe 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.Graphical abstract

Decreased BDNF and impaired TRKB signaling contribute to neurodegeneration in Alzheimer's disease (AD). We have shown previously that coumarin derivative LM-031 enhanced CREB/BDNF/BCL2 pathway. In this study we explored if LM-031 analogs LMDS-1 to -4 may act as TRKB agonists to protect SH-SY5Y cells against Aβ toxicity. By docking computation for binding with TRKB using 7,8-DHF as a control, all four LMDS compounds displayed potential of binding to domain d5 of TRKB. In addition, all four LMDS compounds exhibited anti-aggregation and neuroprotective efficacy on SH-SY5Y cells with induced Aβ-GFP expression. Knock-down of TRKB significantly attenuated TRKB downstream signaling and the neurite outgrowth-promoting effects of these LMDS compounds. Among them, LMDS-1 and -2 were further examined for TRKB signaling. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in Aβ-GFP cells, implicating the neuroprotective effects are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. LMDS-1 and -2 are blood-brain barrier permeable as shown by parallel artificial membrane permeability assay. Our results demonstrate how LMDS-1 and -2 are likely to work as TRKB agonists to exert neuroprotection in Aβ cells, which may shed light on the potential application in therapeutics of AD.

Type 2 diabetes mellitus (T2DM) increases the occurrence and mortality of liver cancer. Insulin growth factor (IGF) plays a crucial role in the development of diabetes and liver cancer, and specifically, IGF-1 may be involved in the development of liver cancer with preexisting T2DM. Autophagy contributes to cancer cell survival and apoptosis. However, the relationship between IGF-1 and autophagy has rarely been evaluated. The purpose of this study was to investigate whether IGF-1 promotes the development of liver cancer in T2DM patients by promoting autophagy.Thirty-three hepatocellular carcinoma (HCC) patients with T2DM and 33 age-matched patients with HCC without T2DM were included in this study. We analyzed the expression of IGF-1 and autophagy-related LC3 and p62 mRNA and the prognosis of two groups. In vitro, we stimulated HepG2 cells with IGF-1 and then detected changes in autophagy and cell proliferation, apoptosis, and migration in the presence/absence of wortmannin, an autophagy inhibitor.IGF-1 promoted autophagy, resulting in inhibition of apoptosis and induction of growth and migration of HepG2 cells. Inhibition of autophagy by wortmannin impaired IGF-1 function. Higher expression of IGF-1 was detected in HCC patients with T2DM. IGF-1 expression was higher in liver cancer tissue compared to paracancerous tissue. Elevated IGF-1 was associated with a poor prognosis in patients with HCC.IGF-1 participates in the development of liver cancer by inducing autophagy. Elevated IGF-1 was a prognostic factor for patients with HCC, especially when accompanied by T2DM.

17β-estradiol (E2) treatment of ovariectomized adult mice stimulates the uterine PI3K-AKT signaling pathway and epithelial proliferation through estrogen receptor 1 (ESR1). However, epithelial proliferation occurs independently of E2/ESR1 signaling in neonatal uteri. Similarly, estrogen-independent uterine epithelial proliferation is seen in adulthood in mice lacking Ezh2, critical for histone methylation, and in wild-type (WT) mice treated neonatally with estrogen. The role of AKT in estrogen-independent uterine epithelial proliferation was the focus of this study. Expression of the catalytically active phosphorylated form of AKT (p-AKT) and epithelial proliferation were high in estrogen receptor 1 knockout and WT mice at postnatal day 6, when E2 concentrations were low, indicating that neither ESR1 nor E2 are essential for p-AKT expression and epithelial proliferation in these mice. However, p-AKT levels and proliferation remained estrogen responsive in preweaning WT mice. Expression of p-AKT and proliferation were both high in uterine luminal epithelium of mice estrogenized neonatally and ovariectomized during adulthood. Increased expression of phosphorylated (inactive) EZH2 was also observed. Consistent with this, Ezh2 conditional knockout mice show ovary-independent uterine epithelial proliferation and high epithelial p-AKT. Thus, adult p-AKT expression is constitutive and E2/ESR1 independent in both model systems. Finally, E2-induced p-AKT expression and normal uterine proliferation did not occur in mice lacking membrane (m)ESR1, indicating a key role for membrane ESR1 in AKT activation. These findings emphasize the importance of AKT activation in promoting uterine epithelial proliferation even when that proliferation is not E2/ESR1 dependent and further indicate that p-AKT can be uncoupled from E2/ESR1 signaling in several experimental scenarios.

Made available in DSpace on 2020-12-12T01:08:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1–4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system. Bioassays and Cell Dynamics Laboratory Department of Chemistry and Biochemistry Bioscience Institute UNESP Department of Biology Dental School University of Taubaté Bioassays and Cell Dynamics Laboratory Department of Chemistry and Biochemistry Bioscience Institute UNESP FAPESP: 2014/22689-3 FAPESP: 2016/22270-8

Complex human diseases such as metabolic disorders, cancer, neurodegenerative diseases, and mitochondrial dysfunctions arise from the biochemical or genetic defects in various cellular processes. Therefore, it is important to understand which metabolic processes are affected by which cellular impairment. Because genome-wide screening of mutant collections (haploid/diploid deletion library) provides important clues for the understanding of conserved biological processes and for finding potential target genes, we screened the haploid mutant collection of Schizosaccharomyces pombe with wortmannin that inhibits phosphatidylinositol-3-kinase signaling. Using genome-wide screening, we determined that 52 mutants were resistant to this chemical. When 52 genes that are deleted in these mutants were grouped in 41 different biological processes, we found that 37 of them have human orthologues and 4 genes were associated with human metabolic disorders. In addition, when we examined the pathways in which these 52 genes function, we determined that 9 genes were related to phosphorylation process. These results might provide new insights for better understanding of certain human diseases.

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.

Phosphoinositide 3-kinase (PI3K) plays a key role in a plethora of physiologic processes and controls cell growth, metabolism, immunity, cardiovascular and neurological function, and more. The discovery of wort-mannin as the first potent PI3K inhibitor (PI3Ki) in the 1990s provided rapid identification of PI3K-dependent processes, which drove the discovery of the PI3K/protein kinase B (PKB/Akt)/target of rapamycin (mTOR) pathway. Genetic mouse models and first PI3K isoform-specific inhibitors pinpointed putative therapeutic applications. The recognition of PI3K as target for cancer therapy drove subsequently drug development. Here we provide a brief journey through the emerging roles of PI3K to the development of preclinical and clinical PI3Ki candidates.

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs – which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

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/β-catenin pathway by increasing phospho-β-catenin levels. XAV939, a Wnt/β-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/β-catenin and/or PI3K/AKT pathways in rDPC, highlighting a candidate nutrient for hair loss treatment.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Leptin is a small molecule protein secreted by adipocytes, which can promote white fat browning through activating the hypothalamic nervous system and inhibiting downstream signaling pathways. Moreover, white fat browning has been proven to alleviate fat tissue fibrosis. This study explores the mechanism of leptin in regulating adipose tissue fibrosis and white fat browning. After treating mice with leptin, we screened out the recombinant integrin alpha 5 (ITGA5) through proteomics sequencing, which may play a role in adipose tissue fibrosis. Through real-time quantitative PCR (qPCR), western blotting (WB), hematoxylin-eosin (HE) staining, Masson’s trichrome, immunofluorescence, immunohistochemistry, etc., the results showed that after leptin treated adipocytes, the expression of fibrosis-related genes and ITGA5 was significantly down-regulated in adipocytes. We constructed fibrosis model through transforming growth factor-β (TGF-β) and a high-fat diet (HFD), and treated with ITGA5 overexpression vector and interference fragments. The results indicated the expression of fibrosis-related genes were significantly down-regulated after interfering with ITGA5. After treating adipocytes with wortmannin, fibrosis-related gene expression was inhibited after overexpression of ITGA5. Moreover, after injecting mice with leptin, we also found that leptin significantly up-regulated the expression of adipose tissue browning-related genes. Overall, our research shows that leptin can inhibit the activation of phosphatidylinositol 3 kinase (PI3K)-protein kinase B (AKT) signaling pathway by reducing the expression of ITGA5, which could alleviate adipose tissue fibrosis, and further promote white fat browning. Our research provides a theoretical basis for further research on the effect of leptin in fibrosis-related adipose tissue metabolism.

Astrocytes are a main target of JC polyomavirus (JCPyV) in the central nervous system (CNS), where the destruction of these cells, along with oligodendrocytes, leads to the fatal disease progressive multifocal leukoencephalopathy (PML). There is no cure currently available for PML, so it is essential to discover antivirals for this aggressive disease. Additionally, the lack of a tractable in vivo models for studying JCPyV infection makes primary cells an accurate alternative for elucidating mechanisms of viral infection in the CNS. This research to better understand the signaling pathways activated in response to JCPyV infection reveals and establishes the importance of the PI3K/AKT/mTOR signaling pathway in JCPyV infection in primary human astrocytes compared to transformed cell lines. Using RNA sequencing and chemical inhibitors to target PI3K, AKT, and mTOR, we have demonstrated the importance of this signaling pathway in JCPyV infection of primary astrocytes not observed in transformed cells. Collectively, these findings illuminate the potential for repurposing drugs that are involved with inhibition of the PI3K/AKT/mTOR signaling pathway and cancer treatment as potential therapeutics for PML, caused by this neuroinvasive virus.

The renin-angiotensin system (RAS) is a paracrine RAS within the central nervous system (CNS) and is closely related to Alzheimer’s disease (AD). The endogenous hexapeptide angiotensin IV (Ang IV), an important component of the brain RAS, was found to rescue cognitive impairment and recover memory in previous studies. In our study, we used different doses of Dihexa, which can be orally administered and cross the BBB in APP/PS1 mice. We found that the amount of AngIV in mouse tissue increased after the administration of Dihexa compared to that in the WT group. Meanwhile, Dihexa restored spatial learning and cognitive functions in the Morris water maze test. Dihexa increased the neuronal cells and the expression of SYP protein in APP/PS1 mice in Nissl staining. Furthermore, Dihexa decreased the activation of astrocytes and microglia, markedly reduced levels of the pro-inflammatory cytokines IL-1β and TNF-α and increased the levels of the anti-inflammatory cytokine IL-10. Dihexa activated the PI3K/AKT signaling pathway, while PI3K inhibitor wortmannin significantly reversed the anti-inflammatory and anti-apoptotic effects of APP/PS1 mice. These findings highlight the brain AngIV/PI3K/AKT axis as a potential target for the treatment of AD.

Cancer stem cells (CSCs) are distinct cancer populations with tumorigenic and self-renewal abilities. CSCs are drivers of cancer initiation, progression, therapeutic failure, and disease recurrence. Thereby, novel compounds targeting CSCs offer a promising way to control cancer. In this study, the hydroquinone 5-O-cinnamoyl ester of renieramycin M (CIN-RM) was demonstrated to suppress lung cancer CSCs. CIN-RM was toxic to lung cancer cells with a half-maximal inhibitory concentration of around 15 µM. CIN-RM suppressed CSCs by inhibiting colony and tumor spheroid formation. In addition, the CSC population was isolated and treated and the CSCs were dispatched in response to CIN-RM within 24 h. CIN-RM was shown to abolish cellular c-Myc, a central survival and stem cell regulatory protein, with the depletion of CSC markers and stem cell transcription factors ALDH1A1, Oct4, Nanog, and Sox2. For up-stream regulation, we found that CIN-RM significantly inhibited Akt and consequently decreased the pluripotent transcription factors. CIN-RM also inhibited mTOR, while slightly decreasing p-GSK3β (Ser9) but rarely affected the protein kinase C (PKC) signal. Inhibiting Akt/mTOR induced ubiquitination of c-Myc and promoted degradation. The mechanism of how Akt regulates the stability of c-Myc was validated with the Akt inhibitor wortmannin. The computational analysis further confirmed the strong interaction between CIN-RM and the Akt protein with a binding affinity of −10.9 kcal/mol at its critical active site. Taken together, we utilized molecular experiments, the CSC phenotype, and molecular docking methods to reveal the novel suppressing the activity of this compound on CSCs to benefit CSC-targeted therapy for lung cancer treatment.

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

Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier–membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to M¯n = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.

Evaluation of the uptake pathways in cells during exposure to nanoparticles (NPs) is key for risk assessment and the development of safer nanomaterials, as the internalisation and fate of NPs is linked to their toxicity and mode of action. Here, we determined the uptake mechanisms activated during the internalisation of 10, 30, and 100 nm AgNPs by embryonic zebrafish cells (ZF4). The uptake results demonstrated an NP size- and time-dependent uptake, showing the highest total silver uptake for the smallest AgNP (10 nm) at the lowest exposure concentration (2.5 μg/mL) after 2 h, while after 24 h, the highest exposure concentration (10 μg/mL) of the 10 nm AgNPs revealed the highest cellular load at 8 pg/cell. Inhibition of the caveolae, clathrin, and macropinocytosis endocytic pathways by pharmaceutical inhibitors (genistein, chlorpromazine, and wortmannin respectively) revealed that uptake was mainly via macropinocytosis for the 10 nm AgNPs and via the caveolae-mediated pathway for the 30 and 100 nm AgNPs. The induction of autophagy was also strongly related to the NP size, showing the highest percentage of induction for the 10 nm (around 3%) compared to naive cells, suggesting that autophagy can be activated along with endocytosis to deal with exposure to NPs. TEM imaging revealed the distribution of NPs across the cytoplasm inside intracellular vesicles. An increase in Early Endosome formation (EE) was observed for the 30 and 100 nm sizes, whereas the 10 nm AgNPs disrupted the activity of EE. The data supports the establishment of adverse outcome pathways by increasing knowledge on the link between a molecular initiating event such as receptor-mediated endocytosis and an adverse outcome, as well as supporting the reduction of animal testing by using alternative testing models, such as fish cell lines.

Phosphatidylinositol may be phosphorylated at either 3- or 4- positions on the inositol ring by PI 3-kinases or PI 4-kinases, respectively.Phosphatidylinositol 3-kinasesPhosphatidylinositol 3-kinases (PI3K, provisional nomenclature) catalyse the introduction of a phosphate into the 3-position of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol 4,5-bisphosphate (PIP2). There is evidence that PI3K can also phosphorylate serine/threonine residues on proteins. In addition to the classes described below, further serine/threonine protein kinases, including ATM (Q13315) and mTOR (P42345), have been described to phosphorylate phosphatidylinositol and have been termed PI3K-related kinases. Structurally, PI3Ks have common motifs of at least one C2, calcium-binding domain and helical domains, alongside structurally-conserved catalytic domains. wortmannin and LY 294002 are widely-used inhibitors of PI3K activities. wortmannin is irreversible and shows modest selectivity between Class I and Class II PI3K, while LY294002 is reversible and selective for Class I compared to Class II PI3K.Class I PI3Ks (EC 2.7.1.153) phosphorylate phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate and are heterodimeric, matching catalytic and regulatory subunits. Class IA PI3Ks include p110α, p110β and p110δ catalytic subunits, with predominantly p85 and p55 regulatory subunits. The single catalytic subunit that forms Class IB PI3K is p110γ. Class IA PI3Ks are more associated with receptor tyrosine kinase pathways, while the Class IB PI3K is linked more with GPCR signalling.Class II PI3Ks (EC 2.7.1.154) phosphorylate phosphatidylinositol to generate phosphatidylinositol 3-phosphate (and possibly phosphatidylinositol 4-phosphate to generate phosphatidylinositol 3,4-bisphosphate). Three monomeric members exist, PI3K-C2α, β and β, and include Ras-binding, Phox homology and two C2 domains.The only class III PI3K isoform (EC 2.7.1.137) is a heterodimer formed of a catalytic subunit (VPS34) and regulatory subunit (VPS15).Phosphatidylinositol 4-kinasesPhosphatidylinositol 4-kinases (EC 2.7.1.67) generate phosphatidylinositol 4-phosphate and may be divided into higher molecular weight type III and lower molecular weight type II forms.

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.