Antidepressant mechanism and active compounds of saffron from network pharmacology study.

Saffron has been applied in depression treatment, but its antidepressant compounds and mechanisms are unclear. In this research, a network pharmacology-based method was proposed to screen the active compounds and the potential mechanisms of saffron for depression treatment. Firstly, the chemical compounds of saffron were collected from literature and filtered by drug-like prediction. Secondly, common targets, by comparing the targets of saffron predicted by Pharm Mapper server with targets associated with depression collected from Genecards, were regarded as the antidepressant targets of saffron. Thirdly, common targets were mapped to KEGG pathways, considered as the pathways related with the antidepressant effects of saffron. Finally, the network of compounds-targets-pathways was constructed and analyzed by cytoscape 3.4.0. Ten compounds including crocetin, picrocrocin, (1R, 5S, 6R)-5-(hydroxymethyl)- 4, 4, 6-trimethyl-7-Oxabicyclo[4.1.0]heptan-2-one and its glycoside were screened as the main antidepressant compounds, some of which were reported for the first time. They might have effective treatment for depression by acting on targets, such as MAP2K1, MAPK1, HRAS, PIK3R1, ALB and AKT1 and pathways related with immune system, signal transduction and so on. This study provided a new insight into the antidepressant mechanism and active compounds of saffron, which also had a guiding effect on later experiments.

Network Pharmacology and Molecular Docking Study on the Potential Mechanism of Yi-Qi-Huo-Xue-Tong-Luo Formula in Treating Diabetic Peripheral Neuropathy.

OBJECTIVE: To investigate the potential mechanism of action of Yi-Qi-Huo-Xue-Tong-Luo formula (YQHXTLF) in the treatment of diabetic peripheral neuropathy (DPN). METHODS: Network pharmacology and molecular docking techniques were used in this study. Firstly, the active ingredients and the corresponding targets of YQHXTLF were retrieved using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform; subsequently, the targets related to DPN were retrieved using GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmgkb, Therapeutic Target Database (TTD) and Drugbank databases; the common targets of YQHXTLF and DPN were obtained by Venn diagram; afterwards, the "YQHXTLF Pharmacodynamic Component-DPN Target" regulatory network was visualized using Cytoscape 3.6.1 software, and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the potential targets using R 3.6.3 software. Finally, molecular docking of the main chemical components in the PPI network with the core targets was verified by Autodock Vina software. RESULTS: A total of 86 active ingredients and 229 targets in YQHXTLF were screened, and 81 active ingredients and 110 targets were identified to be closely related to diabetic peripheral neuropathy disease. PPI network mapping identified TP53, MAPK1, JUN, and STAT3 as possible core targets. KEGG pathway analysis showed that these targets are mostly involved in AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, and MAPK signaling pathway. The molecular docking results showed that the main chemical components of YQHXTLF have a stable binding activity to the core pivotal targets. CONCLUSION: YQHXTLF may act on TP53, MAPK1, JUN, and STAT3 to regulate inflammatory response, apoptosis, or proliferation as a molecular mechanism for the treatment of diabetic peripheral neuropathy, reflecting its multitarget and multipathway action, and providing new ideas to further uncover its pharmacological basis and mechanism of action.

Synergistic efficacy and diminished adverse effect profile of composite treatment of several ADHD medications.

Although attention-deficit/hyperactivity disorder (ADHD) is widely studied, problems regarding the adverse effect risks and non-responder problems still need to be addressed. Combination pharmacotherapy using standard dose regimens of existing medication is currently being practiced mainly to augment the therapeutic efficacy of each drug. The idea of combining different pharmacotherapies with different molecular targets to alleviate the symptoms of ADHD and its comorbidities requires scientific evidence, necessitating the investigation of their therapeutic efficacy and the mechanisms underlying the professed synergistic effects. Here, we injected male ICR mice with MK-801 to induce ADHD behavioral condition. We then modeled a "combined drug" using sub-optimal doses of methylphenidate, atomoxetine, and fluoxetine and investigated the combined treatment effects in MK-801-treated mice. No sub-optimal dose monotherapy alleviated ADHD behavioral condition in MK-801-treated mice. However, treatment with the combined drug attenuated the impaired behavior of MK-801-treated animals. Growth impediment, sleep disturbances, or risk of substance abuse were not observed in mice treated subchronically with the combined drugs. Finally, we observed that the combined ADHD drug rescued alterations in p-AKT and p-ERK1/2 levels in the prefrontal cortex and hippocampus, respectively, of MK-801-treated mice. Our results provide experimental evidence of a possible new pharmacotherapy option in ameliorating the ADHD behavioral condition without the expected adverse effects. The detailed mechanism of action underlying the synergistic therapeutic efficacy and reduced adverse reaction by combinatorial drug treatment should be investigated further in future studies.

Procyanidin A2, a polyphenolic compound, exerts anti-inflammatory and anti-oxidative activity in lipopolysaccharide-stimulated RAW264.7 cells.

Procyandin A2 (PCA2) is a polyphenolic compound which is isolated from grape seeds. It has been reported that PCA2 exhibits antioxidative and anti-inflammatory effects, but its molecular mechanism is still poorly understood. This study tests the hypothesis that PCA2 suppresses lipopolysaccharide (LPS)-induced inflammation and oxidative stress through targeting the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and NF-E2-related factor 2 (Nrf2) pathways in RAW264.7 cells. PCA2 (20, 40, 80 µM) exhibited no significant cytotoxicity in RAW264.7 cells and showed an inhibitory effect on an LPS-induced nitrite level. Pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO), and reactive oxygen species (ROS) were suppressed by PCA2 with a concentration range of 0-80 µM. The mRNA levels of TNF-α and IL-6 were inhibited by PCA2 (80 µM). The hallmark-protein expression of the NF-κB (p-IKKα/ß, p-IκBα, and p-p65) and MAPK (p-p38, p-JNK, and p-ERK) pathways were decreased by PCA2 in LPS-stimulated RAW264.7 cells. In addition, immunofluorescence results indicated that PCA2 (80 µM) promoted the translocation of NF-κB/p65 from the cytoplasm into the nucleus. PCA2 upregulated the expressions of Nrf2 and HO-1 and downregulated the expression of Keap-1. Simultaneously, PCA2 (80 µM) reversed LPS-induced Nrf2 translocation from the nucleus into the cytoplasm. Collectively, PCA2 protect cells against the damage from inflammation and oxidative injury, which suggest a potential therapeutic strategy for inflammatory and oxidative stress through targeting NF-κB, MAPK, and Nrf2 pathways in RAW264.7 cells.

Anti-inflammatory activity of phenylpropyl triterpenoids from Osmanthus fragrans var. aurantiacus leaves.

Osmanthus fragrans var. aurantiacus has been used for the treatment of menopausal pain, foul breath, and intestinal bleeding. Four phenylpropyl triterpenoids, 3-O-trans-p-coumaroyltormentic acid (1), 3ß-trans-p-coumaroyloxy-2α-hydroxyl-urs-12-en-28-oic acid (2), 3ß-cis-p-coumaroyloxy-2α-hydroxyl-urs-12-en-28-oic acid (3), 3-O-cis-coumaroylmaslinic acid (4), were isolated from the leaves of O. fragrans var. aurantiacus and the inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages were evaluated. Among them, compounds 2-4 concentration dependently showed NO production inhibitory effects. To determine the signaling factors involved in the inhibition of NO production by compounds 2-4, we assessed anti-inflammatory activity. Western blot analysis revealed compounds 2-4 significantly decreased the expression of LPS-stimulated protein, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, nuclear factor-kappa B (NF-κB) and phosphorylated extracellular regulated kinase (pERK)1/2. Also, compounds 2-4 downregulated tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-8 levels in LPS-induced macrophages and colonic epithelial cells. This study demonstrated that phenylpropyl triterpenoids 2-4 isolated from O. fragrans var. aurantiacus leaves can be used as potential candidates for the prevention and treatment of inflammatory bowel disease.

Down-Regulation of the Mammalian Target of Rapamycin (mTOR) Pathway Mediates the Effects of the Paeonol-Platinum(II) Complex in Human Thyroid Carcinoma Cells and Mouse SW1736 Tumor Xenografts.

BACKGROUND This study aimed to investigate the effects of the paeonol-platinum(II) (PL-Pt[II]) complex on SW1736 human anaplastic thyroid carcinoma cell line and the BHP7-13 human thyroid papillary carcinoma cell line in vitro and on mouse SW1736 tumor xenografts in vivo. MATERIAL AND METHODS The cytotoxic effects of the PL-Pt(II) complex on SW1736 cells and BHP7-13 cells was measured using the MTT assay. Western blot measured the expression levels of cyclins, cell apoptotic proteins, and signaling proteins. DNA content and apoptosis were detected by flow cytometry. SW1736 cell thyroid tumor xenografts were established in mice followed by treatment with the PL-Pt(II) complex. RESULTS Treatment of the SW1736 and BHP7-13 cells with the PL-Pt(II) complex reduced cell proliferation in a dose-dependent manner, with an IC50 of 1.25 µM and 1.0 µM, respectively, and increased the cell fraction in G0/G1phase, inhibited p53, cyclin D1, promoted p27 and p21 expression, and significantly increased the sub-G1 fraction. Treatment with the PL-Pt(II) complex increased caspase-3 degradation, reduced the expression of p-4EBP1, p-4E-BP1 and p-S6, and reduced the expression of p-ERK1/2 and p-AKT. Treatment with the PL-Pt(II) complex reduced the volume of the SW1736 mouse tumor xenografts on day 14 and day 21, and reduced AKT phosphorylation and S6 protein expression and increased degradation of caspase-3. CONCLUSIONS The cytotoxic effects of the PL-Pt(II) complex in human thyroid carcinoma cells, including activation of apoptosis and an increased sub-G1 cell fraction of the cell cycle, were mediated by down-regulation of the mTOR pathway.

Statins activate the NLRP3 inflammasome and impair insulin signaling via p38 and mTOR.

Statins lower cholesterol and risk of cardiovascular disease. Statins can increase blood glucose and risk of new-onset diabetes. It is unclear why statins can have opposing effects on lipids versus glucose. Statins have cholesterol-independent pleiotropic effects that influence both insulin and glucose control. Statin lowering of isoprenoids required for protein prenylation promotes pancreatic ß-cell dysfunction and adipose tissue insulin resistance. Protein prenylation influences immune function and statin-mediated adipose tissue insulin resistance involves the NLR family pyrin domain-containing 3 (NLRP3) inflammasome and IL-1ß. However, the intracellular cues that statins engage to activate the NLRP3 inflammasome and those responsible for IL-1ß-mediated insulin resistance in adipose tissue have not been identified. We hypothesized that stress kinases or components of the insulin signaling pathway mediated statin-induced insulin resistance. We tested the associations of p38, ERK, JNK, phosphatase, and tensin homolog (PTEN), and mTOR in statin-exposed adipose tissue from WT and IL-1ß-/- mice. We found that statins increased phosphorylation of p38 in WT and IL-1ß-/- mice. Statin activation of p38 upstream of IL-1ß led to priming of this NLRP3 inflammasome effector in macrophages. We found that mTORC1 inhibition with low doses of rapamycin (2 or 20 nM) lowered macrophage priming of IL-1ß mRNA and secretion of IL-1ß caused by multiple statins. Rapamycin (20 nM) or the rapalog everolimus (20 nM) prevented atorvastatin-induced lowering of insulin-mediated phosphorylation of Akt in mouse adipose tissue. These results position p38 and mTOR as mediators of statin-induced insulin resistance in adipose tissue and highlight rapalogs as candidates to mitigate the insulin resistance and glycemic side effects of statins.

Cocaine-induced changes in CX3CL1 and inflammatory signaling pathways in the hippocampus: Association with IL1ß.

Cocaine induces neuroinflammatory response and interleukin-1 beta (IL1ß) is suggested a final effector for many cocaine-induced inflammatory signals. Recently, the chemokine fractalkine (CX3CL1) has been reported to regulate hippocampus-dependent neuroinflammation and synaptic plasticity via CX3C-receptor 1 (CX3CR1), but little is known about the impact of cocaine. This study is mainly focused on the characterization of CX3CL1, IL1ß and relevant inflammatory signal transduction pathways in the hippocampus in acute and repeated cocaine-treated male mice. Complementarily, the rewarding properties of cocaine were also assessed in Cx3cr1-knockout (KO) mice using a conditioned place preference (CPP). We observed significant increases in CX3CL1 and IL1ß concentrations after cocaine, although repeated cocaine produced an enhancement of CX3CL1 concentrations. CX3CL1 and IL1ß concentrations were positively correlated in acute (r = +0.61) and repeated (r = +0.82) cocaine-treated mice. Inflammatory signal transduction pathways were assessed. Whereas acute cocaine-treated mice showed transient increases in p-ERK1/2/ERK1/2 and p-p65/p65 NFκB ratios after cocaine injection, repeated cocaine-treated mice showed transient increases in p-ERK1/2/ERK1/2, p-p38/p38 MAPK, p-NFκB p65/NF-κB p65 and p-CREB/CREB ratios. Baseline p-p38/p38 MAPK and p-CREB/CREB ratios were downregulated in repeated cocaine-treated mice. Regarding the cocaine-induced CPP, Cx3cr1-KO mice showed a notably impaired extinction but no differences during acquisition and reinstatement. These results indicate that cocaine induces alterations in CX3CL1 concentrations, which are associated with IL1ß concentrations, and activates convergent inflammatory pathways in the hippocampus. Furthermore, the CX3CL1/CX3CR1 signaling could mediate the processes involved in the extinction of cocaine-induced CPP.

Effect of metformin and flutamide on insulin, lipogenic and androgen-estrogen signaling, and cardiometabolic risk in a PCOS-prone metabolic syndrome rodent model.

Polycystic ovary syndrome (PCOS) is highly associated with cardiometabolic risk and the metabolic syndrome (MetS), predisposing women to increased risk of developing type 2 diabetes and cardiovascular disease. Metformin is commonly used to treat insulin resistance-glucose intolerance, and flutamide, an androgen receptor (AR) antagonist, is used to target hyperandrogenemia and dyslipidemia. Currently, the physiological mechanism of action of these treatments on androgen, lipidogenic, and insulin signaling pathways remains unclear in PCOS. The aim of this study was to investigate the effects and mechanisms of action of metformin and flutamide on plasma lipid-apolipoprotein (Apo)B-lipoprotein and insulin-glucose metabolism, and endocrine-reproductive indices in a PCOS-prone MetS rodent model. PCOS-prone rodents were treated with metformin (300 mg/kg body wt), flutamide (30 mg/kg body wt), or metformin + flutamide combination treatment for 6 wk. Metformin was shown to improve fasting insulin and HOMA-IR, whereas flutamide and combination treatment were shown to reduce plasma triglycerides, ApoB48, and ApoB100, and this was associated with decreased intestinal secretion of ApoB48/triglyceride. Flutamide and metformin were shown to reduce plasma androgen indices and to improve ovarian primary and preovulatory follicle frequency. Metformin treatment increased hepatic estrogen receptor (ER)α, and metformin-flutamide decreased intestinal AR and increased ERα mRNA expression. Metformin-flutamide treatment upregulated hepatic and intestinal insulin signaling, including insulin receptor, MAPK1, and AKT2. In conclusion, cardiometabolic risk factors, in particular ApoB-hypertriglyceridemia, are independently modulated via the AR, and understanding the contribution of AR and insulin-signaling pathways further may facilitate the development of targeted interventions in high-risk women with PCOS and MetS.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells.

We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Development of RET mutant cutaneous angiosarcoma during BRAF inhibitor therapy.

Treatment with BRAF inhibitors may lead to paradoxical mitogen-activated protein kinase (MAPK) pathway activation and accelerated tumorigenesis in cells with preexisting oncogenic hits. This phenomenon manifests clinically in the development of squamous cell carcinomas (SCCs) and keratoacanthomas (KAs) in patients treated with BRAF inhibitors. Cases of extracutaneous malignancies associated with BRAF inhibitors have also been reported. We present a case of a patient who developed a cutaneous angiosarcoma 6 months after initiation of vemurafenib therapy. Next-generation sequencing (NGS) revealed a mutation in RET, which lies upstream of the MAPK pathway. This case highlights that treatment with BRAF inhibitors may promote the accelerated growth of secondary malignancies. Physician awareness of the spectrum of secondary malignancies associated with BRAF inhibitor treatment will support their early detection and treatment.

Regulation of the macrophage oxytocin receptor in response to inflammation.

It has been demonstrated that the neuropeptide oxytocin (OT) attenuates oxidative stress and inflammation in macrophages. In the current study, we examined the role of inflammation on the expression of the oxytocin receptor (OXTR). We hypothesized that OXTR expression is increased during the inflammation through a nuclear factor-κB (NF-κB)-mediated pathway, thus responding as an acute-phase protein. Inflammation was induced by treating macrophages (human primary, THP-1, and murine) with lipopolysaccharide (LPS) and monitored by expression of IL-6. Expression of OXTR and vasopressin receptors was assessed by qPCR, and OXTR expression was confirmed by immunoblotting. Inflammation upregulated OXTR transcription 10- to 250-fold relative to control in THP-1 and human primary macrophages and increased OXTR protein expression. In contrast, vasopressin receptor-2 mRNA expression was reduced following LPS treatment. Blocking NF-κB activation prevented the increase in OXTR transcription. OT treatment of control cells and LPS-treated cells increased ERK1/2 phosphorylation, demonstrating activation of the OXTR/Gαq/11 signaling pathway. OT activation of OXTR reduced secretion of IL-6 in LPS-activated macrophages. Collectively, these findings suggest that OXTR is an acute-phase protein and that its increased expression is regulated by NF-κB and functions to attenuate cellular inflammatory responses in macrophages.

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours.

PURPOSE: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials. EXPERIMENTAL DESIGN: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules. RESULTS: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma. CONCLUSIONS: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly.

GLP-1 inhibits VEGFA-mediated signaling in isolated human endothelial cells and VEGFA-induced dilation of rat mesenteric arteries.

We investigated the acute effects of glucagon-like peptide-1 (GLP-1), GLP-1(1-36), and GLP-1(7-36) on vascular endothelial growth factor-A (VEGFA)-induced endothelium-dependent signaling and vasodilation. Our hypothesis was that GLP-1 released from intestinal l-cells modulates processes related to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries. In isolated rat mesenteric resistance arteries precontracted with the thromboxane analog U46619 to 80-90% of maximum contraction, VEGFA (25 ng/ml) caused a small and gradual relaxation (28.9 ± 3.9%). Pretreatment of arteries with either GLP-1(1-36) (500 nM) or GLP-1(7-36) (1 nM) abolished the VEGFA-induced relaxation. VEGFA-induced relaxations were also inhibited in endothelial-denuded arteries and in arteries pretreated with the nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester (100 µM). In vivo studies on male Wistar rats also revealed that GLP-1(7-36) inhibited VEGFA-induced vasodilation of the same arteries. In isolated endothelial cells, GLP-1(1-36) and GLP-1(7-36) caused a reduction in VEGFA-induced phosphorylation of PLCγ. Ca2+ imaging of endothelial cells and rat mesenteric resistance arteries using fura-2, revealed that both GLP-1 analogs caused a reduction in VEGFA-induced Ca2+ signaling. GLP-1(1-36) also reduced VEGFA-induced eNOS phosphorylation in HDMEC. In conclusion, GLP-1 reduced relaxation induced by VEGFA in resistance arteries by inhibiting VEGFR2-mediated Ca2+ signaling and endothelial NO synthesis. GLP-1, on its own, also induced phosphorylation of Src and ERK1/2 that can lead to proliferation and is implicated in vessel permeability.

Endoplasmic reticulum stress increases brain MAPK signaling, inflammation and renin-angiotensin system activity and sympathetic nerve activity in heart failure.

We previously reported that endoplasmic reticulum (ER) stress is induced in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN) of heart failure (HF) rats and is reduced by inhibition of mitogen-activated protein kinase (MAPK) signaling. The present study further examined the relationship between brain MAPK signaling, ER stress, and sympathetic excitation in HF. Sham-operated (Sham) and HF rats received a 4-wk intracerebroventricular (ICV) infusion of vehicle (Veh) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10 µg/day). Lower mRNA levels of the ER stress biomarkers GRP78, ATF6, ATF4, and XBP-1s in the SFO and PVN of TUDCA-treated HF rats validated the efficacy of the TUDCA dose. The elevated levels of phosphorylated p44/42 and p38 MAPK in SFO and PVN of Veh-treated HF rats, compared with Sham rats, were significantly reduced in TUDCA-treated HF rats as shown by Western blot and immunofluorescent staining. Plasma norepinephrine levels were higher in Veh-treated HF rats, compared with Veh-treated Sham rats, and were significantly lower in the TUDCA-treated HF rats. TUDCA-treated HF rats also had lower mRNA levels for angiotensin converting enzyme, angiotensin II type 1 receptor, tumor necrosis factor-α, interleukin-1ß, cyclooxygenase-2, and NF-κB p65, and a higher mRNA level of IκB-α, in the SFO and PVN than Veh-treated HF rats. These data suggest that ER stress contributes to the augmented sympathetic activity in HF by inducing MAPK signaling, thereby promoting inflammation and renin-angiotensin system activity in key cardiovascular regulatory regions of the brain.

Tyrosine kinase inhibitor BIBF1000 does not hamper right ventricular pressure adaptation in rats.

BIBF1000 is a small molecule tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR) and is a powerful inhibitor of fibrogenesis. BIBF1000 is very similar to BIBF1120 (nintedanib), a drug recently approved for the treatment of idiopathic pulmonary fibrosis (IPF). A safety concern pertaining to VEGFR, FGFR, and PDGFR inhibition is the possible interference with right ventricular (RV) responses to an increased afterload, which could adversely affect clinical outcome in patients with IPF who developed pulmonary hypertension. We tested the effect of BIBF1000 on the adaptation of the RV in rats subjected to mechanical pressure overload. BIBF1000 was administered for 35 days in pulmonary artery-banded (PAB) rats. RV adaptation was assessed by echocardiography, pressure volume loop analysis, histology, and determination of atrial natriuretic peptide (ANP) expression. BIBF1000 treatment resulted in growth attenuation but had no effects on RV function after PAB, given absence of changes in cardiac index, end-systolic elastance, connective tissue disposition, and capillary density. We conclude that, in this experimental model of increased afterload, combined VEGFR, FGFR, and PDGFR inhibition does not hamper RV adaptation to pressure overload.

A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer.

Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro, and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo. In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo, including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.

Corticotropin releasing hormone and Urocortin 2 activate inflammatory pathways in cultured trophoblast cell lines.

OBJECTIVE: Embryo implantation and parturition are recognized as inflammatory events involving endocrine and immune system. NF-kB and MAPK are two transcription factor families involved in inflammation. A possible role of neuroendocrine mechanism in early pregnancy and delivery was proposed for the neuropeptides related to corticotropin releasing hormones (CRH), named Urocortins (Ucns). Experimental and clinical studies support a role for CRH, Ucn, Ucn2 and Ucn3 in the endocrine/immune modulation of inflammation in human trophoblast; however the intracellular mechanisms are not yet recognized. The aim of the present study was to evaluate which of these neuropeptides modulate NF-kB or MAPKs pathways. STUDY DESIGN: In Jeg-3 placental cell line the effect of CRH, Ucn, Ucn2 or Ucn3 on NF-kB and MAPKs pathways were evaluated using Western blot analysis. RESULTS: CRH induced the phosphorylation of MAPK subunits; Ucn2 was able to induce the phosphorylation of both NF-kB and MAPK subunits. Ucn and Ucn3 had no effects on these pathways. CONCLUSIONS: These data provide novel information on inflammatory process in trophoblast cells: Ucn2 is a potent pro-inflammatory neuropeptide via NF-kB and MAPK pathways and CRH via MAPK, and CRH and Ucn2 network participates in the inflammatory mechanisms of pregnancy and parturition.