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.

Behavioural and biochemical responses to methamphetamine are differentially regulated by mGlu2 and mGlu3 metabotropic glutamate receptors in male mice.

Group II metabotropic glutamate receptors (mGlu2 and mGlu3 receptors) shape mechanisms of methamphetamine addiction, but the individual role played by the two subtypes is unclear. We measured methamphetamine-induced conditioned place preference (CPP) and motor responses to single or repeated injections of methamphetamine in wild-type, mGlu2-/-, and mGlu3-/-mice. Only mGlu3-/-mice showed methamphetamine preference in the CPP test. Motor response to the first methamphetamine injection was dramatically reduced in mGlu2-/-mice, unless these mice were treated with the mGlu5 receptor antagonist, MTEP. In contrast, methamphetamine-induced sensitization was increased in mGlu3-/-mice compared to wild-type mice. Only mGlu3-/-mice sensitized to methamphetamine showed increases in phospho-ERK1/2 levels in the nucleus accumbens (NAc) and free radical formation in the NAc and medial prefrontal cortex. These changes were not detected in mGlu2-/-mice. We also measured a series of biochemical parameters related to the mechanism of action of methamphetamine in naïve mice to disclose the nature of the differential behavioural responses of the three genotypes. We found a reduced expression and activity of dopamine transporter (DAT) and vesicular monoamine transporter-2 in the NAc and striatum of mGlu2-/-and mGlu3-/-mice, whereas expression of the DAT adaptor, syntaxin 1A, was selectively increased in the striatum of mGlu3-/-mice. Methamphetamine-stimulated dopamine release in striatal slices was largely reduced in mGlu2-/-, but not in mGlu3-/-, mice. These findings suggest that drugs that selectively enhance mGlu3 receptor activity or negatively modulate mGlu2 receptors might be beneficial in the treatment of methamphetamine addiction and associated brain damage.

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.

Ghrelin restores memory impairment following olfactory bulbectomy in mice by activating hippocampal NMDA1 and MAPK1 gene expression.

Ghrelin (Ghrl) is an orexigenic peptide with potential roles in the modulation of anxiety- and depressive-like symptoms induced by bilateral olfactory bulbectomy (OB) in rodents. In the present work, we assessed whether intrahippocampal Ghrl could reverse OB-induced depressive-like and amnesic effects by regulating molecular mechanisms related to neuroplasticity. Adult female albino Swiss mice were divided into sham and OB groups, and infused with saline (S) or Ghrl 0.03 nmol/µl, 0.3 nmol/µl, or 3 nmol/µl into the hippocampus before exposition to open-field test (OFT) and tail suspension test (TST) or immediately after training in the object recognition test (ORT). After test phase in ORT, animals were euthanized and their hippocampi were dissected to study the expression of genes related to memory. The OB-S animals presented hyperlocomotion in OFT, increased immobility in TST and memory impairment compared to sham-S (p < 0.05), but acute intrahippocampal infusion of Ghrl 0.3 nmol/µl produced an improvement on these parameters in OB animals (p < 0.05). In addition, this dose of Ghrl reversed OB-induced low expression of NMDA1 and MAPK1 iso1 and up-regulated the expression of CaMKIIa iso1 and iso2, and MAPK1 iso2 (p < 0.05). These results extend the existing literature regarding OB-induced behavioral and neurochemical changes, and provide mechanisms that could underlie the antidepressant effect of Ghrl in this model.

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.

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.

NMDA receptor-mediated CaMKII/ERK activation contributes to renal fibrosis.

BACKGROUND: This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-ß administration. METHODS: Acute renal fibrosis (RF) was induced in mice by unilateral ureteral obstruction (UUO). Histological changes were observed using Masson's trichrome staining. The expression levels of NR1, which is the functional subunit of NMDAR, and fibrotic and epithelial-to-mesenchymal transition markers were measured by immunohistochemical and Western blot analysis. HK-2 cells were incubated with TGF-ß, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were administered for pathway determination. Chronic RF was introduced by sublethal ischemia-reperfusion injury in mice, and NMDAR inhibitor dextromethorphan hydrobromide (DXM) was administered orally. RESULTS: The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-ß1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal-regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic-reperfusion injury. CONCLUSIONS: NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.

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.

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.

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.

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.

Inhibiting myeloperoxidase prevents onset and reverses established high-fat diet-induced microvascular insulin resistance.

A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.

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.

TSH/IGF-1 Receptor Cross-Talk Rapidly Activates Extracellular Signal-Regulated Kinases in Multiple Cell Types.

We previously showed that thyrotropin (TSH)/insulinlike growth factor (IGF)-1 receptor cross-talk appears to be involved in Graves' orbitopathy (GO) pathogenesis and upregulation of thyroid-specific genes in human thyrocytes. In orbital fibroblasts from GO patients, coadministration of TSH and IGF-1 induces synergistic increases in hyaluronan secretion. In human thyrocytes, TSH plus IGF-1 synergistically increased expression of the sodium-iodide symporter that appeared to involve ERK1/2 activation. However, the details of ERK1/2 activation were not known, nor was whether ERK1/2 was involved in this synergism in other cell types. Using primary cultures of GO fibroblasts (GOFs) and human thyrocytes, as well as human embryonic kidney (HEK) 293 cells overexpressing TSH receptors (HEK-TSHRs), we show that simultaneous activation of TSHRs and IGF-1 receptors (IGF-1Rs) causes rapid, synergistic phosphorylation/activation of ERK1 and ERK2 in all three cell types. This effect is partially inhibited by pertussis toxin, an inhibitor of TSHR coupling to Gi/Go proteins. In support of a role for Gi/Go proteins in ERK1/2 phosphorylation, we found that knockdown of Gi(1-3) and Go in HEK-TSHRs inhibited ERK1/2 phosphorylation stimulated by TSH and TSH plus IGF-1. These data demonstrate that the synergistic effects of TSH plus IGF-1 occur early in the TSHR signaling cascade and further support the idea that TSHR/IGF-1R cross-talk is an important mechanism for regulation of human GOFs and thyrocytes.

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.