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Objective: This study aimed to investigate the possible mechanisms by which orphan G protein-coupled receptor GPR41 activation enhances glucose uptake into C2C12 myotubes using a GPR41-selective agonist, AR420626, and to examine the ability of this agent to improve insulin sensitivity and glucose homeostasis in vivo., Methods: Basal and insulin-stimulated glucose uptake and glucose transporter 4 translocations were measured in C2C12 myotubes. Ca 2+ influx into cells was measured and GPR41-mediated signaling by AR420626 was examined. An oral glucose tolerance test was performed, and plasma insulin levels were measured in streptozotocin-treated or high-fat diet-fed diabetic mice. The glycogen content was measured in skeletal muscle tissue., Results: AR420626 increased basal and insulin-stimulated glucose uptake, which was reduced by pertussis toxin, an inhibitor of Gα i -mediated signaling, and treatment with small interfering RNA for GPR41 (siGPR41). AR420626 increased intracellular Ca 2+ influx and phosphorylated Ca 2+ /calmodulin-dependent protein kinase type II, cyclic AMP-responsive element-binding protein, and mitogen-activated protein kinase (p38) in C2C12 myotubes, which were inhibited by treating with pertussis toxin, amlodipine (Ca 2+ channel blocker), and siGPR41. AR420626 increased plasma insulin levels and skeletal muscle glycogen content and improved glucose tolerance in streptozotocin- and high-fat diet-induced diabetic mouse models., Conclusions: GPR41 activation with AR420626 increased glucose uptake mediated by Ca 2+ signaling via GPR41, improving diabetes mellitus., (© 2023 The Obesity Society.)

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF- κ B pathway and proinflammatory cytokine induction in vitro and in vivo . In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF- κ B and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF- κ B pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas., Competing Interests: The authors declare no conflicts of interest., (© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Mulberry leaf (Mori Folium) extract (MLE) is known to have anti-obesity effects. In this study, the enhanced effects of MLE after bioconversion treatment using Pectinex (BMLE) on obesity were explored, and the underlying mechanisms were investigated using the active components, neochlorogenic acid (5-CQA) and cryptochlorogenic acid (4-CQA), whose amounts were increased by bioconversion of MLE. Both MLE and BMLE inhibited lipid accumulation in 3T3-L1 adipocytes without cytotoxicity and suppressed the expression of CCAAT/enhancer-binding protein alpha (C/EBPα). In addition, MLE and BMLE decreased high-fat diet-induced adipose tissue mass expansion. Notably, BMLE significantly increased antiadipogenic and anti-obesity effects compared to MLE in vitro and in vivo. The active ingredients increased by bioconversion, 5-CQA and 4-CQA, inhibited the protein levels of C/EBPα and the mRNA levels of stearoyl-CoA desaturase 1 (Scd1). These findings provide new insights into the therapeutic possibility of using bioconversion of MLE, by which upregulation of 5-CQA and 4-CQA potently inhibits adipogenesis., (© 2022. The Author(s).)

Aim: Endothelial cell (EC) dysfunction initiates atherosclerosis by inducing inflammatory cytokines and adhesion molecules. Herein, we investigated the role of ginsenoside Rh1 (Rh1) in lipopolysaccharide (LPS)-induced EC dysfunction., Main Methods: The inhibitory effect of Rh1 on LPS binding to toll-like receptor 2 (TLR2) or TLR4 was evaluated using an immunofluorescence (IF) assay. Annexin V and cleaved caspase-3-positive EC apoptosis were evaluated by flow cytometry and IF assay. Western blotting and quantitative reverse transcription-PCR were performed to clarify underlying molecular mechanisms. In vivo model, effect of Rh1 on EC dysfunction was evaluated by using en face IF assay on aortas isolated C57BL/6 mice., Key Finding: LPS (500 ng/mL) activated inflammatory signaling pathways, including ERK1/2, STAT3, and NF-κB. Interestingly, Rh1 significantly abolished the binding of LPS to TLR2 and TLR4. Consistently, Rh1 inhibited LPS-induced NF-κB activation and its downstream molecules, including inflammatory cytokines and adhesion molecules. Furthermore, Rh1 alleviated LPS-induced downregulation of eNOS promoter activity. Notably, inactivation of eNOS by 50 μM L-NAME significantly increased NF-κB promoter activity. In addition, Rh1 abolished LPS-mediated cell cycle arrest and EC apoptosis by inhibiting endoplasmic reticulum stress via PERK/CHOP/ERO1-α signaling pathway. Consistent with in vitro experimental data, Rh1 effectively suppressed LPS-induced VCAM-1 and CHOP expression and rescuing LPS-destroyed tight junctions between ECs as indicated in ZO-1 expression on mice aorta., Significance: Rh1 suppresses LPS-induced EC inflammation and apoptosis by inhibiting STAT3/NF-κB and endoplasmic reticulum stress signaling pathways, mediated by blocking LPS binding-to TLR2 and TLR4. Consistently, Rh1 effectively reduced EC dysfunction in vivo model., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6'-sialylactose (6'-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6'-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500 ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6'-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6'-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6'-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6'-SL treatment. Altogether, our findings suggest that 6'-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability., (© 2022. The Pharmaceutical Society of Korea.)

Scope: Garcinia cambogia (G. cambogia) is known to have antiobesity effects. In this study, the therapeutic effects of G. cambogia on glucose homeostasis in obesity-induced diabetes are explored and the underlying mechanisms are investigated., Methods and Results: C2C12 myotubes are treated with G. cambogia; glucose uptake, intracellular Ca 2+ levels, and related alterations in signaling pathways are examined. High-fat diet (HFD)-fed mice are administered G. cambogia for 8 weeks; oral glucose tolerance is evaluated, and the regulation of identified targets of signaling pathways in quadriceps skeletal muscle are examined in vivo. G. cambogia increases glucose uptake in C2C12 myotubes and induces the upregulation of AMPK, ACC, and p38 MAPK phosphorylation. Notably, G. cambogia markedly elevates both intracellular Ca 2+ levels, activating CaMKII, a Ca 2+ -sensing protein, and TBC1D4-mediated GLUT4 translocation, to facilitate glucose uptake. Furthermore, high-glucose-induced inhibition of glucose uptake and signal transduction is reverted by G. cambogia. In an HFD-induced diabetes mouse model, G. cambogia administration results in significant blood glucose-lowering effects, which are attributed to the regulation of targets that have been identified in vitro, in quadricep skeletal muscle., Conclusion: These findings provide new insights into the mechanism by which G. cambogia regulates glucose homeostasis in obesity-induced diabetes., (© 2022 Wiley-VCH GmbH.)

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway.

The overexpansion of adipose tissues leads to obesity and eventually results in metabolic disorders. Garcinia cambogia ( G. cambogia ) has been used as an antiobesity supplement. However, the molecular mechanisms underlying the effects of G. cambogia on cellular processes have yet to be fully understood. Here, we discovered that G. cambogia attenuated the expression of CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), an important adipogenic factor, suppressing its transcription in differentiated cells. In addition, G. cambogia inhibited macroautophagic/autophagic flux by decreasing autophagy-related gene expression and autophagosome formation. Notably, G. cambogia markedly elevated the expression of KLF3 (Kruppel-like factor 3 (basic)), a negative regulator of adipogenesis, by reducing SQSTM1/p62-mediated selective autophagic degradation. Furthermore, increased KLF3 induced by G. cambogia interacted with CTBP2 (C-terminal binding protein 2) to form a transcriptional repressor complex and inhibited Cebpa and Pparg transcription. Importantly, we found that RPS6KA1 and STAT3 were involved in the G. cambogia -mediated regulation of CEBPB and autophagic flux. In an obese animal model, G. cambogia reduced high-fat diet (HFD)-induced obesity by suppressing epididymal and inguinal subcutaneous white adipose tissue mass and adipocyte size, which were attributed to the regulation of targets that had been consistently identified in vitro . These findings provide new insight into the mechanism of G. cambogia -mediated regulation of adipogenesis and suggest molecular links to therapeutic targets for the treatment of obesity. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG: autophagy-related; Baf: bafilomycin A 1 ; BECN1: beclin 1; CEBP: CCAAT/enhancer binding protein (C/EBP); CHX: cycloheximide; CREB: cAMP response element binding protein; CTBP: C-terminal binding protein; EGCG: (-)-epigallocatechin gallate; eWAT: epididymal white; G. cambogia: Garcinia cambogia ; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HFD: high-fat diet; iWAT: inguinal subcutaneous white; KLF: Kruppel-like factor; LAP: liver-enriched transcriptional activating proteins; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; PPARG: peroxisome proliferator activated receptor gamma; qPCR: quantitative real-time PCR; RFP: red fluorescent protein; RPS6KA1: ribosomal protein S6 kinase A1; siRNA: small-interfering RNA; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TEM: transmission electron microscopy.

Background and Purpose: Abnormal vascular smooth muscle cell (VSMC) proliferation and migration lead to neointima formation, which eventually results in cardiovascular hyperplastic diseases. The molecular mechanisms underlying these cellular processes have not been fully understood. Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) has been identified as an anti-apoptotic molecule, but little is known about its target genes and related pathways in VSMC dysfunction or its clinical implication in neointima formation following vascular injury., Experimental Approach: Determination, using loss/gain-of-function approaches by gene delivery, of whether CIAPIN1 modulates VSMC proliferation, migration and neointima formation and the underlying mechanisms was carried out. Balloon injury or ligation and local delivery of lentivirus were performed on rat or mouse carotid arteries. Rat aortic smooth muscle cells, the primary cell, was used as the model to evaluate the effect of CIAPIN1 on proliferation and migration., Key Results: CIAPIN1 was overexpressed in the neointimal region of rat arteries. CIAPIN1 deficiency markedly inhibited injury-induced or ligation-induced intimal hyperplasia and suppressed PDGF-BB-induced VSMC proliferation, migration and cell cycle progression, while overexpression promoted proliferation, migration and neointima formation. CIAPIN1 negatively regulated Tp53 transcription, which promoted cell cycle progression and migration via cyclin E1-CDK2/pRb/PCNA and the MMP2 pathway. CIAPIN1 also increased JAK2 expression, enhancing JAK2 and STAT3 phosphorylation by vascular injury, which forced phenotypic switching from contractile to synthetic state in injured arteries., Conclusions and Implications: These findings provide new insights into the mechanism by which CIAPIN1 regulates vascular remodelling and suggest a novel therapeutic target for treating vascular proliferative diseases., (© 2021 The British Pharmacological Society.)

Breast cancer (BC) a very common cancer in women worldwide. Triple negative breast cancer (TNBC) has been shown to have a poor prognosis with a high level of tumor metastatic spread. Here, the inhibitory effects of ginsenoside-Rh1 (Rh1) on BC metastasis, and its underlying signaling pathway in TNBC were investigated. Rh1-treated MDA-MB-231 cells were analyzed for metastasis using a wound healing assay, transwell migration and invasion assay, western blotting, and qRT-PCR. Rh1 treatment significantly inhibited BC metastasis by inhibiting the both protein and mRNA levels of MMP2, MMP9, and VEGF-A. Further, Rh1-mediated inhibitory effect on BC migration was associated with mitochondrial ROS generation. Rh1 treatment significantly eliminated STAT3 phosphorylation and NF-κB transactivation to downregulate metastatic factors, such as MMP2, MMP9, and VEGF-A. In addition, Mito-TEMPO treatment reversed Rh1 effects on the activation of STAT3, NF-κB, and their transcriptional targets. Rh1 further enhanced the inhibitory effects of STAT3 or NF-κB specific inhibitor, stattic or BAY 11-7082 on MMP2, MMP9, and VEGF-A expression, respectively. In summary, our results revealed the potent anticancer effect of Rh1 on TNBC migration and invasion through mtROS-mediated inhibition of STAT3 and NF-κB signaling.

Propionate is a short-chain fatty acid (SCFA) mainly produced from carbohydrates by gut microbiota. Sodium propionate (SP) has shown to suppress the invasion in G protein-coupled receptor 41 (GPR41) and GPR43-overexpressing breast cancer cells. In this study we investigated the effects of SP on the proliferation, apoptosis, autophagy, and antioxidant production of breast cancer cells. We showed that SP (5-20 mM) dose-dependently inhibited proliferation and induced apoptosis in breast cancer cell lines JIMT-1 (ER-negative and HER2-expressing) and MCF7 (ER-positive type), and this effect was not affected by PTX, thus not mediated by the GPR41 or GPR43 SCFA receptors. Meanwhile, we demonstrated that SP treatment increased autophagic and antioxidant activity in JIMT-1 and MCF7 breast cancer cells, which might be a compensatory mechanism to overcome SP-induced apoptosis, but were not sufficient to overcome SP-mediated suppression of proliferation and induction of apoptosis. We revealed that the anticancer effect of SP was mediated by inhibiting JAK2/STAT3 signaling which led to cell-cycle arrest at G 0 /G 1 phase, and increasing levels of ROS and phosphorylation of p38 MAPK which induced apoptosis. In nude mice bearing JIMT-1 and MCF7 cells xenograft, administration of SP (20 mg/mL in drinking water) significantly suppressed tumor growth by regulating STAT3 and p38 in tumor tissues. These results suggest that SP suppresses proliferation and induces apoptosis in breast cancer cells by inhibiting STAT3, increasing the ROS level and activating p38. Therefore, SP is a candidate therapeutic agent for breast cancer., (© 2020. CPS and SIMM.)

Excessive free fatty acids (FFAs) causes reactive oxygen species (ROS) generation and non-alcoholic fatty liver disease (NAFLD) development. Garcinia cambogia ( G. cambogia ) is used as an anti-obesity supplement, and its protective potential against NAFLD has been investigated. This study aims to present the therapeutic effects of G. cambogia on NAFLD and reveal underlying mechanisms. High-fat diet (HFD)-fed mice were administered G. cambogia for eight weeks, and steatosis, apoptosis, and biochemical parameters were examined in vivo. FFA-induced HepG2 cells were treated with G. cambogia , and lipid accumulation, apoptosis, ROS level, and signal alterations were examined. The results showed that G. cambogia inhibited HFD-induced steatosis and apoptosis and abrogated abnormalities in serum chemistry. G. cambogia increased in NRF2 nuclear expression and activated antioxidant responsive element (ARE), causing induction of antioxidant gene expression. NRF2 activation inhibited FFA-induced ROS production, which suppressed lipogenic transcription factors, C/EBPα and PPARγ. Moreover, the ability of G. cambogia to inhibit ROS production suppressed apoptosis by normalizing the Bcl-2/BAX ratio and PARP cleavage. Lastly, these therapeutic effects of G. cambogia were due to hydroxycitric acid (HCA). These findings provide new insight into the mechanism by which G. cambogia regulates NAFLD progression.

In this study, we investigated the anti-cancer effects of ginsenoside Rg2 (G-Rg2) and its underlying signaling pathways in breast cancer (BC) cells. G-Rg2 significantly induced cytotoxicity and reactive oxygen species (ROS) production in MCF-7 cells among various types of BC cells including HCC1428, T47D, and BT-549. G-Rg2 significantly inhibited protein and mRNA expression of cell cycle G1-S phase regulators, including p-Rb, cyclin D1, CDK4, and CDK6, whereas it enhanced the protein and mRNA expression of cell cycle arrest and apoptotic molecules including cleaved PARP, p21, p27, p53 and Bak through ROS production. These effects were abrogated by the antioxidant N-acetyl-I-cysteine, or NADPH oxidase inhibitors, such as diphenyleneiodonium chloride and apocynin. Interestingly, G-Rg2 induced mitochondrial damage by reducing the membrane potential. G-Rg2 further activated the ROS-sensor protein, AMPK and downstream targets of AMPK activation, including PGC-1α, FOXO1, and IDH2, and downregulated mTOR activation and antioxidant response element-driven luciferase activity. Together, our data demonstrate that G-Rg2 mediates anti-cancer effects by activating cell cycle arrest and signaling pathways related to mitochondrial damage-induced ROS production and apoptosis., (© 2021. The Pharmaceutical Society of Korea.)

The secretion of platelet-derived growth factors (PDGFs) into vascular smooth muscle cells (VSMCs) induced by specific stimuli, such as oxidized low-density lipoprotein (LDL) cholesterol, initially increases the proliferation and migration of VSMCs, and continuous stimulation leads to VSMC apoptosis, resulting in the formation of atheroma. Autophagy suppresses VSMC apoptosis, and statins can activate autophagy. Thus, this study aimed to investigate the mechanism of the autophagy-mediated vasoprotective activity of rosuvastatin, one of the most potent statins, in VSMCs continuously stimulated with PDGF-BB, a PDGF isoform, at a high concentration (100 ng/ml) to induce phenotypic switching of VSMC. Rosuvastatin inhibited apoptosis in a concentration-dependent manner by reducing cleaved caspase-3 and interleukin-1 β (IL-1 β ) levels and reduced intracellular reactive oxygen species (ROS) levels in PDGF-stimulated VSMCs. It also inhibited PDGF-induced p38 phosphorylation and increased the expression of microtubule-associated protein light chain 3 (LC3) and the conversion of LC3-I to LC3-II in PDGF-stimulated VSMCs. The ability of rosuvastatin to inhibit apoptosis and p38 phosphorylation was suppressed by treatment with 3-methyladenine (an autophagy inhibitor) but promoted by rapamycin (an autophagy activator) treatment. SB203580, a p38 inhibitor, reduced the PDGF-induced increase in intracellular ROS levels and inhibited the formation of cleaved caspase-3, indicating the suppression of apoptosis. In carotid ligation model mice, rosuvastatin decreased the thickness and area of the intima and increased the area of the lumen. In conclusion, our observations suggest that rosuvastatin inhibits p38 phosphorylation through autophagy and subsequently reduces intracellular ROS levels, leading to its vasoprotective activity. SIGNIFICANCE STATEMENT: This study shows the mechanism responsible for the vasoprotective activity of rosuvastatin in vascular smooth muscle cells under prolonged platelet-derived growth factor stimulation. Rosuvastatin inhibits p38 activation through autophagy, thereby suppressing intracellular reactive oxygen species levels, leading to the inhibition of apoptosis and reductions in the intima thickness and area. Overall, these results suggest that rosuvastatin can be used as a novel treatment to manage chronic vascular diseases such as atherosclerosis., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Breast cancer (BC) is the leading cause of cancer-related deaths among women worldwide. Ginsenosides exhibit anticancer activity against various cancer cells. However, the effects of ginsenoside Rh1 on BC and the underlying mechanisms remain unknown. Here, we investigated the anticancer effects of Rh1 on human BC MCF-7 and HCC1428 cells and the underlying signaling pathways. The anticancer effects of Rh1 in vitro were evaluated using sulforhodamine B (SRB), 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), clonogenic assay, propidium iodide (PI)/Hoechst staining, Western blotting, flow cytometry, and immunofluorescence analysis. The in vivo effects of Rh1 were determined using a xenograft model via hematoxylin and eosin and the immunohistochemistry staining of tumor tissues. We found that Rh1 exerted cytotoxicity in the cells by increasing cell apoptosis, autophagy, and cell cycle arrest. These effects were further enhanced by a phosphatidylinositol 3-kinase (PI3K) inhibitor but were rescued by the inhibition of reactive oxygen species (ROS). Moreover, enhanced ROS generation by Rh1 inhibited the activation of the PI3K/Akt pathway. Consistently, Rh1 treatment significantly reduced tumor growth in vivo and increased the ROS production and protein expression of LC3B and cleaved caspase-3 but decreased the phosphorylation of Akt and retinoblastoma (Rb) in tumor tissues. Taken together, Rh1 exerted a potential anticancer effect on BC cells by inducing cell cycle arrest, apoptosis, and autophagy via inhibition of the ROS-mediated PI3K/Akt pathway.

Obesity is associated with an increase in adipose tissue, which is mediated by hyperplasia and hypertrophy. Therefore, inhibiting cell proliferation during mitotic clonal expansion (MCE) is one of the major strategies for preventing obesity. The antagonistic effects of Garcinia cambogia (G. cambogia) on obesity have been studied in animal experimental models. However, the effects of G. cambogia extract on MCE, and the underlying molecular mechanisms, are poorly understood. In this study, 3T3-L1 cells were used to investigate whether G. cambogia extract affected cell proliferation during MCE and to identify target molecules for any anti-adipogenic activity. G. cambogia extract suppressed isobutylmethylxanthine and dexamethasone-and-insulin (MDI)-induced adipogenesis at an early stage by attenuating MCE. In G. cambogia extract-treated preadipocytes, MDI-induced cell proliferation and cell cycle progression were inhibited by G 0 /G 1 arrest due to an increase in p21 and p27 expression, and inhibition of cyclin-dependent kinase 2, cyclin E1 expression, and retinoblastoma (Rb) phosphorylation. In addition, the MDI-induced phosphorylation and subsequent translocation into the nucleus of p90 ribosomal S6 kinase (p90RSK) and signal transducer and activator of transcription (Stat) 3 were suppressed. Specific inhibitors of p90RSK (FMK) and Stat3 (stattic) regulated cell proliferation and adipogenesis. In conclusion, this study demonstrated that G. cambogia extract inhibited MCE by regulating p90RSK, Stat3, and cell cycle proteins, leading to G 0 /G 1 arrest. These findings provide new insight into the mechanism by which G. cambogia suppresses adipocyte differentiation and show that p90RSK is critical for adipogenesis as a new molecular target., (© 2020 Wiley Periodicals LLC.)

Aims: Vascular smooth muscle cell (VSMC) phenotype shift is involved in the pathophysiology of vascular injury or platelet-derived growth factor (PDGF)-induced abnormal proliferation and migration of VSMCs. We aimed to investigate the underlying mechanism involved in PDGF-mediated signaling pathways and autophagy regulation followed by VSMC phenotype shift., Main Methods: The proliferation, migration and apoptosis of cultured rat aortic VSMCs were measured, and cells undergoing phenotype shift and autophagy were examined. Specific inhibitors for target proteins in signaling pathways were applied to clarify their roles in regulating cell functions., Key Findings: PDGF-BB stimulation initiated autophagy activation and synthetic phenotype transition by decreasing α-smooth muscle-actin (SMA), calponin and myosin heavy chain (MHC) and increasing osteopontin (OPN) expression. However, U0126, a potent extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, decreased PDGF-BB-induced LC3 expression, while rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), increased it. Furthermore, U0126 decreased the expresseion of autophagy-related genes (Atgs) such as beclin-1, Atg7, Atg5, and Atg12-Atg5 complex, indicating that Erk1/2 is a regulator of PDGF-BB-induced VSMC autophagy. Regardless of autophagy inhibition by U0126 or activation by rapamycin, the PDGF-BB-induced decrease in SMA, calponin and MHC and increase in OPN expression were inhibited. Furthermore, PDGF-BB-stimulated VSMC proliferation, migration and proliferating cell nuclear antigen (PCNA) expression were inhibited by U0126 and rapamycin., Significance: These findings suggest that PDGF-BB-induced autophagy is strongly regulated by Erk1/2, an mTOR-independent pathway, and any approach for targeting autophagy modulation is a potential therapeutic strategy for addressing abnormal VSMC proliferation and migration., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Obesity is a major health concern worldwide, and it is leading to worsening disease morbidity and mortality. Herbal supplements and diet-based therapies have attracted interest in the treatment of obesity. It is known that Garcinia cambogia (GA) and mulberry leaf, which contain polyphenols, have anti-obesity activity. Herein, we developed a combined tablet consisting of GA extract and bioconverted mulberry leaf extract (BMUL) using a statistical design approach. The ratio and amount of sustained polymers were set as factors. In the cell study, the combination of GA and BMUL showed synergistic anti-obesity activity. In a statistical model, the optimized amounts of hydroxypropyl methylcellulose 2208 (HPMC 2208) and polyethylene oxide 303 (POLYOX 303) were 41.02% and 58.98%, respectively. Additionally, the selected ratio of microcrystalline cellulose (MCC) was 0.33. When the release, hardness, and friability of the GABMUL tablet were evaluated, the error percentages of the response were lower than 10%. This indicates that the GABMUL tablet was successfully prepared.

Ginsenosides have been reported to have various biological effects, such as immune regulation and anticancer activity. In this study, we investigated the anti-inflammatory role of a combination of Rg2 and Rh1, which are minor ginsenosides, in lipopolysaccharide (LPS)-stimulated inflammation. In vitro experiments were performed using the RAW264.7 cell line, and an in vivo model of inflammation was established using LPS-treated ICR mice. We employed Griess assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, quantitative reverse transcriptase-polymerase chain reaction, western blotting, immunofluorescence staining, and hematoxylin and eosin staining to evaluate the effect of Rg2 and Rh1. We found that Rg2 and Rh1 significantly decreased LPS-induced major inflammatory mediator production, inducible-nitric oxide synthase expression, and nitric oxide production in macrophages. Moreover, Rg2 and Rh1 combination treatment inhibited the binding of LPS to toll-like receptor 4 (TLR4) on peritoneal macrophages. Therefore, the combination of ginsenoside Rg2 and Rh1 suppressed inflammation by abolishing the binding of LPS to TLR4, thereby inhibiting the TLR4-mediated signaling pathway. The combined ginsenoside synergistically blocked LPS-mediated PKCδ translocation to the plasma membrane, resulting in p38-STAT1 activation and NF-κB translocation. In addition, mRNA levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IFN-β, were significantly decreased by combined ginsenoside treatment. Notably, the 20 mg/kg ginsenoside treatment significantly reduced LPS-induced acute tissue inflammation levels in vivo, as indicated by the tissue histological damage scores and the levels of biochemical markers for liver and kidney function from mouse serum. These results suggest that the minor ginsenosides Rg2 and Rh1 may play a key role in prevention of LPS-induced acute inflammation and tissue damage.

The incidence of rheumatoid arthritis (RA), an autoimmune inflammatory disease, is rapidly increasing in aging societies. In the current study, celecoxib (CXB) micelles were developed to improve the oral absorption and anti-inflammatory effects of CXB in cell studies and λ-carrageenan rat models, and to enhance the therapeutic effects of CXB on RA in complete Freund's adjuvant (CFA)-induced RA rat models. Moreover, CXB micelles and previously developed solid dispersion (SD6) formulations were evaluated. The physical properties of optimal CXB micelles (M3), such as crystallinity, thermal properties, and intramolecular interactions, were altered. Compared with the commercial product (Celebrex®), the M3 and SD6 formulations showed significantly improved anti-inflammatory effects in terms of nitric oxide reduction, 1.5-fold and 2.2-fold, respectively, at the cellular level. The relative bioavailability (BA) of the M3 and SD6 formulations was also significantly improved as oral bioavailability (167.2% and 219.8% respectively), compared with that of Celebrex®. In particular, M3 and SD6 significantly reduced inflammation and edema volume relative to Celebrex® in CFA-induced RA rat models. Moreover, both M3 and SD6 effectively suppressed CFA-induced pro-inflammatory cytokines (TNF-α and IL-1β) in rat splenic tissues. In conclusion, polymeric systems improved the solubility, relative BA (%) and anti-inflammatory effects of CXB. Thus, CXB polymeric systems show potential as therapeutic agents against inflammation and RA and may need to be tested at the clinical level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Angiotensin II (Ang II) has been reported to induce vascular smooth muscle cell (VSMC) proliferation and migration, which are major events that are highly linked to vascular diseases such as atherosclerosis and restenosis. p90 ribosomal S6 kinase (p90RSK), a potential downstream effector of ERK1/2, has been demonstrated to be activated by Ang II in VSMCs. However, the role of p90RSK on Ang II-induced VSMC proliferation and migration and its underlying signaling pathways remain unknown. In this study, we found that the inhibition of p90RSK, using a p90RSK specific inhibitor FMK or transfected cells with a plasmid encoding dominant negative RSK1, inactivated p90RSK kinase action completely and suppressed Ang II-induced rat aortic smooth muscle cell (RASMC) proliferation and migration. Interestingly, inhibition of p90RSK kinase activity abolished the phosphorylation of Akt as well as the protein expression of ICAM-1, VCAM-1, MMP-2, and NF-κB p65 in Ang II-treated RASMCs. Furthermore, the luciferase reporter assay revealed the inhibitory effect of FMK on NF-κB promoter activity induced by Ang II. Notably, using the partial carotid ligation model in mice, FMK was found to attenuate the medial thickness of carotid arteries increased by Ang II. Taken together, these results suggest that p90RSK plays a critical role in Ang II-induced VSMC proliferation and migration by increasing Akt phosphorylation and NF-κB p65 promoter activity associated with up-regulation of adhesion molecules and MMP-2 expression., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cisplatin (Cis-DDP) is one of the most widely used anti-cancer drugs. It is applicable to many types of cancer, including lung, bladder, and breast cancer. However, its use is now limited because of drug resistance. p90 ribosomal S6 kinase (p90RSK) is one of the downstream effectors in the extracellular signalregulated protein kinases 1 and 2 (ERK1/2) pathway and high expression of p90RSK is observed in human breast cancer tissues. Therefore, we investigated the role of p90RSK in the Cis-DDP resistance-related signaling pathway and epithelialmesenchymal transition (EMT) in breast cancer cells. First, we discovered that MDA-MB-231 cells exhibited more Cis-DDP resistance than other breast cancer cells, including MCF-7 and BT549 cells. Cis-DDP increased p90RSK activation, whereas the inactivation of p90RSK using a small interfering RNA (siRNA) or dominant-negative kinase mutant plasmid overexpression significantly reduced Cis-DDP-induced cell proliferation and migration via the inhibition of matrix metallopeptidase (MMP)2 and MMP9 in MDA-MB-231 cells. In addition, p90RSK activation was involved in EMT via the upregulation of mRNA expression, including that of Snail, Twist, ZEB1, N-cadherin, and vimentin. We also investigated NF-κB, the upstream regulator of EMT markers, and discovered that Cis-DDP treatment led to NF-κB translocation in the nucleus as well as its promoter activity. Our results suggest that targeting p90RSK would be a good strategy to increase Cis-DDP sensitivity in triple-negative breast cancers. [BMB Reports 2019; 52(12): 706-711].

Ethnopharmacological Relevance: Acer tegmentosum Maxim (AT), the East Asian stripe maple, is an herb used to treat liver disease and is approved as a functional food in Korea. AT protects against hepatic disorders, atopic dermatitis, and diabetes mellitus., Aim of the Study: We explored the mechanism of the hepatoprotective effects of AT extract in in vitro and in vivo levels., Materials and Methods: AT extract from Acer tegmentosum Maxim was extracted by hot water. Hepatoprotective effects of AT extract were confirmed using carbon tetrachloride (CCl 4 )- or alcohol-induced mouse model, and H 2 O 2 - or alcohol-induced HepG2 (liver hepatocellular carcinoma cell line) cells by measuring GOT, GPT, TG, and MDA levels. Hematoxylin and eosin (H&E) staining was used to observe the pathological analysis. Cytotoxicity or protective effect of AT extract was confirmed using MTT assay in HepG2 cells. Antioxidant effect of AT extract was measured using DPPH or H 2 DCFDA assay. Mechanism study of antioxidant and autophagy was carried out using western blotting and immunofluorescence analysis., Results: AT extract increased the viability of HepG2 cells treated with H 2 O 2 and ethanol, and protected the liver against damage induced by CCl 4 and alcohol. The AT extract increased the levels of nuclear respiratory factor 2 (Nrf2) and heme oxygenase-1 (HO-1). The level of microtubule-associated protein light chain 3 (LC3)-Ⅱ, beclin-1, autophagy-related genes (Atg) such as Atg3 and Atg12-5 as markers of autophagy activation was also increased. Moreover, the AT extract increased activation of mitogen-activated protein kinase (MAPK), which regulated autophagy and HO-1., Conclusion: Therefore, these results indicate that the AT extract has a hepatoprotective effect by increasing antioxidant activity and inducing autophagy., (Copyright © 2019. Published by Elsevier B.V.)

Background: Mulberry is a Korean medicinal herb that shows effective prevention and treatment of obesity and diabetes. Bioconversion is the process of producing active ingredients from natural products using microorganisms or enzymes., Methods: In this study, we prepared bioconverted mulberry leaf extract (BMLE) with Viscozyme L, which we tested in insulin-sensitive cells (i.e., skeletal muscle cells and adipocytes) and insulin-secreting pancreatic β-cells, as well as obese diabetic mice induced by co-administration of streptozotocin (100 mg/kg, IP) and nicotinamide (240 mg/kg, IP) and feeding high-fat diet, as compared to unaltered mulberry leaf extract (MLE)., Results: BMLE increased the glucose uptake in C2C12 myotubes and 3 T3-L1 adipocytes and increased glucose-stimulated insulin secretion in HIT-T15 pancreatic β-cells. The fasting blood glucose levels in diabetic mice treated with BMLE or MLE (300 and 600 mg/kg, PO, 7 weeks) were significantly lower than those of the vehicle-treated group. At the same concentration, BMLE-treated mice showed better glucose tolerance than MLE-treated mice. Moreover, the blood concentration of glycated hemoglobin (HbA 1C ) in mice treated with BMLE was lower than that in the MLE group at the same concentration. Plasma insulin levels in mice treated with BMLE or MLE tended to increase compared to the vehicle-treated group. Treatment with BMLE yielded significant improvements in insulin resistance and insulin sensitivity., Conclusion: These results indicate that in the management of diabetic condition, BMLE is superior to unaltered MLE due to at least, in part, high concentrations of maker compounds (trans-caffeic acid and syringaldehyde) in BMLE.

In this study, 19 known compounds were isolated from mulberry (Morus alba L.) leaves. The lipid accumulation inhibitory activity of the isolated compounds was investigated. Compounds 4 and 12 showed good anti-adipogenic activity based on 3T3-L1 adipocytes with values of 36.6 ± 9.0 and 34.7 ± 4.0%, respectively. In addition, compounds 3, 6 and 15 showed significant inhibitory activity with values from 15.4 to 21.2% and compounds 2, 8-9 and 17-18 exhibited weak activity with values ranging from 2.1 to 10.7% at a concentration of 40.0 μM. These results show the potentiality that mulberry leaf is an excellent inhibitory phytochemical source against lipid accumulation.

Autophagy has been studied as a therapeutic strategy for cardiovascular diseases. However, insufficient studies have been reported concerning the influence of vascular smooth muscle cells (VSMCs) through autophagy regulation. The aim of the present study was to determine the effects of VSMCs on the regulation of autophagy under in vitro conditions similar to vascular status of the equipped microtubule target agent-eluting stent and increased release of platelet-derived growth factor-BB (PDGF-BB). Cell viability and proliferation were measured using MTT and cell counting assays. Immunofluorescence using an anti-α-tubulin antibody was performed to determine microtubule dynamic formation. Cell apoptosis was measured by cleavage of caspase-3 using western blot analysis, and by nuclear fragmentation using a fluorescence assay. Autophagy activity was assessed by microtubule-associated protein light chain 3-II (LC-II) using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured using H 2 DCFDA. The proliferation and viability of VSMCs were inhibited by microtubule regulation. Additionally, microtubule-regulated and PDGF-BB-stimulated VSMCs increased the cleavage of caspase-3 more than only the microtubule-regulated condition, similar to that of LC3-II, implying autophagy. Inhibitory autophagy of microtubule-regulated and PDGF-BB-stimulated VSMCs resulted in low viability. However, enhancement of autophagy maintained survival through the reduction of ROS. These results suggest that the apoptosis of conditioned VSMCs is decreased by the blocking generation of ROS via the promotion of autophagy, and proliferation is also inhibited. Thus, promoting autophagy as a therapeutic target for vascular restenosis and atherosclerosis may be a good strategy., Competing Interests: CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Scope: Momordica charantia (M. charantia) has antidiabetic effects, and cucurbitane-type triterpenoid is one of the compounds of M. charantia. This study aims to investigate whether the new cucurbitane-type triterpenoids affect insulin sensitivity both in vitro and in vivo, and the underlying mechanisms., Methods and Results: Four compounds (C1-C4) isolated from the ethanol extract of M. charantia enhance glucose uptake in C2C12 myotubes via insulin receptor substrate-1 (IRS-1) rather than via adenosine monophosphate-activated protein kinase. The most potent, compound 2 (C2), significantly increases the activation of IRS-1 and downstream signaling pathways, resulting in glucose transporter 4 translocation. Furthermore, these C2-induced in vitro effects are blocked by specific signal inhibitors. We further evaluate the antidiabetic effect of C2 using a streptozotocin (STZ)-induced diabetic mouse model. Consistent with in vitro data, treatment with C2 (1.68 mg kg -1 ) significantly decreases blood glucose level and enhances glycogen storage in STZ-injected mice. These effects appear to be mediated by the IRS-1 signaling pathway in skeletal muscle, not in adipose and liver tissues, suggesting that C2 improves hyperglycemia by increasing glucose uptake into skeletal muscle., Conclusion: Our findings demonstrate that the new cucurbitane-type triterpenoids have potential for prevention and management of diabetes by improving insulin sensitivity and glucose homeostasis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ascorbic acid is one of the most well-known nutritional supplement and antioxidant found in fruits and vegetables. Calcium ascorbate has been developed to mitigate the gastric irritation caused by the acidity of ascorbic acid. The aim of this study was to compare calcium ascorbate and ascorbic acid, focusing on their antioxidant activity and effects on gastric juice pH, total acid output, and pepsin secretion in an in vivo rat model, as well as pharmacokinetic parameters. Calcium ascorbate and ascorbic acid had similar antioxidant activity. However, the gastric fluid pH was increased by calcium ascorbate, whereas total acid output was increased by ascorbic acid. In the rat pylorus ligation-induced ulcer model, calcium ascorbate increased the gastric fluid pH without changing the total acid output. Administration of calcium ascorbate to rats given a single oral dose of 100 mg/kg as ascorbic acid resulted in higher plasma concentrations than that from ascorbic acid alone. The area under the curve (AUC) values of calcium ascorbate were 1.5-fold higher than those of ascorbic acid, and the C max value of calcium ascorbate (91.0 ng/ml) was higher than that of ascorbic acid (74.8 ng/ml). However, their T max values were similar. Thus, although calcium ascorbate showed equivalent antioxidant activity to ascorbic acid, it could attenuate the gastric high acidity caused by ascorbic acid, making it suitable for consideration of use to improve the side effects of ascorbic acid. Furthermore, calcium ascorbate could be an appropriate antioxidant substrate, with increased oral bioavailability, for patients with gastrointestinal disorders., Competing Interests: CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Background and Purpose: The proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) are important steps in cardiovascular diseases, including neointimal lesion formation, myocardial infarction and atherosclerosis. Here, we evaluated the rubiarbonone C-mediated signalling pathways that regulate PDGF-induced VSMC proliferation and migration., Experimental Approach: Cell proliferation and migration were measured in cells treated with rubiarbonone C followed by PDGF BB using the MTT assay, [ 3 H]-thymidine incorporation, flow cytometry and wound-healing migration assay, MMP gelatin zymography, a fluorescence assay for F-actin. Western blotting of molecules including MAPK, focal adhesion kinase (FAK) and STAT3 and an immunofluorescence assay using anti-PCNA and -STAT3 antibodies were performed to evaluate rubiarbonone C signalling pathway(s). The medial thickness of the carotid artery was evaluated using a mouse carotid ligation model., Key Results: Rubiarbonone C inhibited PDGF-induced VSMC proliferation and migration and diminished the ligation-induced increase in medial thickness of the carotid artery. In PDGF-stimulated VSMCs rubiarbonone C decreased the following: (i) levels of cyclin-dependent kinases, cyclins, PCNA and hyperphosphorylated retinoblastoma protein; (ii) levels and activity of MMP2 and MMP9; (iii) activation of MAPK; (iv) F-actin reorganization, by reducing FAK activation; (v) activation of STAT3., Conclusions and Implications: These findings suggest that rubiarbonone C inhibits the proliferation and migration of VSMCs by inhibiting the FAK, MAPK and STAT3 signalling pathways. Therefore, rubiarbonone C could be a good candidate for the treatment of cardiovascular disease., (© 2017 The British Pharmacological Society.)

Atherothrombosis is one of the main underlying cause of cardiovascular diseases. In addition to treating atherothrombosis with antithrombotic agents, there is growing interest in the role of natural food products and biologically active ingredients for the prevention and treatment of cardiovascular diseases. This study aimed to investigate the effect of secolincomolide A ( 3 ) isolated from Lindera obtusiloba Blume on platelet activity and identify possible signaling pathways. In our study, the antiplatelet activities of 3 were measured by collagen-induced platelet aggregation and serotonin secretion in freshly isolated rabbit platelets. Interestingly, 3 effectively inhibited the collagen-induced platelet aggregation and serotonin secretion via decreased production of diacylglycerol, arachidonic acid, and cyclooxygenase-mediated metabolites such as thromboxane B 2 (TXB 2 ), and prostaglandin D 2 (PGD 2 ). In accordance with the antiplatelet activities, 3 prolonged bleeding time and attenuated FeCl 3 -induced thrombus formation in arterial thrombosis model. Notably, 3 abolished the phosphorylation of phospholipase Cγ2 (PLCγ2), spleen tyrosine kinase (Syk), p47, extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt) by inhibiting the activation of the collagen receptor, glycoprotein VI (GPVI). Taken together, our results indicate the therapeutic potential of 3 in antiplatelet action through inhibition of the GPVI-mediated signaling pathway and the COX-1-mediated AA metabolic pathways.

The cucurbitaceous plant Momordica charantia L., named "bitter melon", inhabits Asia, Africa, and South America and has been used as a traditional medicine. The atypical proliferation of vascular smooth muscle cells (VSMCs) plays an important role in triggering the pathogenesis of cardiovascular diseases. Platelet-derived growth factor (PDGF) is regarded as the most powerful growth factor in promoting the intimal accumulation of VSMCs. The current study features the identification of six new cucurbitane-type triterpenoids (1-6) from the fruits of M.  charantia, utilizing diverse chromatographic and spectroscopic techniques. In particular, the 2D structure of 1 was confirmed utilizing the long-range HSQMBC NMR pulse, capable of measuring heteronuclear long-range correlations ( 4-6 J CH ). The cucurbitanes were also assessed for their inhibitory activity against PDGF-induced VSMC proliferation. This current study may constitute a basis for developing those chemotypes into sensible pharmacophores alleviating cardiovascular disorders.

Sobokchukeo-Tang (ST) is a well-known formula that is used for treating primary dysmenorrhea caused by blood stasis syndrome (BSS) in Korea and China. The current study investigated the anti‑inflammatory and anti‑adipogenesis effects of ST on adipocytes and macrophages. The anti‑inflammatory efficacy of ST was evaluated in RAW 264.7 cells and differentiated THP‑1 cells. To induce inflammation, the cells were treated with lipopolysaccharide (LPS; 1 µg/ml). Following the induction of inflammation, the levels of proinflammatory cytokines, interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α) in the cell supernatant were detected using enzyme‑linked immunosorbent assay. 3T3‑L1 preadipocytes differentiated into adipocytes in response to insulin, isobutyl‑1‑methylxanthine and dexamethasone (MDI). To confirm the anti‑adipogenesis efficacy of ST, we investigated Oil Red O staining was performed, triglyceride (TG) and leptin secretion were measured, and the protein expression of lipid metabolism‑associated factors was determined. ST significantly inhibited TNF‑α and IL‑6 production in the LPS‑treated RAW 264.7 cells compared with LPS stimulation alone. In addition, the concentrations of IL‑6 and TNF‑α were significantly inhibited by ST in LPS‑treated THP‑1 cells. Lipid accumulation was reduced by ST, similarly to the positive control treatment, SB203580. In the ST‑treated group, the TG and leptin concentrations were inhibited by up to 50 and 83%, respectively, compared with MDI induction only. The ST‑treated group reduced the protein expression of peroxisome proliferator‑activated receptor‑γ and CCAAT/enhancer‑binding protein α compared with MDI induction only. The results of the present study demonstrated that ST exerts anti‑inflammatory effects on LPS‑treated mouse and human macrophage cell lines. ST inhibited adipogenesis in MDI‑induced 3T3‑L1 adipocytes, as indicated by the significant reduction in TG and leptin concentrations without cytotoxicity. Thus, ST may be useful as a therapeutic agent for preventing lipid‑associated diseases, including obesity and atherosclerosis.

The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are associated with cardiovascular diseases and related complications. Such deleterious proliferation and migration events are triggered by cytokines and growth factors, and among them, platelet-derived growth factor (PDGF) is recognized as the most potent inducer. Despite the genus Rubia being researched to identify valuable commercial and medicinal virtues, Rubia philippinensis has rarely been investigated. Nine arborinane-type triterpenoids (1-9) were identified from this underutilized plant species. In particular, 4 was identified as the first arborinane derivative carrying a ketocarbonyl motif at C-19. The presence of the cyclopentanone moiety and the associated configurational assignment were determined by utilizing NOE and coupling constant analysis. These compounds were assessed for their inhibitory potential on PDGF-induced proliferation and the migration of VSMCs. Treatment with 5 μM compound 5 (62.6 ± 10.7%) and compound 9 (41.1 ± 4.7%) impeded PDGF-stimulated proliferation without exerting cytotoxicity. Compound 7 exhibited antimigration activity in a dose-dependent manner (38.5 ± 3.0% at 10 μM, 57.6 ± 3.2% at 30 μM). These results suggest that the arborinane-type triterpenoids may be a pertinent starting point for the development of cardiovascular drugs capable of preventing the intimal accumulation of VSMCs.

Shaofu Zhuyu decoction (SFZY) or Sobokchugeo-tang, a traditional herbal formula, is used as a treatment for primary dysmenorrhea. We searched four English, seven Korean, three Chinese, and one Japanese database from inception through January 2016 without a language restriction. All randomized controlled trials (RCTs) of SFZY or modified SFZY (MSFZY) were included. Data extraction and risk of bias assessments were performed by two independent reviewers. A total of 51 potentially relevant studies were identified, and 9 RCTs met our inclusion criteria. Seven RCTs tested the effects of SFZY or modified SFZY in treating dysmenorrhea. Three RCTs showed superior effects of (M)SFZY on the response rate, while the other three RCTs failed to do so (n=531, RR: 1.17, 95% CI: 1.09 to 1.26, P<0.0001, I(2)=0%). Three RCTs showed favorable effects of MSFZY for pain reduction compared with conventional drugs (n=340, SMD: -1.39, 95% CI: -2.23 to -0.55, P=0.01). Two RCTs examined the effects of modified SFZY plus conventional drugs and conventional drugs alone. The meta-analysis showed favorable effects of MSFZY (n=206; RR, 1.12; 95% CI 1.08 to 1.36; P=0.0009, I(2)=0%). Our systemic review and meta-analysis provide suggestive evidence of the superiority of SFZY over conventional drugs for treating primary dysmenorrhea. However, the level of evidence is low because of a high risk of bias., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Dysmenorrhea is a common gynecological complaint in adolescent and young females. The purpose of this study is to assess the efficacy of Shaofu Zhuyu (SFZY) decoctions as treatments for primary dysmenorrhea., Methods/design: Fifteen (four English, seven Korean, three Chinese, and one Japanese) databases will be searched from their inception without a language restriction. These include PubMed, AMED, EMBASE, The Cochrane Library, seven Korean Medical Databases (Korean Studies Information, DBPIA, Oriental Medicine Advanced Searching Integrated System, Research Information Service System, KoreaMed, The Town Society of Science Technology, and the Korean National Assembly Library), three Chinese Medical Databases [the Chinese Medical Database (CNKI), Chongqing VIP Chinese Science and Technology Periodical Database (VIP), and WanFang Database], and one Japanese Database (J global). Randomized clinical trials (RCTs) included those that examined an SFZY decoction or a modified SFZY decoction. The control groups include no treatment, placebo, and medication. Trials testing a combination of SFZY decoction and medication compare to the same medication alone will be also included. Data extraction and risk of bias assessments will be performed by two independent reviewers. All statistical analyses will be conducted using Review Manager software (RevMan V.5.3.0). Methodological quality will be assessed with the Cochrane risk of bias tool., Discussion: This systematic review will provide a detailed summary of the available evidence testing the effects of SFZY decoctions for the treatment of primary dysmenorrhea. The review will benefit patients and practitioners in the fields of traditional and complementary medicine., Systematic Review Registration: PROSPERO registration number: CRD42015016386.

Tadalafil is a phosphodiesterase-5 inhibitor indicated for the treatment of erectile dysfunction. In this study, we prepared and evaluated transdermal nanostructured lipid carriers (NLC) to improve the skin permeability of tadalafil. Tadalafil-loaded NLC dispersions were prepared using glyceryl monostearate as a solid lipid, oleic acid as a liquid lipid, and Tween 80 as a surfactant. We characterized the dispersions according to particle size, polydispersity index, zeta potential, encapsulation efficiency, and transmission electron microscopy. In vitro skin permeation studies were carried out using Franz diffusion cells, and cytotoxicity was examined using HaCaT keratinocyte cell lines. Tadalafil skin permeability increased for all tadalafil-loaded NLC formulations. The tadalafil-loaded NLC dispersion with ethanol and limonene as skin permeation enhancers exhibited the highest flux (∼4.8-fold) compared to that observed with tadalafil solution alone. Furthermore, a tadalafil-loaded NLC gel with selected permeation enhancers showed tolerance against toxicity in HaCaT cells. These results suggest that the NLC formulations with ethanol and limonene as skin permeation enhancers could be a promising dermal delivery carrier for tadalafil., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through G0/G1 to S phase of the cell cycle, as measured by [(3)H]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at G0/G1 phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis.

Sesamin, an active ingredient of Asiasarum heterotropoides, is known to exhibit many bioactive functions, but the effect thereof on vascular smooth muscle cell (VSMC) proliferation remains poorly understood. Hence, we explored the antiproliferative action of sesamin on VSMCs and the underlying mechanism thereof, focusing on possible effects of sesamin on cell cycle progression. Sesamin significantly inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation (inhibition percentage at 1, 5, and 10 μM sesamin was 49.8 ± 22.0%, 74.6 ± 19.9%, and 87.8 ± 13.0%, respectively) in the absence of cytotoxicity and apoptosis, and PDGF-induced DNA synthesis; and arrested cell cycle progression in the G0/G1-to-S phase. Sesamin potently inhibited cyclin D1 and CDK4 expression, pRb phosphorylation, and expression of the proliferating cell nuclear antigen (PCNA); and upregulated p27(KIP1), p21(CIP1), and p53. The results thus indicate that the antiproliferative effect of sesamin on PDGF-stimulated VSMCs is attributable to arrest of the cell cycle in G0/G1 caused, in turn, by upregulation of p27(KIP1), p21(CIP1), and p53, and inhibition of cyclin E-CDK2 and cyclin D1-CDK4 expression.

Glucose uptake into insulin-sensitive tissues is important for the regulation of blood glucose. This study has investigated whether the pentacyclic triterpenoids substituted with a carboxylic acid at the C-27 position isolated from Astilbe rivularis can enhance glucose uptake and subsequently to also examine their underlying molecular mechanisms. The structure of the new pentacyclic triterpenoid 1 was assigned by spectroscopic data interpretation. To evaluate the activity of compounds 1 and 2, glucose uptake and glucose transporter 4 (GLUT4) translocation were measured in C2C12 myotubes. The C-27-carboxylated triterpenoids 1 and 2 significantly increased basal and insulin-stimulated glucose uptake and GLUT4 translocation to plasma membrane. Both compounds stimulated the phosphorylation of insulin receptor substrate-1 (IRS-1), protein kinase B (Akt), and extracellular signal-regulated kinase 1/2 (Erk1/2). Pretreatment with the Akt inhibitor triciribine or the Erk1/2 inhibitor U0126 decreased the ability of both compounds to enhance basal- and insulin-stimulated glucose uptake and stimulate GLUT4 translocation. These results indicate that compounds 1 and 2 activated both the IRS-1/Akt and Erk1/2 pathways and subsequently stimulated GLUT4 translocation, leading to enhanced glucose uptake. Thus, these observations suggest that C-27-carboxylated-pentacyclic triterpenoids may serve as scaffolds for development as agents for the management of blood glucose levels in disease states such as diabetes.

Veratrum nigrum is recognized as a medicinal plant used for the treatment of hypertension, stroke, and excessive phlegm. Chemical investigation of the roots and rhizomes led to the isolation of five new steroidal alkaloids, jervine-3-yl formate (1), veramarine-3-yl formate (2), jerv-5,11-diene-3β,13β-diol (3), (1β,3β,5β)-1,3-dihydroxyjervanin-12(13)-en-11-one (4), and veratramine-3-yl acetate (5). Compounds 1 and 5 exhibited potent inhibitory activity (11.3 and 4.7 μM, respectively) against protein tyrosine phosphatase 1B (PTP1B), which has emerged as a viable target for treatment of type 2 diabetes mellitus. On the basis of their PTP1B inhibitory activity, the compounds were evaluated for their potential to enhance glucose uptake in C2C12 skeletal muscle cells. The insulin-stimulated glucose uptake was enhanced upon treatment with compounds 1 and 5 (10 μM) by 49.9 ± 6.5% and 56.0 ± 9.7%, respectively, in a more potent manner than that with the positive control rosiglitazone (47.3 ± 3.4% at 30 μM). These results suggest that steroidal alkaloids serve as practical antidiabetes mellitus leads capable of enhancing glucose uptake.

For the combined delivery of an insulin-sensitizing adipokine; i.e., the ADN gene, and the potent PPARγ agonist rosiglitazone, cationic lipid emulsions were formulated using the cationic lipid DOTAP, helper lipid DOPE, castor oil, Tween 20 and Tween 80. The effect of drug loading on the physicochemical characteristics of the cationic emulsion/DNA complexes was investigated. Complex formation between the cationic emulsion and negatively charged plasmid DNA was confirmed and protection from DNase was observed. The in vitro transfection efficiency and cytotoxicity were evaluated in HepG2 cells. The particle sizes of the cationic emulsion/DNA complex were in the range 230-540 nm and those of the rosiglitazone-loaded cationic emulsion/DNA complex were in the range 220-340 nm. Gel retardation of the complexes was observed when the complexation weight ratios of the cationic lipid to plasmid DNA exceeded 4:1 for both the drug-free and rosiglitazone-loaded complexes. Both complexes stabilized plasmid DNA against DNase. The ADN expression level increased dose-dependently when cells were transfected with the cationic emulsion/DNA complexes. The rosiglitazone-loaded cationic emulsion/DNA complexes showed higher cellular uptake in HepG2 cells depending on the rosiglitazone loading, but not depending on the type of plasmid DNA type such as pVAX/ADN, pCAG/ADN, or pVAX. The drug-loaded cationic emulsion/plasmid DNA complexes were less cytotoxic than free rosiglitazone. Therefore, a cationic emulsion could potentially serve as a co-delivery system for rosiglitazone and the adiponectin gene., (Copyright © 2015 Elsevier B.V. All rights reserved.)

"G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic β-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with 30 µM linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus.

We investigated the antiplatelet activity of indirubin-3'-monoxime (I3O) and the underlying mechanisms. In a rat carotid artery injury model, oral administration (20 mg/kg/day) of I3O for 3 days significantly prolonged occlusion time, and ADP- and collagen-induced platelet aggregation. In washed platelets in vitro, I3O potently inhibited collagen-induced platelet aggregation by suppressing phospholipase Cγ2 (PLCγ2) phosphorylation, subsequently blocking diacylglycerol and arachidonic acid (AA) formation, P-selectin secretion and the production of thromboxane B2. Platelet aggregation induced by phorbol-12-myristate 13-acetate, a protein kinase C (PKC) activator, was inhibited by I3O. Both I3O and U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, markedly reduced collagen-induced phosphorylation of ERK1/2 and p47, resulting in the blockade of cyclooxygenase (COX)-mediated AA metabolite production in AA-treated platelets. I3O suppressed phosphorylation of JNK, p38, GSK-3β, and AKT. I3O inhibited glycoprotein VI (GPVI), as a collagen receptor, by suppressing the phosphorylation of tyrosine kinase Syk of GPVI and the phosphorylation of PLCγ2 and ERK1/2 stimulated by convulxin, as a specific stimulator. Our results indicate that an antiplatelet effect of I3O is due to the suppression of GPVI-mediated signaling pathways. In collagen-stimulated platelets, ERK1/2 phosphorylation is adenylyl cyclase-dependent and leads to the modulation of PKC-p47 signaling and COX-1-mediated AA-metabolic pathways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study was designed to investigate the antiproliferative effect of a novel naphthoquinone derivative, 2-undecylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone (2-undecylsulfonyl-DMNQ), on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) and examine the possible molecular mechanism of its antiproliferative action. 2-Undecylsulfonyl-DMNQ significantly inhibited PDGF-stimulated cell number and DNA synthesis, and arrested the PDGF-stimulated progression through G0/G1 to S phase of cell cycle supported by the suppression of pRb phosphorylation and cyclin D1/E, CDK2/4 and PCNA expressions. 2-Undecylsulfonyl-DMNQ dose-dependently inhibited the PDGF-stimulated phosphorylation of phospholipase Cγ (PLCγ), protein kinase B (Akt/PKB), signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK 1/2). In addition, 2-undecylsulfonyl-DMNQ inhibited PDGF-induced PDGF receptor β (PDGF-Rβ) dimerization and the phosphorylation of Tyr(579/581), Tyr(716), Tyr(751) and Tyr(1021) in PDGF-Rβ. However, 2-undecylsulfonyl-DMNQ has no antiproliferative effect on epidermal growth factor (EGF)- or fetal bovine serum (FBS)-stimulated VSMCs. In conclusion, these findings suggest that the antiproliferative effects of 2-undecylsulfonyl-DMNQ on PDGF-stimulated VSMCs are due to the blockade of receptor dimerization and autophosphorylation on specific tyrosine residues of PDGF-Rβ, which resulted in the subsequent suppression of signaling cascades and a cell cycle arrest. Our observation may explain an important mechanism to block the integration of multiple signals generated by growth factor receptor activation for prevention of VSMC proliferation in cardiovascular diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gαi/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes.

Ulmus macrocarpa Hance (Ulmaceae) has been used as a traditional oriental medicine for the treatment of edema, mastitis, gastric cancer and inflammation. The aim of this study was to investigate the effects of Ulmus macrocarpa extract (UME) on thrombus formation in vivo, platelet activation ex vivo and fibrinolytic activity in vitro. To identify the antithrombotic activity of UME in vivo, we used an arterial thrombosis model. UME delayed the occlusion time by 13.4 and 13.9 min at doses of 300 and 600 mg/kg, respectively. UME significantly inhibited ex vivo platelet aggregation induced by collagen and adenosine 5'-diphosphate (ADP), respectively, but did not affect the coagulation times following activated partial thromboplastin and prothrombin activation. Therefore, to investigate the antiplatelet effect of UME, the effect of UME on collagen and ADP-induced platelet aggregation in vitro was examined. UME exhibited antiplatelet aggregation activity, induced by ADP and collagen. Furthermore, the fibrinolytic activity of UME was investigated. The results showed that UME significantly increased fibrinolysis at 1,000 mg/ml. In conclusion, the results suggested that UME may significantly inhibit artery thrombus formation in vivo, potentially due to antiplatelet activity, and also exhibits potential as a clot‑dissolving agent for thrombolytic therapy.

As the abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis and vascular restenosis, a candidate drug with antiproliferative properties is needed. We investigated the antiproliferative action and underlying mechanism of a newly synthesized naphthoquinone derivative, 5,8-dimethoxy-2-nonylamino-naphthalene-1,4-dione (2-nonylamino-DMNQ), using VSMCs treated with platelet-derived growth factor (PDGF). 2-Nonylamino-DMNQ inhibited proliferation and cell number of VSMCs induced by PDGF, but not epidermal growth factor (EGF), in a concentration-dependent manner without any cytotoxicity. This derivative suppressed PDGF-induced [(3)H]-thymidine incorporation, cell cycle progression from G0/G1 to S phase, and the phosphorylation of phosphor-retinoblastoma protein (pRb) as well as the expression of cyclin E/D, cyclin-dependent kinase (CDK) 2/4, and proliferating cell nuclear antigen (PCNA). Importantly, 2-nonylamino-DMNQ inhibited the phosphorylation of PDGF receptorβ(PDGF-Rβ) enhanced by PDGF at Tyr(579), Tyr(716), Tyr(751), and Tyr(1021) residues. Subsequently, 2-nonylamino-DMNQ inhibited PDGF-induced phosphorylation of STAT3, ERK1/2, Akt, and PLCγ1. Therefore, our results indicate that 2-nonylamino-DMNQ inhibits PDGF-induced VSMC proliferation by blocking PDGF-Rβ autophosphorylation, and subsequently PDGF-Rβ-mediated downstream signaling pathways.