• Biomolecules & Therapeutics
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• 제29권6호
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• pp.615-629
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• 2021

An active compound, triterpene saponin, astersaponin I (AKNS-2) was isolated from Aster koraiensis Nakai (AKNS) and the autophagy activation and neuroprotective effect was investigated on in vitro and in vivo Parkinson's disease (PD) models. The autophagy-regulating effect of AKNS-2 was monitored by analyzing the expression of autophagy-related protein markers in SH-SY5Y cells using Western blot and fluorescent protein quenching assays. The neuroprotection of AKNS-2 was tested by using a 1-methyl-4-phenyl-2,3-dihydropyridium ion (MPP⁺)-induced in vitro PD model in SH-SY5Y cells and an MPTP-induced in vivo PD model in mice. The compound-treated SH-SY5Y cells not only showed enhanced microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and decreased sequestosome 1 (p62) expression but also showed increased phosphorylated extracellular signal-regulated kinases (p-Erk), phosphorylated AMP-activated protein kinase (p-AMPK) and phosphorylated unc-51-like kinase (p-ULK) and decreased phosphorylated mammalian target of rapamycin (p-mTOR) expression. AKNS-2-activated autophagy could be inhibited by the Erk inhibitor U0126 and by AMPK siRNA. In the MPP⁺-induced in vitro PD model, AKNS-2 reversed the reduced cell viability and tyrosine hydroxylase (TH) levels and reduced the induced α-synuclein level. In an MPTP-induced in vivo PD model, AKNS-2 improved mice behavioral performance, and it restored dopamine synthesis and TH and α-synuclein expression in mouse brain tissues. Consistently, AKNS-2 also modulated the expressions of autophagy related markers in mouse brain tissue. Thus, AKNS-2 upregulates autophagy by activating the Erk/mTOR and AMPK/mTOR pathways. AKNS-2 exerts its neuroprotective effect through autophagy activation and may serve as a potential candidate for PD therapy.

• 치위생과학회지
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• 제19권4호
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• pp.254-260
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• 2019

Background: The primary aims of periodontal disease treatment is to remove dental plaque and calculus, the main causes of tooth loss, and restore periodontal tissue destroyed by inflammation. Periodontal disease treatment should also help maintain the alveolar bone, alleviate inflammation, and promote periodontal ligament cell proliferation, which is essential for tissue regeneration. Conventional antibiotics and anti-inflammatories have adverse side effects, especially during long-term use, so there is a need for adjunct treatment agents derived from natural products. The purpose of this study was to investigate whether the herbal flavone baicalein has the osteogenic activity under inflammatory conditions, and assess the involvement of osteoblast immediate early response 3 (IER3) expression. Methods: Human osteoblastic MG-63 cells were cultured with the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β in the presence and absence of baicalein. Proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and expression of IER3 mRNA was assessed using real-time polymerase chain reaction. The expression of IER3 protein levels and activation of associated signal transduction pathways were assessed using western blotting. Results: Baicalein increased IER3 mRNA and protein expression synergistically. In addition, baicalein reversed the suppression of cell proliferation, and the downregulation of osteogenic transcription factor runt-related transcription factor 2 and osterix induced by pro-inflammatory cytokines. Baicalein also upregulated the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK 1/2). The upregulation of IER3 by pro-inflammatory cytokines was blocked by pretreatment with inhibitors of AKT, p38, JNK, and ERK 1/2. Conclusion: Baicalein mitigates the deleterious responses of osteoblasts to pro-inflammatory cytokines. Further, IER3 enhanced the effect of baicalein via activation of AKT, p38, JNK, and ERK pathways.

• The Korean Journal of Physiology and Pharmacology
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• 제14권6호
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• pp.359-364
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• 2010

Many therapeutic roles have been proposed for sigma-1 receptor (Sig-1R), but the involvement of Sig-1R in neuropathic pain has currently not been well explored. The present study aimed to evaluate the anti-nociceptive effect of Sig-1R antagonist (BD1047) in a rat model of chronic compression of the dorsal root ganglion (CCD), which is a model of human foraminal stenosis and radicular pain. When stainless steel rods were inserted into the intervertebral foramen of lumbar vertebrae 4 and 5, the CCD developed reliable mechanical (from 3 day) and cold allodynia (from 1 day) as compared with the sham operation group. The spinal expressions of Sig-1R and phosphorylation of extracellular signal-regulated kinase (pERK) were significantly increased from day 3 to day 14 after CCD surgery, as is consistent with the manifestation of allodynia. The BD 1047 (10, 30, 100 mg/kg) administered on postoperative days 0~5 dose-dependently suppressed both the induction of allodynia and the elevation of the spinal pERK expression in a manner comparable with that of gabapentin (100 mg/kg). At 7 days post-CCD surgery, BD1047 (10, 30, 100 mg/kg) administration also produced anti-nociceptive effects on the mechanical and cold allodynia similar with those of gabapentin (100 mg/kg). Therefore, this data suggested that Sig-1R may play an important role in both the development and maintenance of CCD-induced neuropathy.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2739-2744
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• 2012

Background: Renal-cell carcinoma (RCC) is resistant to almost all chemotherapeutics and radiation therapy. ${\beta}$-Elemene, a promising anticancer drug extracted from a traditional Chinese medicine, has been shown to be effective against various tumors. In the present study, anti-tumor effects on RCC cells and the involved mechanisms were investigated. Methods: Human RCC 786-0 cells were treated with different concentrations of ${\beta}$-elemene, and cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by western blotting. Autophagy was evaluated by transmission electron microscopy. Results: ${\beta}$-Elemene inhibited the viability of 786-0 cells in a dose- and time-dependent manner. The anti-tumor effect was associated with induction of apoptosis. Further study showed that ${\beta}$-elemene inhibited the MAPK/ERK as well as PI3K/Akt/mTOR signalling pathways. Moreover, robust autophagy was observed in cells treated with ${\beta}$-elemene. Combined treatment of ${\beta}$-elemene with autophagy inhibitors 3-methyladenine or chlorochine significantly enhanced the anti-tumor effects. Conclusions: Our data provide first evidence that ${\beta}$-elemene can inhibit the proliferation of RCC 786-0 cells by inducing apoptosis as well as protective autophagy. The anti-tumor effect was associated with the inhibition of MAPK/ERK and PI3K/Akt/mTOR signalling pathway. Inhibition of autophagy might be a useful way to enhance the anti-tumor effect of ${\beta}$-elemene on 786-0 cells.

• The Korean Journal of Physiology and Pharmacology
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• 제21권1호
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• pp.117-124
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• 2017

The present study aimed to show that pro-inflammatory cytokines [tumor necrosis factor (TNF)-${\alpha}$, interferon (IFN)-${\gamma}$, and interleukin (IL)-$1{\beta}$] synergistically induce the production of nitric oxide (NO) production in mouse mesangial cells, which play an important role in inflammatory glomerular injury. We also found that co-treatment with cytokines at low doses (TNF-${\alpha}$; 5 ng/ml, IFN-${\gamma}$; 5 ng/ml, and IL-$1{\beta}$; 1.25 U/ml) synergistically induced NO production, whereas treatment with each cytokine alone did not increase NO production at doses up to 100 ng/ml or 50 U/ml. Silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), attenuates cytokine mixture (TNF-${\alpha}$, IFN-${\gamma}$, and IL-$1{\beta}$)-induced NO production. Western blot and RT-PCR analyses showed that silymarin inhibits inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Silymarin also inhibited extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) phosphorylation. Collectively, we have demonstrated that silymarin inhibits NO production in mouse mesangial cells, and may act as a useful anti-inflammatory agent.

• The Korean Journal of Physiology and Pharmacology
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• 제7권2호
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• pp.97-101
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• 2003

The salivary glands produce 1.5L of fluid per day. As in other exocrine organs, the general mechanism in the salivary glands is that water movement occurs secondary to osmotic driving forces created by active salt transport. Therefore, high water permeability in the salivary glands is expected to have a variety of aquaporin (AQP), a water channel. Although some AQPs have been known to be present in the salivary glands, roles of parasympathetic nerve in AQP expression have not yet been examined. This study was designed to examine the changes of AQPs and extracellular signal-regulated kinase (ERK) in the submandibular glands after parasympathetic denervation. Right chorda-lingual nerve was cut, and each right (experiment) and left (control) submandibular gland was excised at 1, 3, 7, 14, 30 days after denervation. The denervated right submandibular glands were resulted in weight loss and morphologic changes, including cell loss and atrophy, as the time elapsed after parasympathetic denervation increased, whereas there were no histologic alteration in control side. AQP5 which is known to reside in apical membrane and secretory caraliculi of the submandibular acini were gradually underexpressed according, as the time after denervation increased. Expression of AQP4 in submandibular ductal epithelium was down-regulated after denervation. Besides, AQP3 and 8, which is known to be present in basolateral membrane of the glandular acini, were gradually underexpressed after denervation, similar to the pattern of other types. Expression of ERK, a mitogen-activated protein kinase, was downregulated after parasympathetic denervation in the submandibular gland. These results suggest that parasympathetic nervous system regulates the expression of AQPs in salivary glands, and is in part mediated by ERK pathway.

• 대한한방소아과학회지
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• 제20권3호
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• pp.117-127
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• 2006

Objectives : In this study, we investigated the effects of Daehwangmokdanpitang-gagambang(DMG) on treatment for cholecystokinin-octapeptide(CCK)-induced acute pancreatitis(AP) in rats. Methods : Male Wistar rats weighing 200 to 250 g were divided into three groups. The first was normal untreated group, the second was in treatment with DMG group; DMG was administered orally, followed by 75 ${\mu}g/kg$ CCK subcutaneously three times, after 1, 3 and 5 h. This whole procedure was repeated for 5 days. The third was in treatment with saline group, the protocol was the same as in treatment group with DMG. We determined the pancreatic weight/body weight ratio, the levels of pancreatic HSP60, HSP72, ERK, p38 MAPK and the secretion of pro-inflammatory cytokines. Repeated CCK treatment resulted in the typical laboratory and morphological changes of experimentally induced pancreatitis. Results : DMG was significantly decreased the pancreatic weight/body weight ratio in CCKinduced AP and DMG increased HSP60 and HSP72 compared with CCK-induced AP. DMG suppressed ERK and p38 MAPK activation. Additionally, the secretion of $IL-1{\beta}$ and $TNF-{\alpha}$ and the levels of amylase and lipase were lower than that saline. Conclusions : DMG has an effect to treatment for CCK-induced AP.

• 대한한방내과학회지
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• 제31권2호
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• pp.254-263
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• 2010

Objectives : The neuroprotective effects of bee venom (BV) have been demonstrated in many studies, but bee venom has many side effects. So we used sweet bee venom (SBV), which has high molecular elements removed to reduce the side effects. I examined the neuroprotective effect of sweet bee venom in 1-methyl-4-phenylpyridine ($MPP^+$)-induced human neuroblastoma SH-SY5Y cells. Methods : To observe the possible toxicity of SBV itself, SH-SY5Y cells were treated with SBV in various concentrations for 3 h and $MPP^+$ in concentrations (1 and 5mM) for 24h. To investigate the protective effect of SBV against $MPP^+$ toxicity, SH-SY5Y cells were pretreated with vehicle or nontoxic concentrations of SBV for 3h and the cells were not washed, followed by incubation with respective concentrations of SBV and 1 mM $MPP^+$ for 24h. To investigate the protective effect of SBV against $MPP^+$ toxicity, SH-SY5Y cells were pretreated with vehicle or nontoxic concentrations of SBV for 3h and the cells were not washed, followed by incubation with respective of SBV(0.5%), 1 mM $MPP^+$, 5uM AKT inhibitor(LY984002) and 10uM ERK inhibitor(PD98059) for 24 h. The protective effect was measured by cell viability assay. To investigate the degree of apoptosis, caspase-3 enzyme activity was measured in control, $MPP^+$, SBV+$MPP^+$. Results : SBV (0.5%) pretreatment protected the SH-SY5Y cells against $MPP^+$-induced apoptotic cell death. The cell viability was higher in the SH-SY5Y cells that were pretreated with vehicle or nontoxic concentrations of SBV than those not pretreated. The caspase-3 activity was lower in the pretreated groups than these not pretreated. ERK and AKT enzymes have a role in the neuroprotective effects of the sweet bee venom. Conclusions : The results demonstrate that SBV has a protective effect on dopaminergic neurons against $MPP^+$ toxicity. This data suggest that SBV could be a potential therapeutic tool for neurodegenerative diseases such as Parkinson's disease(PD).

• Molecules and Cells
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• 제26권5호
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• pp.468-473
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• 2008

Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that $Cl^-$ channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of $Cl^-$ channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. $Cl^-$ channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated $Cl^-$ concentration, as judged by flow cytometry analysis using MQAE as a $Cl^-$-detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS ($Cl^-$ channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive $Cl^-$-channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

• Biomolecules & Therapeutics
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• 제26권5호
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• pp.487-493
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• 2018

Cluster of differentiation 44 (CD44), a cell surface receptor for hyaluronic acid (HA), is involved in aggressive cancer phenotypes. Herein, we investigated the role of the CD44 standard isoform (CD44s) in hypoxia-inducible $factor-1{\alpha}$ ($HIF-1{\alpha}$) regulation using MCF7 overexpressing CD44s (pCD44s-MCF7). When pCD44s-MCF7 was incubated under hypoxia, levels of $HIF-1{\alpha}$, vascular endothelial growth factor, and the $HIF-1{\alpha}$ response element-derived luciferase activity were significantly increased compared to those in the control MCF7. Incubation of pCD44s-MCF7 cells with HA further increased $HIF-1{\alpha}$ accumulation, and the silencing of CD44s attenuated $HIF-1{\alpha}$ elevation, which verifies the role of CD44s in $HIF-1{\alpha}$ regulation. In addition, the levels of phosphorylated extracellular signal-regulated kinase (ERK) was higher in hypoxic pCD44s-MCF7 cells, and $HIF-1{\alpha}$ accumulation was diminished by the pharmacological inhibitors of ERK. CD44s-mediated $HIF-1{\alpha}$ augmentation resulted in two functional outcomes. First, pCD44s-MCF7 cells showed facilitated cell motility under hypoxia via the upregulation of proteins associated with epithelial-mesenchymal transition, such as SNAIL1 and ZEB1. Second, pCD44s-MCF7 cells exhibited higher levels of glycolytic proteins, such as glucose transporter-1, and produced higher levels of lactate under hypoxa. As a consequence of the enhanced glycolytic adaptation to hypoxia, pCD44s-MCF7 cells exhibited a higher rate of cell survival under hypoxia than that of the control MCF7, and glucose deprivation abolished these differential responses of the two cell lines. Taken together, these results suggest that CD44s activates hypoxia-inducible $HIF-1{\alpha}$ signaling via ERK pathway, and the $CD44s-ERK-HIF-1{\alpha}$ pathway is involved in facilitated cancer cell viability and motility under hypoxic conditions.