• Biomolecules & Therapeutics
• /
• v.29 no.2
• /
• pp.144-153
• /
• 2021

Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Culture-conditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.

• Journal of Life Science
• /
• v.26 no.5
• /
• pp.509-518
• /
• 2016

Asparagus cochinchinensis is a medical plant that has long been used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although several studies have been conducted on the anti-neuroinflammatory effects of A. cochinchinensis, the correlation between these effects and nerve growth factor (NGF) has not yet been examined. In this study, we investigated the effects of an aqueous extract of A. cochinchinensis (AEAC) on the secretion and action mechanism of NGF in neuronal cells. The concentration of the NGF protein in the supernatant collected from cultured cells increased significantly in B35 cells treated with AEAC in comparison with the vehicle-treated group without any specific cytotoxicity. Furthermore, the mRNA expression of NGF showed a very similar pattern to its protein concentration. To examine the bioactivity of NGF secreted from B35 cells, undifferentiated PC12 cells were cultured in an AEAC-conditioned medium and neuritic outgrowth was observed. The dendrite length of PC12 cells in the AEAC-treated group was significantly higher than that in the vehicle-treated group. Moreover, the level of the downstream effectors p-TrkA and p-ERK of the high-affinity NGF receptor was significantly higher in the AEAC-treated group, while the expression of the downstream effectors of the low-affinity NGF receptor was significantly lower in the same group. These results suggest that AEAC may contribute to the regulation of NGF expression and secretion in neuronal cells; it is therefore an excellent candidate for further investigation as a therapeutic drug for neurodegenerative diseases.

• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.6
• /
• pp.781-787
• /
• 2017

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

• Clinical and Experimental Reproductive Medicine
• /
• v.23 no.1
• /
• pp.95-102
• /
• 1996

This study was performed to investigate the effect of human follicular fluid (HFF) on development of mouse embryos, for evaluating the suitability of HFF as a substitutive material of human fetal cord serum in ART program. The various concentrations of HFF were added into the culture medium and the effects of HFF concentrations were examined to identify the optimal concentration of HFF for embryo development. The potency of HFF in improving embryo development was compared to that of other protein supplement. Collected HFFs were classified with the maturity of the containing oocytes; mature, immature, atretic, and then the effects of the classified HFFs on embryo development were examined. Also, HFF was separated into the low (<30,000 Da) and high (>30,000 Da) molecular weight fractions and the effects of the fractions on embryo development were investigated. The highest development rate was found in culture medium supplemented with 20% HFF, bnt this rate was reversely reduced at the concentrations of HFF higher than 20%. The development rates to the blastocyst, hatching blastocyst, attachment and outgrowth cultured in mature HFF was significantly higher than those in immature and atretic HFF, and mean cell number in blastocyst was higher in mature HFF than in immature and atretic HFF. The development rates of mouse embryos according to protein sources were significantly higher in HFF than in fetal cord serum (FCS), maternal serum (MS) and bovine serum albumin (BSA), and mean cell number in blastocyst cultured in HFF was higher than that in FCS, MS and BSA. The development rates of embryo and mean cell number in blastocyst cultured in high molecular weight fraction of HFF were higher than those in low molecular weight fraction, but the results of high molecular weight fraction were lower than those of whole HFF. Therefore, these results indicated that human mature follicular fluid was useful for improving the development of mouse embryos, which suggests a possibility that HFF also may be used efficiently for improving the culture condition in human ART program as a protein supplement.

• Journal of Life Science
• /
• v.16 no.3 s.76
• /
• pp.464-471
• /
• 2006

Peripheral nerve injuries are a commonly encountered clinical problem and often result in severe functional deficits. In the present study, the effects of treadmill exercise on neurotrophin expressions and functional recovery following sciatic crushed nerve injury were investigated. Animals were randomly assigned into four groups: the sciatic nerve injury group, the sciatic nerve injury and 3-day-exercise, the sciatic nerve injury and 7-days-exercise, and the sciatic nerve injury and 14-days-exercise groups. Sciatic nerve injury was caused by crushing the right sciatic nerve for 30 s using a surgical clip. A the light-exercise was applied to each of the exercise group over the respective number of days. In the present results, we identified enhanced axonal re-growth in the distal stump of the sciatic nerve 3-14 days after crush injury with treadmill training. Dorsal root ganglion (DRG) neuron when cultured from animals with nerve injury and treadmill training showed more enhanced neurite outgrowth than that of sedentary animals. Nerve growth factor (NGF) protein levels in low-intensity treadmill training group were highly induced in the injured sciatic nerves 3, 7 and 14 days after injury compared with sedentary group, and brain-derived neurotrophin factor (BDNF) protein levels in treadmill exercise group were highly induced in the injured sciatic nerve 3 days after injury compared with sedentary group. Then, treadmill exercise increased neurotrophic factors induced in the regenerating nerves. We further demonstrate that motor functional recovery after sciatic nerve injury was promoted by treadmill exercise. Thus, the present data provide a new evidence that treadmill exercise enhanced neurotrophins expression and axonal regeneration after sciatic nerve injury in rats.

• Journal of Periodontal and Implant Science
• /
• v.47 no.5
• /
• pp.292-311
• /
• 2017

Purpose: Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance. Methods: Human polyclonal $CD4^+CD25^+CD127^{lo-}$ Tregs (127-Tregs) and $na\ddot{i}ve$ $CD4^+CD25^+CD45RA^+$ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an $NOD/scid/IL-2R{\gamma}^{-/-}$ mouse model of collagen-induced arthritis. Results: Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of $CD4^+CD25^+$ Tregs at the articular joints in a mechanistic and orchestrated way. Conclusions: We propose human $na\ddot{i}ve$ $CD4^+CD25^+CD45RA^+$ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.

• The Journal of Korean Medicine
• /
• v.30 no.3
• /
• pp.1-14
• /
• 2009

Objectives : Alzheimer's disease (AD) is characterized by neuronal loss and extracellular senile plaque. Moreover, the cellular actions of ${\beta}$-amyloid (A${\beta}$ play a causative role in the pathogenesis of AD. This study was designed to determine whether Woo-Gui-Um, a commonly used Korean herbal medicine, has the ability to protect cortical and hippocampal neurons against A${\beta}_{25-35}$ neurotoxicity Methods : In the present study, the authors investigated the preventative effects of the water extract of Woo-Gui-Um in a mouse model of AD. Memory impairment was induced by intraventricularly (i.c.v.) injecting A${\beta}_{25-35}$ peptides into mice. Woo-Gui-Um extract was then administered orally (p.o.) for 14 days. In addition, A${\beta}_{25-35}$ toxicity on the hippocampus was assessed immunohistochemically, by staining for Tau, MAP2, TUNEL, and Bax, and by performing an in vitro study in PC12 cells. Results : Woo-Gui-Um extract had an effect to improve learning ability and memory score in the water maze task. Woo-Gui-Um extract had significant neuroprotective effects in vivo against oxidative damage and apoptotic cell death of hippocampal neurons caused by i.c.v. A${\beta}_{25-35}$. In addition, Woo-Gui-Um extract was found to have a protective effect on A${\beta}_{25-35}$-induced apoptosis, and to promote neurite outgrowth of nerve growth factor (NGF)-differentiated PC12 cells. Conclusions : These results suggest that Woo-Gui-Um extract reduces memory impairment and Alzheimer's dementia via an anti-apoptotic effect and by regulating Tau and MAP2 in the hippocampus.

• The Korean Journal of Physiology and Pharmacology
• /
• v.24 no.3
• /
• pp.241-248
• /
• 2020

Luminespib (AUY922), a heat shock proteins 90 inhibitor, has anti-neoplastic and antitumor effects. However, it is not clear whether AUY922 affects events in vascular diseases. We investigated the effects of AUY922 on the platelet-derived growth factor (PDGF)-BB-stimulated proliferation and migration of vascular smooth muscle cells (VSMC). VSMC viability was detected using the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reagent. To detect the attenuating effects of AUY922 on PDGF-BB-induced VSMCs migration in vitro, we performed the Boyden chamber and scratch wound healing assays. To identify AUY922-mediated changes in the signaling pathway, the phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1/2 was analyzed by immunoblotting. The inhibitory effects of AUY922 on migration and proliferation ex vivo were tested using an aortic ring assay. AUY922 was not cytotoxic at concentrations up to 5 nM. PDGF-BB-induced VSMC proliferation, migration, and sprout outgrowth were significantly decreased by AUY922 in a dose-dependent manner. AUY922 significantly reduced the PDGF-BB-stimulated phosphorylation of Akt and ERK1/2. Furthermore, PD98059 (a selective ERK1/2 inhibitor) and LY294002 (a selective Akt inhibitor) decreased VSMC migration and proliferation by inhibiting phosphorylation of Akt and ERK1/2. Greater attenuation of PDGF-BB-induced cell viability and migration was observed upon treatment with PD98059 or LY294002 in combination with AUY922. AUY922 showed anti-proliferation and anti-migration effects towards PDGF-BB-induced VSMCs by regulating the phosphorylation of ERK1/2 and Akt. Thus, AUY922 is a candidate for the treatment of atherosclerosis and restenosis.

• The Korean Journal of Physiology and Pharmacology
• /
• v.14 no.3
• /
• pp.177-183
• /
• 2010

Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) activity, is known to play important roles in modulating both NO and superoxide production during vascular diseases such as atherosclerosis. However, the role of BH4 in functions of vascular smooth muscle cells is not fully known. In this study, we tested the effects of BH4 and dihydrobiopterin (BH2), a BH4 precursor, on migration and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) in rat aortic smooth muscle cells (RASMCs). Cell migration and proliferation were measured using a Boyden chamber and a 5-bromo-2'-deoxyuridine incorporation assay, respectively, and these results were confirmed with an ex vivo aortic sprout assay. Cell viability was examined by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assays. BH4 and BH2 decreased PDGF-BBinduced cell migration and proliferation in a dose-dependent manner. The inhibition of cell migration and proliferation by BH4 and BH2 was not affected by pretreatment with $N^G$-nitro-L-arginine methyl ester, a NOS inhibitor. Moreover, the sprout outgrowth formation of aortic rings induced by PDGF-BB was inhibited by BH4 and BH2. Cell viability was not inhibited by BH4 and BH2 treatment. The present results suggest that BH4 and BH2 may inhibit PDGF-stimulated RASMC migration and proliferation via the NOS-independent pathway. Therefore, BH4 and its derivative could be useful for the development of a candidate molecule with an NO-independent anti-atherosclerotic function.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.42 no.1
• /
• pp.95-101
• /
• 2016

In the present study, we extracted the hydrosol from flower of Chrysanthemum boreale Makino (CBMF hydrosol) by steam distillation and tested the effect of the CBMF hydrosol on skin regeneration using normal human keratinocytes (HaCaTs). CBMF hydrosol induced proliferation as well as migration in HaCaTs in a dose-dependent manner. Treatment with $1{\mu}g/mL$ CBMF hydrosol increased proliferation to $143.71{\pm}3.37%$ and migration to $139.98{\pm}5.72%$ compared with a control group. CBMF hydrosol also significantly enhanced the phosphorylations of extracellular signal-regulated kinase (Erk) 1/2 and serine/threonine-specific protein kinase (Akt) in HaCaTs. Moreover, CBMF hydrosol dose-dependently induced sprout outgrowth in HaCaTs. These results demonstrate that CBMF hydrosol has skin regeneration and wound healing activity in HaCaTs. Therefore, CBMF hydrosol could be used as a potential cosmetic material.