Estradiol attenuates down-regulation of PEA-15 and its two phosphorylated forms in ischemic brain injury

고필옥 한국실험동물학회 2015 Laboratory Animal Research Vol.31 No.1

Estradiol exerts a neuroprotective effect against focal cerebral ischemic injury through the inhibition ofapoptotic signals. Phosphoprotein enriched in astrocytes 15 (PEA-15) is mainly expressed in brain thatperform anti-apoptotic functions. This study investigated whether estradiol modulates the expression ofPEA-15 and two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in middle cerebral arteryocclusion (MCAO)-induced injury and glutamate exposure-induced neuronal cell death. Adult female ratswere ovariectomized to remove endogenous estradiol and treated with vehicle or estradiol prior toMCAO. Focal cerebral ischemia was induced by MCAO and cerebral cortices were collected 24 h afterMCAO. Western blot analysis indicated that estradiol prevents the MCAO-induced decrease in PEA-15,phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116). Glutamate exposure induced a reduction in PEA-15,phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116) in cultured neurons, whereas estradioltreatment attenuated the glutamate toxicity-induced decrease in the expression of these proteins. It hasbeen known that phosphorylation of PEA-15 is an important step in carrying out its anti-apoptoticfunction. Thus, these findings suggest that the regulation of PEA-15 phosphorylation by estradiolcontributes to the neuroprotective function of estradiol in ischemic brain injury.

Chondrogenesis of Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood

고필옥,조재현,노경환,차윤임,조은혜,김영기,이희천,정태성,연성찬,강경선,이효종 한국임상수의학회 2009 한국임상수의학회지 Vol.26 No.6

In the current study, the mesenchymal stem cells (MSCs) isolated and propagated from the human umbilical cord blood (UCB) were tested for their capabilities of differentiation into chondrocytes in vitro. The mesenchymal progenitor cells (MPCs) collected from UCB were cultured in a low glucose DMEM medium with 10% FBS, Lglutamine and antibiotics. The human MSC colonies were positively stained by PAS reaction. When the immunophenotypes of surface antigens on the MSCs were analyzed by fluorescence-activated cell sorter (FACS) analysis, these cells expressed positively MSC-related antigens of CD 29, CD44, CD 90 and CD105, whereas they did not express antigens of CD14, CD31, CD34, CD45, CD133 and HLA-DR. Following induction these MSCs into chondrocytes in the chondrogenic differentiation medium for 3 weeks or more, the cells were stained positively with safranin O. We clearly confirmed that human MSCs were successfully differentiated into chondrocytes by RT-PCR and immunofluorescent stain of type-II collagen protein. These data also indicate that the isolation, proliferation and differentiation of the hUCB-derived MSCs in vitro can be used for elucidating the mechanisms involved in chondrogenesis. Moreover this differentiation technique can be applied to developing cell-based tissue regeneration or repair damaged tissues

Ferulic Acid Modulates Calbindin-D28K Expression in Focal Cerebral Ischemia and Glutamate-Exposed HT22 Cells

고필옥 경상대학교 농업생명과학연구원 2014 농업생명과학연구 Vol.48 No.3

Calbindin-D28k is a calcium-binding protein that mediates intracellular calcium concentrationsand exerts a neuroprotective effect against ischemic injury. Ferulic acid provides aneuroprotective effect against focal cerebral ischemia through its anti-oxidative andanti-inflammatory mechanisms. In this study, we investigated whether ferulic acid regulatescalbindin-D28k expression during focal cerebral ischemia and glutamate treatment-inducedneuronal cell death. Middle cerebral artery occlusion (MCAO) was performed to induce focalcerebral ischemia. Ferulic acid (100 mg/kg, i.v.) or vehicle was immediately administered afterMCAO, and brain tissues were isolated 24 h after MCAO. RT-PCR and Western blot analysesshowed a decrease in calbindin-D28k in MCAO-operated animals. We found that ferulic acidtreatment prevented the MCAO-induced decrease in calbindin-D28k expression. Glutamateexposure elevated the intracellular calcium levels in cultured hippocampal cells, and ferulic acidprevented the glutamate exposure-induced increase in calcium levels. Moreover, ferulic acid alsoattenuated the glutamate toxicity-induced decrease in calbindin-D28k. Taken together, these invivo and in vitro results demonstrate that ferulic acid regulates calbindin-D28k expression inneuronal cell injury. Therefore, these findings suggest that ferulic acid exerts a neuroprotectiveeffect by modulating calbindin-D28k expression.

Expression of pituitary adenylate cyclase activating polypeptide in the adult rat testis by in situ hybridization and immunohistochemistry

고필옥,곽수동,Koh, Phil-ok,Kwak, Soo-dong The Korean Society of Veterinary Science 2001 大韓獸醫學會誌 Vol.41 No.1

Pituitary adenylate cyclase activating polypeptide (PACAP)는 양의 뇌하수체에서 처음 분리 되었고, 뇌하수체 전엽세포의 cAMP의 생성을 자극하며, 흰쥐고환의 정자형성과 steroid 호르몬 형성에 관련한다고 알려져 있다. 이 연구는 성숙한 흰쥐의 고환에서 PACAP mRNA와 그 단백질의 분포를 조사하여 아래와 같은 결론을 얻었다. PACAP mRNA와 그 단백질은 흰쥐의 정세관에서 정자세포의 생성단계에 따라 특이적으로 발현되었다. 이들은 정세관의 발달단계 중 III~VII 기의 정자세포에서 발현되었고, 특히 V 기에서 초기 VII 기의 원형의 정자세포에서 가장 강하게 발현되었다. 이러한 결과는 흰쥐고환의 발달단계에 있는 정자세포에서 합성된 PACAP이 정자형성에 관련된다는 것을 나타내므로, PACAP이 고환의 기능에 중요한 역할을 하는 것을 암시한다. Pituitary adenyl ate cyclase activating polypeptide (PACAP) was originally isolated from the ovine hypothalamus and stimulated cAMP production in anterior pituitary cells. It is known that PACAP stimulates cAMP accumulation and contributes to the spermatogenesis and steroidogenesis in rat testis. The principal aim of this study is to determinate the distribution of PACAP mRNA and protein in adult rat testis. For this study, we used in situ hybridization and immunohistochemistry techniques in adult rat testis. PACAP mRNA was stage specifically expressed in seminiferous tubules. Positive signals of PACAP mRNA were detected in the developing germ cells at stages HI-VII of the epithelial cycle. The strongest signals of PACAP mRNA and protein were detected in round spermatids at stages V to early VII of the cycle. These results demonstrate that PACAP which is synthesised in the developing germ cells contributes to the spermatogenesis in rat testis. Thus, we suggest that PACAP plays a critical role in the function of testis.

Estradiol Prevents Injury-Induced Reduction of Peroxiredoxin-2 in Brain Ischemia Models

고필옥 한국실험동물학회 2009 Laboratory Animal Research Vol.25 No.4

Peroxiredoxin-2 (Prx-2), an isoform of the antioxidant enzyme family, has been exerts neuroprotectiveeffects against oxidative stress. Estradiol exerts neuroprotective effects against a various stimuli includingexcitotoxic amino acids and oxidative stress. This study investigated whether estradiol regulates Prx-2expression in middle cerebral artery occlusion (MCAO)-induced injury and glutamate exposure-inducedneuronal cell death. Adult female rats were ovariectomized and treated with oil or estradiol prior toMCAO. Brains were collected at 24 hr after MCAO and the cerebral cortices were isolated. Proteomicanalysis and Western blot analysis demonstrated that MCAO induces a decrease in Prx-2, andpretreatment with estradiol prevents MCAO-induced decrease in Prx-2. Moreover, glutamate exposureinduced a decrease in Prx-2 in a hippocampal-derived cell lines, and pretreatment with estradiolprevented the glutamate toxicity-induced decrease in Prx-2. This study suggests that estradiol exertsneuroprotective effects during neuronal cell damage by modulating of Prx-2.

Expression of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in human placenta

고필옥,노해숙,원청길,조경제,Wan-Sung Choi 대한수의학회 2005 Journal of Veterinary Science Vol.6 No.1

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.

Melatonin Prevents the Injury-Induced Decrease of Gamma-Enolase in Brain Tissue and HT22 Cells

고필옥 한국실험동물학회 2008 Laboratory Animal Research Vol.24 No.4

We previously reported that melatonin protects neuronal cells from ischemic brain injury. It is known thatγ-enolase is a glycolytic enzyme and is specifically expressed in neuron. γ-enolase promotes the survivalof neurons and exerts a neuroprotective effect. This study investigated whether melatonin modulates theγ-enolase expression level during ischemic brain injury and glutamate exposure. Adult male rats weretreated with vehicle or melatonin (5 mg/kg) prior to middle cerebral artery occlusion (MCAO). Brainswere collected at 24 hr after MCAO and the cerebral cortices were isolated. Brain injury induced adecrease of γ-enolase, however pretreatment with melatonin prevented the injury-induced a decrease ofγ-enolase. In vitro study, pretreatment with melatonin decreased glutamate toxicity-induced cell death in ahippocampal-derived cell line (HT22). Glutamate exposure induced a decrease of γ-enolase andpretreatment with melatonin prevented the glutamate-induced decrease of γ-enolase. Thus, this studysuggests that melatonin plays a potent neuroprotective role against glutamate-induced toxicity, and theregulation of γ-enolase by melatonin may mediate its neuroprotective effect.

국소적 대뇌허혈시 ferulic acid의 heme oxygenase-1 조절작용

고필옥 경상대학교 농업생명과학연구원 2012 농업생명과학연구 Vol.46 No.6

This study investigated whether ferulic acid modulates the heme oxygenase (HO)-1 and HO-2 expression in middle cerebral artery occlusion (MCAO)-induced brain injury. Rats (Sprague-Dawley, male) were treated with vehicle or ferulic acid (100 mg/kg, i.v.) before MCAO, and cerebral cortex tissues were collected 24 h after MCAO. This study clearly confirmed the protective effects of ferulic acid during MCAO-induced damage using hematoxylin and eosin staining. MCAO induces nuclear chromatin condensations and necrotic changes with scalloped shrunken form. However, ferulic acid prevented MCAO-induced histopathological changes. HO-1 and HO-2 expression levels were measured using reverse-transcription PCR and Western blot analyses. HO-1 levels were decreased in vehicle-treated animals after MCAO, whereas this decrease in HO-1 levels was attenuated by ferulic acid treatment. However, the level of HO-2 was consistently maintained in the cerebral cortex of vehicle- and ferulic acid-treated animals after MCAO. These results demonstrated that ferulic acid regulates HO-1 expression in ischemic brain injury, while ferulic acid do not modulate HO-2 expression in MACO. In conclusion, these findings suggest that ferulic acid exerts a neuroprotective effect by preventing the MCAO-induced decrease of HO-1 expression. 본 연구는 중간대뇌동맥을 폐쇄한 대뇌허혈성 손상모델에서 ferulic acid에 의해 조절되는 HO-1과 HO-2의 발현에 관하여 조사하였다. 흰쥐(Sprague-Dawley, 수컷)에 ferulic acid (100mg/kg) 또는 vehicle을 중간대뇌동맥폐쇄술(MCAO) 후 정맥으로 주사하였고 중간대뇌동맥폐쇄술(MCAO)을 실시한 24시간 후 대뇌피질의 조직을 적출하였다. Hematoxylin과 eosin 염색을 통하여 MCAO로 유도된 뇌 손상시 ferulic acid의 보호효과를 확인하였다. MCAO을 시행한 대뇌피질에서는 응축된 핵과 신경세포의 괴사 소견을 보였으나, ferulic acid 투여군에서는 이들 신경세포의 병변을 현저히 완화시켰다. HO-1과 HO-2의 RNA와 단백질 발현의 변화를 reverse-transcription PCR과 Western blot으로 분석하였다. HO-1 발현은 MCAO 후 vehicle 투여군에서 현저히 감소하였으나, MCAO 후 ferulic acid를 투여한 실험군에서는 이들 감소의 완화를 보였으며, MCAO를 시행하지 않은 실험군의 수준으로 유지되었다. 그러나, HO-2의 발현은 MCAO 후 vehicle 투여군과 ferulic acid 투여군에서 유의적인 차이는 관찰되지 않았고 MCAO를 시행하지 않은 실험군의 수준으로 유지되었다. 따라서, 본 연구의 결과는 허혈성 뇌 손상시 ferulic acid는 HO-1 발현을 조절하였으나, HO-2의 발현에는 영향을 미치지 못함을 확인하였다. 결론적으로, 허혈성 뇌손상시 ferulic acid는 HO-1의 발현을 조절하여 신경세포를 보호하는 역할을 수행한다는 사실을 확인하였다.

Change of Peroxiredoxin-5 Expression by Curcumin Treatment in Focal Cerebral Ischemia

고필옥,박동주,전성준 한국실험동물학회 2016 한국실험동물학회 학술발표대회 논문집 Vol.2016 No.08

흰쥐 난포의 성장과 퇴화에 따른 bcl-2 단백질 발현에 관한 면역조직화학적 연구

고필옥,정성윤,조경제,최완성,곽수동,Koh, Phil-ok,Jeong, Sung-yoon,Cho, Gyeong-jae,Choi, Wan-sung,Kwak, Soo-dong 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.1

In the mammalian ovary, follicular development and atresia continuously occur during the reproductive cycles. Follicular atresia occurs through granulosa cell apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2 and bcl-xLong are known as inhibitors of apoptosis, whereas bax and bcl-xShort are known as inducer of apoptosis. We thought that bcl-2 protein is associated with follicular development and atresia. But it is not known that the distribution of cells containing bcl-2 protein during follicular development and atresia. Therefore, to examine the distribution of cells with bcl-2 protein during ovarian follicular development and atresia, the immunohistochemistry was used in the rat ovary. Bcl-2 immunoreactivity was localized in the interstitial cells, theca externa cells and granulosa cells around of antrum. All positive signals were observed in the cytoplasm of these cells. Positive signals were strongly observed in the interstitial and theca externa cells of growing antral follicles. While, positive signals were weakly observed in these cells from atretic antral follicles. Positive signals were very weakly observed in the granulosa cells of growing and atretic antral follicles. According to these data, we suggested that bcl-2 proteins which were strongly expressed in the interstitial cells and theca externa cells of growing antral follicles inhibit follicular atresia. And we purposed that bcl-2 proteins regulated follicular development and atresia through the action of bcl-2 gene family.