Compositional changes in fecal microbiota in a new Parkinson's disease model: C57BL/6-Tg(NSE-haSyn) mice

김지은,권기천,진유정,설아윤,송희진,노유정,김태렬,박은서,박기호,박지원,정영석,조준용,황대연 한국실험동물학회 2023 Laboratory Animal Research Vol.39 No.4

Background: The gut–brain axis (GBA) in Parkinson's disease (PD) has only been investigated in limited mice models despite dysbiosis of the gut microbiota being considered one of the major treatment targets for neurodegenerative disease. Therefore, this study examined the compositional changes of fecal microbiota in novel transgenic (Tg) mice overexpressing human α-synuclein (hαSyn) proteins under the neuron-specific enolase (NSE) to analyze the potential as GBA model. Results: The expression level of the αSyn proteins was significantly higher in the substantia nigra and striatum of NSE-hαSyn Tg mice than the Non-Tg mice, while those of tyrosine hydroxylase (TH) were decreased in the same group. In addition, a decrease of 72.7% in the fall times and a 3.8-fold increase in the fall number was detected in NSE-hαSyn Tg mice. The villus thickness and crypt length on the histological structure of the gastrointestinal (GI) tract decreased in NSE-hαSyn Tg mice. Furthermore, the NSE-hαSyn Tg mice exhibited a significant increase in 11 genera, including Scatolibacter, Clostridium, Feifania, Lachnoclostridium, and Acetatifactor population, and a decrease in only two genera in Ligilactobacillus and Sangeribacter population during enhancement of microbiota richness and diversity. Conclusions: The motor coordination and balance dysfunction of NSE-hαSyn Tg mice may be associated with compositional changes in gut microbiota. In addition, these mice have potential as a GBA model.

CT26 고형암을 내포하는 BALB/cKorl Syngeneic 마우스에서 Ecklonia cava의 항암효과 및 항염증효과

노유정,김지은,진유정,설아윤,송희진,김태렬,민경선,박은서,박기호,황대연 한국생명과학회 2023 생명과학회지 Vol.33 No.11

염증반응(inflammation)은 발병, 진행, 악성 전이를 포함한 암의 진행과정(tumorigenesis)에서 중요한 역할을 수행하기 때문에 암 치료를 위한 전략으로 고려되고 있다. 감태(Ecklonia cava) 열수추출물(AEC)의 항암활성 동안 나타나는 항염증 반응을 연구하기 위하여, 비만세포(mast cells)의 분포, inducible nitric oxide synthase (iNOS)단백질, cyclooxygenase-2 (COX-2)단백질, nuclear factor (NF)-κB단백질, inflammasome 구성 단백질, inflammatory cytokines 발현의 변화는 AEC를 5주간 경구투여한 CT26 대장암을 내포하는 BALB/ cKorl syngeneic 마우스에서 분석하였다. AEC를 처리한 후, 고형암의 무게와 조직 절편의 괴사 부위가 vehicle처리그룹에 비하여 감소하였다. 비만세포의 수는 vehicle처리그룹에 비하여 AEC처리그룹에서 증가했지만 COX-2와 iNOS의 발현은 AEC처리그룹에서 감소하였다. 또한, NF-κB, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)과 Caspase-1 (Cas-1)단백질의 발현도 유사한 감소가 관찰되었다. 더불어, tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α)와 interleukin-6 (IL-6)의 mRNA 발현이 vehicle처리그룹에 비하여 AEC처리그룹에서 감소하였다. 이러한 결과는 AEC가 CT26 고형암을 내포하는 BALB/cKorl syngeneic 마우스에서 항암활성은 염증반응과 밀접한 관련이 있음을 제시하고 있다. The inflammatory response have been considered as one of important targets for cancer treatment because they play a key role during all steps of tumor development including initiation, promotion, malignant conversion and progression. To investigate the anti-inflammatory response during anti-tumor activity of an aqueous extracts of Ecklonia cava (AEC), alterations on the distribution of mast cells and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, inflammasome compositional protein and inflammatory cytokines were examined in CT26 colon tumor-bearing BALB/cKorl syngeneic mice after administrating AEC for five weeks. After treatment of AEC, total weight of tumor and necrotic region of tumor section were significantly decreased compared to vehicle treated group. The number of infiltered mast cells was higher in AEC treated group than vehicle treated group, while the expression levels of COX-2 and iNOS were decreased in AEC treated group. Also, similar decrease pattern were detected in the expression levels of NF-κB, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 (Cas-1) after AEC treatment although the decrease rate was varied. Furthermore, the mRNA expressions of three inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-6 (IL-6) were remarkably decreased in AEC treated group compared to vehicle treated group. These results suggest that inhibition of inflammatory response may be tightly associated with anti-tumor activity of AEC in CT26 colon tumor-bearing BALB/cKorl syngeneic mice.

Comparison of immunophenotypes between Rag2 knockout mice derived from two different sources

노유정,공정은,김지은,진유정,송희진,설아윤,박주민,임용,황대연 한국실험동물학회 2023 Laboratory Animal Research Vol.39 No.1

Background : Recombination activating gene2 (Rag2) knockout (KO) mice are used widely in various research fields, including vaccine development, transplantation studies, and hematopoiesis research, but few studies have compared their phenotypes. This study examined whether there were differences in the immunophenotypes between Rag2 KO mice derived from different sources. In particular, the changes in the organ weight, histological structure, and subpopulation of T and B cells were compared in the spleen and thymus of C57BL/6-Rag2em1hwl/Korl (Rag2/Korl KO) and B6.Cg-Rag2tm1.1Cgn/J (Rag2/J KO) mice. Results: The weight of the spleen and thymus similarly decreased in the Rag2/Korl and Rag2/J KO mice compared to their wild type (WT) mice, even though the other organs were kept at the same weight. A slight difference between the Rag2/Korl and Rag2/J KO group were detected in the number of white blood cells (WBC), lymphocytes (LYM), red cell distribution width (RDW), and platelets (PLT). In addition, the white pulp of the spleen and the cortex region of the thymus decreased in both Rag2 KO mice compared to WT mice. On the other hand, significant differences in the number of CD8+ T and B cell subpopulations between WT and Rag2 KO mice were observed between Rag2/Korl and Rag2/J KO group, while the CD4+ T subpopulation was maintained similarly in both groups. Conclusions: These results suggest that Rag2/Korl and Rag2/J KO mice exhibit similar immunophenotypes in the spleen and thymus except for the differences in the number of CD8+ T and B cell subpopulations.

Chemosensitivity to doxorubicin in primary cells derived from tumor of FVB/N-Trp53tm1Hw1 with TALEN-mediated Trp53 mutant gene

윤우빈,김지은,진유정,노유정,송희진,설아윤,김태렬,민경선,박은서,박기호,강현구,최연식,황대연 한국실험동물학회 2023 Laboratory Animal Research Vol.39 No.4

Background: To evaluate the chemosensitivity to doxorubicin (DOX) in two primary cells derived from a tumor of FVB/N-Trp53tm1Hw1 knockout (KO) mice with TALEN-mediated Trp53 mutant gene, we evaluated the cell survivability, cell cycle distribution, apoptotic cell numbers and apoptotic protein expression in solid tumor cells and ascetic tumor cells treated with DOX. Results: The primary tumor cells showed a significant (P < 0.05) defect for UV-induced upregulation of the Trp53 protein, and consisted of different ratios of leukocytes, fibroblasts, epithelial cells and mesenchymal cells. The IC50 level to DOX was lower in both primary cells (IC50 = 0.12 μM and 0.20 μM) as compared to the CT26 cells (IC50 = 0.32 μM), although the solid tumor was more sensitive. Also, the number of cells arrested at the G0/G1 stage was significantly decreased (24.7–23.1% in primary tumor cells treated with DOX, P < 0.05) while arrest at the G2 stage was enhanced to 296.8–254.3% in DOX-treated primary tumor cells compared with DOX-treated CT26 cells. Furthermore, apoptotic cells of early and late stage were greatly increased in the two primary cell-lines treated with DOX when compared to same conditions for CT26 cells. However, the Bax/Bcl-2 expression level was maintained constant in the primary tumor and CT26 cells. Conclusions: To the best of our knowledge, these results are the first to successfully detect an alteration in chemosensitivity to DOX in solid tumor cells and ascetic tumor cells derived from tumor of FVB/N-Trp53tm1Hw1 mice TALEN-mediated Trp53 mutant gene.

Improvement of the intestinal epithelial barrier during laxative effects of phlorotannin in loperamide-induced constipation of SD rats

김지은,송희진,최윤주,진유정,노유정,설아윤,Park So Hae,박주민,강현구,황대연 한국실험동물학회 2023 Laboratory Animal Research Vol.39 No.1

Background : Disruptions of the intestinal epithelial barrier (IEB) are frequently observed in various digestive diseases, including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). This study assessed the improvement in the IEB during the laxative activity of phlorotannin (Pt) harvested from Ecklonia cava in constipation by examining the changes in the expression of the regulatory proteins for the tight junction (TJ) and adherens junction (AJ), and inflammatory cytokines in Sprague Dawley (SD) rats with loperamide (Lm)-induced constipation after a Pt treatment. Results : The Pt treatment induced laxative activity, including the improvement of feces-related parameters, gastrointestinal transit rate, and histological structure of the mid colon in Lm-treated SD rats. In addition, significant recovery effects were detected in the histology of IEB, including the mucus layer, epithelial cells, and lamina propria in the mid colon of Lm + Pt treated SD rats. The expression levels of E-cadherin and p120-catenin for AJ and the ZO-1, occludin, and Claudin-1 genes for TJ in epithelial cells were improved remarkably after the Pt treatment, but the rate of increase was different. Furthermore, the Pt treatment increased the expression level of several inflammatory cytokines, such as TNF-α, IL-6, IL-1β, IL-13, and IL-4 in Lm + Pt treated SD rats. Conclusions : These results provide the first evidence that the laxative activity of Pt in SD rats with Lm-induced constipation phenotypes involve improvements in the IEB.

1,2,3-Trichloropropane으로 유도된 SD랫드의 간독성에서 ER 스트레스 반응의 조절

김태렬,진유정,김지은,노유정,송희진,설아윤,박은서,박기호,임수정,왕수하,임용,황대연 한국생명과학회 2024 생명과학회지 Vol.34 No.2

ER (Endoplasmic reticulume) 스트레스반응은 difenoconazole 등과 같은 다양한 독성물질에 의한 독성반응 동안에 유도되지만, 농업 및 산업에서 전반적으로 사용되는 화학물질로 간독성(Hepatotoxicity)을 유도하는 1,2,3-Trichloropropane (TCP)와의 연관성은 연구된 바 없다. 따라서, 본 연구에서는 TCP처리로 유발된 간독성(Hepatotoxicity) 유발과정 동안에 ER스트레스의 유발기전에 대해 연구하기 위하여, TCP로 처리된 SD (Sprague Dawley)랫드에서 간독성, apoptosis 그리고 ER스트레스에 대한 지표들의 변화를 분석하였다. 그 결과, TCP 처리그룹은 Vehicle 처리그룹에 비하여 체중과 식이 섭취량이 감소하였고, 간 조직에서 괴사(Necrosis)와 공포화(Vaculation) 등이 유의적으로 증가하였다. 또한, apoptosis 관련 인자인 Bax/Bcl-2와 Cleaved Caspase-3(Cas-3)/Cas-3의 발현은 Vehicle 처리그룹보다 TCP 처리그룹에서 유의적으로 증가하였다. ER스트레스 반응지표 분석에서, C/EBP homologous protein (CHOP), p-eukaryotic translation initiation factor 2 alpha subunit (eIF2α), p-iniositor-requiring enzyme 1α (IRE1α)의 발현은 TCP100 처리그룹에서만 증가하였다. 하지만 Growth arrest and DNA damage-34 (GADD34)와 X-box binding protein-1 (XBP1)의 전사는 TCP200 처리그룹에서 유의적으로 변화되었다. 따라서, 이러한 결과는 ER스트레스반응은 TCP 처리에 의해 유도된 간독성과정 동안에 unfolded protein response (UPR) pathway의 조절을 통해 성공적으로 유도됨을 제시하고 있다. Endoplasmic reticulum (ER) stress responses are markedly induced during toxic responses caused by various chemical substances, including difenoconazole, but no research has been conducted on 1,2,3-trichloropropane (TCP), a chemical that is generally used in agriculture and industry, which induces hepatotoxicity. Therefore, in this study, the changes in indicators for hepatotoxicity, apoptosis, and ER stress were analyzed in TCP-treated Sprague Dawley (SD) rats to study the regulatory mechanism of ER stress during the hepatotoxicity. The TCP-treated group decreased in body weight and dietary intake compared to the vehicle-treated group, and necrosis and vacuolation increased significantly in liver histology. In addition, the expression of apoptosis-related factors, including Bax/Bcl-2 and cleaved caspase (Cas)-3/Cas-3 increased significantly in the TCP-treated group compared to the vehicle-treated group. In the analysis of ER stress response indicators, the expression of C/EBP homologous protein (CHOP), phospho-eukaryotic translation initiation factor 2 alpha subunit (eIF2α), and phospho-inositol-requiring enzyme 1α (IRE1α) increased only in the TCP100-treated group and decreased in the TCP200-treated group. However, the transcriptions of growth arrest and DNA damage-34 (GADD34) increased in the TCP200-treated group, while Spliced X-box binding protein-1 (XBP1s) and unspliced XBP1(XBP1u) decreased in the same group. These results suggest that the ER stress response is successfully triggered during the hepatotoxicity induced by TCP treatment through the alternative regulation of the unfolded-protein response (UPR) pathway.

3T3-L1 지방세포에서 lipogenesis 저해제와 lipolysis 촉진제로서 Dipterocarpus tuberculatus Roxb.의 새로운 역할

이수진(Su Jin Lee),김지은(Ji Eun Kim),최윤주(Yun Ju Choi),진유정(You Jeong Jin),노유정(Yu Jeong Roh),설아윤(AYun Seol),송희진(Hee Jin Song),황대연(Dae Youn Hwang) 한국생명과학회 2022 생명과학회지 Vol.32 No.11

Dipterocarpus tuberculatus Roxb.의 약리학적 효능은 광노화(photoaging), 염증(inflammation), 간독성(hepatotoxicity), 급성 위염(acute gastritis) 및 골유착(osseointegration)을 포함한 일부 분야에서만 연구되었다. 비만에 대한 D. tuberculatus의 새로운 효능을 규명하기 위해, Dipterocarpus tuberculatus Roxb.의 메탄올 추출물(MED)을 처리한 3T3-L1 지방세포에서 지방축적에 대한 억제효과와 지방분해에 대한 촉진효과를 연구하였다. MDI로 분화를 유도한 3T3-L1 지방세포에 분화 기간동안 MED를 처리했을 때, peroxisome proliferator‐activated receptor (PPAR)γ와 CCAAT‐enhancer binding protein (C/EBP) α의 mRNA 수준 뿐만 아니라 adipocyte fatty acid binding protein 2 (aP2)과 fatty acid synthase (FAS)의 발현을 억제하였다. MDI로 분화를 유도한 3T3-L1 지방세포에 분화 기간 동안 MED를 처리했을 때, Oil red O로 염색된 지방방울(lipid droplets)에서 유사한 감소가 관찰되었다. 더불어, 3T3-L1 지방세포에 MDI로 분화를 유도한 후 MED를 처리했을 때, cAMP농도, free glycerol 농도, lipases의 발현을 포함한 lipolytic target의 증가가 관찰되었다. 이러한 결과는 MED가 MDI로 분화를 유도한 3T3-L1 지방세포에서 lipogenesis 저해제와 lipolysis 촉진제로서 새로운 역할을 갖음을 제시하고 있다. The pharmacological efficacy of Dipterocarpus tuberculatus Roxb. has been verified in only several fields including photoaging, inflammation, hepatotoxicity, acute gastritis and osseointegration. To identify the novel functions of Dipterocarpus tuberculatus Roxb. on anti-obesity, inhibitory effect on lipid accumulation and stimulatory effect on lipolysis were investigated in MDI (3-isobutyl-1-methyl-xanthine, dexamethasone, and insulin) stimulated 3T3-L1 adipocytes treated with methanol extracts of Dipterocarpus tuberculatus Roxb. (MED). Lipogenic targets, including lipid accumulation, level of lipogenic transcription factors, and expression of lipogenic regulators, were downregulated in MDI-stimulated 3T3-L1 adipocytes treated with MED without any significant cytotoxicity. Also, MED treatment inhibited the mRNA levels of adipogenic targets including peroxisome proliferator‐activated receptor (PPAR)γ and CCAAT-enhancer binding protein (C/EBP) α, as well as lipogeic targets including adipocyte fatty acid binding protein 2 (aP2) and fatty acid synthetase (FAS) in MDI-stimulated 3T3-L1 adipocytes. A similar decrease patterns were detected in Oil red O stained lipid droplets of MED treated MDI-stimulated 3T3-L1 adipocytes. Furthermore, several lipolytic targets, such as cAMP concentration, concentration of free glycerol, expression level of lipases, including ATGL, perilipin and HSL, were upregulated in MDI-stimulated 3T3-L1 adipocytes treated with MED. These results show that MED has a novel role as a lipogenesis inhibitor and lipolysis stimulator in MDI-stimulated 3T3-L1 adipocytes.