• Animal Bioscience
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• 제34권10호
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• Molecules and Cells
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• 제47권1호
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• pp.100004.1-100004.11
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• 2024

Insulin is essential for maintaining normoglycemia and is predominantly secreted in response to glucose stimulation by β-cells. Incretin hormones, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide, also stimulate insulin secretion. However, as obesity and type 2 diabetes worsen, glucose-dependent insulinotropic polypeptide loses its insulinotropic efficacy, whereas GLP-1 receptor (GLP-1R) agonists continue to be effective owing to its signaling switch from Gs to Gq. Herein, we demonstrated that endoplasmic reticulum (ER) stress induced a transition from Gs to Gq in GLP-1R signaling in mouse islets. Intriguingly, chemical chaperones known to alleviate ER stress, such as 4-PBA and TUDCA, enforced GLP-1R's Gq utilization rather than reversing GLP-1R's signaling switch induced by ER stress or obese and diabetic conditions. In addition, the activation of X-box binding protein 1 (XBP1) or activating transcription factor 6 (ATF6), 2 key ER stress-associated signaling (unfolded protein response) factors, promoted Gs utilization in GLP-1R signaling, whereas Gq employment by ER stress was unaffected by XBP1 or ATF6 activation. Our study revealed that ER stress and its associated signaling events alter GLP-1R's signaling, which can be used in type 2 diabetes treatment.

• 한국콘텐츠학회논문지
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• 제9권4호
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• pp.355-363
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• 2009

비정상적인 미토콘드리아에 의해 산화 스트레스가 증가하면 세포내 신호전달 및 유전자 발현에 손상을 일으켜 인슐린 저항성이나 당뇨병 등의 여러 질환들을 유발한다. 그런데 자식작용은 산화 스트레스로 기능이 저하된 미토콘드리아를 제거하여 인슐린 저항성 등을 억제해준다. 한편 운동도 미토콘드리아 생합성을 강화시켜 조직의 기능저하나 퇴행을 회복시켜준다. 따라서 운동과 자식작용이 서로 연관되어 미토콘드리아 생합성을 유도하는 신호체계로 작용할 가능성이 있고, 이 연구를 통해 운동 혹은 AICAR (aminoimidazole-4-carboxamide-1-${\beta}$-D-ribofuranoside)처치로 활성 화된 AMPK(5'-AMP- activated protein kinase) 신호전달체계가 미토콘드리아 생합성을 증가시키는 경로에 자식작용이 관여하는지의 여부를 확인하고자 하였다. 연구결과에 따르면, 6시간의 급성운동으로 쥐의 골격근에서 PGC-1(peroxisome proliferator-activated receptor gamma coactivator 1)과 mtTFA (mitochondrial transcription factor A)의 mRNA 발현이 유의하게 증가하였다. 하지만 자식작용 표지제인 LC3(microtubule-associated proteinl light chain 3)의 mRNA 발현은 증가경향을 나타냈지만 유의하지 않았다. 한편 C2C12 근세포에서도 AICAR 처치에 의해 PGC-1, mtTFA mRNA 발현이 모두 증가하였지만, 이러한 증가는 LC3 SiRNA에 의해서 억제되지 않는 것으로 나타났다. 이러한 결과들을 통해 자식작용은 AMPK에 의해 조절되는 신호전달 전달체계와는 다른 경로로 미토콘드리아 생합성에 영향을 미칠 것으로 사료된다.

• BMB Reports
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• 제35권1호
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• pp.116-126
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• 2002

Nitric oxide (NO), synthesized from L-arginine by NO synthases, is a small, lipophilic, diffusible, highly reactive molecule with dichotomous regulatory roles in many biological events under physiological and pathological conditions. NO can promote apoptosis (pro-apoptosis) in some cells, whereas it inhibits apoptosis (anti-apoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as metal ion, thiol, protein tyrosine, and reactive oxygen species. Long-lasting overproduction of NO acts as a pro-apoptotic modulator, activating caspase family proteases through the release of mitochondrial cytochrome c into cytosol, up-regulation of the p53 expression, and alterations in the expression of apoptosis-associated proteins, including the Bcl-2 family. However, low or physiological concentrations of NO prevent cells from apoptosis that is induced by the trophic factor withdrawal, Fas, $TNF{\alpha}$/ActD, and LPS. The anti-apoptotic mechanism is understood on the basis of gene transcription of protective proteins. These include: heat shock protein, hemeoxygenase, or cyclooxygenase-2 and direct inhibition of the apoptotic executive effectors caspase family protease by S-nitrosylation of the cysteine thiol group in their catalytic site in a cell specific way. Our current understanding of the mechanisms by which NO exerts both pro- and anti-apototic action is discussed in this review article.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.645-656
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• 2022

Gossypol, a natural phenolic aldehyde present in cotton plants, was originally used as a means of contraception, but is currently being studied for its anti-proliferative and anti-metastatic effects on various cancers. However, the intracellular mechanism of action regarding the effects of gossypol on pancreatic cancer cells remains unclear. Here, we investigated the anti-cancer effects of gossypol on human pancreatic cancer cells (BxPC-3 and MIA PaCa-2). Cell counting kit-8 assays, annexin V/propidium iodide staining assays, and transmission electron microscopy showed that gossypol induced apoptotic cell death and apoptotic body formation in both cell lines. RNA sequencing analysis also showed that gossypol increased the mRNA levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and activating transcription factor 3 (ATF3) in pancreatic cancer cell lines. In addition, gossypol facilitated the cleavage of caspase-3 via protein kinase RNA-like ER kinase (PERK), CHOP, and Bax/Bcl-2 upregulation in both cells, whereas the upregulation of ATF was limited to BxPC-3 cells. Finally, a three-dimensional culture experiment confirmed the successful suppression of cancer cell spheroids via gossypol treatment. Taken together, our data suggest that gossypol may trigger apoptosis in pancreatic cancer cells via the PERK-CHOP signaling pathway. These findings propose a promising therapeutic approach to pancreatic cancer treatment using gossypol.

• 생명과학회지
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• 제30권3호
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• pp.285-290
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• 2020

구제역바이러스(FMDV)는 Picornaviridae 과의 Aphthovirus 속의 한 종류이며, 야생과 가축의 소와 돼지에 감염한다. FMDV는 감염 조직에서 중증의 염증반응을 포함한 다양한 임상적 증후들을 일으킨다. FMDV 게놈 RNA는 약 8.3 kb 길이의 양성-단일 가닥을 가지고 있으며, 하나의 긴 단백질 번역틀(ORF)을 만든다. 이 ORF는 바이러스의 단백질가수분해효소에 의해서 구조단백질과 비구조단백질로 나누어진다. FMDV의 FMDV 2C 단백질은 FMDV 유전자에서 만들어지는 비구조단백질로서 염증과 세포사를 포함한 FMD 병리 과정과 바이러스 복제에서 중요한 역할을 한다. 이 연구에서 우리는 FMDV 2C가 염증 유도 사이토카인인 tumor necrosis factor alpha (TNFα)의 세포내 발현을 유도하는 가능성을 검토하였다. FMDV 2C의 돼지 세포인 IBRS-2 세포내 발현은 TNFα의 유전자 발현 조절 부위인 프로모터의 활성화를 이용하여 전사수준에서 TNFα의 mRNA와 단백질 생성을 증가시켰다. 추가적으로, 소포체 스트레스를 감소시키는 화학물질인 4-phenylbutyric acid (4-PBA) 처리는 FMDV 2C에 의해 유도된 TNFα 발현을 감소시켰다. 소포체 스트레스 반응을 매개하는 전사인자의 한 종류인 ATF4는 TNFα 프로모터의 활성을 유도하고, TNFα의 mRNA와 단백질 발현을 증가시켰다. 하지만, ATF4의 기능 결핍 돌연변이체 단백질의 발현은 FMDV 2C에 의한 TNFα 생성을 유도하지 못하였다. 이들 결과들은 FMDV FMDV 2C 단백질이 ATF4-매개 TNFα 발현을 통해 임상적 염증반응을 증가시키고, 이는 소포체 스트레스의 유도와 연관되어있음을 제시한다.

• 대한한의학회지
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• 제32권2호
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• pp.1-13
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• 2011

Background and Objective: Coptidis Rhizoma is a medicinal herb known for its antioxidant and anti-inflammatory effect. The purpose of this study was to examine the effects of CR on the experimental burn elicitation in vitro and in vivo. Material and Methods: In order to know the antioxidant effect on skin cell of mice after burn elicitation, superoxide dismutase (SOD) activity was measured. In vitro, the RAW 264.7 macrophage cells were treated with lipopolysaccharides for experimental inflammation. iNOS mRNA expression was observed after CR-treatment. In order to know effects on the skin regeneration in the burned mice, we counted the nitric oxide (NO) in blood. We also observed the histological structure in the epidermal basal layer and the dermal section, and we studied changes of angiogenesis in the capillaries surrounding the basal layer and dermal papilla. The changes of transcription of iNOS mRNA (inducible nitric oxide synthase mRNA) and changes of NF-${\kappa}$B (nuclear factor ${\kappa}$B) p65 positive reaction were also observed to investigate the changes of the stress in the skin. Results: The results indicated that CR has significant effects on the antioxidant effect on skin cells of mice after burn elicitation by increasing SOD activity in the in vitro test. It seemed that CR decreased the amount of NF-${\kappa}$B which induced the iNOS mRNA dose-dependently and suppress activating NO and angiogenesis. Furthermore, CR facilitated the process of skin recovery after experimental burn. Conclusion: CR can be applied for burned skin via antioxidant effect and skin regeneration.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.350-356
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• 2017

We previously reported that the CopA3 peptide (LLCIALRKK, ${\small{D}}-form$) originally isolated from the Korean dung beetle has antimicrobial and immunosuppressive effects. However, the high cost of producing the synthetic peptide, especially the ${\small{D}}-form$, has limited the development of CopA3 for therapeutic purposes. Here, we investigated whether the CopA3 deletion derivative, CopA5, which is composed of only five amino acids (LLCIA) and has the ${\small{L}}-form$ structure, could inhibit the lipopolysaccharide (LPS)-induced activation of macrophages. Peritoneal exudate macrophages (PEM) were isolated from mice and exposed to LPS in the presence or absence of CopA5, and biomarkers of macrophage activation were measured. Our results revealed that LPS-induced nitric oxide (NO) production, tumor necrosis factor $(TNF)-{\alpha}$ secretion, and phagocytic activity of PEM were significantly inhibited by CopA5 treatment. Similar to CopA3, the structurally modified CopA5 peptide had no cell toxicity (as assessed by measurement of cell viability loss and apoptosis) in PEM. Moreover, the LPS-induced upregulation of the activating phosphorylation of signal transducer and activator of transcription 1 (STAT1) was markedly inhibited by CopA5 treatment. These results suggest that, similar to CopA3, CopA5 inhibits macrophage activation by inhibiting STAT1 phosphorylation and blocking the release of NO and $TNF-{\alpha}$. CopA5 may therefore prove therapeutically useful in the realm of immune suppression.

• 동의생리병리학회지
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• 제33권4호
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• pp.191-197
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• 2019

This study aims to evaluate the effects of the root bark of Morus alba (RMA) on the regulation of the Hippo-YAP pathway. Hippo-YAP signaling is a critical regulator in controlling organ size and tissue homeostasis. Hippo, the serine/threonine kinase phosphorylate the LATS. Phosphorylated LATS then phosphorylates and inactivates the YAP and TAZ, which are two closely related transcriptional co-activator. Here we report RMA activates the Hippo signaling, thereby inhibits the YAP/TAZ activity. First, we examine the cytotoxic effects of RMA by MTT assay. RMA was cytotoxic at concentrations higher than $50{\mu}g/ml$ in HEK293A cells. The reporter gene assay was performed to measure the activity of TEAD, a key transcription factor that controls cell growth and proliferation. RMA significantly suppressed the luciferase activity. By phos-taq gel shift assay, and western blotting, we showed that RMA enhanced the phosphorylation of YAP in wild type cells, but not in LATS1/2 knock out cells, which means RMA activates classical Hippo pathway. RMA induced the cytoplasmic sequestration of YAP. RMA also suppressed the mRNA expression of CTGF and CYR61; the two major YAP dependent genes. Taken together, RMA is considered to be a good candidate for proliferative disease such as cancer, by facilitating cell death through activating the Hippo signaling pathway.

• Journal of Ginseng Research
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• 제46권2호
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• pp.304-314
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• 2022

Background: Ginsenosides are biologically active components of ginseng and have various functions. In this study, we investigated the immunomodulatory activity of a ginseng product generated from ginseng powder (GP) via enzymatic bioconversion. This product, General Bio compound K-10 mg solution (GBCK10S), exhibited increased levels of minor ginsenosides, including ginsenoside-F1, compound K, and compound Y. Methods: The immunomodulatory properties of GBCK10S were confirmed using mice and a human natural killer (NK) cell line. We monitored the expression of molecules involved in immune responses via enzyme-linked immunosorbent assay, flow cytometry, NK cell-targeted cell destruction, quantitative reverse-transcription real-time polymerase chain reaction, and Western blot analyses. Results: Oral administration of GBCK10S significantly increased serum immunoglobulin M levels and primed splenocytes to express pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ. Oral administration of GBCK10S also activated NK cells in mice. Furthermore, GBCK10S treatment stimulated a human NK cell line in vitro, thereby increasing granzyme B gene expression and activating STAT5. Conclusion: GBCK10S may have potent immunostimulatory properties and can activate immune responses mediated by B cells, Th1-type T cells, and NK cells.