• Biomolecules & Therapeutics
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• 제29권2호
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• pp.175-183
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• 2021

The mitogen-activated protein kinase (MAPK) pathway controls intestinal epithelial barrier permeability by regulating tight junctions (TJs) and epithelial cells damage. Heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal epithelial barrier function, but the molecular mechanism is not yet clarified. MAPK activation and barrier permeability were studied using monolayers of Caco-2 cells treated with tissue necrosis factor α (TNF-α) transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. Intestinal mucosal barrier permeability and MAPK activation were also investigated using carbon tetrachloride (CCl4) administration with CoPP (a HO-1 inducer), ZnPP (a HO-1 inhibitor), CO releasing molecule 2 (CORM-2), or inactived-CORM-2-treated wild-type mice and mice with HO-1 deficiency in intestinal epithelial cells. TNF-α increased epithelial TJ disruption and cleaved caspase-3 expression, induced ERK, p38, and JNK phosphorylation. In addition, HO-1 blocked TNF-α-induced increase in epithelial TJs disruption, cleaved caspase-3 expression, as well as ERK, p38, and JNK phosphorylation in an HO-1-dependent manner. CoPP and CORM-2 directly ameliorated intestinal mucosal injury, attenuated TJ disruption and cleaved caspase-3 expression, and inhibited epithelial ERK, p38, and JNK phosphorylation after chronic CCl4 injection. Conversely, ZnPP completely reversed these effects. Furthermore, mice with intestinal epithelial HO-1 deficient exhibited a robust increase in mucosal TJs disruption, cleaved caspase-3 expression, and MAPKs activation as compared to the control group mice. These data demonstrated that HO-1-dependent MAPK signaling inhibition preserves the intestinal mucosal barrier integrity by abrogating TJ dysregulation and epithelial cell damage. The differential targeting of gut HO-1-MAPK axis leads to improved intestinal disease therapy.

• Molecules and Cells
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• 제45권4호
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• pp.257-272
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• 2022

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

• 한국자원식물학회지
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• 제32권6호
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• pp.633-643
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• 2019

Long-term ultraviolet (UV) exposure accelerates the phenomenon of skin photo-aging by activating collagenase and elastase. In this study, we aimed to investigate the effects of a combination of grapefruit and rosemary extracts (cG&Re) on UVB-irradiated damage in HaCaT cells and dorsal mouse skin. In HaCaT cells, cG&Re recovered UVB-reduced cell viability and inhibited protein expression of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (p-Erk), c-Jun N-terminal kinases (p-JNK), and a class of MAPKs (p-P38). Also, cG&Re suppressed UVB-induced collagen and elastin degradation by decreasing matrix metalloproteinases (MMPs) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) expression, which is a transcription factor. Similar results were observed in dorsal mouse skin. Taken together, our data indicate that cG&Re prevent UVB-induced skin photo-aging due to collagen/elastin degradation via activation of MAPKs, MMPs, and the NF-κB signaling pathway in vitro and in vivo.

• Asian Pacific Journal of Cancer Prevention
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• 제15권1호
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• pp.167-174
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• 2014

Tumor angiogenesis, growth and metastasis are three closely related processes. We therefore investigated the effects of barbigerone on all three in the B16F10 tumor model established in both zebrafish and mouse models, and explored underlying molecular mechanisms. In vitro, barbigerone inhibited B16F10 cell proliferation, survival, migration and invasion and suppressed human umbilical vascular endothelial cell migration, invasion and tube formation in concentration-dependent manners. In the transgenic zebrafish model, treatment with $10{\mu}M$ barbigerone remarkably inhibited angiogenesis and tumor-associated angiogenesis by reducing blood vessel development more than 90%. In vivo, barbigerone significantly suppressed angiogenesis as measured by H and E staining of matrigel plugs and CD31 staining of B16F10 melanoma tumors in C57BL/6 mice. Furthermore, it exhibited highly potent activity at inhibiting tumor growth and metastasis to the lung of B16F10 melanoma cells injected into C57BL/6 mice. Western blotting revealed that barbigerone inhibited phosphorylation of AKT, FAK and MAPK family members, including ERK, JNK, and p38 MAPKs, in B16F10 cells mainly through the MEK3/6/p38 MAPK signaling pathway. These findings suggested for the first time that barbigerone could inhibit tumor-angiogenesis, tumor growth and lung metastasis via downregulation of the MEK3/6/p38 MAPK signaling pathway. The findings support further investigation of barbigerone as a potential anti-cancer drug.

• 한국축산식품학회지
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• 제40권2호
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• pp.274-285
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• 2020

In this study, ovalbumin (OVA) hydrolysates were prepared using various proteolytic enzymes and the anti-inflammatory activities of the hydrolysates were determined. Also, the potential application of OVA as a functional food material was discussed. The effect of OVA hydrolysates on the inhibition of nitric oxide (NO) production was evaluated via the Griess reaction, and their effects on the expression of inducible NO synthase (inducible nitric oxide synthase, iNOS) were assessed using the quantitative real-time PCR and Western blotting. To determine the mechanism by which OVA hydrolysates activate macrophages, pathways associated with the mitogen-activated protein kinase (MAPK) signaling were evaluated. When the OVA hydrolysates were added to RAW 264.7 cells without lipopolysaccharide (LPS) stimulation, they did not affect the production of NO. However, both the OVA-Protex 6L hydrolysate (OHPT) and OVA-trypsin hydrolysate (OHT) inhibited NO production dose-dependently in LPS-stimulated RAW 264.7 cells. Especially, OHT showed a strong NO-inhibitory activity (62.35% at 2 mg/mL) and suppressed iNOS production and the mRNA expression for iNOS (p<0.05). Also, OHT treatment decreased the phosphorylation levels of Jun amino-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK) in the MAPK signaling pathway. These findings suggested that OVA hydrolysates could be used as an anti-inflammatory agent that prevent the overproduction of NO.

• Mycobiology
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• 제43권4호
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• pp.450-457
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• 2015

Medicinal mushrooms have been used worldwide to treat cancer and modulate the immune system. Over the last several years, there has been increasing interest in isolating bioactive compounds from medicinal mushrooms and evaluating their health beneficial effects. Fomes fomentarius is used in traditional oriental medicine and is known to possess antioxidant, antiinflammatory, antidiabetic, and antitumor effects. In the present study, we isolated fomentariol from Fomes fomentarius and investigated its anti-inflammatory effect in murine macrophages (RAW264.7 cells) stimulated with lipopolysaccharides. Fomentariol inhibited the production of nitric oxide and intracellular reactive oxygen species triggered by lipopolysaccharides. Interestingly, fomentariol differentially regulated cytokine production triggered by lipopolysaccharides. Fomentariol effectively suppressed the production of interleukin-$1{\beta}$ and interleukin-6 but not tumor necrosis factor-${\alpha}$. The inhibitory effect of fomentariol against nitric oxide, interleukin-$1{\beta}$, and interleukin-6 production was possibly mediated by downregulation of the extracellular signal-regulated kinase signaling pathway. Taken together, our results suggest that fomentariol differentially modulated inflammatory responses triggered by lipopolysaccharides in macrophages and is one of the bioactive compounds that mediate the physiological effects of Fomes fomentarius.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.775-783
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• 2021

Sea cucumber, Holothuria scabra, is a well-known traditional Asian medicine that has been used for suppressing inflammation, promoting wound healing, and improving immunity. Moreover, previous studies demonstrated that the extract from H. scabra contains many bioactive compounds with potent inhibitory effect on tumor cell survival and progression. However, the effect of the methanolic extract from the body wall of H. scabra (BWMT) on human prostate cancer cells has not yet been investigated. In this study, we aimed to investigate the effects and underlying mechanism of BWMT on prostate cancer cell viability and metastasis. BWMT was obtained by maceration with methanol. The effect of BWMT on cell viability was assessed by MTT and colony formation assays. The intracellular ROS accumulation was evaluated using a DCFH-DA fluorescence probe. Hoechst 33342 staining and Annexin V-FITC/PI staining were used to examine the apoptotic-inducing effect of the extract. A transwell migration assay was performed to determine the anti-metastasis effect. BWMT significantly reduced cell viability and triggered cellular apoptosis by accumulating intracellular ROS resulting in the upregulation of JNK and p38 signaling pathways. In addition, BWMT also inhibited the invasion of PC3 cells by downregulating MMP-2/-9 expression via the ERK pathway. Consequently, our study provides BWMT from H. scabra as a putative therapeutic agent that could be applicable against prostate cancer progression.

• IMMUNE NETWORK
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• 제16권4호
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• pp.242-248
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• 2016

Thymic atrophy is a complication that results from exposure to many environmental stressors, disease treatments, and microbial challenges. Such acute stress-associated thymic loss can have a dramatic impact on the host's ability to replenish the necessary naïve T cell output to reconstitute the peripheral T cell numbers and repertoire to respond to new antigenic challenges. We have previously reported that treatment with the orexigenic hormone ghrelin results in an increase in the number and proliferation of thymocytes after dexamethasone challenge, suggesting a role for ghrelin in restraint stress-induced thymic involution and cell apoptosis and its potential use as a thymostimulatory agent. In an effort to understand how ghrelin suppresses thymic T cell apoptosis, we have examined the various signaling pathways induced by receptor-specific ghrelin stimulation using a restraint stress mouse model. In this model, stress-induced apoptosis in thymocytes was effectively blocked by ghrelin. Western blot analysis demonstrated that ghrelin prevents the cleavage of pro-apoptotic proteins such as Bim, Caspase-3, and PARP. In addition, ghrelin stimulation activates the Akt and Mitogen-activated protein kinases (MAPK) signaling pathways in a time/dose-dependent manner. Moreover, we also revealed the involvement of the FoxO3a pathway in the phosphorylation of Akt and ERK1/2. Together, these findings suggest that ghrelin inhibits apoptosis by modulating the stress-induced apoptotic signal pathway in the restraint-induced thymic apoptosis.

• Journal of Ginseng Research
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• 제45권2호
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• pp.354-360
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• 2021

Background: Protopanaxatriol (PPT) is a secondary intestinal metabolite of ginsenoside in ginseng. Although the effects of PPT have been reported in various diseases including cancer, diabetes and inflammatory diseases, the skin protective effects of PPT are poorly understood. Methods: HaCaT cells were treated with PPT in a dose-dependent manner. mRNA and protein levels which related to skin barrier and hydration were detected compared with retinol. Luciferase assay was performed to explore the relative signaling pathway. Western blot was conducted to confirm these pathways and excavated further signals. Results: PPT enhanced the expression of filaggrin (FLG), transglutaminase (TGM)-1, claudin, occludin and hyaluronic acid synthase (HAS) -1, -2 and -3. The mRNA expression levels of FLG, TGM-1, HAS-1 and HAS-2 were suppressed under NF-κB inhibition. PPT significantly augmented NF-κB-luc activity and upregulated Src/AKT/NF-κB signaling. In addition, PPT also increased phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 and upstream MAPK activators (MEK and MKK). Furthermore, transcriptional activity of AP-1 and CREB, which are downstream signaling targets of MAPK, was enhanced by PPT. Conclusion: PPT improves skin barrier function and hydration through Src/AKT/NF-κB and MAPK signaling. Therefore, PPT may be a valuable component for cosmetics or treating skin disorders.

• 대한소아치과학회지
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• 제29권3호
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• pp.337-344
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• 2002

조골 세포의 분화에 중요한 역할을 하는 전사 인자인 Runx2는 그 역할은 많이 알려져 있지만, 이를 조절하는 신호 전달체계에 대해서는 많이 알려지지 않았다. 이에 본 연구에서는 조골 세포의 분화 및 증식에 영향을 미친다고 알려진 PKC 및 MAPK pathway가 Runx2에 미치는 영향을 알아보고자 하였다. PKC활성화에 따른 Runx2의 전사 활성 및 발현 양상을 관찰하기 위해 6XOSE2-C2C12 cell에 PKC 활성제를 처리하여 luciferase assay와 Northern blot analysis를 시행하였다. MAPK 활성화에 따른 Runx2의 전사 활성을 관찰하기 위해 MAPK 활성제를 6XOSE2-C2C12 cell에 처리하여 luciferase assay를 시행하였다. 두 신호 전달 체계의 활성화에 따른 골 표지 유전자의 전사 양상을 관찰하기 위해 osteocalcin과 osteopontin을 transient transfection한 C2C12 cell에 각 신호 전달 체계의 활성제를 처리하여 luciferase assay를 시행하였다. 또한 각 신호 전달 체계가 상호 작용하는지 알아보기 위하여 MAPK 억제제를 전처리하여 MAPK pathway를 차단한 1 시간 뒤 PKC 활성제를 처리하고 luciferase assay를 시행하여 Runx2의 전사 활성을 관찰하였다. 이상의 실험으로 다음과 같은 결론을 얻었다. - PKC pathway의 활성화는 Runx2의 전사 활성 및 발현을 증가시키고 이로 인해 그의 영향을 받는 골 표지 유전자 (osteopontin, osteocalcin)의 전사도 증가한다. - MAPK pathway의 활성화는 Runx2 및 골 표지 유전자 (osteopontin, osteocalcin)의 전사활성을 증가시킨다. - PKC pathway는 MAPK pathway를 경유하여 Runx2의 전사 활성을 조절한다.