• The Korean Journal of Physiology and Pharmacology
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• 제22권5호
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• pp.577-584
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• 2018

Bladder dysfunction is a common complication of diabetes mellitus (DM). However, there have been a few studies evaluating bladder smooth muscle contraction in DM in the presence of pharmacological inhibitors. In the present study, we compared the contractility of bladder smooth muscle from normal rats and DM rats. Furthermore, we utilized pharmacological inhibitors to delineate the mechanisms underlying bladder muscle differences between normal and DM rats. DM was established in 14 days after using a single injection of streptozotocin (65 mg/kg, intraperitoneal) in Sprague-Dawley rats. Bladder smooth muscle contraction was induced electrically using electrical field stimulation consisting of pulse trains at an amplitude of 40 V and pulse duration of 1 ms at frequencies of 2-10 Hz. In this study, the pharmacological inhibitors atropine (muscarinic receptor antagonist), U73122 (phospholipase C inhibitor), DPCPX (adenosine $A_1$ receptor antagonist), udenafil (PDE5 inhibitor), prazosin (${\alpha}_1$-receptor antagonist), verapamil (calcium channel blocker), and chelerythrine (protein kinase C inhibitor) were used to pretreat bladder smooth muscles. It was found that the contractility of bladder smooth muscles from DM rats was lower than that of normal rats. In addition, there were significant differences in percent change of contractility between normal and DM rats following pretreatment with prazosin, udenafil, verapamil, and U73122. In conclusion, we suggest that the decreased bladder muscle contractility in DM rats was a result of perturbations in $PLC/IP_3$-mediated intracellular $Ca^{2+}$ release and PDE5 activity.

• Biomolecules & Therapeutics
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• 제28권4호
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• pp.328-336
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• 2020

Diabetes mellitus affects the colonic motility developing gastrointestinal symptoms, such as constipation. The aim of the study was to examine the role of intracellular signaling pathways contributing to colonic dysmotility in diabetes mellitus. To generate diabetes mellitus, the rats were injected by a single high dose of streptozotocin (65 mg/kg) intraperitoneally. The proximal colons from both normal and diabetic rats were contracted by applying an electrical field stimulation with pulse voltage of 40 V in amplitude and pulse duration of 1 ms at frequencies of 1, 2, 4, and 6 Hz. The muscle strips from both normal rats and rats with diabetes mellitus were pretreated with different antagonists and inhibitors. Rats with diabetes mellitus had lower motility than the control group. There were significant differences in the percentage of inhibition of contraction between normal rats and rats with diabetes mellitus after the incubation of tetrodotoxin (neuronal blocker), atropine (muscarinic receptor antagonist), prazosin (α₁ adrenergic receptor antagonist), DPCPX (adenosine A1 receptor antagonist), verapamil (L-type Ca²⁺ channel blocker), U73122 (PLC inhibitor), ML-9 (MLCK inhibitor), udenafil (PDE₅ inhibitor), and methylene blue (guanylate cyclase inhibitor). The protein expression of p-MLC and PDE₅ were decreased in the diabetic group compared to the normal group. These results showed that the reduced colonic contractility resulted from the impaired neuronal conduction and decreased muscarinic receptor sensitivity, which resulted in decreased phosphorylation of MLC via MLCK, and cGMP activity through PDE₅.

• Natural Product Sciences
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• 제15권1호
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• pp.27-31
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• 2009

The present study was performed to investigate the binding affinity of the ethanol extract from the leaves of Morus alba (EMA) and some EMA related plant materials (EMA-D, EMA-DM) for melanin-concentrating hormone-1 receptor (MCH-1) and also to examine the antagonistic effect of them for the recombinant MCH-1 receptor expressed in CHO cells. EMA, dichloromethane fraction (EMA-D) and EMA-DM exhibited high affinity for mammalian MCH receptor in receptor binding assays ($IC_{50}$ value: 2.3, 1.6 and $1.0{\mu}g/ml$, respectively). Other plant materials (MMA-D, MMA-DM) obtained from methanol extracts from the leaves of Morus alba (MMA) also exhibited high affinity for mammalian MCH receptor, even though the $IC_{50}$ values of them were lower than those of EMA-D and EMA-DM. In Chinese hamster ovary (CHO) cells expressing human MCH-1, EMA-DM and EMA-D significantly inhibited MCH-induced intracellular $Ca^{2+}$ increase ($IC_{50}$ values: 16.5 and $22.7{\mu}g/ml$, respectively). These results clearly indicate that the ethanol extract from the leaves of Morus alba (EMA) and some EMA related plant materials (EMA-D, EMA-DM) are novel selective MCH-1 receptor antagonist, respectively.

• ALGAE
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• 제36권4호
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• pp.315-326
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• 2021

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (K_v), tandem K_v, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5-trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H⁺/Cl^- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of G-protein coupled receptors- and Ca²⁺-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

• Journal of Ginseng Research
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• 제45권4호
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• pp.490-497
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• 2021

Background and objective: Synthetic ginsenoside compounds G-Rp (1,3, and 4) and natural ginsenosides in Panax ginseng 20(S)-Rg3, Rg6, F4 and Ro have inhibitory actions on human platelets. However, the inhibitory mechanism of ginsenoside Rk1 (G-Rk1) is still unclear thus, we initiated investigation of the anti-platelet mechanism by G-Rk1 from Panax ginseng. Methodology: Our study focused to investigate the action of G-Rk1 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin α_IIbβ₃ using flow cytometry, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane B2 secretion. Thrombin-induced clot retraction was also observed in human platelets. Key Results: Collagen, thrombin, and U46619-stimulated human platelet aggregation were dose-dependently inhibited by G-Rk1, while it demonstrated a more effective suppression on collagen-stimulated platelet aggregation using human platelets. Moreover, G-Rk1 suppressed collagen-induced elevation of Ca²⁺ release from endoplasmic reticulum, granule release, and α_IIbβ₃ activity without any cytotoxicity. Conclusions and implications: These results indicate that G-Rk1 possess strong anti-platelet effect, proposing a new drug candidate for treatment and prevention of platelet-mediated thrombosis in cardiovascular disease.

• 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.

• 공업화학
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• 제34권2호
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• pp.121-125
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• 2023

카세인(casein)은 포유류의 우유에서 발견되는 단백질로 우유에서는 80% 이상 함유되어 있다. 사람의 모유에는 약 20~45%가 포함되어 있으며 생체 적합성이 높아 의료 및 산업 소재로 사용되고 있다. 카세인은 양친매성 구조로 내부는 소수성이기 때문에 수용액에서 마이셀로 자가 조립이 가능하여 난용성 약물을 봉입할 수 있다. 또한, 단백질 고분자 소재로 생분해성을 갖고 있어 약물의 전달체로서 적합한 특징을 가진다. 본 연구에서는 칼슘 이온 외에 마그네슘, 아연, 철 등 생체 내 존재하는 다양한 금속 이온들을 사용하여 각각 효과적인 카세인 나노입자 형성 조건을 규명하였다. 동적 광산란 측정기와 제타 전위 측정을 통해 150 nm 이하의 균일한 사이즈를 유지하고 음전하를 띠는 나노입자가 형성됨을 확인하였다. 또한, 각각의 카세인 나노입자가 HeLa 세포주에서 80% 이상의 생존율을 나타내 낮은 세포 독성을 확인하였고, 카세인 나노입자 내부에 시험 약물로서 나일 레드를 봉입하여 세포 내부로 효과적으로 유입됨을 공초점 현미경으로 입증하였다. 본 실험들을 통해 제조된 카세인 나노입자의 약물 전달체로서의 가능성을 확인하였다.

• 공업화학
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• 제29권6호
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• pp.772-781
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• 2018

본 연구에서는 범부채 뿌리 50% 에탄올 추출물 및 에틸아세테이트 분획물을 제조하고 이들의 항산화 및 항균 활성, 세포 보호 효능을 평가하였다. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) 자유라디칼 소거 활성($FSC_{50}$) 측정 결과, 50% 에탄올 추출물은 $621.5{\mu}g/mL$, 에틸아세테이트 분획물은 $253.0{\mu}g/mL$이었다. Luminol 발광법을 이용한 총 항산화능($OSC_{50}$)은 추출물과 분획물에서 각각 13.6 및 $3.0{\mu}g/mL$이었다. 항균 활성 측정에서 Staphylococcus aureus 및 Candida albicans에 대한 에틸아세테이트 분획물의 최소저해농도(minimum inhibitory concentration, MIC)는 각각 156 및 $1,250{\mu}g/mL$으로 나타났으며, 화장품에 사용하는 기존 방부제인 methyl paraben보다 유사하거나 더 높은 활성을 보여주었다. $^1O_2$로 유도된 세포 손상에 대한 보호 효과(${\tau}_{50}$)에서 50% 에탄올 추출물은 $4{\sim}64{\mu}g/mL$ 농도 범위에서 농도 의존적으로 세포 보호 활성을 나타냈다. $16{\mu}g/mL$ 농도에서 50% 에탄올 추출물, 에틸아세테이트 분획물 및 (+)-${\alpha}$-tocopherol의 ${\tau}_{50}$은 각각 36.4, 45.0 및 45.8 min이었으며, 에틸아세테이트 분획물은 $^1O_2$로 유도된 세포 손상에서 (+)-${\alpha}$-tocopherol과 유사한 세포 보호 활성을 나타냈다. UVB로 유도된 HaCaT 세포 손상에서 에틸아세테이트 분획물은 $8{\mu}g/mL$에서 세포 내 활성산소종(reactive oxygen species, ROS)을 최대 45.9%까지 감소시켰다. 과산화수소로 유도된 HaCaT 세포 손상에서도 에틸아세테이트 분획물은 $0.5{\sim}8.0{\mu}g/mL$에서 세포 생존율을 유의적으로 증가시켰다. 범부채 뿌리 에틸아세테이트 분획물의 성분 분석 결과, irisflorentin, irigenin, tectorigenin, resveratrol, iridin 및 tectoridin 등의 플라보노이드 및 폴리페놀 성분이 확인되었다. 결론적으로 범부채 뿌리 추출물 및 분획물은 화장품의 천연 항산화 및 항균 소재로서 적용 가능성이 있음을 시사한다.

• 대한화장품학회지
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• 제49권3호
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• pp.193-201
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• 2023

H₂O₂는 세포에서 산화 스트레스를 유발하여 세포 성장, 증식, 노화 및 사멸에 영향을 미치는 일종의 활성산소종(ROS)이다. 본 연구의 목적은 H₂O₂의 세포 독성을 완화시키는 활성 펩타이드를 찾는 것이다. 잠재적인 활성 펩타이드의 서열을 예측하기 위해서 위치 스캐닝 합성 테트라펩타이드 조합 라이브러리를 탐색하였다. H₂O₂로 유도된 인간 각질형성세포(HaCaT 세포)의 사멸에 대한 펩타이드 풀들의 완화 효과를 비교한 결과, 다양한 활성 펩타이드의 시퀀스가 예측되었다. 특히 시스테인(C) 잔기를 함유한 펩타이드가 활성이 있을 것으로 예측되었다. 후속 실험에서 C-NH₂, CC-NH₂, CCC-NH₂, CCCC-NH₂ 등의 길이가 다른 시스테인 함유 펩타이드들의 세포 독성과 활성을 조사하였다. C-NH₂ 및 CC-NH₂는 1.0 mM 이하에서 유의한 세포 독성이 없었지만 CCC-NH₂ 및 CCCC-NH₂는 상대적으로 강한 세포 독성을 보였다. C-NH₂와 CC-NH₂는 H₂O₂로 유도된 세포독성을 완화시켰다. CC-NH₂는 C-NH₂, C, N-아세틸 시스테인(NAC) 및 글루타티온(GSH)보다 세포 보호 효과가 높았다. 유세포 분석법으로 세포 내 ROS를 측정하였을 때, H₂O₂는 ROS의 생성을 증가시켰다.H₂O₂ 노출조건에서 CC-NH₂는 C-NH₂보다는 더 효과적으로 ROS 생성을 억제하였고, C, NAC, GSH 만큼 효과적이었다. 본 연구의 결과는 다양한 시스테인 함유 펩타이드 중 특히 CC-NH₂가 H₂O₂로 유도된 세포 독성과 ROS 생성을 안전하고 효과적으로 완화시키는 항산화 특성이 있음을 시사한다.

• Journal of Ginseng Research
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• 제35권4호
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• pp.471-478
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• 2011

Ginseng, the root of Panax ginseng, is one of the oldest herbal medicines. It has a variety of physiological and pharmacological effects. Recently, we isolated a subset of glycolipoproteins that we designated gintonin, and demonstrated that it induced transient change in intracellular calcium concentration $([Ca^{2+}]_i)$ in cells via G-protein-coupled receptor signaling pathway(s). The previous method for gintonin isolation included multiple steps using methanol, butanol, and other organic solvents. In the present study, we developed a much simple method for the preparation of gintonin from ginseng root using 80% ethanol extraction. The extracted fraction was designated edible gintonin. This method produced a high yield of gintonin (0.20%). The chemical characteristics of gintonin such as molecular weight and the composition of the extract product were almost identical as the gintonin prepared using the previous extraction regimen involving various organic solvents. We also examined the physiological effects of edible gintonin on endogenous $Ca^{2+}$-activated $Cl^-$ channel activity of Xenopus oocytes. The 50% effective dose was $1.03{\pm}0.3\;{\mu}g$/mL. Finally, since gintonin preparation through ethanol extraction is easily reproducible, gintonin could be commercially applied for ginseng-derived functional health food and/or drug following the confirmations of in vitro and in vivo physiological and pharmacological effects of gintonin.