• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.304-310
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• 2012

The present study showed that Transforming growth factor beta 1 (TGF-${\beta}_1$) can induce apoptosis of bovine mammary epithelial cells. This apoptosis was also observed with phosphorylation of Smad2/3 within 0.5-2 h. Afterwards the signal transferred into the nucleus. Moreover, intracellular free $Ca^{2+}$ concentration was significantly elevated as well as Caspase-3 activated and DNA lysised, thereby inducing the programmed cell death. This signaling pathway of TGF-${\beta}_1$ was blocked by SB-431542 ($10^{-2}{\mu}M$) via inhibiting ALK-5 kinase activity, which thus reversed the anti-proliferation and apoptosis effect of TGF-${\beta}_1$ in mammary epithelial cells. These results indicated that TGF-${\beta}_1$ induced apoptosis of bovine mammary epithelial cells through the ALK-5-Smad2/3 pathway, which plays an important role in inhibiting survival of mammary epithelial cells. Moreover, intracellular $Ca^{2+}$ also played a critical role in TGF-${\beta}_1$-induced cell apoptosis.

• The Korea Journal of Herbology
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• v.31 no.1
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• pp.61-67
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• 2016

Objectives : The present study aimed to investigate the anti-inflammatory effects of the water extracts of Portulacae Herba (PH).Methods : We measured the effects of the water extracts of Portulacae Herba (PH) on the cell viability of mouse macrophage RAW 264.7 cells, the intracellular calcium production, and the proinflammatory mediators including nitric oxide (NO), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, which are induced by the lipopolysaccharides (LPS), and obtained the results shown below.Results : After the cultivation of the PH extracts along with the mouse macrophages, the cell survival rate did not decrease with the MTT assay. However, the PH extracts did significantly suppress the production of NO by the mouse macrophages induced by LPS at the concentrations of 25, 50 and 100 ㎍/mL. The PH extract also significantly suppressed the VEGF, PDGF-BB and intracellular calcium production of the mouse macrophages by LPS at concentrations of 25, 50 and 100 ㎍/mL. As shown in the results above, the PH extracts do not have a toxic effect on the macrophages, but still have an anti-inflammatory effect that significantly reduces the intracellular calcium production as well as the production of NO, VEGF and PDGF-BB at concentrations above 25 ㎍/mL.Conclusions : In conclusion, the inhibitory anti-inflammatory effects of the PH extract can be used for a new treatment of anti-inflammatory diseases.

• Journal of Medicine and Life Science
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• v.15 no.2
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• pp.67-71
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• 2018

Neuronal excitotoxicity induces mitochondrial dysfunction and the release of proapoptotic proteins. Excitotoxicity, the process by which the overactivation of excitatory neurotransmitter receptors leads to neuronal cell death. Neuronal death by excitotoxicity was related to neuronal degenerative disorders and hypoxia, results from excessive exposure to excitatory neurotransmitters, such as glutamate. Glutamate acts at NMDA receptors in cultured neurons to increase the intracellular free calcium concentration. Therefore endogenous glutamate may be a key factor to regulate neuronal cell death via activating $Ca^{2+}$ signaling. For this issue, we tested some conditions to alter intracellular $Ca^{2+}$ level in dissociated hippocampal neurons of rats. Cultured hippocampal neuron were treated by KCl (20 mM), $CaCl_2$ (3.8 mM) and glutamate ($5{\mu}M$) for 24 hrs. Interestingly, The Optical Density of hippocampal neurons was increased by high KCl application in MTT assay data. This enhanced response by high KCl was dependent on synaptic $Ca^{2+}$ influx but not on intracellular $Ca^{2+}$ level. However, the number of neurons seemed to be not changed in Hoechst 33342 staining data. These results suggest that enhancement of synaptic activity plays a key role to increase mitochondrial signaling in hippocampal neurons.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.2
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• pp.117-126
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• 2022

Objective: This study examined whether the addition of triple antioxidants (3A)-10 µM acetyl-L-carnitine, 10 µM N-acetyl-L-cysteine, and 5 µM α-lipoic acid-in freezing-thawing medium during human sperm cryopreservation using the sucrose vitrification (SuV) and liquid nitrogen vapor (Vapor) techniques could improve post-thaw survival of spermatozoa. Methods: We analyzed 30 samples from healthy human sperm donors. Each sample was allocated into one of five groups: fresh control, SuV, SuV+3A, Vapor, and Vapor+3A. The sperm motility, morphology, viability, intracellular and extracellular reactive oxygen species (ROS) levels, and sperm DNA fragmentation (SDF) were evaluated. Results: The cryopreserved spermatozoa had significantly reduced percentages of motility (p<0.05) and viability (p<0.05). Antioxidant supplementation non-significantly improved these parameters (p>0.05). No significant differences were found in sperm morphology between the fresh and frozen-thawed groups (p>0.05). After freezing, the extracellular ROS levels in the frozen-thawed groups were significantly higher (p<0.05) than in the fresh group. However, we did not find any differences in intracellular ROS parameters among these groups (p>0.05). The SDF was higher in the SuV and Vapor groups than in the fresh group, but without statistical significance (p=0.075 and p=0.077, respectively). Conclusion: Cryopreservation had detrimental effects on sperm motility, viability, and extracellular ROS levels, without changing the morphology or intracellular ROS levels. Antioxidant supplementation was slightly effective in preventing SDF in frozen-thawed spermatozoa.

• Animal Bioscience
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• v.36 no.7
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• pp.1120-1129
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• 2023

Objective: This study aimed to determine the protective efficacy of Buddha's Temple (BT) extract against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in Gallus gallus chicken embryo fibroblast cell line (DF-1) and its effects on the cell lipid metabolism. Methods: In this experimental study, Gallus gallus DF-1 fibroblast cells were pretreated with BT 10^-7 for 24 hours, followed by their six-hour exposure to t-BHP (100 μM). Water-soluble tetrazolium salt-8 (WST-8) assays were performed, and the growth curve was computed. The intracellular gene expression changes caused by BT extract were confirmed through quantitative polymerase chain reaction (qPCR). Flow cytometry, oil red O staining experiment, and thin-layer chromatography were performed for the detection of intracellular metabolic mechanism changes. Results: The WST-8 assay results showed that the BT pretreatment of Gallus gallus DF-1 fibroblast cell increased their cell survival rate by 1.08%±0.04%, decreased the reactive oxygen species (ROS) level by 0.93%±0.12% even after exposure to oxidants, and stabilized mitochondrial activity by 1.37%±0.36%. In addition, qPCR results confirmed that the gene expression levels of tumor necrosis factor α (TNFα), TIR domain-containing adapter inducing IFN-beta (TICAM1), and glucose-regulated protein 78 (GRP78) were regulated, which contributed to cell stabilization. Thin-layer chromatography and oil red O analyses showed a clear decrease in the contents of lipid metabolites such as triacylglycerol and free fatty acids. Conclusion: In this study, we confirmed that the examined BT extract exerted selective protective effects on Gallus gallus DF-1 fibroblast cells against cell damage caused by t-BHP, which is a strong oxidative inducer. Furthermore, we established that this extract significantly reduced the intracellular ROS accumulation due to oxidative stress, which contributes to an increase in poultry production and higher incomes.

• Journal of Microbiology and Biotechnology
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• v.32 no.9
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• pp.1126-1133
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• 2022

This study investigated the contribution of lipoxygenase (LOX) inhibitors, nordihydroguaiaretic acid (NDGA), tetra-O-methyl nordihydroguaiaretic acid (M₄N) and zileuton (ZIL), and thromboxane A2 (TXA₂) inhibitor 4,5-diphenylimidazole (DPI) in the proliferation of Brucella abortus infection. None of the compounds affected the uptake of Brucella into the macrophages. We determined the effect of neutralizing leukotriene B4 (LTB4) receptor and showed that the uptake of the bacteria was inhibited at 30 min post-infection. M₄N treatment attenuated intracellular survival of Brucella at 2 h post-incubation but it was not observed in the succeeding time points. DPI treatment showed reduced survival of Brucella at 24 h post-incubation while blocking LTB4 receptor was observed to have a lower intracellular growth at 48 h post-incubation suggesting different action of the inhibitors in the course of the survival of Brucella within the cells. Reduced proliferation of the bacteria in the spleens of mice was observed in animals treated with ZIL or DPI. Increased serum cytokine level of TNF-α and MCP-1 was observed in mice treated with M₄N or ZIL while a lower IFN-γ level in ZIL-treated mice and a higher IL-12 serum level in DPI-treated mice were observed at 7 d post-infection. At 14 d post-infection, ZIL-treated mice displayed reduced serum level of IL-12 and IL-10. Overall, inhibition of 5-LOX or TXA₂ or a combination therapy promises a potential alternative therapy against B. abortus infection. Furthermore, strong ligands for LTB4 receptor could also be a good candidate for the control of Brucella infection.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.373-377
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• 1995

We investigated the survival, $\beta$-galactosidase activity and cellular permeability of lactic acid bacteria such as Lactobacillus acidophilus ATCC 4356, Lactobacillus casei subsp. casei IFO 3533, Streptococcus thermophilus KCTC 2185, Lactobacillus delbrueckii subsp. lactis ATCC 4797, and Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 in anaerobic condition of pH 1.5-3.5 range. Numbers of all tested viable cells did not decrease at pH 3.5, but decreased rapidly at pH 1.5 and pH 2.5 during 2 hour incubation at modified EG medium. Immediately after 2 hour incubation, the decrease in population at pH 1.5 and pH 2.5 was about 6-8 and 5-7 log cycles/ml, respectively. Lactobacillus acidophilus ATCC 4356 showed the higest survival of all tested bacteria. The $\beta$-galactosidase activity from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Streptococcus thermophilus KCTC 2185 decreased rapidly at pH 1.5 and 2.5, but there was a little decrease at pH 3.5. The cellular permeability that was measured by the leakage of intracellular materials increased with decrease of pH. These results suggest that the ingested lactic acid bacteria may be destroyed in contact with low pH of gastric acid.

• CELLMED
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• v.4 no.2
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• pp.12.1-12.12
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• 2014

Epinephrine is a critical drug for patients at risk for anaphylaxis. Here, we suggest moxibustion as an alternative method to reduce anaphylaxis. Moxibustion was applied to the Shimen (CV5) acupoint and found to attenuate compound 48/80-induced mortality. Capsazepine, a transient receptor potential vanilloid (TRPV) 1 antagonist, significantly improved overall survival rates compared to groups treated with moxibustion or 2-aminoethoxydiphenyl borate (an activator of TRPV1, 2, and 3). Probenecid (a TRPV2 agonist) also increased survival rate and reduced histamine levels. Survival rates increased by moxibustion and probenecid were completely inhibited by ruthenium red (a TRPV2 and 3 antagonist) and gadolinium chloride (general TRPV antagonist), respectively. Passive cutaneous anaphylaxis and ear swelling were significantly reduced by moxibustion and probenecid (p < 0.05). In cardiomyocytes, TRPV2 was over-expressed by compound 48/80 and histamine but this increased TRPV2 expression decreased to baseline with moxibustion and probenecid treatment. In addition, intracellular calcium levels increased by compound 48/80 were reduced by probenecid. Overall, these findings suggest that the reduction of anaphylaxis caused by moxibustion could represent a new mechanism of moxibustion related to the regulation of TRPV2 activation and promotion of epinephrine secretion.

• Biomolecules & Therapeutics
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• v.24 no.1
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• pp.99-107
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• 2016

Triclosan is an antimicrobial or sanitizing agent used in personal care and household products such as toothpaste, soaps, mouthwashes and kitchen utensils. There are increasing evidence of the potentially harmful effects of triclosan in many systemic and cellular processes of the body. In this study, we investigated the effects of triclosan in the survivability of cultured rat neural stem cells (NSCs). Cortical cells from embryonic day 14 rat embryos were isolated and cultured in vitro. After stabilizing the culture, triclosan was introduced to the cells with concentrations ranging from $1{\mu}M$ to $50{\mu}M$ and in varied time periods. Thereafter, cell viability parameters were measured using MTT assay and PI staining. TCS decreased the cell viability of treated NSC in a concentration-dependent manner along with increased expressions of apoptotic markers, cleaved caspase-3 and Bax, while reduced expression of Bcl2. To explore the mechanisms underlying the effects of TCS in NSC, we measured the activation of MAPKs and intracellular ROS. TCS at $50{\mu}M$ induced the activations of both p38 and JNK, which may adversely affect cell survival. In contrast, the activities of ERK, Akt and PI3K, which are positively correlated with cell survival, were inhibited. Moreover, TCS at this concentration augmented the ROS generation in treated NSC and depleted the glutathione activity. Taken together, these results suggest that TCS can induce neurodegenerative effects in developing rat brains through mechanisms involving ROS activation and apoptosis initiation.

• Journal of Ginseng Research
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• v.42 no.2
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• pp.165-174
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• 2018

Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of $eIF2{\alpha}$ and protein levels of GRP78/BiP, XBP-1S, and $IRE1{\alpha}$ in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular $Ca^{2+}$ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular $Ca^{2+}$ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.