• Archives of Pharmacal Research
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• 제26권1호
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• pp.70-75
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• 2003

The objective of the present study was to investigate the uptake process of 4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide), a hydrophilic and collagenase-labile pentapeptide, by isolated hepatocytes. For comparison, the uptake of Pz-peptide by Caco-2 cells and colonic cells, two known paracellular routes of Pz-peptide, was also evaluated. A simple and sensitive reversed-phase HPLC assay method using UV detection has been developed. The coefficient of variation for all the criteria of validation were less than 15%. The method was, therefore, considered to be sutable for measuring the concentration of Pz-peptide in the biological cells. Pz-peptide was extensively uptaked into hepatocytes. The initial velocity of Pz-peptide uptake assessed from the initial slope of the curve was plotted as Eadie-Hofstee plots. The maximum velocity ($V_{max}$) and the Michaelis constant ($K_m$) were 0.190$\pm$0.020 $nmol/min/10^6$ cells and 12.1$\pm$3.23 $\mu$M, respectively. The permeability-surface area product ($PS{influx}$) was calculated to be 0.0157 ml/min/10^6$ cells. $V_{max}$ and $K_m$ values for Caco-2 cells were calculated to be 6.22$\pm$0.930 pmol/min/10^6$ cells and 82.8$\pm$8.37 $\mu$M, respectively, being comparable with those of colonocytes (6.04$\pm$1.03 pmol/min/10^6$ cells and 87.8$\pm$13.2 $\mu$M, respectively). $PS_{influx}$ values for Caco-2 cells and colonocytes were calculated to be 0.0751 $\mu$l/min/10^6$ cells and 0.0688 $\mu$l/min/10^6$ cells, respectively. The more pronounced uptake of Pz-peptide by hepatocytes, when compared with Caco-2 cells and colonocytes, is probably due to its specific transporter. In conclusion, Pz-peptide, a paracellularly transported pentapeptide in the intestine and ocular epithelia, was uptaked into hepatocytes extensively. Although Pz-peptide is able to be uptaked into the Caco-2 cells and colonocytes, it is less pronounced when compared with hepatocytes. $PS_{influx}$ values of Caco-2 cells and colonocytes for unbound Pz-peptide under linear conditions were less than 0.4% when compared with that of hepatocytes.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 춘계학술대회
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• pp.121-121
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• 2019

In Islamic dietary guidelines, Halal foods are allowed as edible blessed food. Most foods were categorized within halal for Muslims. The main point of Halal food is that foods are clean in every process and based on Halal standard which might be different in each country. Most pancreatic ${\beta}$ cells synthetize, store, and release insulin. Specific molecular, functional as well as ultrastructural traits of pancreatic ${\beta}$ cells could control their insulin secretion properties and survival phentoype. Insulin-secreting pancreatic ${\beta}$-cells are essential regulators of mammalian metabolism. In addition, the pancreatic ${\beta}$ cell plays an important role in the pathogenesis of type 1 and type 2 diabetes as improving glucose homeostasis by preserving, expanding and improving the function of this key cell type. However, the pharmacological effect of halal food has not been unclear yet, especially food habit-dependent diabetes. The aim of the this study was to determine the preventive effect of Iran plants extract (Almond, Garlic, Cumin, Ginkgo biloba, Holy basil, Psyllium, Satureja khuzistanica, Fenugreek, Green tea, Ipomoea betatas, Blueberry) on RINm5F cells and MIN6 cells as pancreatic ${\beta}$ cell line. The cytotoxicity of the extracts of Iran plants on RINm5F cells and MIN6 cells were measured by using MTT assays. The preventive effects of Iran plant extracts were measured by WST-8 cell proliferation assay on streptozotocin (STZ)-induced cell death in MIN6 cells. In presented result showed that all extract of Iran plants (0.01-10mg/ml) did not show cytotoxicity in RINm5F cells and MIN6 cells. Among non-cytotoxic extract, the protective effects could be detect in high dose concentration. These results suggest that the extract of Iran plants may serve as a potential therapy for diabetes.

• The Korean Journal of Physiology and Pharmacology
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• 제7권3호
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• pp.163-168
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• 2003

The reactive oxygen species (ROS) are considered to be an important mediator in pancreatic ${\beta}$ cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, HIV-1 Tat-mediated transduction of Cu,Zn-superoxide dismutase (SOD) was investigated to evaluate its protective potential against streptozotocin (STZ)-induced cytotoxicity in insulin-producing MIN6N cells. Tat-SOD fusion protein was successfully delivered into MIN6N cells in a dose-dependent manner and the transduced fusion protein was enzymatically active for 48 h. The STZ induced-cell destruction, superoxide anion radical production, and DNA fragmentation of MIN6N cells were significantly decreased in the cells pretreated with Tat-SOD for 1 h. Furthermore, the transduction of Tat-SOD increased Bcl-2 and heat shock protein 70 (hsp70) expressions in cells exposed to STZ, which might be partly responsible for the effect of Tat-SOD. These results suggest that an increased of free radical scavenging activity by transduction of Tat-SOD enhanced the tolerance of the cell against oxidative stress in STZ-treated MIN6N cells. Therefore, this Tat-SOD transduction technique may provide a new strategy to protect the pancreatic ${\beta}$ cell destruction in ROS-mediated diabetes.

• Parasites, Hosts and Diseases
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• 제54권2호
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• pp.147-154
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• 2016

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.

• 대한의생명과학회지
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• 제18권3호
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• pp.193-200
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• 2012

We examined the effects of polyamines such as putrescine and cadaverine on the biosynthesis and secretion of insulin in the mouse pancreatic ${\beta}$-cell line, MIN-6. Basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) from the MIN-6 cells were significantly increased by 20 min- or 24 h-treatment with micromolar concentrations of polyamines. To determine whether the enhancement was due to increase of insulin production by polyamines, we investigated the insulin mRNA and protein production. Both insulin mRNA and protein production were found to be not significantly affected by the polyamine treatment. Next, we examined the expression of several transcription factors (TFs) related to insulin synthesis and secretion in order to identify upstream events responsible for the promotion of insulin secretion of MIN6 cells by polyamines. Of the 6 TFs tested, MafA was induced by treatment of polyamines. MafA mRNA and protein expressions increased with treatment of polyamines. Overall results suggest that cadaverine and putrescine promote the insulin secretion process rather than the insulin biosynthesis from MIN6 cells. Also MafA may be involved in the enhanced insulin secretion process. Further studies are needed to elucidate the underlying mechanisms for promotion of insulin secretion by polyamines.

• The Korean Journal of Physiology and Pharmacology
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• 제16권6호
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• pp.463-468
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• 2012

Type 1 diabetes (T1D) is caused by dysregulation of the immune system in the pancreatic islets, which eventually leads to insulin-producing pancreatic ${\beta}$-cell death and destabilization of glucose homeostasis. One of the major characteristics of T1D pathogenesis is the production of inflammatory mediators by macrophages that result in destruction or damage of pancreatic ${\beta}$-cells. In this study the inflammatory microenvironment of T1D was simulated with RAW264.7 cells and MIN6 cells, acting as macrophages and pancreatic ${\beta}$-cells respectably. In this setting, peroxiredoxin-1, an anti-oxidant enzyme was knocked down to observe its functions in the pathogenesis of T1D. RAW264.7 cells were primed with lipopolysaccharide and co-cultured with MIN6 cells while PRX-1 was knocked down in one or both cell types. Our results suggest that hindrance of PRX-1 activity or the deficiency of this enzyme in inflammatory conditions negatively affects pancreatic ${\beta}$-cell survival. The observed decrease in viability of MIN6 cells seems to be caused by nitric oxide production. Additionally, it seems that PRX-1 affects previously reported protective activity of IL-6 in pancreatic ${\beta}$ cells as well. These results signify new, undiscovered roles for PRX-1 in inflammatory conditions and may contribute toward our understanding of autoimmunity.

• Molecular & Cellular Toxicology
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• 제5권1호
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• pp.1-6
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• 2009

Microglial cells constitute the first line of defense against infection and injury in the brain. This study was conducted to evaluate the protective mechanisms of Agrimonia pilosa (AP) on LPS-induced activation of BV-2 microglial cells. The effects of AP on gene expression profiles in activated BV-2 microglial cells were evaluated using microarray analysis. BV-2 microglial cells were cultured in a 100 mm dish ($1{\times}10^7/mL$) for 24 hr and then pretreated with 1 g/mL AP or left untreated for 30 min. Next, 1 g/mL LPS was added to the samples and the cells were reincubated at $37^{\circ}C$ for 30 min, 3 hr and 6 hr. The gene expression profiles of the BV-2 microglial cells varied depending on the AP. The microarray analysis revealed that MAPK signaling pathway-related genes were down-regulated and IL10 gene was up-regulated in AP-treated BV-2 microglial cells. AP can affect the inflammatory response and MAPK pathway in BV-2 microglial cells.

• Animal cells and systems
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• 제11권1호
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• pp.17-22
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• 2007

The effects of antimetabolite 6-AN (6-amino-nicotinamide) on viability and morphology of L6 myoblast cells have been investigated. 6-AN ($100{\mu}M$) induced a time-dependent decrease in cell viability with respect to the untreated control cells. Following 6-AN administration the viability rate started to decline sharply, reaching about 23% of the untreated control cells at 48 h. Inverted phase-contrast microscopy revealed that 6-AN caused characteristic morphological changes such as irregularly elongated and stellate shape of cells, round-shaped nucleus, cytoplasmic vacuolization, irregular cell arrangements and formation of large spaces among cell clusters. The concentrations of ATP and $NAD^{+}$ in the 6-AN treated cells were significantly lower (p < 0.01) than those of the untreated control cells. In contrast, the concentration of AMP was significantly increased by the 6-AN treatment. Activities of catalase, superoxide dismutase and glutathione peroxidase in 6-AN treated cells were significantly higher (p < 0.01) than those of the untreated control cells. The activities of glyceraldehyde-3-phosphate dehydrogenase in 6-AN treated cells were significantly lower (p < 0.01) than those of the untreated control cells. The results suggest that 6-AN caused marked reduction of cell viability and alterations of some important metabolites and enzymes.

• 대한물리치료과학회지
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• 제7권2호
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• pp.513-521
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• 2000

This study was carried out to investigate the effects of electrical stimulation on the change Tyrosine-Hydroxylase immunoreactive(TH-IR) cells in the Central gray and retrorubral field of the male SD rats. 9 healthy and normal rats were divided into three groups, 3 SD rat in each group. The one group has been stimulated by electroacupuncture(EA, 2Hz) for 30min and the other group by EA for 1hr 30min and control group has been stimulated. TH-IR cells were found in the Central gray(CG) and Retrorubral field(RF). The numbers of TH-IR cells of CG and RF were significantly increased after 30 min (CG $6.2{\pm}0.83$, RF $1.4{\pm}0.55$)as compared with control group(CG $24{\pm}3.16$, RF $6.4{\pm}0.55$) and were also significantly increased after 1hr 30min(CG $75.6{\pm}4.51$, RF $18.8{\pm}0.89$) than 30min. These results show that TH is related into CG and RF in response to electrical stimulation.

• Journal of Pharmaceutical Investigation
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• 제26권1호
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• pp.33-41
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• 1996

In order to study the effect of phenobarbital(PB) on the hepatic transport of diltiazem(DTZ), $Ca^{2+}$ channel blocker, we used isolated hepatocytes of rat which was intraperitoneally pretreated with phenobarbital sodium(75 mg/kg) for four days once a day. For the isolation of rat liver cells, a modification of the two step procedure of Seglen was used. DTZ was dissolved in incubation buffer to the final DTZ concentrations of 200, 400, 600, 800 and 1000 ng/ml in order to elucidate the uptake characteristics of DTZ by hepatocytes. Reactions were stopped at 10, 20, 30, 45, 60, 90, 120 and 300 sec. The initial velocity was determined by disappearance of diltiazem in the hepatocyte suspension. On the other hand, to determine the effect of PB on the in vitro hepatic intrinsic clearance of DTZ we obtained the metabolism rates of DTZ in the control and the PB-pretreated rat hepatocyte at various time intervals. According to pretreatment with PB, the size of hepatocyte and the amount of protein per $10^6$ cells were significantly (p<0.01) increased from $26.92{\pm}0.1364\;m$ to $35.31{\pm}1.00\;m$ and from $468{\pm}6.5\;{\mu}g/10^6$ cells to $628.8{\pm}12.1{\mu}g/10^6$ cells, respectively. In the case or hepatic uptake of diltiazem, $K_m$ was not different in the normalization by cell numbers and increased from $2.90\;{\mu}M\;to\;13.89\;{\mu}M$ in the normalization by protein amount. $V_max$ was increased regardless of normalization by protein amount and cell numbers, from $1.21\;{\mu}mole/min \;{\cdot}\;mg\;protein\;to\;3.96\;{\mu}mole/min\;{\cdot}\;mg\;protein\;and\;from\;2.38\;{\mu}mole/min\;{\cdot}\;10^6\;cells\;to\;2.83\;{\mu}mole/min\;{\cdot}\;10^6\;cells$, respectively. The in vitro hepatic intrinsic clearance of DTZ was significantly (p<0.01) increased from $0.640{\pm}0.038\;ml/mim\;{\cdot}\;10^6\;cells\;to\;2.385{\pm}0.212\;ml/min\;{\cdot}\;10^6\;cells$ due to PB-pretreatment. These results suggest that the uptake of DTZ by hepatocyte is extremely fast and PB enhances the hepatic intrinsic metabolic clearance of DTZ.