• International Journal of Oral Biology
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• v.34 no.1
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• pp.7-14
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• 2009

Nitric oxide (NO) acts as an intracellular messenger at the physiological level but can be cytotoxic at high concentrations. The cells within periodontal tissues, such as gingival and periodontal fibroblasts, contain nitric oxide syntheses and produce high concentrations of NO when exposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-induced cytotoxicity in periodontal tissues are unclear at present. In our current study, we examined the NO-induced cytotoxic mechanisms in human gingival fibroblasts (HGF). Cell viability and the levels of reactive oxygen species (ROS) were determined using a MTT assay and a fluorescent spectrometer, respectively. The morphological changes in the cells were examined by Diff-Quick staining. Expression of the Bcl-2 family and Fas was determined by RT-PCR or western blotting. The activity of caspase-3, -8 and -9 was assessed using a spectrophotometer. Sodium nitroprusside (SNP), a NO donor, decreased the cell viability of the HGF cells in a dose- and time-dependent manner. SNP enhanced the production of ROS, which was ameliorated by NAC, a free radical scavenger. ODQ, a soluble guanylate cyclase inhibitor, did not block the SNP-induced decrease in cell viability. SNP also caused apoptotic morphological changes, including cell shrinkage, chromatin condensation, and DNA fragmentation. The expression of Bax, a member of the proapoptotic Bcl-2 family, was upregulated in the SNP-treated HGF cells, whereas the expression of Bcl-2, a member of the anti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from the mitochondria into the cytosol and enhanced the activity of caspase-8, -9, and -3. SNP also upregulated Fas, a component of the death receptor assembly. These results suggest that NO induces apoptosis in human gingival fibroblast via ROS and the Bcl-2 family through both mitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is not involved in NO-induced apoptosis.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.354-364
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• 2015

Background: Intracellular $Ca^{2+}$($[Ca^{2+}]_i$) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of $[Ca^{2+}]_i$mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the $Ca^{2+}$-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods: We investigated the $Ca^{2+}$-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I ($IP_3RI$) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results: The inhibition of $[Ca^{2+}]_i$ mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-BrcAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)- dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) ($Thr^{197}$) by KRG-TS. The phosphorylation of $IP_3RI$ ($Ser^{1756}$) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-BrcGMPS. These results suggest that the inhibitory effect of $[Ca^{2+}]_i$ mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion: These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits $[Ca^{2+}]_i$ mobilization in thrombin-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

• Archives of Pharmacal Research
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• v.29 no.5
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• pp.375-383
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• 2006

The anti platelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen $(10{\mu}g/mL)$, thrombin (0.05 U/mL), arachidonic acid $(100{\mu}M)$, a thromboxane (TX) $A_2$ mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin $F_2,\;1{\mu}M$) and a $Ca^{2+}$ ATPase inhibitor thapsigargin $(0.5{\mu}M)$ ($IC_{50}$ values: $13.8{\pm}1.8,\;26.3{\pm}1.2,\;8.5{\pm}0.9,\;4.3{\pm}1.7\;and\;49.8{\pm}1.4{\mu}M$, respectively). KR-32570 inhibited the collagen-induced liberation of $[^3H]$arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at $50{\mu}M$. The $TXA_2$ synthase assay showed that KR-32570 also inhibited the conversion of the substrate $PGH_2$ to $TXB_2$ at all concentrations. Furthermore, KR-32570 significantly inhibited the $[Ca^{2+}]_i$ mobilization induced by collagen at $50{\mu}M$, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen $(10{\mu}g/mL)$induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, $TXA_2$ synthase, the mobilization of cytosolic $Ca^{2+}$ and NHE-1.

• Journal of Life Science
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• v.20 no.5
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• pp.736-744
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• 2010

The root of Playtcodon grandiflorum, called Platycodi radix, has been a favorite edible plant in Asia and contains a large amount of saponins. Melanoma cells (B16F10) were used to investigate the inhibitory effect of aged black Platycodi radix extract (ABPRE) on oxidative stress and matrix metalloproteinases (MMPs). Platycodon radix has been known to have a variety of medicinal effects such as prevention of gastric ulcers, antiallergenic activities, histamine release inhibition, and antioxidant effects. However, the mechanism of its action remains unclear in humans. ABPRE was prepared using ethanol extraction of aged black Platycodi radix. In an antioxidant effect study of ABPRE, it was observed that ABPRE specifically exhibited the scavenging activity of DPPH radical, but did not inhibit the production of malondialdehyde from lipid peroxidation. DNA oxidation was also blocked in the presence of ABPRE. In addition, ABPRE decreased the expression and activation of MMP-2 stimulated by phenazine methosulfate. Furthermore, ABPRE revealed the inhibitory effect on melanin production induced by L-dopa via antioxidant effect and the reduction of tyrosinase expression. Especially, the expression of antioxidant enzymes such as SOD-1 and SOD-2 regulated by Nrf2 was increased in the presence of ABPRE. Therefore, it appears that ABPRE may be a possible chemopreventive agent for the prevention of metastasis related to oxidative stress.

• Korean Journal of Clinical Pharmacy
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• v.12 no.1
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• pp.22-28
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• 2002

Aceclofenac, 2-[(2',6'-dichlorphenyl)amino]phenylacetoxiacetic acid, is a new nonsteroidal anti-inflammatory drug that belongs to the family of phenylacetic acids. It shows good tolerance and potent analgesic/antiinflammatory properties, and acts on cartilaginous chondriocytes, stimulating their repair mechanism. The purpose of the present study was to evaluate the bioequivalence of two aceclofenac products, $Airtal^{TM}$ tablet (Daewoong Pharmaceutical Co.) and $Acer^{TM}$ capsule (Kyungdong Pharmaceutical Co.), according to the guideliner of Korea Food and Drug Administration (KFDA). The aceclofenac release from the two aceclofenac products in vitro was tested using KP VII Apparatus II method at pH 7.8 dissolution media. Sixteen normal male volunteers, $23.13\pm2.03$ years in age and $66.33\pm7.08$ kg in body weight, were divided into two groups and a randomized $2\times2$ cross-over study was employed. After one tablet or capsule containing 100 mg of aceclofenac was orally administered, blood was taken at predetermined time intervals and the concentrations of aceclofenac in serum were determined using HPLC with UV detector. The dissolution profiles of the two aceclofenac products were very similar at pH 7.8 dissolution media. The pharmacokinetic parameters such as $AUC_t,\;C_{max}\;and\;T_max$ were calculated and ANOVA test was utilized for the statistical analysis of the parameters. The results showed that the differences in $AUC_t,\;C_{max}\;and\;T_{max}$ between two products were $6.50\%,\;-1.06\%\;and\;11.96\%$ respectively, when calculated against the $Airtal^{TM}$ tablet. The powers $(1-\beta)\;for\;AUC_t,\;C_{max}\;were\;89.82\%\;and\;82.84\%$, respectively. Minimum detectable differences $(\Delta)\;at\;\alpha=0.05\;and\;1-\beta=0.8$ were less than $20\%\;(e.g.,\;17.51\%\;and\;19.30\%\;for\;AUC_t,\;C_{max}$, ). The $90\%$ confidence intervals were within $\pm20\%\;(e.g.,\;-3.73\%\sim16.73\%\;and\;-12.34\%\sim10.22\%\;for\;AUC_t,\;C_{max},\;respectively)$. Two parameters met the criteria of KFDA for bioequivalence, indicating that $Acer^{TM}$ capsule is bioequivalent to $Airtal^{TM}$ tablet.

• Journal of Korean Society for Atmospheric Environment
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• v.24 no.1
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• pp.16-29
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• 2008

Nitrous acid (HONO) can be produced by heterogenous reactions of nitrogen dioxide on surface materials and direct emission from combustion sources. However, a little is known of indoor HONO levels or the relationship between residential HONO, NO, and $NO_2$ concentrations in occupied houses. Therefore, we measured simultaneously NO, $NO_2$, and HONO concentrations in living room of an apartment using continuous analyzers to study the production of HONO (June $22{\sim}30$, 2006). The 4-min average concentrations of indoor NO, $NO_2$, and HONO were 4.3 (range: $0.4{\sim}214.3$), 10.3 ($2.0{\sim}87.3$), and 1.8 ppb ($0.3{\sim}7.7$), respectively. Peak levels of HONO up to 7.7 ppb and 24-hr averages as high as 1.7 ppb were measured. In agreement with previous studies, indoor HONO concentrations increased during operation of an unvented gas range. Examination of the observed kinetics suggests that the secondary production of indoor HONO, possibly as a result of heterogeneous reactions involving $NO_2$ and $H_2O$ is associated with $[NO_2]^2[H_2O]\;(r^2=0.88)$ rather than with $[NO][NO_2][H_2O]\;(r^2=0.75)$. Three combustion experiments at nighttime were also carried out to investigate the effects of vented combustion on the HONO, NO, and $NO_2$ concentrations. It was found to release HONO for $10{\sim}15$ minutes after NO and $NO_2$ source was turned off, and peak values were finally attained. Compared to unvented combustion, peak $NO_2$ and HONO concentrations were 3.2 and 2.0 times lower at weak vented combustion (air flow: $340\;m^3/hr$) and 4.9 and 2.4 times lower at strong vented combustion (air flow: $540\;m^3/hr$), respectively, emphasizing importance of operating ventilation hood fan during combustion to improve indoor air quality.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.1
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• pp.47-54
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• 2000

Transition metal ions including $Se^{2+},\;Cd^{2+},\;Hg^{2+}\;or\;Mn^{2+}$ have been thought to disturb the bone metabolism directly. However, the mechanism for the bone lesion is unknown. In this study, we demonstrated that MC3T3E1 osteoblasts, exposed to various transition metal ions; selenium, cadmium, mercury or manganese, generated massive amounts of reactive oxygen species (ROS). The released ROS were completely quenched by free radical scavengers-N-acetyl cysteine (NAC), reduced glutathione (GSH), or superoxide dismutase (SOD). First, we have observed that selenium $(10\;{\mu}M),$ cadmium $(100\;{\mu}M),$ mercury $(100\;{\mu}M)$ or manganese (1 mM) treatment induced apoptotic phenomena like DNA fragmentation, chromatin condensation and caspase-3-like cysteine protease activation in MC3T3E1 osteoblasts. Concomitant treatment of antioxidant; N-acetyl-L-cysteine (NAC), reduced-form glutathione (GSH), or superoxide dismutase (SOD), prevented apoptosis induced by each of the transition metal ions. Catalase or dimethylsulfoxide (DMSO) has less potent inhibitory effect on the apoptosis, compared with NAC, GSH or SOD. In line with the results, nitroblue tetrazolium (NBT) stain shows that each of the transition metals is a potent source of free radicals in MC3T3E1 osteoblast. Our data show that oxidative damage is associated with the induction of apoptosis in MC3T3E1 osteoblasts following $Se^{2+},\;Cd^{2+},\;Hg^{2+}\;or\;Mn^{2+}$ treatment.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.3
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• pp.181-186
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• 2012

Fenofibrate is a selective peroxisome proliferator-activated receptor ${\alpha}$ ($PPAR{\alpha}$) activator and is prescribed to treat hyperlipidemia. The mechanism through which $PPAR{\alpha}$ agonists reduce food intake, body weight, and adiposity remains unclear. One explanation for the reduction of food intake is that fenofibrate promotes fatty acid oxidation and increases the production of ketone bodies upon a standard experimental dose of the drug (100~300 mg/kg/day). We observed that low-dose treatment of fenofibrate (30 mg/kg/day), which does not cause significant changes in ketone body synthesis, reduced food intake in Long-Evans Tokushima (LETO) rats. LETO rats are the physiologically normal controls for Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which are obese and cholecystokinin (CCK)-A receptor deficient. We hypothesized that the reduced food intake by fenofibrate-treated LETO rats may be associated with CCK production. To investigate the anorexic effects of fenofibrate in vivo and to determine whether CCK production may be involved, we examined the amount of food intake and CCK production. Fenofibrate-treated OLETF rats did not significantly change their food intake while LETO rats decreased their food intake. Treatment of fenofibrate increased CCK synthesis in the duodenal epithelial cells of both LETO and OLETF rats. The absence of a change in the food intake of OLETF rats, despite the increase in CCK production, may be explained by the absence of CCK-A receptors. Contrary to the OLETF rats, LETO rats, which have normal CCK receptors, presented a decrease in food intake and an increase in CCK production. These results suggest that reduced food intake by fenofibrate treatment may be associated with CCK production.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.18 no.8
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• pp.378-385
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• 2017

This study investigated the design of a snap fit, which is widely used for fastening plastic parts. We analyzed the assembly mechanism of a lock snapfit, measured the assembly force and separation force based on the design of experiments, and derived a regression equation through an analysis of variance. The response surface methodology was also used. Polybutylene terephthalate was used to fabricate specimens, and the assembly force and separation force were measured using a micro-tensile tester. The length, width, thickness, and interference were considered as factors. A second-order regression model was used to derive the regression equation. The assembly force decreased with increasing length and width, but it increased with increasing thickness and interference. The finite element method was used to analyze the assembly mechanics. The width decreased the assembly force by increasing the ductility. The influences of the factors for low assembly force and high release force were shown to be opposite to each other. It was necessary to design a structure that minimized the assembly force while maintaining an appropriate level of separation force.

• Journal of Applied Biological Chemistry
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• v.57 no.2
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• pp.103-111
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• 2014

A1E is an extract from traditional Asian medicinal plants that has therapeutic activities against cancers, metabolic disease, and other intractable conditions. However, its mechanism of action on cervical cancer has not been studied. In order to ascertain if A1E would have pronounced anti-cervical cancer effect, cervical cancer cells were incubated with A1E and apoptosis was detected by nuclear morphological changes, annexin V-FITC/PI staining, cell cycle analysis, western blotting, Reverse-transcription polymerase chain reaction, and measurement of mitochondrial membrane potential. Expression of human papiloma virus E6 and E7 oncogenes was down-regulated in A1E-treated cervical cancer cells, while p53 and retinoblastoma protein levels were enhanced. A1E also perturbed cell cycle progression at sub-G1 and altered cell cycle regulatory factors in SiHa cervical cancer cells. A1E activated apoptotic intrinsic pathway markers such as caspase-9, caspase-3 and poly ADP-ribose polymerase, and down-regulated expression of Bcl-2 and Bcl-xl. A1E induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited phosphatidylinositol 3-kinase (PI3K)/Akt, key factors involved in cell survival signaling. Taken all these results, A1E induced apoptosis via activation of the intrinsic pathway and inhibition of the PI3K/Akt survival-signaling pathway in SiHa cervical cancer cells. In conclusion, A1E exerts anti-proliferative action growth inhibition on cervical cancer cells through apoptosis which demonstrates its anti-cervical cancer properties.