• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.3
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• pp.603-608
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• 2006

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, water extracts from the fruit of Chaenomeles sinensis, the root of Polygonum cuspidatum and Boswellia carterii inhibited the LPS-induced NO production in a parallel dose-dependent manner. To investigate the mechanism by which those extracts inhibits NO production, we examined the expression of iNOS and COX-2 in both mRNA and protein levels. We observed a significant change in the iNOS expression between LPS alone and LPS plus those extracts-treated cells. However, water extracts from Chaenomeles sinensis, Polygonum cuspidatum and Boswellia carterii did not inhibit COX-2 expression which was induced by LPS treatment in Raw 264.7 cells. These data suggest that water extracts from Chaenomeles sinensis, Polygonum cuspidatum and Boswellia carterii can modulate anti-inflammatory immune response, which may be in part associated with the regulation of NO synthesis through the regulation of iNOS expression in mouse macrophage cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.31 no.4
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• pp.220-225
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• 2017

Lithospermi Radix (LR) is known to have an anti-inflammatory effect. However, the mechanisms are not well known. In this study, LPS-induced mouse RAW 264.7 macrophage cells were treated with LR to investigate the time-dependent inflammation response of LR. RAW 264.7 cells were treated with various concentrations of LR for 24 hours, followed by MTS assay. Cell viability was increased at all experimental concentrations. The mRNA expression levels of $IL-1{\beta}$, $TNF-{\alpha}$ and iNOS were increased by treatment of RAW 264.7 cells with LR at a concentration of $200{\mu}g/ml$ for 6 hours and 24 hours. Treatment of LR with $200{\mu}g/ml$ concentration for 6 hours promoted mRNA expression levels of $IL-1{\beta}$, $TNF-{\alpha}$ and iNOS in LPS-induced RAW 264.7 cells. However, $IL-1{\beta}$, $TNF-{\alpha}$ and iNOS mRNA expression was suppressed by treatment of LR with $200{\mu}g/ml$ concentration for 24 hours in LPS-induced RAW 264.7 cells. These results suggest that the effect on inflammation of LR is promptly promoted and then to rapidly alleviate the inflammatory reaction. This study proposes that the time-dependent activities of herbal medicine is a very important factor in analyzing the anti-inflammatory effect of various herbal medicines including LR.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.33 no.1
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• pp.1-11
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• 2020

Objectives : The purpose of this study was to investigate the anti-inflammatory and anti-oxidative effects of Osung-tang(OST) extract. Methods : MTT assay was performed to confirm the survival rate of RAW 264.7 cells treated with OST extract(50-400㎍/㎖) and the production of NO from LPS-induced RAW 264.7 cells was confirmed. The mRNA expression of iNOS, COX-2, and NF-κB were measured by real-time PCR. The protein expression of iNOS, COX-2 and p-IκB were measured by western blot and the anti-oxidant activity of OST extract(50-400㎍/㎖) was investigated by measuring DPPH scavenging activity. Results : OST extract showed a cell survival rate of 90% or more at 50-400㎍/㎖. The NO production was inhibited in a dose-dependent manner in RAW 264.7 cells treated with LPS on OST treated group. mRNA expression levels of iNOS, COX-2 and NF-κB decreased in a concentration-dependent manner after treatment with OST(50-400㎍/㎖). Protein expression levels of iNOS, COX-2 and p-IκB decreased in a concentration-dependent manner after treatment with OST(50-400㎍/㎖). It was found that OST has a high free-radical scavenging ability. Conclusions : These results suggest that OST extract can be used as a treatment for various skin diseases by demonstrating its anti-inflammatory and anti-oxidant effects.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2151-2157
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• 2016

Scabraside D, a sulfated triterpene glycoside, was extracted from the sea cucumber Holothuria scabra. It shows anti-proliferation in many of cancer cell lines, but the function and mechanisms of action of scabraside D in human cholangiocarcinoma (HuCCA) have not previously determined. In this study, we investigated the activity of scabraside D on HuCCA cell apoptosis, lymphangiogenesis and metastasis in a nude mouse model. Scabraside D induced signs of apoptosis, such as cell shrinkage, nuclear condensation, nuclear fragmentation and DNA fragmentation on TUNEL assays, while effectively decreasing expression of BCl-2 but increasing caspase-3 gene level expression. Immunohistochemistry revealed that scabraside D significantly reduced lymphatic vessel density (LVD). Moreover, scabraside D treatment significantly decreased VEGF-C, MMP-9 and uPA gene expression, which play important roles in the lymphangiogenesis and invasion of cancer cells in metastasis processes. Quantitative real-time PCR showed that scabraside D significantly decreased iNOS and STAT-3 gene expression. This study demonstrated that scabraside D plays a role in activation of HuCCA tumor apoptosis and inhibition of lymphangiogenesis, invasion and metastasis through decreasing BCl-2, MMP-9, uPA and VEGF-C and increasing caspase-3 expression by suppression of iNOS and STAT-3 expression. Therefore, scabraside D could be a promising candidate for cholangiocarcinoma treatment.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.2
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• pp.278-283
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• 2010

Inducible nitric oxide synthase (iNOS) are important inflammation enzyme and severe up-nitric oxide (NO) production by this enzyme has been intricate with pathogenesis of atopy dermatitis. The present study was designed in order to determine whether Lonicera japonica could inhibit atopy dermatitis through modulation of iNOS by NF-${\kappa}B$ suppression. We found that IKK mRNA and iNOS mRNA expression in RAW 264.7 macrophages stimulated with lipopolysaccharide dose-dependantly decreased by Lonicera japonica (0.4 - 1.0 mg/$m{\ell}$) and NO production decreased. The distribution of NF-${\kappa}B$ p65 and iNOS positive reacted cell in NC/Nga mice with atopy dermatitis were decreased by Lonicera japonica (45 mg/kg/day) and apoptosis were increased. These data likely indicate that Lonicera japonica may act as inflammatory regulator for atopy dermatitis through iNOS modulation by NF-${\kappa}B$B suppression and may be possible to develop useful agent for chemoprevention of NO intricate inflammatory diseases.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.571-576
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• 2016

In this study, the anti-inflammatory activities of the 80% ethanol extract of Dystaenia takeshimana (DT) were investigated using Raw 264.7 cells treated with lipopolysaccharide (LPS). The effect of DT extract on the production of pro-inflammatory factors (iNOS, COX-2) in LPS-stimulated Raw 264.7 macrophages was examined. The cytotoxic effect of DT extract on macrophage cells (Raw 264.7) was examined by the 3-[4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Treatment with DT extract showed 100% or more cell viability at the concentration $1,000{\mu}g/ml$. The inhibitory effect of DT extract on protein expression of inducible NOS (iNOS) and cyclooxygenase-2 (COX-2) was measured by western blotting using the concentrations 50, 100, and $500{\mu}g/ml$, with ${\beta}-actin$ used as the positive control. Consequently, the protein expression of iNOS, and COX-2 as observed by western blotting, was decreased by 56%, 61.6%, respectively with $500{\mu}g/ml$ DT extract. Inhibition of iNOS and COX-2 mRNA expression was measured by reverse transcription- polymerase chain reaction (PCR) using DT extract concentrations 50, 100, and $500{\mu}g/ml$, with GAPDH used as a positive control. Consequently, the mRNA expression of iNOS and COX-2 as observed by reverse-transcription-PCR was decreased by 77.9% and 83.3%, respectively at $500{\mu}g/ml$ concentration of DT extract. In conclusion, DT extract may affect inflammatory factors as a potential anti-inflammatory agent.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.5
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• pp.1233-1240
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• 2006

We have recently demonstrated that Siegesbeckia glabrescens(SG), a herbal medicine, induces apoptosis via nitric oxide(NO) production in human aortic smooth muscle cells(HASMCS). However, the molecular pathways involved in SG-mediated apoptosis are not fully understand. In the present study, we investigated the cellular mechanisms of SG-induced apoptosis in HASMCS. SG induced NO production through inducible nitric oxide synthase(iNOS) induction. The apoptotic effect of SG was attenuated by L-NNA, a NOS inhibitor. In the presence of L-NNA, the degradation of procaspase-3 by SG was inhibited. SG treatment induced a decrease in Bcl-2 expression but did not affect the expression of Bax. In addition, SG treatment evoked both down-regulation of PKC ${\alpha}$ and inhibition of PKC ${\alpha}$ phosphorylation. These downregulations were reversed by addition of L-NNA. It seems likely to De a downregulation of PKC${\alpha}$ due to long term treatment with PMA. Taken together, these results suggest that apoptotic effects of SG may be due to NO production via iNOS mRNA expression. Furthermore, Bcl-2 and PKC${\alpha}$ downregulation, and caspase-3 activation may be involved in the mechanisms for apoptotic effects by SG.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.5
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• pp.626-631
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• 2005

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) is involved in cell apoptosis, which contributes to luteal regression and luteolysis in some species. In large domestic animals, no direct evidence for the relationship between NO and cell apoptosis in the process of corpus luteum regression is reported. The present study was conducted to investigate the localization of iNOS on porcine corpora lutea (CL) during the oestrus cycle and its relation to cell DNA fragmentation and CL regression. According to morphology, four luteal phases throughout the estrous cycle were defined as CL1, CL2, CL3 and CL4. By isoform-specific antibody against iNOS, the immunochemial staining was determined. Luteal cell DNA fragmentation was determined by flow cytometry. The results showed that no positive staining for iNOS was in CL1 and that iNOS was produced but limited to the periphery of CL2, while in the CL3, the spreading immunochemical staining was found inside the CL. No iNOS positive staining was detected in CL4. Meanwhile, DNA fragmentation increased dramatically when CL developed from CL2 to CL3 (p<0.05). In CL4, higher proportion of luteal cells still had fragmented DNA than that of luteal cells from CL1 or CL2 (p<0.05). These results indicate that iNOS expression is closely related to luteal cell apoptosis and then to luteal regression.

• Journal of Life Science
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• v.33 no.1
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• pp.50-55
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• 2023

This study was designed to determine the activities of Rosa davurica Pall. leaf extract and their regulatory mechanisms in macrophage inflammation. Anti-inflammatory potential of Rosa davurica Pall. leaf extract was evaluated by measuring the nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) synthesis in lipopolysaccharide (LPS)-treated macrophage Raw 264.7 cells. Rosa davurica Pall. leaf extract potently inhibited LPS-induced NO release in a dose dependent manner. However, cell viability decreased to about 50% at high dose of 500 ㎍/ml, resulting in cytotoxicity. LPS-induced iNOS protein expression was also reduced significantly after treatment with Rosa davurica Pall. leaf extract. Furthermore, extract of Rosa davurica Pall. attenuated LPS-mediated phosphorylation of IκB and nuclear factor (NF-κB). Suppression of NF-κB signaling by treatment with PDTC, an NF-κB specific inhibitor, accelerated the inhibition of NO production and iNOS protein expression. These results suggest that Rosa davurica Pall. exhibits the anti-inflammatory potential in LPS-induced macrophage inflammation, partly through inhibition of NO production by down-regulation of NF-κB signaling.

• Journal of Acupuncture Research
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• v.29 no.1
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• pp.15-24
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• 2012

Objectives : In recent years, many studies have been widely researching anti-inflammation effect of various medicinal plants. $Cinnamomi$ $Cortex$ was not enough in researching of the anti-inflammation. Moreover, there is no comparative study about extraction methods. Therefore, we investigated the inhibitory effects of $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction on Nitric oxide(NO), Prostaglandin E2(PGE2) production, Cyclooxygenase(COX)-2, inducible NOS(iNOS) expression and extracellular signal regulate kinase(ERK)1/2 phosphorylation in lipopolysaccharide(LPS) induced RAW 264.7 macrophage cell. Methods : $Cinnamomi$ $Cortex$ was extracted by EtOH and Hot water. RAW 264.7 macrophage cell viability was measured by MTT assay. Effect of $Cinnamomi$ $Cortex$ pharmacopuncture on NO and PGE2 production in LPS induced macrophages was accessed by Griess assay and enzyme-linked immunospecific assay(ELISA), respectively. Inhibition effect on COX-2, iNOS expression and ERK1/2 phosphorylation was examined by Immunoblotting assay. Results : 1. Cytotoxic effect of $Cinnamomi$ $Cortex$ pharmacopuncture by Hot water extraction in RAW 264.7 macrophages was not appeared, except $3125{\mu}g/m{\ell}$. And cytotoxic effect was not appeared in EtOH extraction method. 2. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited NO production in LPS induced macrophages significantly. 3. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited PGE2 production in LPS induced macrophages significantly. 4. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited COX-2, iNOS expression in LPS induced macrophages. Especially, it has been confirmed that COX-2, iNOS expression were effectively inhibited in Hot water extraction. 5. $Cinnamomi$ $Cortex$ pharmacopuncture by EtOH and Hot water extraction inhibited ERK1/2 phosphorylation in LPS induced macrophages. Especially, it has been confirmed that ERK1/2 phosphorylation was effectively inhibited in Hot water extraction. Conclusions : According to the results, $Cinnamomi$ $Cortex$ pharmacopuncture suppresses NO, PGE2 production, COX-2, iNOS expression and ERK1/2 phosphorylation in LPS induced macrophages. It has a potential for treating various inflammatory diseases, and Hot water extraction method could be used more extensively than EtOH extraction method.