• The Korea Journal of Herbology
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• v.31 no.5
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• pp.1-6
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• 2016

Objectives : Nardostachys jatamansi (NJ) is a medicinal herb that has been reported in various traditional systems of medicine for its use in antispasmodic, a digestive stimulant, skin diseases. Previous studies have already reported that NJ effectively protects against inflammation. However, the active compound in NJ is unknown. Therefore, in the present study, we analyzed effects of a compound, 8α-hydroxy pinoresinol (HP), isolated from NJ against lipopolysaccharide (LPS) induced inflammation in RAW 264.7 cells.Methods : To examine the anti-inflammatory effect of HP against LPS, intraperitoneally pre-treat the HP (100, 200, 500 and 1,000 nM) 1 h prior to LPS challenges. LPS was stimulated with 500 ng/ml in RAW 264.7 cells. To identify the anti-inflammatory effect of HP, we measured inflammatory mediators such as inducible nitric oxide synthase (iNOS) and its derivative nitric oxide (NO), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2). Also we evaluated molecular mechanisms including mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB) activation by western blot.Results : The HP inhibited production of inflammatory mediators, such as iNOS and its derivative NO, COX-2 and PGE2 in LPS- induced inflammationin RAW 264.7 cells. Additionally, HP also inhibited activation of p38 pathway signaling but not extracellularsignal-regulatedkinase (ERK), c-jun NH2-terminal kinase (JNK), and NF-κB.Conclusion : Our results suggest that HP has anti-inflammatory functions through the dephosphorylation of p38 and HP can provide beneficial strategy for prevention and therapy of inflammation.

• Archives of Pharmacal Research
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• v.29 no.10
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• pp.890-897
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• 2006

In inflammatory responses, induction of cytokines and other immune regulator genes in macrophages by pathogen-associated signal such as lipopolysaccharide (LPS) plays a crucial role. In this study, the gene expression profile changes by LPS treatment in the macrophage/monocyte lineage cell line RAW264.7 was investigated. A 60-mer oligonucleotide microarray of which probes target 32381 mouse genes was used. A reverse transcription-in vitro translation labeling protocol and a chemileuminescence detection system were employed. The mRNA expression levels in RAW264.7 cells treated for 6 h with LPS and the control vehicle were compared. 747 genes were up-regulated and 523 genes were down-regulated by more than 2 folds. 320 genes showing more than 4-fold change by LPS treatment were further classified for the biological process, molecular function, and signaling pathway. The biological process categories that showed high number of increased genes include the immunity and defense, the nucleic acid metabolism, the protein metabolism and modification, and the signal transduction process. The chemokine-cytokine signaling, interleukin signaling, Toll receptor signaling, and apoptosis signaling pathways involved high number of genes differentially expressed in response to LPS. These expression profile data provide more comprehensive information on LPS-target genes in RAW264.7 cells, which will be useful in comparing gene expression changes induced by extracts and compounds from anti-inflammatory medicinal herbs.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.365-370
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• 2015

Background: The beneficial effects of ginsenoside species have been well demonstrated in a number of studies. However, the function of ginsenoside Ro (GRo), an oleanane-type saponin, has not been sufficiently investigated. Thus, the aim of the present study was to investigate the anti-inflammatory effects of GRo in vitro using the Raw 264.7 mouse macrophage cell line treated with lipopolysaccharide (LPS), and to clarify the possible mechanism of GRo involving heme oxygenase-1 (HO-1), which itself plays a critical role in self-defense in the presence of inflammatory stress. Methods: Raw 264.7 cells were pretreated with GRo (up to $200{\mu}M$) for 1 h before treatment with 1 mg/mL LPS, and both cell viability and inflammatory markers involving HO-1 were evaluated. Results: GRo significantly increased cell viability in a dose dependent manner following treatment with LPS, and decreased levels of reactive oxygen species and nitric oxide. GRo decreased inflammatory cytokines such as nitric oxide synthase and cyclooxygenase-2 induced by LPS. Moreover, GRo increased the expression of HO-1 in a dose dependent manner. Cotreatment of GRo with tin protoporphyrin IX, a selective inhibitor of HO-1, not only inhibited upregulation of HO-1 induced by GRo, but also reversed the anti-inflammatory effect of GRo in LPS treated Raw 264.7 cells. Conclusion: GRo induces anti-inflammatory effects following treatment with LPS via upregulation of HO-1.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.5
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• pp.395-401
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• 2021

Extended inflammation and cytokine production pathogenically contribute to a number of inflammatory disorders. Formosanin C (FC) is the major diosgenin saponin found in herb Paris formosana Hayata (Liliaceae), which has been shown to exert anti-cancer and immunomodulatory functions. In this study, we aimed to investigate anti-inflammatory activity of FC and the underlying molecular mechanism. RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) or pretreated with FC prior to being stimulated with LPS. Thereafter, the macrophages were subjected to analysis of the expression levels of pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, as well as two relevant enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). The analysis revealed that FC administration blunted LPS-induced production of NO and PGE in a dose-dependent manner, while the expression of iNOS and COX-2 at both mRNA and protein levels was inhibited in LPS-stimulated macrophages pre-treated with FC. Moreover, LPS stimulation upregulated mRNA expression and medium release of TNF-α, IL-1β, and IL-6, whereas this effect was blocked upon FC pre-administration. Mechanistic studies showed that inhibitory effects of FC on LPS-induced inflammation were associated with a downregulation of IκB kinase, IκB, and p65/NF-κB pathway. Taken together, these data suggest that FC possesses an inflammation-suppressing activity, thus being a potential agent for the treatment of inflammation-associated disorders.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.2
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• pp.105-110
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• 2021

Objective: Uropathogenic Escherichia coli is known to cause urinary tract infections, and the endotoxin (lipopolysaccharide [LPS]) of this bacterium may cause deficiencies of sperm quality and morphology. In the present study, the effects of LPS on mouse sperm were studied, and the levels of interleukin (IL)-17A and possible changes in testis tissue were evaluated. Methods: LPS of uropathogenic E. coli was extracted using the methanol-chloroform method, followed confirmation using sodium dodecyl sulfate-polyacrylamide electrophoresis. Purified LPS (100 ㎍/kg) or phosphate-buffered saline was injected intraperitoneally into BALB/c mice for 7 days consecutively in the test and control groups, mice were sacrificed on days 3, 7, and 42 after the first injection. Blood was tested for levels of IL-17A using the enzyme-linked immunosorbent assay method. Testis tissue and sperm were collected from each mouse and were studied according to standard protocols. Results: The mean sperm count and motility significantly decreased (p=0.03) at 3, 7, and 42 days after the injections. The level of IL-17A in the test groups increased, but not significantly (p=0.8, p=0.11, and p=0.15, respectively). Microscopic studies showed no obvious changes in the morphology of the testis tissue; however, significant changes were observed in the cellular parenchyma on day 42. Conclusion: LPS can stimulate the immune system to produce proinflammatory cytokines, resulting in an immune response in the testis and ultimately leading to deficiency in sperm parameters and testis tissue damage. In addition, the presence of LPS could significantly impair sperm parameters, as shown by the finding of decreased motility.

• The Korea Journal of Herbology
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• v.27 no.6
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• pp.23-28
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• 2012

Objectives : Samhwangsashim-tang(SHSST), a mixture of Rhei radix et rhizoma, Scutellariae radix, and Coptidis rhizoma, has been regarded as being able to treat bleeding, gastric discomfort, dry mouth, insomnia and purpura due to Blood Heat. Currently, this herbal formula is applied to gastritis, gastric ulcer, hypertension, atherosclerosis or other types of vascular inflammatory disorders. Methods : We extracted this herbal mixture with 30% ethanol and examined for its effects on systemic inflammatory responses and in vitro macrophage activity. Mice were orally given to SHSST for 7 days and then lipopolysaccharide(LPS) was intraperitoneally injected. Tumor necrosis factor-${\alpha}$(TNF-${\alpha}$) levels in serum were measured 1 h after LPS challenge. Peritoneal macrophages were isolated from thioglycollate-injected mice and used for in vitro cellular activity. Cell death was measured using the MTT method and annexin V/propidium iodide staining. LPS-stimulated signaling molecules necessary for TNF-${\alpha}$ expression were determined by Western blotting. Results : Oral administration of SHSST for 7 days resulted in a significant reduction in LPS-stimulated TNF-${\alpha}$ release into serum. In vitro treatment of SHSST was cytotoxic in a concentration-dependent manner. However, SHSST caused a concentration-dependent reduction in necrosis and increase in apoptosis in mouse peritoneal macrophages. SHSST inhibited the activation of NF-${\kappa}B$, p38 and JNK signaling molecules in response to LPS. Conclusion : Taken together, our results demonstrated that SHSST was effective in lowering LPS-stimulated TNF-${\alpha}$ serum levels, possibly through its modulation of NF-${\kappa}B$, p38 and JNK in macrophages.

• Tuberculosis and Respiratory Diseases
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• v.69 no.5
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• pp.337-347
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• 2010

Background: Transglutaminase-2 (TG-2) has been reported to play an important role in the process of fibrosis. However, TG-2 studies on fibroproliferation of acute lung injury (ALI) are absent. The purpose of this study was to investigate the role of TG-2 in the fibroproliferation of lipopolysaccharide (LPS)-induced ALI. Methods: The male C57BL/6 mice of 5 weeks age were divided into 3 groups; control group (n=30) in which $50{\mu}L$ of saline was given intratracheally (IT), LPS group (n=30) in which LPS 0.5 mg/kg/$50{\mu}L$ of saline was given IT, and LPS+Cyst group treated with intraperitoneal 200 mg/kg of cystamine, competitive inhibitor of TG-2, after induction of ALI by LPS. TG-2 activity and nuclear factor $(NF)-{\kappa}B$ were measured in lung tissue homogenate. Tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-$1{\beta}$, IL-6, myeloperoxidase (MPO), and transforming growth factor (TGF)-${\beta}1$ were measured using bronchoalveolar lavage fluids. Histopathologic ALI score and Mallory's phosphotunistic acid hematoxylin (PTAH) for collagen and fibronectin deposition were performed. Results: The TG-2 activities in the LPS group were significantly higher than the control and LPS+Cyst groups (p<0.05). The TNF-${\alpha}$ and IL-$1{\beta}$ concentrations and $NF-{\kappa}B$ activity were lower in the LPS+Cyst group than the LPS group (p<0.05). The LPS+Cyst group showed lower MPO, ALI score, TGF-${\beta}1$ concentration, and Mallory's PTAH stain than the LPS group, but the differences were not significant (p>0.05). Conclusion: Inhibition of TG-2 activity in the LPS-induced ALI prevented early inflammatory parameters, but had limited effects on late ALI and fibroproliferative parameters.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.3
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• pp.183-192
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• 2022

Lipopolysaccharide (LPS) is an endotoxin factor present in the cell wall of Gram-negative bacteria and induces various immune responses to infection. Recent studies have reported that LPS induces cellular stress in various cells including oocytes and embryos. Melatonin (N-acetyl-5-methoxytryptamine) is a regulatory hormone of circadian rhythm and a powerful antioxidant. It has been known that melatonin has an effective function in scavenging oxygen free radicals and has been used as an antioxidant to reduce the cytotoxic effects induced by LPS. However, the effect of melatonin on LPS treated early embryonic development has not yet been confirmed. In this study, we cultured mouse embryos in medium supplemented with LPS or/and melatonin up to the blastocyst stage in vitro and then evaluated the developmental rate. As a result of the LPS-treatment, the rate of blastocyst development was significantly reduced compared to the control group in all the LPS groups. Next, in the melatonin only treated group, there was no statistical difference in embryonic development and no toxic effects were observed. And then we found that the treatment of melatonin improved the rates of compaction and blastocyst development of LPS-treated embryos. In addition, we showed that melatonin treatment decreased ROS levels compared to the LPS only treated group. In conclusion, we demonstrated the protective effect of melatonin on the embryonic developmental rate reduced by LPS. These results suggest a direction to improve reproduction loss that may occur due to LPS exposure and bacterial infection through the using of melatonin during in vitro culture.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.8
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• pp.1150-1156
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• 2014

This trail was conducted to study the effect of L-proline on the growth performance, and blood parameter in the weaned lipopolysaccharide (LPS)-challenged pigs. Thirty six pigs ($9.13{\pm}0.85$ kg) were assigned randomly to dietary treatments in a $2{\times}3$ factorial arrangement in a 20-d growth assay. Factors were intraperitoneal injection with saline or LPS, and three dietary L-proline supplement levels (0%, 0.5%, or 1.0%). On d 10, blood samples were collected at 3 h after LPS (100 ${\mu}g$ LPS/kg body weight [BW]) or saline injection. On d 20 of the trial, all pigs were orally administrated D-xylose (0.1 g/kg BW) at 2 h, and blood samples were collected at 3 h after LPS or saline injection. As a result, dietary supplementation with 0.5% proline had a tendency to increase average daily gain (ADG) in piglets during d 10 to 20 (p = 0.088). Without LPS challenge, dietary supplementation with 1.0% proline had no effect on growth hormone (GH) concentrations on d 10 (p>0.05), but decreased it after LPS challenge (p<0.05). There was LPS challenge${\times}$proline interaction for GH concentrations on d 10 (p<0.05). Dietary supplementation with 1.0% proline decreased glucagon concentration on d 10 after LPS challenge (p<0.05). In addition, dietary supplementation with proline increased superoxide dismutase (SOD) activity significantly on d 10 and 20 (p<0.05), and 1.0% proline increased heat shock proteins-70 concentration on d 10 (p<0.05). Moreover, proline supplementation increased diamine oxidase (DAO) concentrations after LPS challenge (p<0.05). There was LPS challenge${\times}$proline interaction for DAO (p<0.05). Furthermore, dietary supplementation with 1.0% proline increased the D-xylose level when no LPS challenge (p<0.05). These results indicate that proline supplementation could improve growth performance, increase SOD activities, and has a positive effect on the gastrointestinal tract digestibility in early weaned pigs.

• Journal of Veterinary Clinics
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• v.33 no.4
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• pp.194-199
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• 2016

The objective of this study was to examine the effect of trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) on the expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathway in lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs). t10c12-CLA was treated with different concentrations in culture medium of LPS$na{\ddot{i}}ve$ and LPS-stimulated PBMCs. The mRNA expressions of prostaglandin $E_2$ ($PGE_2$)-synthase, COX-2 and 5-LOX were measured using quantitative real-time PCR. In addition, the production levels of $PGE_2$ and 5-LOX in culture supernatant from PBMCs with or without LPS were assessed by ELISA. In LPS$na{\ddot{i}}ve$ PBMCs, treatment of t10c12-CLA significantly (p < 0.05) increased the mRNA expressions of PGE2 synthase and 5-LOX compared to vehicle control. Expression of COX-2 mRNA did not show significant difference compared to vehicle control by t10c12-CLA treatment in LPS$na{\ddot{i}}ve$ PBMCs. However, the addition of LPS in PBMCs markedly (p < 0.05) increased the mRNA expression of COX-2, $PGE_2$ synthase and 5-LOX, and also significantly (p < 0.05) enhanced the production of $PGE_2$ and 5-LOX relative to LPS$na{\ddot{i}}ve$ PBMCs, respectively. However, the addition of t10c12-CLA significantly (p < 0.01) suppressed the LPS-induced excessive expression of COX-2, $PGE_2$ synthase, and 5-LOX compared to those of PBMCs treated with LPS alone. The production levels of $PGE_2$ and 5-LOX in culture supernatant from LPS-stimulated PBMCs were also significantly (p < 0.05) inhibited by the treatment of t10c12-CLA compared to LPS alone. These results suggested that t10c12-CLA has an anti-inflammatory effect via dual inhibition of COX-2 and 5-LOX with gene expression and production level in LPS-stimulated porcine PBMCs. Therefore, it was thought that t10c12-CLA can attenuate the inflammatory response by down-regulation of eicosanoids production.