• Title/Summary/Keyword: inhibition ELISA

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Inhibition of gene expression and production of iNOS and $TNF-{\alpha}$ in LPS-stimulated microglia by On-Bi-Tang (오령산가온비탕의 BV2에서 LPS로 유도된 nitric oxide 및 $TNF-{\alpha}$ 생성에 미치는 영향)

  • Son, Hye-Young;Kim, Sang-Woon;Jung, Sun-Ju;Jung, Hyo-Won;Yoon, Cheol-Ho;Park, Yong-Ki
    • The Korea Journal of Herbology
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    • v.21 no.2
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    • pp.135-142
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    • 2006
  • Objectives : On-Bi-Tang(OB) has been prescribed Chinese traditional medicine for the treatment of inflammatory disease such as chronic renal failure. In this study, we investigated the anti-inflammatory effect of OB extract in the BV2 murine microglial cells. Methods : After the water extract of OB was treated in BV2 cells, murine microglial line, the cell viability was measured by MTT assay. The production of nitric oxide (NO) and $TNF-{\alpha}$ was determined based on Griess reagent and enzyme linked immunosorbant assay (ELISA). mRNA expression of inducible nitric oxide synthase (iNOS) and $TNF-{\alpha}$ was analyzed by RT-PCR. Results : OB extract significantly inhibited the LPS-induced production of NO and TNF-a in BV2 cells. OB extract also suppressed the mRNA expression of iNOS and $TNF-{\alpha}$ in BV2 cells activated with LPS. Conclusion : These data suggests that OB extract may have the anti-inflammatory effect through the modulation of NO production and inflammatory cytokine such as $TNF-{\alpha}$.

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Immune and Anti-oxidant Functions of Ethanol Extracts of Scutellaria baicalensis Georgi in Mice Bearing U14 Cervical Cancers

  • Peng, Yong;Guo, Cong-Shan;Li, Pan-Xia;Fu, Zhan-Zhao;Gao, Li-Ming;Di, Ya;Ju, Ya-Kun;Tian, Ru;Xue, Jia-Jia
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.10
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    • pp.4129-4133
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    • 2014
  • Background: The objective was to study the effect of Scutellaria baicalensis Georgi ethanol extracts (SBGE) on immune and anti-oxidant function in U14 tumor-bearing mice. Materials and Methods: U14 tumor-bearing mice were randomly divided into eight groups: a control group, a cyclophosphamide (CTX) group, three dose groups of SBGEI (high, medium, low), and three dose groups of SBGEII (high, medium, low). After two weeks, the thymus and spleen weight indices of mice bearing U14 cervical cancer were calculated. Enzyme linked immunosorbent assays (ELISA) was used to determine the levels of serum IL-2, TNF-${\alpha}$, IL-8, and PCNA. MDA activity and SOD activity in plasma were measured with detection kits. Results: In the SBGE groups, thymus weight and spleen weight indices of U14 tumor-bearing mice were significantly higher than in the control group or CTX group (p<0.05). Compared to control group, the levels of serum IL-2 and TNF-${\alpha}$ in U14 tumor-bearing mice increased significantly, whereas the contents of serum IL-8 and PCNA decreased (p<0.05). The activity of SOD increased with the growing dose of SBGE, while the activity of MDA decreased significantly in the highe-rdose groups of SBGE. Conclusions: These findings suggested that SBGE, especially at high dose, 1000 mg/kg, showed significant immune and anti-oxidant effects infU14 tumor-bearing mice, which might be the mechanisms of SBGE inhibition of tumor growth.

Inhibitory effects of Sam-Myo-San on the LPS-induced production of nitric oxide and $TNF-{\alpha}$ in RAW 264.7 cells and BV-2 Microglia cells (삼묘환(三妙丸)의 LPS에 의해 활성화된 RAW 264.7 cells과 BV-2 Microglia cells로부터 생성되는 nitric oxide 및 $TNF-{\alpha}$의 생성억제효과)

  • Lee, Jae-Hyun;Jung, Hyo-Won;Park, Yong-Ki
    • The Korea Journal of Herbology
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    • v.21 no.4
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    • pp.59-67
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    • 2006
  • Objectives : Sam-Myo-Whan(SMW) has been known traditional prescription with anti- anthritis activities. We investigated inhibitory effects of SMW on lipopolysaccharide (LPS)-induced nitric oxide(NO), $TNF-{\alpha}$ and inducible nitric oxide synthase(iNOS) production from RAW264.7 cells and BV-2 Microglia cells. Methods : SMW, which had been extracted with 70% MeOH, concentrated and freeze-dried was used for this experiment. After BV2 mouse brain macrophages and RAW264.7 mouse peritoneal macrophages were pretreated with increasing concentrations of SMW extract for 30min, and then activated with LPS. To investigate cytotoxicity of SMW extract, cell viability was measured by MTT assay. NO production was measured in each culture supernatant by Griess reaction. mRNA expression of iNOS in two type cells was investigated by RT-PCR. $TNF-{\alpha}$ production was measured in each culture supernatant by ELISA. Results : SMW extract significantly inhibited LPS-induced NO and $TNF-{\alpha}$ production in BV2 cells and RAW264.7 cells dose-dependently. SMW extract also greatly suppressed mRNA expression of iNOS in both type cells activated with LPS. Conclusion : These data suggests that SMW extract may have an anti-inflammatory effect through the inhibition of iNOS expression.

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MiR-886-5p Inhibition Inhibits Growth and Induces Apoptosis of MCF7 Cells

  • Zhang, Lei-Lei;Wu, Jiang;Liu, Qiang;Zhang, Yan;Sun, Zhu-Lei;Jing, Hong
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.4
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    • pp.1511-1515
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    • 2014
  • Background and Aims: To explore the molecular mechanisms of miR-886-5p in breast cancer., we examined roles in inhibiting growth and migration of MCF-7 cells. Methods: MiR-886-5p mimics and inhibitors were used to express or inhibit MiR-886-5p, respectively, and MTT and clone formation assays were used to determine the survival and proliferation. Hoechst 33342/ PI double staining was applied to detect apoptosis. The expression of caspase-3, caspase-8, caspase-9, MT1-MMP, VEGF-C and VEGF-D was detected by Western blotting, and the levels of MMP2 and MMP9 secreted from MCF-7 cells were assessed by ELISA. MCF-7 cell migration was determined by wound healing and Transwell assays. Results: We found that the growth of MCF-7 cells was inhibited upon decreasing miR-886-5p levels. Inhibiting miR-866-5p also significantly induced apoptosis and decreased the migratory capacity of these cells. The expression of VEGF-C, VEGF-D, MT1-MMP, MMP2, and MMP9 was also found to be decreased as compared to controls. Conclusions: Our data show that downregulation of miR-886-5p expression in MCF-7 cells could significantly inhibit cell growth and migration. This might imply that inhibiting miR-886-5p could be a therapeutic strategy in breast cancer.

Inhibition of Leptin and Leptin Receptor Gene Expression by Silibinin-Curcumin Combination

  • Nejati-Koshki, Kazem;Akbarzadeh, Abolfazl;Pourhasan-Moghaddam, Mohammad;Abhari, Alireza;Dariushnejad, Hassan
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.11
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    • pp.6595-6599
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    • 2013
  • Leptin and its receptor are involved in breast carcinogenesis as mitogenic factors. Therefore, they could be considered as targets for breast cancer therapy. Expression of the leptin receptor gene could be modulated by leptin secretion. Silibinin and curcumin are herbal compounds with anti-cancer activity against breast cancer. The aim of this study was to assess their potential to inhibit of expression of the leptin gene and its receptor and leptin secretion. Cytotoxic effects of the two agents on combination on T47D breast cancer cells was investigated by MTT assay test after 24h treatment. With different concentrations the levels of leptin, leptin receptor genes expression were measured by reverse-transcription real-time PCR. Amount of secreted leptin in the culture medium was determined by ELISA. Data were statistically analyzed by one-way ANOVA test. The silibinin and curcumin combination inhibited growth of T47D cells in a dose dependent manner. There were also significant difference between control and treated cells in leptin expression and the quantity of secreted leptin with a relative decrease in leptin receptor expression. In conclusion, these herbal compounds inhibit the expression and secretion of leptin and it could probably be used as drug candidates for breast cancer therapy through leptin targeting in the future.

Down-Regulation of Mcl-1 by Small Interference RNA Induces Apoptosis and Sensitizes HL-60 Leukemia Cells to Etoposide

  • Karami, Hadi;Baradaran, Behzad;Esfehani, Ali;Sakhinia, Masoud;Sakhinia, Ebrahim
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.2
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    • pp.629-635
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    • 2014
  • Background: Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. Materials and Methods: siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Results: Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time-dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Conclusions: Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

Effect of Mixture extracted from Bupleuri Radix and Physalidis Herba on the LPS-induced depression in Rats (시호(柴胡)와 산장(酸漿) 복합제가 LPS로 유도된 우울증에 미치는 효과)

  • Lee, Won-Haeng;Kim, Do-Hoon;Lee, Tae Hee
    • The Korea Journal of Herbology
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    • v.30 no.3
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    • pp.69-76
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    • 2015
  • Objectives : The effect of mixture extracted from Bupleuri Radix and Physalidis Herba(BR+RH) on the LPS-induced Depression in rats was investigated. Methods : Rats were administered intragastrically BR+PH after injectio of LPS to induce deprssion. Immobility was examined using Tail Suspension Test(TST), Forced Swimming Test(FST). The level of plasma corticosterone was measured by an Enzyme-Linked Immunosorbent Assay(ELISA) method. The expressions of c-Fos, Corticotropin Releasing Factor(CRF), NADPH-d in the Paraventricular nucleus(PVN) and TH in the Locus coeruleus(LC) were measured by immunohistochemical method. Results : In the effect of BR + PH on TST, immobility was significantly decreased comparing with the LPS group. In FST, immobility was shown decrease tendency in the BR+PH group. The expression of c-Fos in the PVN was significantly decreased at BR + PH400 group, comparing with the LPS group. The expression of CRF in PVN was shown dto have the decrease tendency in the BR+PH group, comparing with the LPS group. The expression of NADPH-d in PVN was not significantly decreased at BR+PH groups, comparing with the LPS group. The expression of TH in the LC was shown to have the decrease tendency at BR + RH groups, but not significantly, comparing with the LPS group. Conclusions : Anti-depressant effect of mixture after extracted from Bupleuri Radix and Physalidis Herba was through the anti-inflammatory effect via inhibition of HPA axis. NO and catecholamine system is not involved.

Anti-inflammatory effects of proanthocyanidin-rich red rice extract via suppression of MAPK, AP-1 and NF-κB pathways in Raw 264.7 macrophages

  • Limtrakul, Pornngarm;Yodkeeree, Supachai;Pitchakarn, Pornsiri;Punfa, Wanisa
    • Nutrition Research and Practice
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    • v.10 no.3
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    • pp.251-258
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    • 2016
  • BACKGROUND/OBJECTIVES: Several pharmacological properties of red rice extract have been reported including anti-oxidant, anti-tumor, and reduced cancer cell invasion. This study was conducted to evaluate the anti-inflammatory effects of red rice extract on the production of inflammatory mediators in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages. MATERIALS/METHODS: Pro-inflammatory cytokines including tumor necrosis factor-${\alpha}$ and interleukin-6 were determined by ELISA and cyclooxygenase-2 and inducible nitric oxide synthase expression was evaluated using western blot analysis. In addition, the signaling pathway controlling the inflammatory cascade such as nuclear factor kappa B ($NF-{\kappa}B$), activator proteins-1 (AP-1), and mitogen-activated protein kinase (MAPK) was determined. RESULTS: Our results showed that red rice polar extract fraction (RR-P), but not non-polar extract fraction, inhibited interleukin-6, tumor necrosis factor-${\alpha}$, and nitric oxide production in LPS-induced Raw 264.7 cells. RR-P also reduced the expression of inflammatory enzymes, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, activation of AP-1 and $NF-{\kappa}B$ transcription factor in the nucleus was abrogated by RR-P. RR-P inhibited the phosphorylation of extracellular signaling-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 MAPK signaling responsible for the expression of inflammatory mediators in LPS-stimulated Raw 264.7 cells. Based on chemical analysis, high amounts of proanthocyanidin and catechins were detected in the RR-P fraction. However, only proanthocyanidin reduced $NF-{\kappa}B$ and AP-1 activation in LPS-activated Raw 264.7 cells. CONCLUSION: These observations suggest that the anti-inflammatory properties of RR-P may stem from the inhibition of pro-inflammatory mediators via suppression of the AP-1, $NF-{\kappa}B$, and MAPKs pathways.

Anti-inflammatory and utero-relaxant effect of α-bisabolol on the pregnant human uterus

  • Munoz-Perez, Victor Manuel;Ortiz, Mario I.;Ponce-Monter, Hector A.;Monter-Perez, Vicente;Barragan-Ramirez, Guillermo
    • The Korean Journal of Physiology and Pharmacology
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    • v.22 no.4
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    • pp.391-398
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    • 2018
  • The aim of this study was to evaluate the in vitro anti-inflammatory and utero-relaxant effect of ${\alpha}$-bisabolol on the pregnant human myometrium. Samples from the pregnant human myometrium were used in functional tests to evaluate the inhibitory effect of ${\alpha}$-bisabolol (560, 860, 1,200 and $1,860{\mu}M$) on spontaneous myometrial contractions. The intracellular cyclic adenosine monophosphate (cAMP) levels generated in response to ${\alpha}$-bisabolol in human myometrial homogenates were measured by ELISA. The anti-inflammatory effect of ${\alpha}$-bisabolol was determined through the measurement of two pro-inflammatory cytokines, tumor necrosis factor-${\alpha}$ ($TNF{\alpha}$) and interleukin $(IL)-1{\beta}$, and the anti-inflammatory cytokine IL-10, in pregnant human myometrial explants stimulated with lipopolysaccharide (LPS). Forskolin was used as a positive control to evaluate the cAMP and cytokine levels. ${\alpha}$-Bisabolol was found to induce a significant inhibition of spontaneous myometrial contractions at the highest concentration level (p<0.05). ${\alpha}$-Bisabolol caused a concentration-dependent decrease in myometrial cAMP levels (p<0.05) and a concentration-dependent decrease in LPS-induced $TNF{\alpha}$ and $IL-1{\beta}$ production, while IL-10 production did not increase significantly (p>0.05). The anti-inflammatory and utero-relaxant effects induced by ${\alpha}$-bisabolol were not associated with an increase in cAMP levels in pregnant human myometrial samples. These properties place ${\alpha}$-bisabolol as a potentially safe and effective adjuvant agent in cases of preterm birth, an area of pharmacological treatment that requires urgent improvement.

The Effect of Herbs on Inhibition of HBeAg Production in HepG2.2.15 Cell line (수종의 한약재가 HepG 2.2.15 Cell의 HBeAg발현 억제에 미치는 효과(效果))

  • Woo, Hong-Jung;Lee, Jang-Hoon;Kim, Young-Chul
    • The Journal of Internal Korean Medicine
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    • v.20 no.1
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    • pp.122-132
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    • 1999
  • Purpose : Hepatitis B virus DNA transfected cell line(HepG2.2.15) was cultured to evaluate the effect of herbs on the expression of HBeAg and the replication of HBV. HepG2.2.15 produces HBV particles as well as viral proteins into cell culture media. Methods : Extracts of herbs were adminitered to the cells on the proper concentration. Culture media was collected 48 hours after the herbal administration and HBeAg level in the media was examined by ELISA method. To confirm that the anti-viral effect was not due to direct cytotocixity of the extracts, normal cell proliferation was shown by cell counting. And as of the interference in protein synthesis of HepG2.2.15 by herb-extracts, we used the result of study that we performed before by ${\alpha}FP$ assay using EIA method. Results& Conclusion : Herb medicines like 地楡(Sanguisorbae Radix) and 覆盆子(Rubi Frusctus) showed significant inhibitory effect on HBeAg expression at p<0.01 and 五味子(Acanthopanacis Cortex) at p<0.05. Whereas, though some herbs such as ?草根(Rubiae Radix), 山査(Crataegii Fructus), 白芍藥(Paeoniae Radix Alba), and 大黃(Rhei Radix et Rhizoma) showed the tendecy to suppress HBeAg. most of them were not significant statistically. From the above, we could conclude that those herb medicines can be applied to patients effectively and further studies on effective fraction of some herbs are thought to be needed.

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