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Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF- κ B and JNK Activation in LPS-Stimulated BV2 Microglia

View Article: PubMed Central - PubMed

ABSTRACT

Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1β, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1β and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti-neuroinflammatory activities by inhibiting pro-inflammatory mediator expression and production, upregulating HO-1, GCLM and NQO1, blocking NF-κB and modulating JNK signaling pathways. They may offer therapeutic potential for suppressing overactivated microglia and alleviating neurodegeneration.

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Effects of ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET) on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and activation. BV2 cells were pre-treated for 0.5 h with polymyxin B (PMB, 10 µg/mL), vehicle (0.1% ethanol), or the indicated concentration of extract, and then stimulated with LPS (10 or 100 ng/mL) for 20 h. (a,b) The nitrite production in supernatant was determined by the Griess reagent; (c,d) The cell viability was analyzed by MTT assay. Data are represented as the mean ± SD (n = 3). Statistical differences are presented ** p < 0.01 compared with the vehicle control (without LPS) and ## p < 0.01 compared with the LPS-treated vehicle; (e) Representative images of BV2 microglia incubated for 20 h with vehicle (0.1% ethanol), LPS (10 ng/mL) plus vehicle (0.1% ethanol), LPS (10 ng/mL) + CR (0.05 mg/mL) or LPS (10 ng/mL) + ET (0.05 mg/mL). Images were acquired with Nikon Eclipse Ti-E inverted microscope (Scale bar, 50 µm).
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ijms-17-01420-f001: Effects of ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET) on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and activation. BV2 cells were pre-treated for 0.5 h with polymyxin B (PMB, 10 µg/mL), vehicle (0.1% ethanol), or the indicated concentration of extract, and then stimulated with LPS (10 or 100 ng/mL) for 20 h. (a,b) The nitrite production in supernatant was determined by the Griess reagent; (c,d) The cell viability was analyzed by MTT assay. Data are represented as the mean ± SD (n = 3). Statistical differences are presented ** p < 0.01 compared with the vehicle control (without LPS) and ## p < 0.01 compared with the LPS-treated vehicle; (e) Representative images of BV2 microglia incubated for 20 h with vehicle (0.1% ethanol), LPS (10 ng/mL) plus vehicle (0.1% ethanol), LPS (10 ng/mL) + CR (0.05 mg/mL) or LPS (10 ng/mL) + ET (0.05 mg/mL). Images were acquired with Nikon Eclipse Ti-E inverted microscope (Scale bar, 50 µm).

Mentions: To test whether CR and ET can function as inhibitors for NO release, BV2 cells were pre-treated with vehicle (0.1% ethanol), CR, or ET for 30 min followed by LPS (10 or 100 ng/mL) insult for a further 20 h. Polymyxin B (PMB, 10 µg/mL), a cyclic cationic polypeptide antibiotic, which binds to lipid A, served as a control LPS inhibitor. To set the optimal concentrations of CR and ET, we started with various concentrations of CR and ET ranging from 0.025–0.2 mg/mL with 1:2 serial dilutions. Our preliminary data showed that CR and ET at 0.025 mg/mL did not exert anti-NO activity significantly, while at 0.2 mg/mL caused significant cell death. As a result, 0.05 and 0.1 mg/mL of CR and ET were chosen for the experiments. Figure 1a,b shows that 10 and 100 ng/mL LPS plus vehicle stimulated NO production from basal levels (0.7–1.3 µM) to 25.0 ± 0.4 and 33.0 ± 0.8 µM, respectively. CR and ET (0.05–0.1 mg/mL) dose-dependently decreased 10 ng/mL LPS-induced NO production by 72%–93% and 43%–75%, respectively (p < 0.01). Slightly weaker inhibition (67%–93% for CR and 36%–57% for ET) was noted against 100 ng/mL LPS (p < 0.01). In comparison, PMB (10 µg/mL) almost completely inhibited 10 and 100 ng/mL LPS-mediated NO production.


Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF- κ B and JNK Activation in LPS-Stimulated BV2 Microglia
Effects of ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET) on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and activation. BV2 cells were pre-treated for 0.5 h with polymyxin B (PMB, 10 µg/mL), vehicle (0.1% ethanol), or the indicated concentration of extract, and then stimulated with LPS (10 or 100 ng/mL) for 20 h. (a,b) The nitrite production in supernatant was determined by the Griess reagent; (c,d) The cell viability was analyzed by MTT assay. Data are represented as the mean ± SD (n = 3). Statistical differences are presented ** p < 0.01 compared with the vehicle control (without LPS) and ## p < 0.01 compared with the LPS-treated vehicle; (e) Representative images of BV2 microglia incubated for 20 h with vehicle (0.1% ethanol), LPS (10 ng/mL) plus vehicle (0.1% ethanol), LPS (10 ng/mL) + CR (0.05 mg/mL) or LPS (10 ng/mL) + ET (0.05 mg/mL). Images were acquired with Nikon Eclipse Ti-E inverted microscope (Scale bar, 50 µm).
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ijms-17-01420-f001: Effects of ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET) on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and activation. BV2 cells were pre-treated for 0.5 h with polymyxin B (PMB, 10 µg/mL), vehicle (0.1% ethanol), or the indicated concentration of extract, and then stimulated with LPS (10 or 100 ng/mL) for 20 h. (a,b) The nitrite production in supernatant was determined by the Griess reagent; (c,d) The cell viability was analyzed by MTT assay. Data are represented as the mean ± SD (n = 3). Statistical differences are presented ** p < 0.01 compared with the vehicle control (without LPS) and ## p < 0.01 compared with the LPS-treated vehicle; (e) Representative images of BV2 microglia incubated for 20 h with vehicle (0.1% ethanol), LPS (10 ng/mL) plus vehicle (0.1% ethanol), LPS (10 ng/mL) + CR (0.05 mg/mL) or LPS (10 ng/mL) + ET (0.05 mg/mL). Images were acquired with Nikon Eclipse Ti-E inverted microscope (Scale bar, 50 µm).
Mentions: To test whether CR and ET can function as inhibitors for NO release, BV2 cells were pre-treated with vehicle (0.1% ethanol), CR, or ET for 30 min followed by LPS (10 or 100 ng/mL) insult for a further 20 h. Polymyxin B (PMB, 10 µg/mL), a cyclic cationic polypeptide antibiotic, which binds to lipid A, served as a control LPS inhibitor. To set the optimal concentrations of CR and ET, we started with various concentrations of CR and ET ranging from 0.025–0.2 mg/mL with 1:2 serial dilutions. Our preliminary data showed that CR and ET at 0.025 mg/mL did not exert anti-NO activity significantly, while at 0.2 mg/mL caused significant cell death. As a result, 0.05 and 0.1 mg/mL of CR and ET were chosen for the experiments. Figure 1a,b shows that 10 and 100 ng/mL LPS plus vehicle stimulated NO production from basal levels (0.7–1.3 µM) to 25.0 ± 0.4 and 33.0 ± 0.8 µM, respectively. CR and ET (0.05–0.1 mg/mL) dose-dependently decreased 10 ng/mL LPS-induced NO production by 72%–93% and 43%–75%, respectively (p < 0.01). Slightly weaker inhibition (67%–93% for CR and 36%–57% for ET) was noted against 100 ng/mL LPS (p < 0.01). In comparison, PMB (10 µg/mL) almost completely inhibited 10 and 100 ng/mL LPS-mediated NO production.

View Article: PubMed Central - PubMed

ABSTRACT

Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1&beta;, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-&kappa;B signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1&beta; and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-&kappa;B phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-&kappa;B inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti-neuroinflammatory activities by inhibiting pro-inflammatory mediator expression and production, upregulating HO-1, GCLM and NQO1, blocking NF-&kappa;B and modulating JNK signaling pathways. They may offer therapeutic potential for suppressing overactivated microglia and alleviating neurodegeneration.

No MeSH data available.


Related in: MedlinePlus