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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 signaling inhibitors on the anti-IL-6 effects of CR and ET. (a) BV2 cells were pretreated for 30 min with inhibitor (SP600125, 10 μM; Bay 11-7082, 10 μM) followed by CR or ET (0.1 mg/mL) for 30 min before LPS (10 ng/mL) insult for 16 h. (a) Supernatant was recovered for IL-6 analysis; (b) 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 LPS-treated vehicle and ## p < 0.01 compared with relative no inhibitor group.
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ijms-17-01420-f007: Effects of signaling inhibitors on the anti-IL-6 effects of CR and ET. (a) BV2 cells were pretreated for 30 min with inhibitor (SP600125, 10 μM; Bay 11-7082, 10 μM) followed by CR or ET (0.1 mg/mL) for 30 min before LPS (10 ng/mL) insult for 16 h. (a) Supernatant was recovered for IL-6 analysis; (b) 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 LPS-treated vehicle and ## p < 0.01 compared with relative no inhibitor group.

Mentions: To determine whether modulation of JNK or NF-κB activation relates to the anti-inflammatory activity of CR or ET, BV2 cells were pretreated with inhibitor of each pathway, SP600125, a JNK inhibitor, and Bay 11-7082, an inhibitor of IκB-α phosphorylation, for 30 min and then incubated with 0.1 mg/mL CR or ET for 30 min followed by LPS (10 ng/mL) for 16 h before analyzing IL-6 production and cell viability. Figure 7a shows that SP600125 and Bay 11-7082 (10 µM) significantly reduced LPS-mediated IL-6 production by about 39% and 67%, respectively, indicating that activation of the JNK and NF-κB pathways is involved in IL-6 production. Furthermore, the inhibitory effect of CR on IL-6 production was significantly enhanced by Bay 11-7082, but not by SP600125, as compared with no inhibitor control (p < 0.01). This result indicates that the attenuation of NF-κB activation by CR, but not the modulation of JNK signaling, participates in its anti-inflammatory action. In contrast, the inhibitory effect of ET on IL-6 production was augmented by the addition of both SP600125 and Bay 11-7082, indicating suppressed NF-κB activation and JNK phosphorylation many contribute to ET’s anti-inflammatory effects. Figure 7b shows that SP600125 and Bay 11-7082 (10 μM) alleviated LPS-mediated cytotoxicity in all test groups significantly (p < 0.01). This result indicates the involvements of JNK and NF-κB signaling in the LPS-mediated cytotoxicity in BV2 cells.


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 signaling inhibitors on the anti-IL-6 effects of CR and ET. (a) BV2 cells were pretreated for 30 min with inhibitor (SP600125, 10 μM; Bay 11-7082, 10 μM) followed by CR or ET (0.1 mg/mL) for 30 min before LPS (10 ng/mL) insult for 16 h. (a) Supernatant was recovered for IL-6 analysis; (b) 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 LPS-treated vehicle and ## p < 0.01 compared with relative no inhibitor group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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ijms-17-01420-f007: Effects of signaling inhibitors on the anti-IL-6 effects of CR and ET. (a) BV2 cells were pretreated for 30 min with inhibitor (SP600125, 10 μM; Bay 11-7082, 10 μM) followed by CR or ET (0.1 mg/mL) for 30 min before LPS (10 ng/mL) insult for 16 h. (a) Supernatant was recovered for IL-6 analysis; (b) 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 LPS-treated vehicle and ## p < 0.01 compared with relative no inhibitor group.
Mentions: To determine whether modulation of JNK or NF-κB activation relates to the anti-inflammatory activity of CR or ET, BV2 cells were pretreated with inhibitor of each pathway, SP600125, a JNK inhibitor, and Bay 11-7082, an inhibitor of IκB-α phosphorylation, for 30 min and then incubated with 0.1 mg/mL CR or ET for 30 min followed by LPS (10 ng/mL) for 16 h before analyzing IL-6 production and cell viability. Figure 7a shows that SP600125 and Bay 11-7082 (10 µM) significantly reduced LPS-mediated IL-6 production by about 39% and 67%, respectively, indicating that activation of the JNK and NF-κB pathways is involved in IL-6 production. Furthermore, the inhibitory effect of CR on IL-6 production was significantly enhanced by Bay 11-7082, but not by SP600125, as compared with no inhibitor control (p < 0.01). This result indicates that the attenuation of NF-κB activation by CR, but not the modulation of JNK signaling, participates in its anti-inflammatory action. In contrast, the inhibitory effect of ET on IL-6 production was augmented by the addition of both SP600125 and Bay 11-7082, indicating suppressed NF-κB activation and JNK phosphorylation many contribute to ET’s anti-inflammatory effects. Figure 7b shows that SP600125 and Bay 11-7082 (10 μM) alleviated LPS-mediated cytotoxicity in all test groups significantly (p < 0.01). This result indicates the involvements of JNK and NF-κB signaling in the LPS-mediated cytotoxicity in BV2 cells.

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