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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

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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 and E. thymifolia on the mRNA expression of iNOS and COX-2 in LPS-treated BV2 cells. BV2 cells were cultured with indicated reagent in six-well plates for 4 h followed by RNA extraction and RT-Q-PCR for measuring mRNA levels of iNOS (a) and COX-2 (b), as described in Materials and Methods. 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 < 0.05; ## p < 0.01 compared with the LPS-treated vehicle.
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ijms-17-01420-f003: Effects of ethanol extracts of C. rutidosperma and E. thymifolia on the mRNA expression of iNOS and COX-2 in LPS-treated BV2 cells. BV2 cells were cultured with indicated reagent in six-well plates for 4 h followed by RNA extraction and RT-Q-PCR for measuring mRNA levels of iNOS (a) and COX-2 (b), as described in Materials and Methods. 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 < 0.05; ## p < 0.01 compared with the LPS-treated vehicle.

Mentions: To investigate how CR and ET affect mRNA levels of iNOS and COX-2 in LPS-treated BV2 cells, RT-Q-PCR were employed. Transcriptome analysis shows that most of inflammatory genes were overexpressed 4 h after LPS stimulation in BV2 cells [33]. Similarly, we found that treatment of BV2 cells with LPS (10 ng/mL) for 4 h significantly upregulated the iNOS transcript level by 322-fold (Figure 3a). CR and ET dose-dependently inhibited iNOS mRNA expression. The iNOS mRNA levels correlated well with protein levels, indicating that CR and ET inhibited LPS-mediated nitric oxide production via repression of iNOS expression, predominantly at the transcription stage 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 ethanol extracts of C. rutidosperma and E. thymifolia on the mRNA expression of iNOS and COX-2 in LPS-treated BV2 cells. BV2 cells were cultured with indicated reagent in six-well plates for 4 h followed by RNA extraction and RT-Q-PCR for measuring mRNA levels of iNOS (a) and COX-2 (b), as described in Materials and Methods. 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 < 0.05; ## p < 0.01 compared with the LPS-treated vehicle.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037699&req=5

ijms-17-01420-f003: Effects of ethanol extracts of C. rutidosperma and E. thymifolia on the mRNA expression of iNOS and COX-2 in LPS-treated BV2 cells. BV2 cells were cultured with indicated reagent in six-well plates for 4 h followed by RNA extraction and RT-Q-PCR for measuring mRNA levels of iNOS (a) and COX-2 (b), as described in Materials and Methods. 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 < 0.05; ## p < 0.01 compared with the LPS-treated vehicle.
Mentions: To investigate how CR and ET affect mRNA levels of iNOS and COX-2 in LPS-treated BV2 cells, RT-Q-PCR were employed. Transcriptome analysis shows that most of inflammatory genes were overexpressed 4 h after LPS stimulation in BV2 cells [33]. Similarly, we found that treatment of BV2 cells with LPS (10 ng/mL) for 4 h significantly upregulated the iNOS transcript level by 322-fold (Figure 3a). CR and ET dose-dependently inhibited iNOS mRNA expression. The iNOS mRNA levels correlated well with protein levels, indicating that CR and ET inhibited LPS-mediated nitric oxide production via repression of iNOS expression, predominantly at the transcription stage 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