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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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Induction of Nrf2 target gene expression by ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET). (a) BV2 cells were cultured with indicated reagent for 16 h and level of HO-1 expression was determined by Western blotting; (b) Band intensities were quantified by ImageJ software and indicated as relative fold of HO-1/α-tubulin; (c,e,f) HO-1, GCLM and NQO-1 mRNA expression levels were determined at 4 h after LPS treatment by RT-Q-PCR. 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; (d) BV2 cells were pre-treated with Znpp (5 μM) for 30 min, followed by PMB (10 μg/mL), CR or ET (0.05 and 0.1 mg/mL) treatment for further 30 min, prior to LPS (10 ng/mL) challenge for 20 h. NO production was determined by the Griess reagent. 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 Znpp-untreated group.
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ijms-17-01420-f005: Induction of Nrf2 target gene expression by ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET). (a) BV2 cells were cultured with indicated reagent for 16 h and level of HO-1 expression was determined by Western blotting; (b) Band intensities were quantified by ImageJ software and indicated as relative fold of HO-1/α-tubulin; (c,e,f) HO-1, GCLM and NQO-1 mRNA expression levels were determined at 4 h after LPS treatment by RT-Q-PCR. 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; (d) BV2 cells were pre-treated with Znpp (5 μM) for 30 min, followed by PMB (10 μg/mL), CR or ET (0.05 and 0.1 mg/mL) treatment for further 30 min, prior to LPS (10 ng/mL) challenge for 20 h. NO production was determined by the Griess reagent. 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 Znpp-untreated group.

Mentions: Heme oxygenase 1 (HO-1), displaying established antioxidant and anti-inflammatory properties, is a known Nrf2 target gene [41]. Accumulating evidence indicates that HO-1 upregulation works against inflammatory responses, especially iNOS expression, in macrophages and microglia [9,11,42,43]. Figure 5a,b shows that LPS (10 ng/mL) slightly induced HO-1 protein expression by 1.9-fold, and co-treatment with 0.1 mg/mL CR and ET significantly enhanced HO-1 upregulation. However, a lower concentration of CR or ET (0.05 mg/mL) did not affect HO-1 expression.


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
Induction of Nrf2 target gene expression by ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET). (a) BV2 cells were cultured with indicated reagent for 16 h and level of HO-1 expression was determined by Western blotting; (b) Band intensities were quantified by ImageJ software and indicated as relative fold of HO-1/α-tubulin; (c,e,f) HO-1, GCLM and NQO-1 mRNA expression levels were determined at 4 h after LPS treatment by RT-Q-PCR. 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; (d) BV2 cells were pre-treated with Znpp (5 μM) for 30 min, followed by PMB (10 μg/mL), CR or ET (0.05 and 0.1 mg/mL) treatment for further 30 min, prior to LPS (10 ng/mL) challenge for 20 h. NO production was determined by the Griess reagent. 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 Znpp-untreated group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037699&req=5

ijms-17-01420-f005: Induction of Nrf2 target gene expression by ethanol extracts of C. rutidosperma (CR) and E. thymifolia (ET). (a) BV2 cells were cultured with indicated reagent for 16 h and level of HO-1 expression was determined by Western blotting; (b) Band intensities were quantified by ImageJ software and indicated as relative fold of HO-1/α-tubulin; (c,e,f) HO-1, GCLM and NQO-1 mRNA expression levels were determined at 4 h after LPS treatment by RT-Q-PCR. 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; (d) BV2 cells were pre-treated with Znpp (5 μM) for 30 min, followed by PMB (10 μg/mL), CR or ET (0.05 and 0.1 mg/mL) treatment for further 30 min, prior to LPS (10 ng/mL) challenge for 20 h. NO production was determined by the Griess reagent. 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 Znpp-untreated group.
Mentions: Heme oxygenase 1 (HO-1), displaying established antioxidant and anti-inflammatory properties, is a known Nrf2 target gene [41]. Accumulating evidence indicates that HO-1 upregulation works against inflammatory responses, especially iNOS expression, in macrophages and microglia [9,11,42,43]. Figure 5a,b shows that LPS (10 ng/mL) slightly induced HO-1 protein expression by 1.9-fold, and co-treatment with 0.1 mg/mL CR and ET significantly enhanced HO-1 upregulation. However, a lower concentration of CR or ET (0.05 mg/mL) did not affect HO-1 expression.

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