International Journal of
Molecular Sciences Article
Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia Hsiou-Yu Ding 1,† , Pei-Shan Wu 2,† and Ming-Jiuan Wu 2, * 1 2
* †
Department of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan; [email protected] Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan; [email protected] Correspondence: [email protected]; Tel.: +886-6-266-4911 (ext. 2520) These authors contributed equally to this work.
Academic Editors: Paula Andrade and Patrícia Valentão Received: 29 June 2016; Accepted: 22 August 2016; Published: 27 August 2016
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. Keywords: Cleome rutidosperma; Euphorbia thymifolia; microglial cells; HO-1; JNK; NF-κB
Int. J. Mol. Sci. 2016, 17, 1420; doi:10.3390/ijms17091420
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 1420
2 of 15
1. Introduction Inflammation is a complex response that presents as series of vascular and cellular reactions triggered by injury or infection. Resident macrophages belong to the innate immune system responsible for the first line of defense against injury and infection. Microglia, the resident macrophage cells in the central nervous system (CNS), effectively phagocyte plaques, damaged or unnecessary neurons and synapses, and infectious agents to maintain neuronal homeostasis [1,2]. In response to pathophysiological brain insults, microglia become activated as characterized by shape changes and production of immune modulators in order to regulate tissue repair and recovery [3]. Sustained activation of microglial cells cause overproduction of various pro-inflammatory cytokines and mediators, which could lead to neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) [1]. NO (nitric oxide) is a very potent activator of Keap1-Nrf2 (NF-E2-related factor 2), which induces the expression of Phase II detoxification enzymes to adapt to oxidative stress conditions [4]. The Phase II enzymes include heme oxygenase (HO)-1, glutamate cysteine ligase (GCL), NAD(P)H quinone dehydrogenase 1 (NQO1) and other antioxidant enzymes [5]. The HOs are the rate-limiting enzymes in the degradation of excess heme and generation of biliverdin, ferrous ion, and carbon monoxide (CO) [6]. HO-1 is normally expressed at low levels but is induced in response to a variety of stimuli to protect cells [7]. A substantial body of evidence demonstrates that the induction of HO-1 is a vital step in the cellular adaptation to pathological stress [8]. Many inducers of HO-1 have been reported to have anti-inflammatory properties [8–12], possibly due to the antioxidant functions of enzymatic products. Mitogen-activated protein kinases (MAPK) are protein kinases involved in relaying a diverse array of stimuli from the cell membrane to the nucleus by means of a cascade of phosphorylation events [13]. The extracellular signal-regulated kinases (ERKs) are widely expressed and involved in the regulation of proliferation, differentiation and cell cycle progression [14]. Phospho-c-Jun N-terminal kinases (JNKs) are activated primarily by cytokines and exposure to environmental stress [15]. The p38 MAPK is activated in immune cells by inflammatory cytokines and is critical for normal immune and inflammatory response [14]. The nuclear factor (NF)-κB is a critical component in regulation of inflammatory responses. NF-κB can form different dimers, but a p50/p65 heterodimer is the most common one [16]. The central mechanism for NF-κB activation is phosphorylation of inhibitors of NF-κB (IκBs) by IκB kinase (IκKs) and leads to the proteasomal degradation of IκBs and allowing NF-κB to enter the nucleus. Besides the phosphorylation and degradation of IκBs, post-translational phosphorylation of NF-κB is also important for its activity [17]. Special attention has been paid to phosphorylation of p65 at Ser536 , which is involved in regulation of transcriptional activity, nuclear import and protein stability [18]. There is a growing interest in searching for natural products with anti-inflammatory activities [19]. Cleome rutidosperma DC. is an herb native to West Africa that has spread and naturalized in various parts of the world, and is used in Indian medicine to treat paralysis, epilepsy, convulsions, spasm, pain and skin disease [20], with its antiplasmodial, antimicrobial, diuretic, antioxidant, analgesic, anti-pyretic, anti-arthritic and wound healing activities having been reported in the literature [20–25]. Euphorbia thymifolia L. has many synonyms such as Anisophyllum thymifolium (L.) Haw., Aplarina microphylla (Lam.) Raf., Chamaesyce microphylla (Lam.) Soják, C. rubrosperma (Lotsy) Millsp., C. thymifolia f. suffrutescens (Boiss.) Hurus., E. afzelii N.E.Br., E. botryoides Noronha, E. rubicunda Blume, E. rubrosperma Lotsy, E. thymifolia f. laxifoliata Chodat and Hassl., and so on [26]. It is a heat-clearing remedy in the Chinese medicine and commonly used for the treatment of acute enteritis, diarrhea, atopic dermatitis and inflammatory diseases. Previous studies reveal that it has various kinds of bioactivities, including antioxidant [27,28], anti-viral [27,29,30], anti-microbial [31], and anti-earthworm [32]. However, the molecular pharmacological mechanism underlying their anti-inflammatory activities has never been reported. Immortalized microglia BV2 stimulated with lipopolysaccharide (LPS) is a suitable model for evaluating neuroinflammatory responses [33–36]. Because C. rutidosperma and E. thymifolia share
Int. J. Mol. Sci. 2016, 17, 1420
3 of 15
similar are used in treating fever or inflammatory diseases in folk medicine,3the Int.bioactivities J. Mol. Sci. 2016, and 17, 1420 of 15aim of this study is to investigate their anti-neuroinflammatory effects and the underlying molecular of this in study to investigate their anti-neuroinflammatory effects and the underlying molecular mechanism BV2iscells. mechanism in BV2 cells.
2. Results 2. Results
2.1. Ethanol Extracts of C. rutidosperma (CR) and E. thymifolia (ET) Inhibited Nitric Oxide (NO) Production 2.1. Ethanol of C. rutidosperma (CR) and BV2 E. thymifolia and Activation inExtracts Lipopolysaccharide (LPS)-Treated Cells (ET) Inhibited Nitric Oxide (NO) Production and Activation in Lipopolysaccharide (LPS)-Treated BV2 Cells
To test whether CR and ET can function as inhibitors for NO release, BV2 cells were pre-treated To test whether CR and ETor canET function inhibitors for NO pre-treated with vehicle (0.1% ethanol), CR, for 30asmin followed by release, LPS (10BV2 or cells 100 were ng/mL) insult for with vehicle (0.1% ethanol), CR, or ET for 30 min followed by LPS (10 or 100 ng/mL) insult for a a further 20 h. Polymyxin B (PMB, 10 µg/mL), a cyclic cationic polypeptide antibiotic, which further 20 h. Polymyxin B (PMB, 10 µg/mL), a cyclic cationic polypeptide antibiotic, which binds to binds to lipid A, served as a control LPS inhibitor. To set the optimal concentrations of CR and lipid A, served as a control LPS inhibitor. To set the optimal concentrations of CR and ET, we started ET, we started withconcentrations various concentrations of CR and ET ranging from 0.025–0.2 1:2 serial with various of CR and ET ranging from 0.025–0.2 mg/mL withmg/mL 1:2 serialwith dilutions. dilutions. Our preliminary data showed that CR and ET at 0.025 mg/mL did not exert anti-NO activity 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 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 and 100 100ng/mL ng/mL LPS plus vehicle and ET were chosen for the experiments. Figure1a,b 1a,bshows shows that that 10 and LPS plus vehicle stimulated NO production from basal levels (0.7–1.3µM) µM)toto25.0 25.0± ± 0.4 µM,µM, respectively. stimulated NO production from basal levels (0.7–1.3 0.4and and33.0 33.0± 0.8 ± 0.8 respectively. CRET and(0.05–0.1 ET (0.05–0.1 mg/mL) dose-dependently decreased ng/mL LPS-induced NO production by CR and mg/mL) dose-dependently decreased1010 ng/mL LPS-induced NO production 72%–93% and 43%–75%, respectively (p < 0.01). Slightly weaker inhibition (67%–93% for CR and by 72%–93% and 43%–75%, respectively (p < 0.01). Slightly weaker inhibition (67%–93% for CR and 36%–57% forwas ET) was noted against ng/mLLPS LPS(p(p
Molecular Sciences Article
Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia Hsiou-Yu Ding 1,† , Pei-Shan Wu 2,† and Ming-Jiuan Wu 2, * 1 2
* †
Department of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan; [email protected] Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan; [email protected] Correspondence: [email protected]; Tel.: +886-6-266-4911 (ext. 2520) These authors contributed equally to this work.
Academic Editors: Paula Andrade and Patrícia Valentão Received: 29 June 2016; Accepted: 22 August 2016; Published: 27 August 2016
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. Keywords: Cleome rutidosperma; Euphorbia thymifolia; microglial cells; HO-1; JNK; NF-κB
Int. J. Mol. Sci. 2016, 17, 1420; doi:10.3390/ijms17091420
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 1420
2 of 15
1. Introduction Inflammation is a complex response that presents as series of vascular and cellular reactions triggered by injury or infection. Resident macrophages belong to the innate immune system responsible for the first line of defense against injury and infection. Microglia, the resident macrophage cells in the central nervous system (CNS), effectively phagocyte plaques, damaged or unnecessary neurons and synapses, and infectious agents to maintain neuronal homeostasis [1,2]. In response to pathophysiological brain insults, microglia become activated as characterized by shape changes and production of immune modulators in order to regulate tissue repair and recovery [3]. Sustained activation of microglial cells cause overproduction of various pro-inflammatory cytokines and mediators, which could lead to neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) [1]. NO (nitric oxide) is a very potent activator of Keap1-Nrf2 (NF-E2-related factor 2), which induces the expression of Phase II detoxification enzymes to adapt to oxidative stress conditions [4]. The Phase II enzymes include heme oxygenase (HO)-1, glutamate cysteine ligase (GCL), NAD(P)H quinone dehydrogenase 1 (NQO1) and other antioxidant enzymes [5]. The HOs are the rate-limiting enzymes in the degradation of excess heme and generation of biliverdin, ferrous ion, and carbon monoxide (CO) [6]. HO-1 is normally expressed at low levels but is induced in response to a variety of stimuli to protect cells [7]. A substantial body of evidence demonstrates that the induction of HO-1 is a vital step in the cellular adaptation to pathological stress [8]. Many inducers of HO-1 have been reported to have anti-inflammatory properties [8–12], possibly due to the antioxidant functions of enzymatic products. Mitogen-activated protein kinases (MAPK) are protein kinases involved in relaying a diverse array of stimuli from the cell membrane to the nucleus by means of a cascade of phosphorylation events [13]. The extracellular signal-regulated kinases (ERKs) are widely expressed and involved in the regulation of proliferation, differentiation and cell cycle progression [14]. Phospho-c-Jun N-terminal kinases (JNKs) are activated primarily by cytokines and exposure to environmental stress [15]. The p38 MAPK is activated in immune cells by inflammatory cytokines and is critical for normal immune and inflammatory response [14]. The nuclear factor (NF)-κB is a critical component in regulation of inflammatory responses. NF-κB can form different dimers, but a p50/p65 heterodimer is the most common one [16]. The central mechanism for NF-κB activation is phosphorylation of inhibitors of NF-κB (IκBs) by IκB kinase (IκKs) and leads to the proteasomal degradation of IκBs and allowing NF-κB to enter the nucleus. Besides the phosphorylation and degradation of IκBs, post-translational phosphorylation of NF-κB is also important for its activity [17]. Special attention has been paid to phosphorylation of p65 at Ser536 , which is involved in regulation of transcriptional activity, nuclear import and protein stability [18]. There is a growing interest in searching for natural products with anti-inflammatory activities [19]. Cleome rutidosperma DC. is an herb native to West Africa that has spread and naturalized in various parts of the world, and is used in Indian medicine to treat paralysis, epilepsy, convulsions, spasm, pain and skin disease [20], with its antiplasmodial, antimicrobial, diuretic, antioxidant, analgesic, anti-pyretic, anti-arthritic and wound healing activities having been reported in the literature [20–25]. Euphorbia thymifolia L. has many synonyms such as Anisophyllum thymifolium (L.) Haw., Aplarina microphylla (Lam.) Raf., Chamaesyce microphylla (Lam.) Soják, C. rubrosperma (Lotsy) Millsp., C. thymifolia f. suffrutescens (Boiss.) Hurus., E. afzelii N.E.Br., E. botryoides Noronha, E. rubicunda Blume, E. rubrosperma Lotsy, E. thymifolia f. laxifoliata Chodat and Hassl., and so on [26]. It is a heat-clearing remedy in the Chinese medicine and commonly used for the treatment of acute enteritis, diarrhea, atopic dermatitis and inflammatory diseases. Previous studies reveal that it has various kinds of bioactivities, including antioxidant [27,28], anti-viral [27,29,30], anti-microbial [31], and anti-earthworm [32]. However, the molecular pharmacological mechanism underlying their anti-inflammatory activities has never been reported. Immortalized microglia BV2 stimulated with lipopolysaccharide (LPS) is a suitable model for evaluating neuroinflammatory responses [33–36]. Because C. rutidosperma and E. thymifolia share
Int. J. Mol. Sci. 2016, 17, 1420
3 of 15
similar are used in treating fever or inflammatory diseases in folk medicine,3the Int.bioactivities J. Mol. Sci. 2016, and 17, 1420 of 15aim of this study is to investigate their anti-neuroinflammatory effects and the underlying molecular of this in study to investigate their anti-neuroinflammatory effects and the underlying molecular mechanism BV2iscells. mechanism in BV2 cells.
2. Results 2. Results
2.1. Ethanol Extracts of C. rutidosperma (CR) and E. thymifolia (ET) Inhibited Nitric Oxide (NO) Production 2.1. Ethanol of C. rutidosperma (CR) and BV2 E. thymifolia and Activation inExtracts Lipopolysaccharide (LPS)-Treated Cells (ET) Inhibited Nitric Oxide (NO) Production and Activation in Lipopolysaccharide (LPS)-Treated BV2 Cells
To test whether CR and ET can function as inhibitors for NO release, BV2 cells were pre-treated To test whether CR and ETor canET function inhibitors for NO pre-treated with vehicle (0.1% ethanol), CR, for 30asmin followed by release, LPS (10BV2 or cells 100 were ng/mL) insult for with vehicle (0.1% ethanol), CR, or ET for 30 min followed by LPS (10 or 100 ng/mL) insult for a a further 20 h. Polymyxin B (PMB, 10 µg/mL), a cyclic cationic polypeptide antibiotic, which further 20 h. Polymyxin B (PMB, 10 µg/mL), a cyclic cationic polypeptide antibiotic, which binds to binds to lipid A, served as a control LPS inhibitor. To set the optimal concentrations of CR and lipid A, served as a control LPS inhibitor. To set the optimal concentrations of CR and ET, we started ET, we started withconcentrations various concentrations of CR and ET ranging from 0.025–0.2 1:2 serial with various of CR and ET ranging from 0.025–0.2 mg/mL withmg/mL 1:2 serialwith dilutions. dilutions. Our preliminary data showed that CR and ET at 0.025 mg/mL did not exert anti-NO activity 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 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 and 100 100ng/mL ng/mL LPS plus vehicle and ET were chosen for the experiments. Figure1a,b 1a,bshows shows that that 10 and LPS plus vehicle stimulated NO production from basal levels (0.7–1.3µM) µM)toto25.0 25.0± ± 0.4 µM,µM, respectively. stimulated NO production from basal levels (0.7–1.3 0.4and and33.0 33.0± 0.8 ± 0.8 respectively. CRET and(0.05–0.1 ET (0.05–0.1 mg/mL) dose-dependently decreased ng/mL LPS-induced NO production by CR and mg/mL) dose-dependently decreased1010 ng/mL LPS-induced NO production 72%–93% and 43%–75%, respectively (p < 0.01). Slightly weaker inhibition (67%–93% for CR and by 72%–93% and 43%–75%, respectively (p < 0.01). Slightly weaker inhibition (67%–93% for CR and 36%–57% forwas ET) was noted against ng/mLLPS LPS(p(p
