International Journal of
Molecular Sciences Article
Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells Dong-Cheol Kim 1,† , Chi-Su Yoon 1,† , Tran Hong Quang 1,2,† , Wonmin Ko 1 , Jong-Su Kim 1 , Hyuncheol Oh 1 and Youn-Chul Kim 1, * 1
2
* †
Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan 570–749, Korea;
[email protected] (D.-C.K.);
[email protected] (C.-S.Y.);
[email protected] (T.H.Q.);
[email protected] (W.K.);
[email protected] (J.-S.K.);
[email protected] (H.O.) Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi 10000, Vietnam Correspondence:
[email protected]; Tel.: +82-63-850-6823; Fax: +82-63-852-8837 These authors contributed equally to this study.
Academic Editor: Chang Won Choi Received: 8 December 2015; Accepted: 5 February 2016; Published: 19 February 2016
Abstract: In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE2 in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE2 in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation. Keywords: Cudrania tricuspidata; cudraflavanone D; microglia; neuroinflammation; nuclear factor-κB; mitogen-activated protein kinase
1. Introduction Cudrania tricuspidata, a deciduous broadleaf thorny tree belonging to the Moraceae family, is spread throughout East Asia in Japan, Korea and China. According to Korean literature, this species has been used in oriental medicine for the treatment of poor health, impotency, and insomnia [1]. In addition, the root bark and cortex of this species have been used in traditional medicine for the therapy of inflammation and neuritis [2]. Previous phytochemical research has shown that C. tricuspidata contains several components such as xanthones [3], flavonoids [4], and glycoproteins [5]. In recent studies of the pharmacological effects of this plant, C. tricuspidata extracts have been shown to have various biological effects, including hepatoprotective [6], antioxidant [7], monoamine oxidase-A inhibitory [8], neuroprotective [9], anti-atherosclerotic, and anti-inflammatory activities [10]. Microglia have been traditionally defined as cerebral macrophages that may play important roles in neuroinflammation [11–13]. Moreover, microglia are a primary factor of the cerebral immune system [14]. When microglia are activated by a stimulus they produce neurotoxic mediators and Int. J. Mol. Sci. 2016, 17, 255; doi:10.3390/ijms17020255
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pro-inflammatory cytokines including prostaglandin E2 (PGE2 ), nitric oxide (NO), interleukin-6 (IL-6), Int. J. Mol. Sci. 2016, 17, 255 of 12 interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and superoxide anions [15,16]. In2addition, microglial activation hascytokines been researched for its etiological in neurodegenerative diseases and pro-inflammatory including prostaglandin E2 (PGErole 2), nitric oxide (NO), interleukin-6 (e.g., (IL-6), Alzheimer’s disease and ischemia etc.) [17,18]. Therefore, control of activated microglia would interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and superoxide anions [15,16]. In be an effective therapeutic approach for the range of neurodegenerative diseases. addition, microglial activation has been researched for its etiological role in neurodegenerative diseases factor-κB (e.g., Alzheimer’s disease andfactor ischemia [17,18]. of Therefore, controlNF-κB of activated Nuclear (NF-κB) is a crucial in theetc.) responses inflammation. is activated microglia would be (LPS) an effective therapeutic approach for the range of neurodegenerative diseases. by lipopolysaccharides through the phosphorylation of inhibitor of kappa B-α (IκB-α). As a result, Nuclear factor-κB (NF-κB) is a crucial factor in the responses of inflammation. NF-κB is the NF-κB translocates to the nucleus through separating from IκB-α [19,20]. Once NF-κB reaches activated by lipopolysaccharides (LPS) through the phosphorylation of inhibitor of kappa B-α nucleus, this molecule binds to specific sites on DNA and regulates the transcription of its target genes; (IκB-α). As a result, NF-κB translocates to the nucleus through separating from IκB-α [19,20]. Once this leads to the production of pro-inflammatory mediators and cytokines such as cyclooxygenase-2 NF-κB reaches the nucleus, this molecule binds to specific sites on DNA and regulates the (COX-2), inducible NO synthase (iNOS), PGE2 , NO, TNF-α, IL-6, and IL-1β [21,22]. transcription of its target genes; this leads to the production of pro-inflammatory mediators and Mitogen-activated protein kinases (MAPKs) consistNO of synthase threonine-/serine-specific proteinIL-6, kinases, cytokines such as cyclooxygenase-2 (COX-2), inducible (iNOS), PGE2, NO, TNF-α, including extracellular signal-regulated kinase (ERK) 1/2 (p44/p42), c-Jun N-terminal kinase (JNK) and IL-1β [21,22]. and p38. These factors haveprotein important roles in cellconsist differentiation, death, and proliferation [23] and are Mitogen-activated kinases (MAPKs) of threonine-/serine-specific protein kinases, including extracellular signal-regulated kinase (ERK) 1/2 (p44/p42), c-Jun N-terminal kinase (JNK) and stimulated in signal transduction cascades by the phosphorylation of tyrosine/threonine residues. p38. These factors have important roles in cell differentiation, death, and proliferation [23] and In this study, the anti-inflammatory activity of C. tricuspidata was investigated as partare of our signal transduction the phosphorylation of tyrosine/threonine widerstimulated efforts toindiscover medicinalcascades plants by which have anti-inflammatory effects. residues. We found that In this study, the anti-inflammatory of C. tricuspidata investigated as part of our seven prenylated flavonoids isolated fromactivity C. tricuspidata exertedwas anti-neuroinflammatory effects wider efforts to discover medicinal plants which have anti-inflammatory effects. We found that in LPS-induced microglial cells. Furthermore, the prenylated flavonoid cudraflavanone D (1) was seven prenylated flavonoids isolated from C. tricuspidata exerted anti-neuroinflammatory effects in selected to investigate the mechanism underlying these anti-neuroinflammatory activities, and the LPS-induced microglial cells. Furthermore, the prenylated flavonoid cudraflavanone D (1) was results suggested that it the targeted COX-2 and iNOS through theactivities, MAPK and andthe NF-κB selected to investigate mechanism underlying theseexpression anti-neuroinflammatory signaling pathways. results suggested that it targeted COX-2 and iNOS expression through the MAPK and NF-κB signaling pathways.
2. Results
2. Results
2.1. Structures of Compounds 1–7 and Cell Viability in BV2 Microglial Cells 2.1. Structures of Compounds 1–7 and Cell Viability in BV2 Microglial Cells
The structures of cudraflavanone D (1), cudraflavanone B (2), euchrestaflavanone C (3), The structures (4), of cudraflavanone D cudraflavone (1), cudraflavanone (2),kuwanon euchrestaflavanone C (3), (+)-dihydrokaempferol steppogenin (5), C (6), Band C (7) (Figure 1) were (+)-dihydrokaempferol (4), steppogenin (5), cudraflavone C (6), and kuwanon C (7) (Figure 1) were identified in a previous study [24]. Before investigating the anti-neuroinflammatory potential of these identifiedthe in cytotoxicity a previous study [24]. Before1–7 investigating the anti-neuroinflammatory of MTT compounds, of compounds in BV2 microglial cells was estimatedpotential by using these compounds, the cytotoxicity of compounds 1–7 in BV2 microglial cells was estimated by using assay. The individual compounds showed different cytotoxic effects and the non-toxic concentration MTT assay. The individual compounds showed different cytotoxic effects and the non-toxic range was determined for each compound (Figure S1). Non-toxic concentrations of compounds 1–7 concentration range was determined for each compound (Figure S1). Non-toxic concentrations of were compounds tested and 1–7 are were described testedbelow. and are described below. HO HO
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Figure 1. The structures of compounds 1–7.
Figure 1. The structures of compounds 1–7.
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2.2. Effects of Compounds 1–7 on NO Production in LPS-Stimulated BV2 Microglial Cells 2.2. Effects of Compounds 1–7 on NO Production in LPS-Stimulated BV2 Microglial Cells 2.2.To Effects of Compounds 1–7 on NO Production in LPS-Stimulated BV2 Microglial determine the anti-neuroinflammatory effects of compounds 1–7Cells on LPS-induced BV2 To determine the anti-neuroinflammatory effects of compounds 1–7 on LPS-induced BV2 microglial cells, the concentrations of the pro-inflammatory NO1–7 wereonassessed in the absence To determine the anti-neuroinflammatory effects of mediator compounds LPS-induced BV2 microglial cells, the concentrations of the pro-inflammatory mediator NO were assessed in the microglial the concentrations of the pro-inflammatory mediator NO were assessed in the and presencecells, of non-cytotoxic concentrations of each compound. BV2 microglial cells were pretreated absence and presence of non-cytotoxic concentrations of each compound. BV2 microglial cells were absence and presence of non-cytotoxic concentrations of eachwith compound. BV2 microglial cells were 2, with the indicated compound for 3 h, followed by activation LPS (1 µg/mL) for 24 h. In Figure pretreated with the indicated compound for 3 h, followed by activation with LPS (1 µg/mL) for 24 h. pretreated with the indicated compound for eight-fold 3 h, followed byinactivation with LPS (1 µg/mL) for 24 h. LPS treatment triggered an approximately rise the nitrite concentration of the culture In Figure 2, LPS treatment triggered an approximately eight-fold rise in the nitrite concentration of In Figure 2, LPS treatment triggered an approximately eight-fold rise in the nitrite concentration of media rathermedia than the untreated Compounds 1–3 and1–3 5 inhibited the production of NO the culture rather than the cells. untreated cells. Compounds and 5 inhibited the production of in culture media rather thanmanner, the untreated cells. Compounds 1–3 and 5 inhibited the production of a the concentration-dependent with IC values of 6.28 ˘ 0.31, 19.83 ˘ 0.99, 24.42 ˘ 1.22, 50 IC50 values of 6.28 ± 0.31, 19.83 ± 0.99, 24.42 ± 1.22, NO in a concentration-dependent manner, with NO43.55 in a concentration-dependent manner, with IC50 values 4, of 6, 6.28 ± 0.31, 19.83 ± 0.99, 24.42 ± 1.22, and ˘ 2.17, respectively. In contrast, compounds and 7 exhibited weak effects or and 43.55 ± 2.17, respectively. In contrast, compounds 4, 6, and 7 exhibited weak effects or no no and 43.55 ± 2.17, respectively. In contrast, compounds 4, 6,control and 7 [25]. exhibited weak effects or no inhibitory Following these findings, inhibitoryeffect effectatat80 80µM. µM. Butein Butein was was used used as as a a positive positive control [25]. Following these findings, inhibitory effect at 80 µM. Butein was used as a positive control [25]. Following these findings, cudraflavanone D (1) the strongest inhibitory effect and wasand selected subsequent investigation cudraflavanone D had (1) had the strongest inhibitory effect was for selected for subsequent cudraflavanone D (1) had the strongest inhibitory effect and was selected for subsequent of investigation the underlying mechanism. of the underlying mechanism. investigation of the underlying mechanism.
Nitrite (µM) Nitrite (µM)
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Figure 2. Theeffects effects ofcompounds compounds 1–7 1–7 on on nitrite nitrite production ininBV2 microglial cells stimulated with Figure productionin BV2 microglial cells stimulated with Figure2.2.The The effectsof of compounds 1–7 on nitrite production BV2 microglial cells stimulated with LPS. The cells were pre-treated for 3 h with the indicated concentrations of compounds 1–7 and then LPS. h with withthe theindicated indicatedconcentrations concentrations compounds LPS.The Thecells cellswere werepre-treated pre-treated for for 33 h of of compounds 1–71–7 andand thenthen stimulated for 24 h with LPS (1 µg/mL).The Theconcentrations concentrations of of nitrite nitrite were were determined determined as described stimulated for 24 h with LPS (1 µg/mL). as described stimulated for 24 h with LPS (1 µg/mL). The concentrations of nitrite were determined as described in in the Materials and Methods section. The data represent the mean values ± SD of three experiments. the andand Methods section. the mean meanvalues values± ˘ three experiments. in Materials the Materials Methods section.The Thedata datarepresent represent the SDSD of of three experiments. * p < 0.05, as compared with cells treated with LPS only. * p* < 0.05, as compared with cells treated with LPS only. p < 0.05, as compared with cells treated with LPS only.
2.3. Effects of Cudraflavanone D (1) on TNF-α, IL-1β, IL-12, and IL-6 mRNA Expression in LPS-Stimulated 2.3.Effects EffectsofofCudraflavanone Cudraflavanone D D (1) on TNFα, IL-1 β, IL-12, mRNA Expression in LPS-Stimulated 2.3. TNF-α, IL-1β, IL-12,and andIL-6 IL-6 mRNA Expression in LPS-Stimulated BV2 Microglial Cells BV2 BV2Microglial MicroglialCells Cells We investigated the inhibitory effects of cudraflavanone D (1) on the production of We effects of of cudraflavanone cudraflavanoneD D(1)(1)ononthetheproduction production We investigated investigated the the inhibitory inhibitory effects of of pro-inflammatory cytokines (TNF-α, IL-1β, IL-12, and IL-6) in BV2 microglial cells. Cells were pro-inflammatory cytokines cytokines (TNF-α, (TNF-α, IL-1β, Cells were pro-inflammatory IL-1β, IL-12, IL-12,and andIL-6) IL-6)ininBV2 BV2microglial microglialcells. cells. Cells were pre-treated with different doses of cudraflavanone D (1) induced by LPS for 12 h. As shown in pre-treatedwith with different different doses induced by by LPSLPS for for 12 h.12As in pre-treated doses of of cudraflavanone cudraflavanoneD D(1)(1) induced h. shown As shown Figure 3A–D, cudraflavanone D (1) decreased TNF-α, IL-1β, IL-12, and IL-6 production in a D D (1) (1) decreased TNF-α, IL-1β, IL-12, and and IL-6 IL-6 production in a in inFigure Figure3A–D, 3A–D,cudraflavanone cudraflavanone decreased TNF-α, IL-1β, IL-12, production dose-dependent manner. dose-dependent manner. a dose-dependent manner. B B
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Int. J. Mol. Sci. 2016, 17, 255 Int. J. Mol. Sci. 2016, 17, 255 Int. J. Mol. Sci. 2016, C 17, 25510
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Figure 3. The effects of cudraflavanone D (1) on TNF-α (A), IL-1β (B), IL-12 (C), and IL-6 (D) mRNA expression in BV2 microglial cells stimulated with LPS. Cells (B), were pre-treated for (D) 3 hmRNA with the Figure3.3.The The effects ofcudraflavanone cudraflavanone D IL-12 (C), and IL-6 Figure effects of D (1) (1) on onTNF-α TNF-α(A), (A),IL-1β IL-1β (B), IL-12 (C), and IL-6 (D) mRNA indicated concentrations of cudraflavanone D (1) and then stimulated for 12 h with LPS (1 µg/mL). expression in BV2 microglial cells stimulated with LPS. Cells were pre-treated for 3 h with the the expression in BV2 microglial cells stimulated with LPS. Cells were pre-treated for 3 h with The concentrations of TNF-α (A), IL-1β (B), D IL-12 (C), then and IL-6 (D) were determined as(1described in indicated concentrations of cudraflavanone (1) and stimulated for 12 h with LPS µg/mL). indicated concentrations of cudraflavanone D (1) and then stimulated for 12 h with LPS (1 µg/mL). The concentrations of TNF-α IL-1β (B), IL-12 and IL-6as(D) were determined as described in Materials and Methods. RNA (A), quantification was (C), performed described in Materials and Methods The concentrations of TNF-α (A), IL-1β (B), IL-12 (C), and IL-6 (D) were determined as described in Materials and Methods. RNA quantification was performed are as described in Materials and Methods and representative blots of three independent experiments shown. The data represent the mean Materials and Methods. RNA quantification was performed as described in Materials and Methods and representative blots of ± three independent experiments are the shown. The data with represent the mean values of three experiments SD. * p < 0.05, as compared with cells treated LPS only. and representative blots of three independent experiments are shown. The data represent the mean values of three experiments ± SD. * p < 0.05, as compared with the cells treated with LPS only. values of three experiments ˘ SD. * p < 0.05, as compared with the cells treated with LPS only.
2.4. Effects of Cudraflavanone D (1) on PGE2 Production and iNOS and COX-2 Protein Expression in 2.4. Effects of Cudraflavanone D (1) on PGE2 Production and iNOS and COX-2 Protein Expression in LPS-Stimulated BV2 Microglial 2.4. Effects of Cudraflavanone DCells (1) on PGE2 Production and iNOS and COX-2 Protein Expression in LPS-Stimulated BV2 Microglial Cells LPS-Stimulated BV2 Microglial Cells We investigated the inhibitory effect of cudraflavanone D (1) on LPS-stimulated PGE2 We investigated the inhibitory effect of cudraflavanone D (1) on LPS-stimulated PGE2 production, and on the iNOS and COX-2 protein expressionD (Figure 4). BV2 microglial cells were We investigated inhibitory effect of cudraflavanone (1) on LPS-stimulated PGE2 production, production, and on iNOS and COX-2 protein expression (Figure 4). BV2 microglial cells were challenged LPS (1 µg/mL) in the(Figure absence andmicroglial presencecells of were non-cytotoxic of and on iNOSwith and COX-2 protein expression 4). BV2 challengeddoses with LPS challenged with LPS (1 µg/mL) in the absence and presence of non-cytotoxic doses of cudraflavanone D (1) ranging from 1.25–10 µM. BV2 microglial cells pre-treated with (1cudraflavanone µg/mL) in the absence presence cudraflavanone D (1) ranging from D (1) and ranging fromof non-cytotoxic 1.25–10 µM. doses BV2 of microglial cells pre-treated with cudraflavanone Dmicroglial (1) for 24 cells h resulted in decreased iNOS expression (Figure 4B) and reduced PGE2 1.25–10 µM. BV2 pre-treated with cudraflavanone D (1) for 24 h resulted in decreased cudraflavanone D (1) for 24 h resulted in decreased iNOS expression (Figure 4B) and reduced PGE2 production derived from (Figure 4A). In the same conditions, cudraflavanone D (1) iNOS expression (Figure 4B)COX-2 and reduced derived from COX-2 (Figure 4A). 2 production production derived from COX-2 (FigurePGE 4A). In the same conditions, cudraflavanone D In (1) the suppressed COX-2 expression (Figure 4B). same conditions, cudraflavanone D (1)4B). suppressed COX-2 expression (Figure 4B). suppressed COX-2 expression (Figure
Figure 4.(A) (A)The Theeffects effects of cudraflavanone cudraflavanone D (1) on protein expression ofofiNOS and COX-2 (B)(B) in in Figure protein expression iNOS COX-2 Figure 4. 4. (A) The effects ofofcudraflavanone DD(1)(1) onon protein expression of iNOS andand COX-2 (B) in BV2 BV2 microglial cells stimulated with LPS. Cells were pre-treated for 3 h with the indicated BV2 microglial cells stimulated with LPS. Cells were pre-treated h with concentrations the indicated microglial cells stimulated with LPS. Cells were pre-treated for 3 h withfor the3indicated concentrations of cudraflavanone D (1) and then stimulated for 24 h with LPS (1 µg/mL). The concentrations of cudraflavanone (1) and then for 24 h with LPS µg/mL). The of cudraflavanone D (1) and thenDstimulated for stimulated 24 h with LPS (1 µg/mL). The(1concentrations concentrations of iNOS and COX-2 (B) were determined as described in Materials and Methods. concentrations of iNOS COX-2 (B) were describedand in Materials Methods. of iNOS and COX-2 (B)and were determined as determined described inasMaterials Methods. and Western blot Western blot analyses were performed as described in Materials and Methods and representative Western blot analyses were performed as described in Materials and Methods and representative analyses were performed as described in Materials and Methods and representative blots of blots of three independent experiments are shown. Band intensity was quantified by densitometry blots three independent experiments are shown. intensity was quantified by densitometry three of independent experiments are shown. Band Band intensity was quantified by densitometry and and normalized to β-actin; the values are presented below each band. Relative data represent the and normalized to β-actin; the values are presented below each band. Relative data represent the normalized to β-actin; the values are presented below each band. Relative data represent the mean mean values of three experiments ± SD. * p < 0.05, as compared to the cells treated with LPS only. mean of experiments three experiments * p < as 0.05, as compared the treated cells treated withonly. LPS only. valuesvalues of three ˘ SD. ±* SD. p < 0.05, compared to thetocells with LPS
2.5. Effects of Cudraflavanone D (1) on IκB-α Levels, NF-κB Nuclear Translocation, and NF-κB DNA 2.5. Effects of D (1) on IκBα Levels, Levels,Cells NF-κBNuclear NuclearTranslocation, Translocation,and andNF-κB NF-κBDNA DNABinding 2.5. EffectsActivity of Cudraflavanone Cudraflavanone IκB-α NF-κB Binding in LPS-Stimulated BV2 Microglial Activity Activity in LPS-Stimulated BV2 Microglial Cells Cells Binding in LPS-Stimulated BV2 Microglial NF-κB activation stimulates expression of the iNOS and COX-2 proteins. NF-κB is inactive NF-κB activation stimulates expression ofof thethe iNOS and COX-2 proteins. NF-κB is inactive when NF-κB activation stimulates expression iNOS and COX-2 proteins. NF-κB is inactive when bound to its inhibitor, IκB, in the cytoplasm. In response to external signals, NF-κB is bound to itsfrom inhibitor, IκB, subsequently in the In response tonucleus external signals, NF-κB is further separated when bound to its IκB,cytoplasm. in thetranslocates cytoplasm. response to[26,27]. external signals, NF-κB is separated IκBinhibitor, and toInthe Therefore, from IκB and translocates to the nucleus [26,27]. Therefore, investigation separated fromsubsequently IκB conducted and subsequently translocates to the nucleus [26,27]. Therefore, further investigation was to determine whether cudraflavanone D (1) further suppressed IκB-α was conducted to determine whether cudraflavanone D (1) suppressed IκB-α degradation and degradation and thus suppressing the cudraflavanone NF-κB (p50 and p65) translocation to the investigation was phosphorylation, conducted to determine whether D (1) suppressed IκB-α phosphorylation, suppressing the NF-κB (p50 andthe p65)NF-κB translocation to p65) the nucleus. In Figure 5A, degradation and thus phosphorylation, thus suppressing (p50 and translocation to the
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nucleus. Indegraded Figure 5A, IκB-α was degraded after exposure of BV2 cells to LPS for with 1 h. IκB-α was after exposure of BV2 microglial cells to LPS for 1microglial h. However, pretreatment However, pretreatment withD1.25–10 cudraflavanone D (1)this forLPS-stimulated 3 h markedly degradation suppressed this 1.25–10 µM cudraflavanone (1) for 3µM h markedly suppressed and LPS-stimulated phosphorylation of IκB-α in thus a concentration-dependent phosphorylationdegradation of IκB-α in aand concentration-dependent manner, suppressing p50 and p65manner, nuclear thus suppressing p505B,C). and Furthermore, p65 nuclear fluorescence translocationmicroscopy (Figure 5B,C). Furthermore, fluorescence translocation (Figure identified that cudraflavanone D microscopy identified cudraflavanone D (1)-treated cells reduced NF-κB nuclear translocation (1)-treated cells reducedthat NF-κB nuclear translocation as compared with untreated microglia (Figure 5D). as with untreated microglia (Figure 5D). activity We further experimented aboutBV2 NF-κB DNA Wecompared further experimented about NF-κB DNA binding in nuclear extracts from microglial binding activitybyinLPS nuclear extracts from BV2induced microglial cells activated 10-fold by LPSrise forin1the h. DNA This cells activated for 1 h. This processing an approximately processing induced an approximately 10-fold rise the DNA binding of NF-κB, which was binding activity of NF-κB, which was inhibited by in cudraflavanone D (1)activity in a concentration-dependent inhibited by cudraflavanone D (1) in a concentration-dependent manner (Figure 5E). manner (Figure 5E).
Figure Figure 5. 5. The Theeffects effectsof ofcudraflavanone cudraflavanoneD D(1) (1)on on IκB-α IκB-α phosphorylation phosphorylationand anddegradation degradation(A), (A),NF-κB NF-κB activation activation(B,C), (B,C),NF-κB NF-κBlocalization localization(D), (D),and andNF-κB NF-κBDNA DNAbinding bindingactivity activity(E) (E)ininBV2 BV2microglial microglialcells. cells. Cells Cells were were pre-treated pre-treated for for 33 hh with with the the indicated indicated concentrations concentrations of of cudraflavanone cudraflavanoneD D (1), (1), and and then then stimulated stimulatedfor for11hhwith with LPS LPS (1 (1 µg/mL). µg/mL).Western Westernblot blotanalyses analysesof ofIκB-α IκB-αand andphosphorylated phosphorylated(p)-IκB-α (p)-IκB-α in in the the cytoplasm cytoplasm (A), (A), and and NF-κB NF-κB in in the the cytoplasm cytoplasm (B) (B) and and nucleus nucleus (C), (C), and and immunofluorescent immunofluorescent analysis performed as as described describedin inMaterials Materialsand andMethods. Methods.Band Bandintensity intensity was quantified analysis (E), (E), were performed was quantified by by densitometry normalized to β-actin PCNA, the values are presented each densitometry andand normalized to β-actin andand PCNA, and and the values are presented belowbelow each band. band. Relative data represent thevalues mean values three experiments * p as < 0.05, as compared to Relative data represent the mean of threeofexperiments ˘ SD. * ±p SD. < 0.05, compared to the cells the cells with treated LPS only. treated LPSwith only.
2.6. 2.6.Effects EffectsofofCudraflavanone CudraflavanoneD D(1) (1)on onMAPK MAPK Phosphorylation Phosphorylation in in LPS-Stimulated LPS-Stimulated BV2 BV2 Microglial Microglial Cells Cells To the MAPK MAPK pathway–mediated pathway–mediatedsuppression suppressionofofinflammation inflammationbybycudraflavanone cudraflavanone To investigate investigate the D D (1), we assessed its effect on the LPS-stimulated phosphorylation of p38, JNK and ERK in BV2 (1), we assessed its effect on the LPS-stimulated phosphorylation of p38, JNK and ERK in BV2 microglial microglial cells.6,Inphosphorylation Figure 6, phosphorylation p38, JNK and ERK after was being raisedinduced after being cells. In Figure of p38, JNKofand ERK was raised withinduced LPS for with LPS for 1 pre-treatment h. However, pre-treatment with 2.5–10 µM cudraflavanone D (1) forsuppressed 3 h markedly 1 h. However, with 2.5–10 µM cudraflavanone D (1) for 3 h markedly the suppressed the LPS-stimulated phosphorylation of JNK and p38 in a concentration-dependent LPS-stimulated phosphorylation of JNK and p38 in a concentration-dependent manner (Figure 6B,C); manner (Figure 6B,C); however, ERK was not changed. of Protein expressions of were p38, however, ERK phosphorylation was phosphorylation not changed. Protein expressions p38, JNK and ERK JNK and ERK were unconverted by LPS. These data represented that cudraflavanone D (1)-mediated inflammatory responses by inhibiting the p38 and JNK MAPK signaling pathways.
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unconverted by LPS. These data represented that cudraflavanone D (1)-mediated inflammatory responses inhibiting the p38 and JNK MAPK signaling pathways. Int. J. Mol. Sci.by 2016, 17, 255 6 of 12
Figure 6. The effects of cudraflavanone D (1) on ERK, JNK, and p38 MAPK protein expression and Figure 6. The effects of cudraflavanone D (1) on ERK, JNK, and p38 MAPK protein expression and phosphorylation. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone phosphorylation. Cells were pre-treated for 3 h with the indicated concentrations of cudraflavanone D D (1) and stimulated for 1 h with LPS (1 µg/mL) (A–C). The levels of (A) phosphorylated-ERK (1) and stimulated for 1 h with LPS (1 µg/mL) (A–C). The levels of (A) phosphorylated-ERK (p-ERK), (p-ERK), (B) phosphorylated-JNK (p-JNK), and (C) phosphorylated-p38 MAPK (p-p38 MAPK) were (B) phosphorylated-JNK (p-JNK), and (C) phosphorylated-p38 MAPK (p-p38 MAPK) were determined determined Western blotting. Representative blotsindependent from three independent experiments are by Western by blotting. Representative blots from three experiments are shown. Band shown. Band intensity was quantified by densitometry and normalized to β-actin; the values are intensity was quantified by densitometry and normalized to β-actin; the values are presented below presented eachdata band. Relativethe data represent meanexperiments. values of three experiments. each band.below Relative represent mean values the of three
3. Discussion 3. Discussion A literature survey revealed that several studies had reported the anti-inflammatory effects of A literature survey revealed that several studies had reported the anti-inflammatory effects C. tricuspidata. An ethyl acetate fraction of this plant was investigated to suppress the production of of C. tricuspidata. An ethyl acetate fraction of this plant was investigated to suppress the production of NO NO in LPS-induced RAW264.7 macrophages [28]. Similar effects were also observed using a in LPS-induced RAW264.7 macrophages [28]. Similar effects were also observed using a chloroform chloroform fraction of this plant [29]. With respect to the components of the plant responsible for fraction of this plant [29]. With respect to the components of the plant responsible for its its effects on inflammation, a glycoprotein isolated from C. tricuspidata modulated activities of effects on inflammation, a glycoprotein isolated from C. tricuspidata modulated activities of inflammatory signals in LPS-stimulated RAW264.7 cells [5]. Additionally, cudratricusxanthone A, inflammatory signals in LPS-stimulated RAW264.7 cells [5]. Additionally, cudratricusxanthone a xanthone isolated from C. tricuspidata, inhibited inflammatory responses in LPS-stimulated A, a xanthone isolated from C. tricuspidata, inhibited inflammatory responses in LPS-stimulated RAW264.7 cells via effects on the expression of the heme oxygenase 1 enzyme [30]. Some flavonoids RAW264.7 cells via effects on the expression of the heme oxygenase 1 enzyme [30]. Some isolated from this plant were also reported to have anti-inflammatory effects. For example, flavonoids isolated from this plant were also reported to have anti-inflammatory effects. Parks et al. reported that three flavonoids (cudraflavone B, cudraflavanone D and 2’,5,7-trihidroxyFor example, Parks et al. reported that three flavonoids (cudraflavone B, cudraflavanone D 4’,5’-(2,2-dimethylchromeno)-8-(3-hydroxy-3-methylbutyl)flavanone) produced anti-inflammatory and 2’,5,7-trihidroxy-4’,5’-(2,2-dimethylchromeno)-8-(3-hydroxy-3-methylbutyl)flavanone) produced effects by suppressing the protein expression of iNOS in LPS-stimulated RAW264.7 cells [10]. In anti-inflammatory effects by suppressing the protein expression of iNOS in LPS-stimulated RAW264.7 addition, the anti-neuroinflammatory effect of methylalpinumisoflavone isolated from C. tricuspidata cells [10]. In addition, the anti-neuroinflammatory effect of methylalpinumisoflavone isolated from has been demonstrated to be mediated through the MAPK and NF-κB signaling pathways in C. tricuspidata has been demonstrated to be mediated through the MAPK and NF-κB signaling LPS-induced microglial cells [31]. Cudraflavanone D (1) is a prenylated flavonoid from C. Tricuspidata pathways in LPS-induced microglial cells [31]. Cudraflavanone D (1) is a prenylated flavonoid that has been reported to have cytotoxic effects [32] and neuraminidase inhibitory activity [33]. from C. Tricuspidata that has been reported to have cytotoxic effects [32] and neuraminidase In addition, an anti-inflammatory effect mediated via suppression of iNOS protein expression in inhibitory activity [33]. In addition, an anti-inflammatory effect mediated via suppression of LPS-stimulated RAW264.7 cells has been reported [10]. However, the anti-neuroinflammatory effects of cudraflavanone D (1) and other related flavonoids on microglia, and the molecular mechanism involved, are unknown. In this study, anti-neuroinflammatory effects of the prenylated flavonoids from C. tricuspidata on NO expression were identified in LPS-induced BV2 microglial cells. Therefore, further investigation was conducted to explore these effects in more detail, as well as the possible mechanisms involved.
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iNOS protein expression in LPS-stimulated RAW264.7 cells has been reported [10]. However, the anti-neuroinflammatory effects of cudraflavanone D (1) and other related flavonoids on microglia, and the molecular mechanism involved, are unknown. In this study, anti-neuroinflammatory effects of the prenylated flavonoids from C. tricuspidata on NO expression were identified in LPS-induced BV2 microglial cells. Therefore, further investigation was conducted to explore these effects in more detail, as well as the possible mechanisms involved. Inflammation is an integrated response of an organism to various pathological changes, and it involves rapid upregulation and activation of many genes. This complicated process is modulated by pro-inflammatory mediators and cytokines, including NO, IL-1β, PGE2, IL-6, IL-12 and TNF-α, in immune cells. The suppression of various mediators is critical during the treatment of inflammation. Recent researches have reported that inflammation is generated by iNOS and NO [34]. NO is synthesized by NOS, and inflammation is correlated with the level of iNOS [35]. Therefore, the suppression of iNOS and NO overproduction can be used to determine anti-inflammatory effects. We investigated whether prenylated flavonoids suppressed the production of NO during LPS-induced neuroinflammation in BV2 microglial cells. Some compounds had inhibitory effects on NO (Figure 2). PGE2 , one of the inflammatory mediators, is generated at the inflammatory position by COX-2, an enzyme isoform that is stimulated in response to a variety of stimulants and that exacerbates inflammatory diseases [36,37]. Therefore, PGE2 and COX-2 are suggested as key enzymes for anti-inflammatory treatments. We investigated whether cudraflavanone D (1), a prenylated flavonoid component of C. tricuspidata, inhibited the expression of pro-inflammatory mediators, cytokines, and enzymes in LPS-stimulated inflammatory conditions in BV2 microglial cells. LPS stimulated COX-2 and iNOS expression in BV2 microglial cells, and these processes were suppressed by pre-treatment with cudraflavanone D (1) (Figure 5). Cudraflavanone D (1) also suppressed levels of the COX-2 product PGE2 , and of the mRNA expression of the pro-inflammatory cytokines IL-12, IL-1β, IL-6, and TNF-α (Figures 2 and 3). LPS triggers its inflammatory effects by activating NF-κB, a transcription molecule that controls the expression of several genes such as IL-1β, COX-2, iNOS, and TNF-α. NF-κB heterodimers such as those composed of p65 and p50 are normally combined with IκB-α in the cytoplasm while NF-κB is regulated by various proteins such as TRAF6, TAK1, TRIF and CD14 which have a crucial role in translocation to the DNA binding site and phosphorylation of IκB [38]. Moreover, translocated NF-κB regulates the expression of pro-inflammatory proteins including TNF-α, COX-2, iNOS, IL-6, IL-12, and IL-1β, which are associated with neuroinflammation. [39]. However, in the existence of a pro-inflammatory stimulus, IκB-α is degraded and phosphorylated and NF-κB translocates to the nucleus, where it combines with target sites and induces the pro-inflammatory mediators [40]. We investigated the effects of cudraflavanone D (1) on IκB-α which degraded and phosphorylated, and on translocation of NF-κB heterodimers. After treatment with cudraflavanone D (1), LPS-stimulated IκB-α degradation and NF-κB activation were suppressed in BV2 microglial cells (Figure 5A–C). Furthermore, cudraflavanone D (1) decreased the NF-κB DNA binding activity (Figure 5E). Many researchers have reported that MAPK intracellular signaling pathways are related to the regulation of inflammatory mediators [41,42]. We therefore investigated whether the anti-inflammatory effects of cudraflavanone D (1) in LPS-induced microglia involved the altered expression of MAPKs. Our results demonstrated that cudraflavanone D (1) was a strong inhibitor of the MAPK activation stimulated by LPS stimulation of BV2 microglial cells (Figure 6), indicating that these anti-inflammatory effects involved suppression of the MAPK signaling pathway. In summary, prenylated flavonoid derivatives isolated from C. tricuspidata were identified to have inhibitory effects against NO expression in LPS-induced BV2 microglial cells. In the further study of the anti-neuroinflammatory effects of these metabolites, cudraflavanone D (1) was shown to suppress expression of LPS-stimulated COX-2 and iNOS at the protein level. In addition, 1 reduced the expression of pro-inflammatory cytokines. For the evaluation of the molecular mechanisms under the anti-inflammatory effects of 1, the compound was found to suppress the NF-κB and MPAK
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signaling pathways in BV2 microglial cells stimulated with LPS. This regulation of the production of inflammatory molecules by cudraflavanone D (1) may have therapeutic potential against the range of neuroinflammatory and neurodegenerative diseases. 4. Experimental Section 4.1. Plant Materials The root barks of Cudrania tricuspidata were purchased in May 2014 at Daerim Korean crude drug store, Kumsan, Chungnam Province, Korea, and identified by Dr. Kyu-Kwan Jang, Botanical Garden, Wonkwang University. A voucher specimen (No. WP-2014-12) was deposited at the Herbarium of the College of Pharmacy, Wonkwang University (Iksan, Korea). Prenylated flavonoids (Figure 1) were isolated from the methanol extract of C. tricuspidata (Moraceae) by various chromatographic methods, and the structures were determined mainly by analysis of MS and NMR data [24,43]. 4.2. Chemicals and Reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies, including mouse/goat/rabbit anti-COX-2, iNOS, β-actin, IκB-α, phosphorylated (p)-IκB-α, p50, p65, proliferating cell nuclear antigen (PCNA), and secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), while p-ERK, ERK, p-JNK, JNK, p-p38, and p38 antibodies were obtained from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA) [44]. 4.3. Cell Culture and Viability Assay BV2 microglial cells were received from Prof. Hyun Park at Wonkwang University (Iksan, Korea). BV2 microglial cells were maintained at 5 ˆ 106 cells/dish in 100-mm dishes in DMEM supplemented with 10% heat-inactivated FBS, penicillin G (100 units/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM), and incubated at 37 ˝ C in a humidified atmosphere containing 5% CO2 and 95% air. For the determination of cell viability, 2 ˆ 104 cells/well in 96-well plates were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at a final concentration of 0.5 mg/mL for 3 h, and the formazan formed was dissolved in acidic 2-propanol. Optical density was measured at 590 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The optical density of the formazan formed in control (untreated) cells was considered to represent 100% cell viability [43,44]. 4.4. Quantitative Reverse-Transcription Polymerase Chain Reaction (qPCR) Total RNA was isolated from the cells using Trizol (Invitrogen), in accordance with the manufacturer’s recommendations, and quantified spectrophotometrically at 260 nm. Total RNA (1 µg) was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). The cDNA was then amplified using the SYBR Premix Ex Taq kit (TaKaRa Bio, Shiga, Japan) in a StepOnePlus Real-Time PCR system (Applied Biosystems). Briefly, each 20 µL reaction volume contained 10 µL SYBR Green PCR Master Mix, 0.8 µM of each primer, and diethyl pyrocarbonate-treated water. The primer sequences were designed using PrimerQuest (Integrated DNA Technologies, Cambridge, MA, USA). The primer sequences were: 5’-CCA GAC CCT CAC ACT CAC AA-3’ (forward) and 5’-ACA AGG TAC AAC CCA TCG GC-3’ (reverse) for TNF-α; 5’-AAT TGG TCA TAG CCC GCA CT-3’ (forward) and 5’-AAG CAA TGT GCT GGT GCT TC-3’ (reverse) for IL-1β; 5’-ACT TCA CAA GTC GGA GGC TT-3’ (forward) and 5’-TGC AAG TGC ATC ATC GTT GT-3’ (reverse) primers for IL-6; and 5’-AGT GAC ATG TGG AAT GGC GT-3’ (forward) and 5’-CAG TTC AAT GGG CAG GGT CT-3’ (reverse) for IL-12. The qPCR conditions were established by following the manufacturer’s instructions. The data were analyzed using StepOne software (Applied Biosystems)
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and the cycle numbers at the linear amplification threshold (Ct ) values for the endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the target gene were recorded [43]. 4.5. DNA Binding Activity of NF-κB Microglia were pretreated for 3 h with the indicated concentrations of cudraflavanone D (1) and then stimulated for 1 h with LPS (1 µg/mL). The DNA-binding activity of NF-κB in nuclear extracts was measured using the TransAM kit (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s instructions [45]. 4.6. Preparation of Cytosolic and Nuclear Fractions BV2 microglial cells were homogenized in PER-Mammalian Protein Extraction Buffer (1:20, w:v) (Pierce Biotechnology, Rockford, IL, USA) containing freshly added protease inhibitor cocktail I (EMD Biosciences, San Diego, CA, USA) and 1 mM phenylmethylsulfonylfluoride (PMSF). The cytosolic fraction of the cells was prepared by centrifugation at 16,000ˆ g for 5 min at 4 ˝ C. The nuclear and cytoplasmic cell extracts were prepared with NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL, USA), respectively [44]. 4.7. Nitrite Determination The nitrite concentration in the medium, an indicator of NO production, was measured using the Griess reaction. Each supernatant (100 µL) was mixed with an equal volume of Griess reagent (Sigma; Solution A: 222488; Solution B: S438081), and the absorbance of the mixture at 525 nm was determined using a microplate reader [44]. 4.8. Western Blot Analysis BV2 microglial cells were harvested and pelleted by centrifugation at 16,000 rpm for 15 min. The cells were then washed with phosphate-buffered saline (PBS) and lysed with 20 mM Tris–HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM PMSF, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). The protein concentration was determined using a Lowry protein assay kit (P5626; Sigma). An equal amount of protein from each sample was resolved using 7.5% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and sequentially incubated with the appropriate primary antibody (Santa Cruz Biotechnology, CA, USA) and horseradish peroxidase-conjugated secondary antibody, followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA) [43]. 4.9. NF-κB Localization and Immunofluorescence To study NF-κB localization, cells were grown on Lab-Tek II chamber slides and treated with 10 µM cudraflavanone D (1) for 0–60 min. Cells were then fixed in formalin and permeabilized with cold acetone. The cells were probed with p50 antibody followed by fluorescein isothiocyanate (FITC)-labeled secondary antibody (Alexa Fluor 488, Invitrogen). To visualize the nuclei, cells were then treated with 1 µg/mL 4’,6-diamidino-2-phenylindole (DAPI) for 30 min, washed with PBS for 5 min, and treated with 50 µL of VectaShield (Vector Laboratories, Burlingame, CA, USA). Stained cells were visualized using a Zeiss fluorescence microscope and photographed (Provis AX70, Olympus Optical Co., Tokyo, Japan) [46]. 4.10. Statistical Analysis The data are expressed as the mean ˘ standard deviation (SD) of at least three independent experiments. To compare three or more groups, one-way analysis of the variance was used, followed
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by Tukey’s multiple comparison tests. The statistical analysis was performed with GraphPad Prism software, version 3.03 (GraphPad Software Inc., San Diego, CA, USA) [46]. 5. Conclusions Treatment of BV2 microglial cells with cudraflavanone D (1) displayed a markedly dose-dependent suppression of lipopolysaccharide-induced NO production. In addition, cudraflavanone D (1) decreased the levels of pro-inflammatory cytokines. Cudraflavanone D (1) suppressed the expression of p38, and the JNK, MAPK, and NF-κB pathways in lipopolysaccharide-induced BV2 microglial cells. This study indicated that cudraflavanone D (1) represents a potential drug candidate for the treatment of neuroinflammation. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/ 17/2/255/s1. Acknowledgments: This research was supported in part by the Korea Research Foundation of Korea Grant funded by the Korean Government (NRF-2014R1A2A1A11050034). Author Contributions: Jong-Su Kim, Hyuncheol Oh, and Youn-Chul Kim conceived the study and designed the experiments; Dong-Cheol Kim, Chi-Su Yoon, Tran Hong Quang, and Wonmin Ko performed the experiments; Jong-Su Kim, Hyuncheol Oh, Youn-Chul analyzed the data with suggestion; Dong-Cheol Kim and Chi-Su Yoon wrote the manuscript. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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