Boehmeria nivea attenuates LPS-induced ...

http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2013; 51(9): 1131–1136 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.781196

ORIGINAL ARTICLE

Boehmeria nivea attenuates LPS-induced inflammatory markers by inhibiting p38 and JNK phosphorylations in RAW264.7 macrophages Mi Jeong Sung, Munkhtugs Davaatseren, Sung Hee Kim, Min Jung Kim, and Jin-Taek Hwang

Abstract

Keywords

Context: Boehmeria nivea (Linn.) Gaudich (Urticaceae), a natural herb, has a long history of treating several diseases including wound healing. However, the anti-inflammatory effect of B. nivea has not been investigated. Objective: We investigated whether the 70% ethanol extract of B. nivea (Ebn) can exert antiinflammatory activity. Several phenolic compounds of extracts were determined to provide further information on the correlation between anti-inflammatory effects and phenolic compounds. Materials and methods: We prepared a 70% ethanol extract of B. nivea leaves and evaluated its anti-inflammatory activity (200, 400, 800, 1200 mg/mL) by measuring the secretions of nitric oxide (NO), tumor necrosis factor alpha (TNF-a) and interleukin 6 (IL-6), which were stimulated by lipopolysaccharide (LPS) in RAW264.7 macrophages. The total phenolic compounds were determined by the Folin–Ciocalteu method and major compounds were determined by HPLC. Results: Ebn was able to abolish the LPS-induced secretions of NO, TNF-a and IL-6. It also decreased the protein levels (IC50 ¼ 186 mg/mL) of LPS-induced inducible nitric oxide synthase (iNOS). The LPS stimulated p38, JNK and ERK phosphorylations significantly more than the controls. Surprisingly, although Ebn reduced p38 and JNK phosphorylations, it did not influence ERK phosphorylation. We found that Ebn revealed several major compounds such as chlorogenic acid (1.96 mg/100 g), rutin (46.48 mg/100 g), luteolin-7-glucoside (11.29 mg/100 g), naringin (1.13 mg/100 g), hesperidin (23.69 mg/100 g) and tangeretin (1.59 mg/100 g). Discussion and conclusion: Boehmeria nivea exerts an anti-inflammatory effect on macrophages by inhibiting p38 and JNK, suggesting that it may be used as a functional ingredient against inflammation.

Anti-inflammation, Boehmeria nivea (L.) Gaudich, lipopolysaccharide, mitogen-activated protein kinase, RAW 264.7 macrophage

Introduction Inflammation is a multi-complex biological response to stimuli such as pathogenic infections, and presents a major obstacle in maintaining a high quality of life globally. Lipopolysaccharide (LPS) is a component of the bacteria cell wall that is widely used as a stimulus for activating macrophage cells (Arenzana-Seisdedos & Virelizier, 1983). Pro-inflammatory cytokines such as nitric oxide (NO), tumor necrosis factor alpha (TNF-a, and interleukin 6 (IL-6) are produced by LPS in macrophages, and trigger the pathogenesis of several diseases, including rheumatoid arthritis (O’Shea et al., 2002). It has been suggested that cellular signaling molecules are important mediators for the production of pro-inflammatory cytokine responses to LPS (Zhao et al., 2005). Mitogen-activated protein kinases (MAPKs) such as ERK, JNK and p38 are signaling molecules that act as considerable mediators in the production of pro-inflammatory cytokines. Specifically, MAPKs regulate the LPS-induced

Correspondence: Jin-Taek Hwang, Korea Food Research Institute, 516 Baekhyundong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-746, Republic of Korea. E-mail: [email protected]

History Received 11 November 2012 Revised 7 January 2013 Accepted 24 February 2013 Published online 10 June 2013

production of NO, TNF-a, IL-6 and iNOS expression in macrophages (Ajizian et al., 1999; Zhao et al., 2005). In addition, it is well established that MAPKs are also involved in the regulation of the nuclear factor kappa b (NF-kB), which is a transcription factor for increasing the expression of inflammatory genes (Dong et al., 1993). Recently, several functional ingredients have been shown to combat inflammatory diseases and influence signaling molecules such as MAPKs. For example, medicinal plants such as Sanguisorba officinalis L. (Rosaceae), Eriobotrya japonica (Thunb.) Lindl., and Lilium lancifolium Thunb. (Liliaceae) exert anti-inflammatory effects via the inhibition of JNK, p38 and ERK (Kwon et al., 2010; Uto et al., 2010; Yu et al., 2011). Boehmeria nivea (Linn.) Gaudich (Urticaceae) is a plant that is widely grown in Asian countries such as China, Korea, the Philippines and Thailand. It has traditionally been used to treat several diseases, including wound healing. Although B. nivea has been shown to have antifungal and antioxidant properties (Lin et al., 1998; Xu et al., 2011), its other biological functions with respect to disease control are poorly understood. Thus, we first evaluated the effects of B. nivea on LPS-induced secretions of nitrate, TNF-a and IL-6 in

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Korea Food Research Institute, Seongnam, Republic of Korea


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RAW264.7 macrophages. Moreover, phosphorylation changes due to B. nivea were also investigated in signals of the MAPK family (i.e., p38, JNK and ERK), which were also measured in LPS-stimulated RAW264.7 macrophages. Our results demonstrated that the 70% ethanol extracts of B. nivea (Ebn) reduce LPS-induced secretions of inflammatory markers such as nitrate, TNF-a and IL-6 via the inhibition of p38 and JNK.

in 24-well plates and were transferred to the serum-free medium for 24 h. Cells were pretreated with Ebn for 1 h and then exposed to 1 mg/mL LPS in the serum-free condition. After 24 h, the medium was collected and NO production was measured using the Griess reagent. The production of TNF-a and IL-6 was analyzed from the medium using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Materials and methods

Western blotting

Materials

Western blotting was performed as described previously (Yang et al., 2012). Briefly, after harvesting cells with the lysis buffer [50 mM Tris HCl, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM sodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 1 mM NaF and 0.2% protease inhibitor cocktail; pH 7.2], equal amounts of proteins were separated using 10% SDSPAGE and were transferred onto a nitrocellulose membrane. Protein phosphorylation or expression was detected using specific antibodies.

RAW264.7 macrophage cells were purchased from the American Type Culture Collection (Manassas, VA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from WelGene Inc. (Daegu, Republic of Korea). Anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38, anti-ERK, anti-JNK, anti-iNOS and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LPS was purchased from Sigma (St. Louis, MO). Authenticated B. nivea was provided by public officer in the Seocheon County Office (Seocheon, Republic of Korea), where a voucher specimen was deposited. Extraction of B. nivea and HPLC analysis B. nivea leaves were ground in a blender, and the powders were extracted with 70% ethanol by shaking for 24 h at 25 C. The precipitates were centrifuged at 8000g for 30 min (Beckman Coulter Inc., Brea, CA). The supernatants were then lyophilized using a freeze-dryer (Ilshin, Dongduchun, Republic of Korea). Chromatographic analyses were performed with (JASCO, Tokyo, Japan) a high-performance liquid chromatography (HPLC) system equipped with a PU-980 pump and a Jasco AS-2057 automatic sampler with a 20 mL loop. Detection was performed with a Jasco model UV-975 UV detector. Chromatographic separation of the compounds was achieved with a YMC ODS-AM (5 mm, 250 4.60 mm) column (Kyoto, Japan), operating at 35 C. The eluent was a gradient of 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B) at a flow rate of 1 mL min1, with a linear gradient as follows: 0 min, 12% B; 18 min, 22% B; 28 min, 28% B and 35 min, 38% B. A UV detector at 260 nm was used for the quantification based on the internal standard method. Determination of total phenolic contents The phenolic contents in the extracts were determined by the Folin–Ciocalteu method, using gallic acid as the standard (Yang et al., 2012). Briefly, 1 mL of the sample was added to 0.1 mL of 2 N Folin–Ciocalteu reagent. This mixture was allowed to stand for 5 min before the addition of 0.4 mL of 10% (w/w) sodium carbonate (Na2CO3). After incubation for 1 h in a dark room at 22–24 C, the absorbance was measured at 760 nm in a UV spectrophotometer (JASCO). The results were expressed as mg gallic acid equivalents (GAE) g1.

Cytotoxicity test Cells (4 104/well) were seeded in 96-well plates for 24 h and then starved with DMEM containing 1% FBS overnight before treatment. Cells were treated with extract for 24 h. Cell viability determined by using Cell proliferation reagent WST1 (Roche Applied Science, Indianapolis, IN) by the procedure of the manufacturer. Statistical analyses Statistical analyses were conducted using SPSS 9.0 (SPSS Inc., Chicago, IL). Statistical differences were evaluated by one-way analysis of variance (ANOVA) followed by the Bonferroni test. All data are expressed as mean standard deviation (SD) values. The significance was set to p50.05.

Results The phenolic composition of B. nivea As it is well known that polyphenols derived from natural herbs exhibit powerful beneficial health properties as an antiinflammatory effect (Chandrasekara & Shahidi, 2011; de la Rosa et al., 2011), we first investigated the phenolic compound content of Ebn. As shown in Figure 1 and Table 1, total phenolic contents are 3640 mg/100 g. Rutin (46.48 mg/100 g) (peak 2) was the predominant polyphenol in Ebn. Several phenolic compounds, such as chlorogenic acid (1.96 mg/100 g) (peak 1), luteolin-7-glucoside (11.29 mg/100 g) (peak 3), naringin (1.13 mg/100 g) (peak 4), hesperidin (23.69 mg/100 g) (peak 5) and tangeretin (1.59 mg/100 g) (peak 6), were also found in Ebn.

Measurement of NO, TNF-a and IL-6 production

Ebn inhibits NO production and iNOS expression in LPS-induced RAW264.7 macrophage cells

NO assay was performed as described previously with minor modifications (Sung et al., 2009). Briefly, cells were cultured

Nitric oxide (NO) acts as an important mediator in the inflammatory response that is produced by iNOS (Kanwar

Anti-inflammatory effect of B. nivea

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Figure 1. HPLC analysis of several phenolic compounds. The peaks shown in the HPLC chromatograms correspond to the following compounds: (1) chlorogenic acid, (2) rutin, (3) luteolin-7-glucoside, (4) naringin, (5) hesperidin, and (6) tangertin. All experiments were repeated at least three times.

Table 1. Several major compounds content, expressed as mg/100 g of Ebn.

Ebn

Total phenolic content

Chlorogenic acid

Rutin

Luteolin-7-glucoside

Naringin

Hesperidin

Tangeretin

3640 0.19

1.96 0.21

46.48 8.64

11.29 0.85

1.13 0.14

23.69 0.68

1.59 0.46

All experiments were repeated at least three times.

et al., 2009). Thus, we first investigated the effect of Ebn on NO production and iNOS expression in LPS-induced RAW264.7 macrophage cells. As shown in Figure 2 (A and B), 1 mg/mL of LPS significantly increased NO production and iNOS expression in RAW264.7 macrophage cells. Ebn inhibited NO production and iNOS expression in a dosedependent manner under LPS-treated conditions. On the other hand, cytotoxic effects of Ebn did not appear until the concentration of 1200 mg/mL (Figure 2C). These results demonstrate that Ebn has inhibitory activity on LPS-induced NO production and iNOS expression in RAW264.7 macrophage cells. Effect of Ebn on TNF-a and IL-6 production in LPS-induced RAW264.7 macrophage cells As it is well known that TNF-a and IL-6 are produced by activated macrophages (O’Shea et al., 2002), we next examined whether Ebn influences LPS-induced TNF-a and IL-6 production. As shown in Figure 3 (A and B), Ebn decreased the production of TNF-a and IL-6 in LPS-induced RAW264.7 macrophage cells. JNK and P38 phosphorylations were inhibited by Ebn in LPS-induced RAW264.7 macrophage cells In order to determine the precise mechanisms operating in the Ebn treatments, we measured the phosphorylations of several MAPK proteins such as p38, JNK and ERK using western blot analysis. A number of studies have proposed that p38, JNK and ERK signaling pathways are involved in LPSinduced inflammatory cytokine productions (Kwon et al., 2010; Uto et al., 2010; Yu et al., 2011). As shown in Figure 4 ,

1 mg/mL LPS significantly increased the phosphorylation of p38, JNK and ERK. Pretreatment of Ebn significantly reduced the phosphorylation of JNK and p38 in LPS-induced RAW264.7 macrophage cells. However, ERK phosphorylation was not inhibited by Ebn treatment in LPS-induced RAW264.7 macrophage cells. These results suggest that Ebn inhibits LPS-induced NO, TNF-a and IL-6 production via the inhibition of JNK and p38 phosphorylations in RAW264.7 macrophage cells.

Discussion Boehmeria nivea, a perennial herbaceous plant, is grown as a fiber crop that consists mainly of cellulose. It has traditionally been used to make cloth, and in Asian countries it has also been used for a long time as a functional material against inflammation, and bacterial and fungal infections. As B. nivea has a long history of use, it is considered relatively safe when ingested. A recent study on the safety of B. nivea reported that the plant showed no embryotoxicity, and produced no fetal external or skeletal malformations. In the present study, we similarly showed that an ethanol extract of B. nivea (Ebn) did not induce cytotoxicity in a cell culture system at concentrations of 200, 400, 800 and 1200 mg/ml (Figure 2C). We accordingly speculate that B. nivea extract may be safe to use as a functional ingredient. In this study, we found that Ebn contains several polyphenolic compounds such as chlorogenic acid, rutin, luteolin-7-glucoside, naringin, hesperidin and tangeretin. It is well established that the biological activities of functional foods are based on their polyphenolic composition (Chandrasekara & Shahidi, 2011; de la Rosa et al., 2011). Of all the polyphenolic compounds detected in the Ebn, rutin


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Figure 2. Inhibitory effect of Ebn on NO production and iNOS expression in LPS-induced RAW264.7 macrophage cells. Cells were pretreated with Ebn in a dose-dependent manner for 1 h, and they were then exposed to 1 mg/mL LPS in serum-free conditions for 24 h. NO production was measured using Griess reagent (A). Cells were pretreated with Ebn in a dose-dependent manner for 1 h and then exposed to 1 mg/mL LPS in serum-free conditions for 6 h. iNOS expression was analyzed by western blotting (B). The cytotoxicity was measured by WST-1 assay as described in ‘‘Material and methods’’ (C).

was the most abundant. Previous studies have suggested that rutin has an inhibitory effect on LPS-induced pro-inflammatory cytokines due to its inhibitory action on NO and TNF-a production (Chen et al., 2001; Guruvayoorappan & Kuttan, 2007). Similarly, another study proposed that several flavonoids, including rutin, could inhibit LPS-induced NO production and iNOS gene expression (Chen et al., 2001). In addition to rutin, chlorogenic acid, luteolin-7-glucoside, naringin, hesperidin and tangeretin have also been proposed to be effective for suppressing inflammatory responses (Choi et al., 2007; Jung et al., 2012; Kanno et al., 2006; Kim et al., 2010; Sakata et al., 2003). Therefore, we speculated that Ebn exerts an anti-inflammatory action, at least in part, because of a

combination of polyphenolic compounds, even though rutin is the most abundant. We next showed that Ebn significantly inhibited LPSinduced production of inflammatory markers such as NO, TNF-a and IL-6. Moreover, we also found that JNK and p38 inhibition was involved in the anti-inflammatory action of Ebn. Inflammation is a multi-complex processes that involves various molecular mechanisms in response to extracellular stimuli, such as bacteria (Arenzana-Seisdedos & Virelizier, 1983). Cytokines are well-known pro-inflammatory markers that promote the pathogenesis of inflammatory diseases such as rheumatoid arthritis, cerebral malaria and autoimmune diseases (Sung et al., 2009). NO, TNF-a and IL-6 are

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Figure 3. Effect of Ebn on TNF-a and IL-6 production in LPS-induced RAW264.7 macrophage cells. Cells were pretreated with Ebn in a dose-dependent manner for 1 h, and they were then exposed to 1 mg/mL LPS in serum-free conditions for 24 h. TNF-a and IL-6 production was measured by ELISA.

Figure 4. Effect of Ebn on JNK, p38 and ERK phosphorylation induced by LPS. Cells were pretreated with Ebn in a dose-dependent manner for 1 h and then exposed to 1 mg/mL LPS for 20 min. The phosphorylation levels of JNK, p38 and ERK were measured by western blot analysis.

well-characterized pro-inflammatory cytokines that are produced by LPS (a component of the bacteria cell wall) to trigger pathogenesis of inflammatory diseases (O’Shea et al., 2002). In the present study, we showed that LPS induced both NO production and iNOS expression in RAW264.7 macrophage cells. In addition, TNF-a and IL-6 also increased the production due to LPS stimulation in RAW264.7 macrophage cells. Interestingly, Ebn decreased the production of NO, TNF-a IL-6 and iNOS. Therefore, we demonstrated that Ebn has anti-inflammatory abilities, which is at least in part due to decreasing NO, TNF-a IL-6 and iNOS. However, we did not investigate the effect of Ebn on the production of various cytokines related to inflammation. Further studies should be conducted in order to determine what kinds of cytokines are regulated by Ebn. On the other hand, various protein signal molecules are necessary for the induction of cytokines that function as mediators between external stimuli and cytokine productions, such as LPS. A number of studies have suggested that MAPKsignaling pathways are considerable targets for controlling inflammation since JNK, p38 and ERK are activated by LPS; they also induce cytokine production (Dong et al., 1993; Kwon et al., 2010; Uto et al., 2010; Yu et al., 2011). In addition, MAPKs have been reported to mediate LPS-induced iNOS and KF-kB activations (Lu et al., 2012). In the present study, we have confirmed that Ebn significantly inhibits JNK and p38 phosphorylation stimulated by LPS in RAW264.7 macrophage cells. These results are consistent with previous

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data showing that LPS induces JNK and p38 phosphorylation, which is inhibited by Ebn. However, Ebn did not alter the ERK signaling pathway in LPS-induced RAW264.7 macrophage cells. Therefore, Ebn demonstrates anti-inflammatory activity in LPS-induced macrophage cells, which is due, at least in part, to the inhibition of JNK and p38, but not ERK.


Conclusion The present study demonstrates that a 70% ethanol extract of B. nivea exerts anti-inflammatory effects by inhibiting JNK and p38 phosphorylation in LPS-induced RAW264.7 macrophage cells. On the basis of our findings, we suggest that B. nivea may be useful for preventing inflammation. Further, we expect that our results will contribute to the development of functional foods that prevent inflammation, and will facilitate an understanding of the mechanism of action, which will provide a basis for further investigations using B. nivea.

Declaration of interest There are no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This research was supported by grants from the Seocheon County Office and the Korea Food Research Institute, Republic of Korea. References Ajizian SJ, English BK, Meals EA. (1999). Specific inhibitors of p38 and extracellular signal regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma. J Infect Dis 179:939–44. Arenzana-Seisdedos F, Virelizier J. (1983). Interferons as macrophageactivating factors. II. Enhanced secretion of interleukin 1 by lipopolysaccharide-stimulated human monocytes. Eur J Immunol 13: 437–40. Chandrasekara N, Shahidi F. (2011). Effect of roasting on phenolic content and antioxidant activities of whole cashew nuts, kernels, and testa. J Agric Food Chem 59:5006–14. Chen YC, Shen SC, Lee WR, et al. (2001). Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages. J Cell Biochem 82:537–48. Choi SY, Ko HC, Ko SY, et al. (2007). Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits. Biol Pharm Bull 30:772–8. de la Rosa LA, Alvarez-Parrilla E, Shahidi F. (2011). Phenolic compounds and antioxidant activity of kernels and shells of Mexican pecan (Carya illinoinensis). J Agric Food Chem 59:152–62.

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