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1,6-0, 0-diacetylbritannilactones inhibits I[kappa]B kinase [beta]-dependent NF-[kappa]B activation.

Abstract

To determine the chemical constituents responsible for pharmacological effects of Inula britannica-F., three specific sesquiterpene lactones in Inula britannica were isolated from chloroform extract and identified, including britannilactone (BL), 1-O-acetylbritannilactone (ABLO), and 1, 6-0, 0-diacetylbritannilactone (ABLOO). Electro-phoretic mobility shift assay (EMSA) was performed to detect the nuclear translocation of nuclear factor-KB (NF-kB) p65. The expressions of IkB[alpha], pIKB[alpha], inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IkB kinase [alpha]/[beta] (IKK[alpha]/[beta]) and NF-kB kinase (NIK) were detected by Western blot and RT-PCR. We found that acetyl side groups enhanced the inhibitory action of the agents on LPS/IFN-[gamma]-induced iNOS and COX-2 expression. Their inhibiting activity was positive correlation with the acetyl side group number. The effects of LPS/IFN-7 were reversed by ABLOO, and BL without acetyl side groups showed only a weak inhibitory action. Further study indicated that ABLOO markedly inhibited the phosphorylation of IKK[beta] down to based level, but not IKK[alpha], corresponding with decreased in IkB[alpha] degradation and phosphorylation induced by LPS/IFN-7, resulting in the suppression of NF-kB nuclear translocation and activity. These results suggest that the acetyl moieties add to the lipophilicity, and consequently enhance cellular penetration, so that ABLOO possess the most anti-inflammatory effect and may be a potent lead structure for the development of therapeutic and cytokine-suppressing remedies valuable for the treatment of various inflammatory diseases. [C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Sesquiterpene lactones; Anti-inflammatory activity; Britannilactone; Nuclear factor-KrB; IkB kinase; RAW264.7 cells

Introduction

Sesquiterpene lactones (SQLs) are the active constituents of many medicinal plants from the Aster-aceac family and possess a variety of biological effects (Schmidt, 2000). In particular, their potent antiinflammatory and cytotoxic properties have received considerable attention. One of the main mechanisms of SQLs anti-inflammatory effects is the inhibition of the transcription factor NF-KB (Hehner et al., 1999; Bremner and Heinrich, 2002). SQLs most often possess [alpha], [beta]-unsaturated carbonyl structures, such as a-methylene-y-lactones or [alpha], [beta]-unsaturated cyclopentenones. The presence of these functional groups strongly enhances the inhibitory effect of SQLs.

1-O-acetylbritannilactone (ABLO) is a sesquiterpene lactone abundant in Inula britannica-F., which is used to treat bronchitis and inflammation (Fig. 1). ABLO does not only inhibit the activation of NF-kB but also influences the NF-KB-dependent gene expression and inhibits prostaglandin release (Han et al., 2004; Liu et al., 2007, 2008). The mechanism of activation of ABLO has been demonstrated in RAW 264.7 macrophages and vascular smooth muscle cells (VSMCs) to prevent IKB[alpha] phosphorylation and degradation (Liu et al., 2007). However, the other two SQLs, britannilactone (BL) and 1, 6-0, 0-diacetylbritannilactone (ABLOO), which are different from ABLO in 1, 6-acetyl side groups, have been identified from Inula brilannica-F. (Fig. 1). Because the acetyl moieties add to the lipophilicity and consequently enhance cellular penetration, we presumed that ABLOO possess the most anti-inflammatory effect. The present study demonstrated that ABLOO blocked the phosphorylation and degradation of IKB[alpha] induced by LPS/IFN-[gamma], resulting in the suppression of NF-KB activation by the blockade of IKK[beta] phosphorylation. Therefore, ABLOO may be a potent lead structure for the development of therapeutic and cytokine-suppres-sing remedies valuable for the treatment of various inflammatory diseases.

[FIGURE 1 OMITTED]

Materials and methods

Drugs and reagents

Three SQLs, BL, ABLO, ABLOO (purity > 99%), were isolated from Inula britannica-F. var chinensis Regel and the structures were determined spectro-scopically, as has previously been reported (Wang et al., 2005) (Fig. 1). Dulbecco's modified Eagle's medium (DMEM) with high glucose and other reagents for cell culture were obtained from Gibco-BRL Life Biotechnologies (Rockville, MD). Anti-COX-2, anti-iNOS, anti-NF-KB p65, anti-IKB[alpha], anti-NIK (RelA), anti-phospho-NIK antibodies and ECL detection system were obtained from Santa Cruz Co. (Biotechnology, CA); anti-phospho-IKB[alpha], anti-IKK[alpha], anti-IKK[beta], antiphospho-IKK[alpha]/[beta] antibodies were purchased from Cell Signaling Technology (Danvers, MA). LPS and IFN-y were purchased from Sigma Chemical Co. (Aldrich, MO). Murine expression plasmids, pcDNA3.1-IKK[beta] (wild-type IKK[beta]), was kindly provided by Dr. Ballard D. (NIH/NIDCR, Bethesda, MD, USA).

Cell culture and transfection

Murine RAW264.7 macrophage cells (Manassa, VA, USA) were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 5% [CO.sub.2], 37[degrees]C. The cells grown up to 70% confluence were transiently transfected with 1[micro]g pcDNA3.1-IKK[beta] using ESCORT[TM] Transfection Reagent (Sigma) according to manufacturer's protocols. For stimulation, the medium was replaced with fresh DMEM, and the cells were then stimulated with LPS (10[micro]g/ml)/IFN-y (20 IU) following pretreatment by three SQLs (10 [micro]M) for 60 min, respectively.

Western blotting

Equal cytosolic protein (50 [micro]g) after being subjected to polyacrylamide gel electrophoresis were transferred to PVDF membrane, and then the membrane was incubated with primary antibodies, followed by an alkaline phosphatase-conjugated antibody and chemilu-minescent detection. All Western blots were repeated three times from three different experiments.

Electrophoretic mobility shift assay (EMSA)

Nuclear proteins (5 [micro]g) were incubated with 0.05 pmol [[.sup.32]P]-labeled NF-kB oligonucleotides, and resolved on 6% nondenaturing polyacrylamide gels. Specificity was conformed with 100-fold excess unlabeled probe (Han et al, 2004).

Statistical analysis

Results are expressed as means [+ or -] S.D., and an analysis of variance with Bonferroni's test was used for the statistical of multiple comparisons of date. P values of 0.05 or less were considered statistically significant.

Results

Acetylation enhances the inhibitory effects of britannilactones on inflammatory gene expression induced by LPS/IFN-[gamma]

iNOS and COX-2 expressions are the key markers in activated macrophages. We first determined the effects of three SQLs on the expression of iNOS and COX-2 in RAW264.7 cells exposed to LPS/IFN-[gamma] for 16h by Western blot analysis. Fig. 2A showed that three SQLs inhibited LPS/IFN-[gamma]-induced iNOS and COX-2 protein and mRNA expression, and the inhibitory effects of ABLOO were the strongest, abolishing the expression of iNOS and COX-2 proteins induced by LPS/IFN-[gamma]. Compared to the dramatic and potent inhibition elicited by ABLO and ABLOO, BL showed a weak potency for inhibiting LPS/IFN-[gamma]-induced iNOS and COX-2 expression. The inhibitory of three SQLs activity was positive correlation with the acetyl side groups number, Thus, suggesting that the acetyl structure moiety may augment the anti-inflammatory effects, but they did not affect cell viability as measured by MTT method (data not shown).

[FIGURE 2 OMITTED]

Acetylation enhances the inhibitory effect of the britannilactones on NF-kB activation and DNA-binding activity

RAW264.7 cells were treated with BL, ABLO and ABLOO for 60min, respectively, before LPS/IFN-[gamma] stimulation. NF-KB activity in nuclear extract was analyzed by EMSA. Three homologue SQLs acetyl-dependently reduced the intensity of specific shifted-bands of nuclear extracts bound with NF-kB oligonucleotides (Fig. 2B), indicting that ABLOO blocks NF-kB DNA-binding activity. ABLOO also inhibited NF-kB activation through preventing the IkB[alpha] from phosphorylation and degradation, and consequently NF-kB p65 nuclear translocation induced by LPS/ IFN-[gamma] in RAW264.7 cells, while BL and ABLO mildly inhibited NF-kB activation (Fig. 2A). These findings suggest that the anti-inflammatory activity of britannilactones depends partly on the number of acetyl side group.

ABLOO blocks NF-kB activation through inhibiting IKK[beta] phosphorylation

Two distinct pathways of NF-KB activation are, respectively, the canonical pathway by IKK[beta] activation and then IkB phosphorylation and degradation, and the noncanonical pathway based on NF-KB-inducing kinase (NIK) and IKK[alpha] activation. To determine the upstream mechanism of ABLOO inhibiting NF-kB activation, IKK[alpha]/[beta]phosphorylation in RAW264.7 cells was analyzed by Western blotting. There is almost no phosphorylated IKK found in unstimulated cells, whereas LPS/IFN-[gamma] strongly induced IKK phosphorylation in RAW264.7 cells (Fig. 3A). ABLOO markedly inhibited LPS/IFN-[gamma]-induced phosphorylation of IKK[beta] down to based level, but not IKK[alpha], corresponding with a decrease in IKB[alpha] degradation and phosphorylation in the dose of 10 [micro]M, which was not toxic to the cells (Fig. 3A). The IC50 of ABLOO was 9.12 [micro]M. The inhibitory effect of ABLOO was further demonstrated by immunoprecipitation using anti-phospho-Ser antibody and Western blotting by anti-IKK[alpha]/[beta] antibodies (Fig. 3B). RAW264.7 cells transfected with pcDNA 3.1-IKK[beta] highly expressed exogenous IKK[beta] (not shown). The overexpression of IKK[beta] abolished the inhibition of IKK[beta] phosphorylation and subsequently IKB[alpha] degradation by ABLOO (Fig. 3C). However, ABLOO did not inhibit NIK phosphorylation induced with LPS/IFN-[gamma], and did not effect the phosphorylation of IKK[beta] and IkB[alpha] in resting cells (not shown). These results suggest that IKK[beta] may serve as a molecular target for ABLOO anti-inflammation.

[FIGURE 3 OMITTED]

Discussion

We have demonstrated that ABLO, as one of SQLs from Inula britannica-F., is an NF-[kappa]B activation inhibitor (Han et al., 2004). However, the role of the acetyl side groups in their structure is still not clear. In the present study, by comparing the anti-inflammatory activities of three britannilactones with different number of acetyl side groups, we found that acetyl side groups enhanced the inhibitory action of the agent on LPS/IFN-[gamma]-induced NF-[kappa]B activation, and inflammatory gene iNOS and COX-2 expression. ABLOO with two acetyl side groups blocked the phosphorylation and degradation of I[kappa]B[alpha] induced by LPS/IFN-[gamma], resulting in the suppression of NF-[kappa]B nuclear translocation and activity. The effects of LPS/IFN-[gamma] were abolished by ABLOO, while BL without acetyl side groups showed only a weak inhibitory action. The anti-inflammatory activity increased with a higher number of acetyl group. These results indicate that the presence of the acetyl moieties enhance the anti-inflammatory action of SQLs.

Inflammatory response is a frequent consequence of a severe microbial infection or trauma. The release of microbial products (e.g., LPS) and other proinflammatory mediators (e.g., IFN-[gamma]) from the affected tissue results in the activation of circulating monocytes/macrophages (Chang et al., 2001; Guslandi, 1998; Simons et al., 1996). NF-[kappa]B activation and nuclear translocation is a key step in inflammatory cascade effects, leading to amplifying the inflammatory state (Simons et al., 1996; Bonizzi et al., 2000; Collins et al., 1995). In activated monocytes/macrophages, I[kappa]B[alpha] is phosphorylated, ubiquitinated and subsequently degraded by the proteasome pathway. The loss of I[kappa]B[alpha] allows NF-[kappa]B to enter the nucleus and initiate the transcription of relevant pro-inflammatory genes, including COX-2 and iNOS (Lee et al., 2007; Franco et al., 2004). In previous report, many SQLs compounds were found to possess antimicrobial, antitumor and anti-inflammatory activities (Robles et al., 1995; Li et al., 2005; Rios and Recio, 2005). It has been reported that ABLO induces the apoptosis activity of kidney cell line BRK-p53 (Rafi et al., 2005) and anti-inflammatory action (Han et al., 2004). As SQLs are a lipophilic agent, the acetylation may add to the lipophilicity, and hence, possibly enhances cellular penetration, and consequently augments the anti-inflammatory activity (Choi et al., 1999).

The key regulatory step in NF-[kappa]B activation is the activation of the upstream kinase IKK. In the present study, we provided the experimental evidence that ABLOO inhibits LPS/IFN-[gamma]-induced NF-[kappa]B activation through a complete blockade of IKK[beta] phosphorylation. These findings are consistent with previous data from the literature, indicating that IKK[beta] represents a key target of various NF-[kappa]B inhibitors, including cyclopen-tone prostaglandins, arsenite, and NO (Liu et al., 2003). ABLOO specially blocked LPS/IFN-[gamma]-induced IKK[beta] phosphorylation, but did not affect IKK[alpha] phosphorylation. In keeping with the findings that ABLOO blocked IKK[beta]-dependent NF-[kappa]B activation, we found that it also markedly reduced the transcription of NF-[kappa]B-dependent iNOS and COX-2 genes in RAW264.7 cells. The inhibition of IKK[beta]-dependent NF-[kappa]B activation by ABLOO further resulted in a complete inhibition of the transcription of NF-[kappa]B-dependent genes. These findings offer new perspectives in understanding the relationships between ABLOO and anti-inflammation.

The present study is the first report on the increase in anti-inflammatory activity of britannilactones with a high number of acetyl groups. Our findings demonstrate that the acetyl groups' structural requirement may be essential for potent anti-inflammatory activity, and that the inhibition of NF-[kappa]B activation by ABLOO is related to the blockade of IKK[beta] phosphorylation, resulting in the inactivation of the kinase and downstream signaling. This inhibitory effect has important implications for the development of anti-inflammatory drugs and strategies to limit pathological inflammation. These results suggest that ABLOO may serve as lead structures for the development of anti-inflammatory drugs to treat various inflammatory diseases.

Acknowledgements

This research was supported by a grant from National Nature Science Foundation of PR China (no. 305706614 and 30670845) and a grant from Hebei Province Natural Science Foundation of PR China (no. C2007000831).

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Yue-Ping Liu (a), (c), Jin-Kun Wen (a), Yi-Bing Wu (b), Jia Zhang (a), Bin Zheng (a), Di-Qun Zhang (b), Mei Han (a), *

(a) Department of Biochemistry and Molecular Biology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, PR China

(b) Department of Pharmaceutical Chemistry, College of Pharmacy, Hebei Medical University, Shijiazhuang 050017, PR China

(c)Department of Pathology, The Forth Hospital of Hebei Medical University, Shijiazhuang 050017, PR China

* Corresponding author. Tel.: +86 31186265563; fax: +8631186266180.

E-mail address: hanmei@hebmu.edu.cn (M. Han).
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Title Annotation:SHORT COMMUNICATION; nuclear factor kappa-light-chain-enhancer of activated B cells
Author:Liu, Yue-Ping; Wen, Jin-Kun; Wu, Yi-Bing; Zhang, Jia; Zheng, Bin; Zhang, Di-Qun; Han, Mei
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Mar 1, 2009
Words:2735
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