Methyl salicylate 2-O-β- D -lactoside, a novel salicylic acid analogue, acts as an anti-inflammatory agent on microglia and astrocytes
© Lan et al; licensee BioMed Central Ltd. 2011
Received: 7 April 2011
Accepted: 11 August 2011
Published: 11 August 2011
Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer's disease (AD). Activation of microglia and astrocytes is a characteristic of brain inflammation. Epidemiological studies have shown that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) delays the onset of AD and suppresses its progression. Methyl salicylate-2-O-β-D-lactoside (DL0309) is a new molecule chemically related to salicylic acid. The present study aimed to evaluate the anti-inflammatory effects of DL0309.
Our studies show that DL0309 significantly inhibits lipopolysaccharide (LPS)-induced release of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α; and the expression of the inflammation-related proteins iNOS, COX-1, and COX-2 by microglia and astrocytes. At a concentration of 10 μM, DL0309 prominently inhibited LPS-induced activation of NF-κB in glial cells by blocking phosphorylation of IKK and p65, and by blocking IκB degradation.
We demonstrate here for the first time that DL0309 exerts anti-inflammatory effects in glial cells by suppressing different pro-inflammatory cytokines and iNOS/NO. Furthermore, it also regulates the NF-κB signaling pathway by blocking IKK and p65 activation and IκB degradation. DL0309 also acts as a non-selective COX inhibitor in glial cells. These studies suggest that DL0309 may be effective in the treatment of neuroinflammatory disorders, including AD.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the elderly characterized by global deficits in cognition ranging from loss of memory to impaired judgment. It has been hypothesized that early microglial activation in AD delays disease progression by promoting clearance of beta amyloid peptide (Aβ) before formation of senile plaques [1–3]. Microglia are antigen-presenting cells that, upon activation, are capable of phagocytosis and the production of various pro-inflammatory molecules such as nitric oxide (NO) and interleukin-1β (IL-1β) . These molecules are able to destroy pathogens, but can also induce toxicity in neurons, which are compromised in AD. Furthermore, in aged human brain, many microglia are dystrophic, showing morphological features indicative of senescence such as fragmented cytoplasmic processes . Like microglia, chronically activated astrocytes are believed to contribute to AD through production of NO and of various pro-inflammatory cytokines and chemokines. Apart from this, astrocytes become activated around plaques to take up Aβ and neuronal debris . Not only that, activated astrocytes are also involved in plaque formation. Therefore, agents that block the activation of microglia and astrocytes may be effective in the treatment of AD.
A recent study showed that the administration of non-steroidal anti-inflammatory drugs (NSAIDs) could delay progression of AD, most likely because of their ability to reduce microglial activation and cytokine release. The presence of inflammatory processes in AD brains suggests that anti-inflammatory agents like ibuprofen may be beneficial in this disease . NSAIDs have been well studied, both in vitro and in vivo, and have been observed to ameliorate inflammation related to Aβ deposition in AD. Several in vitro studies have shown that NSAIDs like aspirin might have anti-aggregation activity for Aβ by blocking the NF-κB signaling pathway .
Primary rat glia cells were obtained through a modification of McCarthy and deVellis's protocol . Primary cells were cultured in DMEM/F12 medium containing 10% FBS (Gibco), 1.4 M L-glutamine, 100 U/mL penicillin, and 0.1 mg/ml streptomycin; and were found to be of 95% purity as determined by immunocytochemical staining with ox42 and anti-glial fibrillary acidic protein (GFAP) antibody.
To investigate the anti-inflammatory actions of DL0309, microglia and astrocytes were incubated with DL0309 (0.1, 1.0 or 10 μM) in the presence or absence of LPS (0.5 μg/ml) for 24 h. Pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) levels in the culture medium were measured by ELISA. The production of NO-derivative nitrite was determined by the Griess reaction as described previously .
Western blot analysis was carried out evaluating the expression of iNOS, COX-1, COX-2, and NF-κB pathway-relevant proteins such as IκB-α, total/phosphorylated IKK and NF-κB-p65. Cells were plated overnight in 100 mm dishes and pre-treated with DL0309 at concentrations of 0.1, 1.0 or 10 μM for 1 h. After exposing the cells to LPS (0.5 μg/ml) for either 10 min or 45 min, cytosolic protein extracts were prepared. COX-1 and COX-2 antibodies were obtained from Abcam (Cambridge, UK). Other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Western blotting results were quantified using Quantity One software (Bio-Rad).
To determine the direct inhibitory effects of DL0309 on COX-1 and COX-2 enzymatic activities, primary rat glial cells were pre-incubated with LPS (0.5 μg/ml) for 24 h. Medium was then removed, and DL0309 (0.1, 1.0 or 10 μM) was added for 1 h. Cells were treated with arachidonic acid, 30 μM, for another 20 min, and PGE2 levels in the medium were then measured by ELISA [13, 14]. To investigate the effect of DL0309 on COX-1 enzymatic activity, cells were not treated with LPS in order to express only the COX-1 isoform . Thus, measured PGE2 levels represent COX-1 activity alone.
At least three independent experiments were used for data analysis. All data are presented as mean ± S.E.M. Values were compared using a t-test (two groups) or one-way ANOVA with post-hoc Student-Newman-Keuls test (multiple comparisons).
In recent years, a number of mechanisms have been proposed to account for the protective effects of aspirin [17, 18], ibuprofen  and other anti-inflammatory agents in AD. Studies have shown various degrees (risk reductions of up to 50%) of benefit from the use of NSAIDs on onset of disease and on dementia, with increased duration of NSAIDs use having increased protective effect against AD . The best characterized action of these anti-inflammatory agents is to suppress neuroinflammation, primarily through their ability to inhibit COX, leading to reduced biosynthesis of pro-inflammatory molecules in glial cells. Several studies have shown that the onset of AD may be apparently suppressed or delayed by mixed COX-1 and COX-2 inhibitors .
In summary, we demonstrate that DL0309 is capable of acting as a non-selective inhibitor of COX-1 and COX-2. DL0309 also regulates the NF-κB signaling pathway not only by blocking degradation of IκB, but also by restraining pIKK and pNF-κB-p65 activity. Therefore, this agent can suppress proteins that are regulated by the NF-κB pathway, including iNOS, NO and the cytokines IL-1β, IL-6 and TNF-α. These studies suggest that DL0309 may be an effective agent in the treatment of neuroinflammatory disorders, including AD.
This work was supported by National Scientific & Technological Major Project for "Significant New Drugs Creation" (No.2009ZX09102-034), the International S&T Cooperation Projects (No.2009DFA32010) and National Natural Science Foundation (No. 81073120) by China government. We are grateful to Dr. Xin Wang (Manchester University, UK) and Prof. Humphrey Rang (London College University, UK) for the revision of manuscript.
- Maragakis NJ, Rothstein JD: Mechanisms of Disease: astrocytes in neurodegenerative disease. Nat Clin Pract Neurol. 2006, 2: 679-689.View ArticlePubMedGoogle Scholar
- Wyss-Coray T, Loike JD, Brionne TC, Lu E, Anankov R, Yan F, Silverstein SC, Husemann J: Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat Med. 2003, 9: 453-457. 10.1038/nm838.View ArticlePubMedGoogle Scholar
- Wyss-Coray T: Inflammation in Alzheimer disease: driving force, bystander or beneficial response?. Nat Med. 2006, 12: 1005-1015.PubMedGoogle Scholar
- Benveniste EN: Cytokines: influence on glial cell gene expression and function. Chem Immunol. 1997, 69: 31-75.View ArticlePubMedGoogle Scholar
- Streit WJ, Sammons NW, Kuhns AJ, Sparks DL: Dystrophic microglia in the aging human brain. Glia. 2004, 45: 208-212. 10.1002/glia.10319.View ArticlePubMedGoogle Scholar
- Allaman I, Belanger M, Magistretti PJ: Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci. 2011, 34: 76-87. 10.1016/j.tins.2010.12.001.View ArticlePubMedGoogle Scholar
- Pinnen F, Sozio P, Cacciatore I, Cornacchia C, Mollica A, Iannitelli DAE, Cataldi A, Zara S, Nasuti C, Di Stefano A: Ibuprofen and Glutathione Conjugate as a Potential Therapeutic Agent for Treating Alzheimer's Disease. Arch Pharm (Weinheim). 2010Google Scholar
- Ray B, Lahiri DK: Neuroinflammation in Alzheimer's disease: different molecular targets and potential therapeutic agents including curcumin. Curr Opin Pharmacol. 2009, 9: 434-444. 10.1016/j.coph.2009.06.012.View ArticlePubMedGoogle Scholar
- Zhang D, Liu R, Sun L, Huang C, Wang C, Zhang DM, Zhang TT, Du GH: Anti-inflammatory activity of methyl salicylate glycosides isolated from Gaultheria yunnanensis (Franch.) Rehder. Molecules. 2011, 16: 3875-3884. 10.3390/molecules16053875.View ArticlePubMedGoogle Scholar
- Zhang B, He XL, Ding Y, Du GH: Gaultherin, a natural salicylate derivative from Gaultheria yunnanensis: towards a better non-steroidal anti-inflammatory drug. Eur J Pharmacol. 2006, 530: 166-171. 10.1016/j.ejphar.2005.11.030.View ArticlePubMedGoogle Scholar
- McCarthy KD, de Vellis J: Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 1980, 85: 890-902. 10.1083/jcb.85.3.890.View ArticlePubMedGoogle Scholar
- Drew PD, Chavis JA: The cyclopentone prostaglandin 15-deoxy-Delta(12,14) prostaglandin J2 represses nitric oxide, TNF-alpha, and IL-12 production by microglial cells. J Neuroimmunol. 2001, 115: 28-35. 10.1016/S0165-5728(01)00267-3.View ArticlePubMedGoogle Scholar
- Fiebich BL, Lieb K, Kammerer N, Hull M: Synergistic inhibitory effect of ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary rat microglia. J Neurochem. 2003, 86: 173-178.View ArticlePubMedGoogle Scholar
- Candelario-Jalil E, de Oliveira AC, Graf S, Bhatia HS, Hull M, Munoz E, Fiebich BL: Resveratrol potently reduces prostaglandin E2 production and free radical formation in lipopolysaccharide-activated primary rat microglia. J Neuroinflammation. 2007, 4: 25-10.1186/1742-2094-4-25.PubMed CentralView ArticlePubMedGoogle Scholar
- Akundi RS, Candelario-Jalil E, Hess S, Hull M, Lieb K, Gebicke-Haerter PJ, Fiebich BL: Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia. Glia. 2005, 51: 199-208. 10.1002/glia.20198.View ArticlePubMedGoogle Scholar
- Brambilla R, Bracchi-Ricard V, Hu WH, Frydel B, Bramwell A, Karmally S, Green EJ, Bethea JR: Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury. J Exp Med. 2005, 202: 145-156. 10.1084/jem.20041918.PubMed CentralView ArticlePubMedGoogle Scholar
- Thomas T, Nadackal TG, Thomas K: Aspirin and non-steroidal anti-inflammatory drugs inhibit amyloid-beta aggregation. Neuroreport. 2001, 12: 3263-3267. 10.1097/00001756-200110290-00024.View ArticlePubMedGoogle Scholar
- Cornelius C, Fastbom J, Winblad B, Viitanen M: Aspirin, NSAIDs, risk of dementia, and influence of the apolipoprotein E epsilon 4 allele in an elderly population. Neuroepidemiology. 2004, 23: 35-143.View ArticleGoogle Scholar
- Iwata Y, Nicole O, Zurakowski D, Okamura T, Jonas RA: Ibuprofen for neuroprotection after cerebral ischemia. J Thorac Cardiovasc Surg. 2010, 139: 489-493. 10.1016/j.jtcvs.2009.05.049.PubMed CentralView ArticlePubMedGoogle Scholar
- Zandi PP, Breitner JC, Anthony JC: Is pharmacological prevention of Alzheimer's a realistic goal?. Expert Opin Pharmacother. 2002, 3: 365-380. 10.1517/146565220.127.116.115.View ArticlePubMedGoogle Scholar
- Ferrera P, Arias C: Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells. Neurochem Int. 2005, 47: 589-596. 10.1016/j.neuint.2005.06.012.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.