Indirubin-3′-oxime inhibits inflammatory activation of rat brain microglia

Neuroscience Letters 487 (2011) 139–143

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Indirubin-3 -oxime inhibits inflammatory activation of rat brain microglia Hoon-Ji Jung a , Kyong Nyon Nam a , Min-Sook Son a , Hee Kang a , Joung-Woo Hong a , Jong Woo Kim b , Eunjoo H. Lee a,∗ a b

Graduate School of East-West Medical Science, Kyung Hee University, 1 Seochun, Yongin-si 446-701, Republic of Korea East-West Neo Medical Center, Kyung Hee University, 149 Sangil-dong, Seoul 134-727, Republic of Korea

a r t i c l e

i n f o

Article history: Received 9 July 2010 Received in revised form 24 September 2010 Accepted 4 October 2010 Keywords: Indirubin-3 -oxime Microglia NF-␬B Nitric oxide Organotypic hippocampal slice culture TNF-␣

a b s t r a c t Microglial cells play critical roles in the immune and inflammatory responses of the brain. Under pathological conditions, the activation of microglia helps to restore brain homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. As such, regulators of microglial activation have been considered as potential therapeutic candidates to reduce the risk of neurodegeneration associated with neurodegenerative diseases, including Alzheimer’s and, Parkinson’s diseases. Indirubin-3 -oxime, a potent inhibitor of cyclindependent kinases and glycogen synthase kinase-3␤, has been shown to have neuroprotective potential. The specific aim of this study was to examine the efficacy of indirubin-3 -oxime in the repression of microglial activation. Indirubin-3 -oxime was shown to effectively inhibit lipopolysaccharide (LPS)induced nitric oxide release from cultured rat brain microglia. This compound reduced the LPS-stimulated productions of tumor necrosis factor-␣, interleukin-1␤, prostaglandin E2 , and intracellular reactive oxygen species and also effectively reduced LPS-elicited NF-␬B activation. In organotypic hippocampal slice cultures, indirubin-3 -oxime blocked LPS-related hippocampal cell death. These results suggest that indirubin-3 -oxime provides neuroprotection by reducing the productions of various neurotoxic molecules in activated microglia. © 2010 Elsevier Ireland Ltd. All rights reserved.

Immune and inflammatory responses in the central nervous system (CNS) are principally mediated by the microglia. These responses are activated during neuropathological conditions and restore CNS homeostasis [8]. The activation of microglia involves proliferation, migration to the injury site, increased expression of immunomodulators, and transformation into phagocytes [8,29]. Activated microglia also promotes neuronal injury through the release of proinflammatory and cytotoxic factors, including tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, nitric oxide (NO), and reactive oxygen species (ROS) [29]. Chronic microglial activation has been implicated in neuronal destruction associated with various neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases [26]. Thus, activation of counter-regulatory mechanisms to reduce the activation of microglial cells is essential to avoid the escalation of CNS inflammatory processes [24]. The identification of agents that target over-activated microglial cells is essential to reducing the neuronal destruction associated with neurodegenerative diseases.

∗ Corresponding author. E-mail address: [email protected] (E.H. Lee). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.10.009

Indirubin was initially identified in traditional Chinese medicine, and used in the treatment of various diseases, including leukemias [9]. Indirubin-3 -oxime, a synthetic analogue of indirubin, is a potent inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK)-3␤ [6,17]. CDK2/5 and GSK-3␤ are responsible for abnormal hyperphosphorylation of the microtubule-associated protein, tau, which is involved in the pathogenesis of Alzheimer’s disease [7]. As these kinases play important roles in neuronal survival and apoptosis [13,31], indirubin-3 -oxime has been investigated as a potential rescue mechanism for neurons enduring diverse injurious stimuli. Indirubin-3 -oxime inhibits neurotoxicity caused by amyloid-␤, MPTP, and potassium withdrawal [30,32,35]. In a recent study using a mouse model of Alzheimer’s disease, indirubin-3 -oxime was effective for reducing spatial memory deterioration and attenuating diverse amyloid-␤-associated neuropathological symptoms [4]. Based on these studies, indirubin-3 -oxime has been proposed to act as a neuroprotective agent. CDK5 and GSK-3␤ are also involved in peripheral inflammation [5,23]. However, the efficacy of this compound for controlling neuroinflammation has been largely unexplored. The purpose of this study was to examine the scope of indirubin-3 -oxime that is able to control inflammatory responses of the brain microglia, and the protective potential of indirubin-3 oxime for reducing inflammation-induced neurotoxicity.

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Fig. 1. Effect of indirubin-3 -oxime on nitrite production and cell viability in microglial cells. Primary microglial cells were incubated in the absence (control) or presence of LPS. The cells were pretreated with the indicated amounts of indirubin-3 -oxime (IMX) for 30 min prior to the addition of LPS. Twenty-four hours after LPS was administered, the cultures were subjected to a nitrite assay (A) and a cell viability assay (B). As a reference, cells were treated with indirubin-3 -oxime only for 24 h, and the cultures were subjected to nitrite quantification (C) and a cell viability assay (D). Data are expressed as means ± SEM from triplicate assays. ## p < 0.001, # p < 0.05 vs. control group; **p < 0.001 vs. LPS-only treated group. LPS has been reported to cause activation-induced cell death in microglia [18]. Thus, cell viability, as measured using the MTT assay, was often reduced by LPS.

All cell culture products were purchased from Invitrogen (Carlsbad, CA, USA). Escherichia coli lipopolysaccharide (LPS), indirubin-3 -oxime, and other chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies against phosphop44/42 MAPK, p44/42 MAPK, phospho-SAPK/JNK, SAPK/JNK, phospho-GSK-3␤ (pY216), phospho-GSK-3␤ (pS9), and GSK-3␤ were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary microglial cells were prepared from the cerebral cortices of one-day-old Sprague–Dawley rats (Orient, Kyunggido, Korea) as described in previous work [19,21]. All animal experiments were performed in accordance with approved institutional animal care guidelines. BV2 mouse microglial cells were cultured as described in previous work [20]. The cells were pretreated with indirubin-3 -oxime in fresh medium containing 0.1% fetal bovine serum (FBS) for 30 min prior to the addition of LPS. The nitrite in the culture supernatants was measured using Griess reagents (Molecular Probes, Eugene, OR, USA), and cell viability was determined using MTT solution (Sigma), as described in previous work [21]. Cytokine levels in the culture supernatants were assayed using PGE2 , TNF-␣, and IL-1␤ immunoassay kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The presence of intracellular ROS was measured using a nonfluorescent dye of 2 ,7 -dichlorofluorescein (DCFH-DA; Molecular Probes) as described in previous work [20]. DCF fluorescence was measured using a Wallac 1420 fluorometer (Perkin-Elmer, Finland) at 485 nm for excitation and 530 nm for emission. NF-␬B activity was measured with a luciferase assay as previously described [12]. The cells in 12-well plates were co-transfected with 0.5 ␮g of NF-␬B responsive luciferase reporter plasmid containing four ␬B sites (pNF␬B-Luc; Clontech Laboratories, Mountain View, CA, USA) and 0.2 ␮g of pSV-␤-galatosidase control vector (Promega, Madison, WI, USA). After 24 h, the cells were pretreated with indirubin-3 -oxime for 30 min, and then stimulated with LPS

for 4 h. NF-␬B luciferase activities were analyzed using a luminometer and normalized to ␤-galactosidase activity. For statistical analysis of cell culture experiments, data were expressed as means ± SEM from three independent experiments. Student’s t-test was used for statistical analyses and only p-values <0.05 were reported as significant. For Western blot analysis, cells were lysed on ice in RIPA buffer containing phosphatase inhibitors cocktails (Sigma). Lysate samples containing 30 ␮g of protein were fractionated by SDS-10% polyacrylamide gel electrophoresis and then electroblotted onto nitrocellulose membranes. The membranes were probed with primary antibodies, and immunoreactivity was detected with ECL Reagent (Amersham Biosciences, Piscataway, NJ, USA). Organotypic hippocampal slice cultures were prepared from male Sprague–Dawley rats (seven-days-old; Orient) using the method described by Stoppini and colleagues [25,33]. Neurotoxicity was evaluated through the uptake of the fluorescent dye, propidium iodide (PI), as described in previous studies [28,33]. LPS (10 ␮g/ml) was applied to hippocampal cultures with or without pretreatment with indirubin-3 -oxime. At the end of the LPS treatment, the culture medium was replaced with fresh serum-free medium containing 5 ␮g/ml of PI. Neuronal death was observed within 30–60 min of the addition of PI. PI-stained images were captured using a laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany) and the observed PI uptake areas were measured using confocal microscopy with LSM 510 software (release 3.2; Carl Zeiss). All data were background subtracted by using the fluorescence emission origination from a region on the insert containing no tissue. The data were evaluated for statistical significance with one-way ANOVA followed by Duncan’s multiple range test. The effects of pretreatment with indirubin-3 -oxime on microglial activation were tested. Indirubin-3 -oxime dosedependently suppressed LPS-induced nitrite release from microglial cells (Fig. 1A). Cell viability, as measured using the

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MTT assay, was not reduced at up to 2 ␮M. However, indirubin-3 oxime showed a degree of cytotoxicity to activated microglia at 4 ␮M (Fig. 1B). Indirubin-3 -oxime alone had no effects on basal NO release up to 2 ␮M but had a slight increase at 4 ␮M (Fig. 1C). Based on MTT assay, indirubin-3 -oxime alone seemed to reduce microglial cell viability at 4 ␮M but not at up to 2 ␮M (Fig. 1D). Thus, 2 ␮M was selected as the optimal dose of indirubin-3 -oxime for the remainder of the experiments. The range of doses tested was similar to that tested in previous studies [32,35]. In studies of SH-SY5Y neural cells, treatment with a dose of more than 3 ␮M resulted in a significant reduction in cell viability [35]. In studies with cerebellar granule neurons, indirubin-3 -oxime was shown to inhibit JNKs with a half inhibition dose of 0.8–1.4 ␮M [32]. To test the effects of indirubin-3 -oxime on the production of proinflammatory molecules, the amounts of TNF-␣, PGE2 , and IL-1␤ in the culture media of the primary microglia were measured using an ELISA. Indirubin-3 -oxime considerably reduced the LPS-induced production of these mediators (Fig. 2A). Intracellular ROS act as second messengers in the regulation of the LPSstimulated production of neurotoxic factors in microglia [22]. The ROS levels measured using DCFH-DA revealed that pretreatment with indirubin-3 -oxime decreased LPS-induced ROS production in microglia (Fig. 2B). Multiple signaling pathways, such as those involving mitogenactivated protein kinases (MAPKs), have been reported to be involved in LPS-induced signal transduction, which leads to the activation of NF-␬B and the induction of proinflammatory gene expression [1,11,15]. Several inflammatory stimuli, including bacterial LPS, activate the transcription factor NF-␬B in microglia [2]. NF-␬B is crucial in controlling the expression of many proinflammatory genes, and NF-␬B has been recognized as a potentially important drug target for the treatment of inflammatory diseases [3]. We found that indirubin-3 -oxime moderately inhibited the LPS-enhanced tyrosine phosphorylation of GSK-3␤, with a bare inhibition of p44/42 MAPK and pSAPK/JNK (Fig. 3A). LPS caused GSK-3␤ dephosphorylation at serine 9, which was not influenced by indirubin-3 -oxime (Fig. 3A). Additionally, indirubin-3 -oxime considerably inhibited the LPS-stimulated induction of NF-␬B activity in microglial cells (Fig. 3B). Phosphorylation at tyrosine 216 is responsible for activating regulation of GSK-3␤ whereas phosphorylation at serine 9 is critical for inactivation of GSK-3␤ [14,27]. Phosphorylation of GSK-3␤ at tyrosine 216 is increased in the brain in mouse models of Alzheimer’s disease, and this increase may be reduced by an injection of indirubin-3 -oxime [4]. GSK-3␤ has been implicated in NF-␬B activation of LPS-stimulated RAW264.7 mouse macrophage cells [16]. The CDK5/NF-␬B pathway has also been implicated in the LPS-induced expression of iNOS, IL-1␤, IL-6, and COX2 in RAW264.7 cells [5]. Taken together, our data suggest that the anti-inflammatory action of indirubin-3 -oxime in microglia is, in part, mediated by the inhibition of the GSK-3␤ signaling pathway, which enhances NF-␬B activation. Indirubin-3 -oxime, with antiapoptotic action, potently suppresses JNKs in cerebellar granule neurons [32]. Although more work is needed to determine whether indirubin-3 -oxime suppresses LPS-induced JNK activity by measuring substrate phosphorylation level, we observed no effects on phosphorylation of JNKs in the present study. Our recent work has demonstrated the effect of LPS exposure on neuronal damage in organotypic hippocampal slice cultures [21]. In comparison to untreated control slices, slice cultures exposed to LPS for 72 h displayed strong PI uptake in the hippocampus (Fig. 4A). PI uptake was markedly blocked in the presence of indirubin-3 oxime (Fig. 4B). This result indicates that indirubin-3 -oxime has protective effects against LPS-induced neurotoxicity. According to a recent study, GSK-3 inhibitors, including indirubin-3 -oxime, attenuate microglial migration and inflam-

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Fig. 2. Effect of indirubin-3 -oxime on the secretion of cytokines and the production of ROS. Primary microglial cultures were prepared in triplicate and stimulated with LPS (100 ng/ml) with or without pretreatment with indirubin-3 -oxime (IMX) at 2 ␮M. (A) After 24 h of incubation, the culture supernatants were assayed for TNF-␣, PGE2 , and IL-1␤ using ELISA. (B) After 30 min or 2 h of LPS incubation with or without pretreatment with the indicated concentrations of IMX, the levels of intracellular ROS in microglia were determined using DCFH-DA. Data are expressed as means ± SEM from triplicate assays. ## p < 0.001, # p < 0.05 vs. Control group; **p < 0.001, *p < 0.05 vs. LPS-only treated group.

mation, which is consistent with the results of this study [34]. With indirubin-3 -oxime, there is a discrepancy related to the effects of this compound as an inhibitor of activated microglial cells. Yuskaitis and colleagues reported that indirubin-3 -oxime was effective at suppressing the expression of iNOS, IL-6, but not COX-2, in BV2 microglial cells. By contrast, we observed significant suppression in the production of PGE2 , one of the major COX-2

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Fig. 3. Effect of indirubin-3 -oxime on signaling molecules in LPS-stimulated microglial cells. (A) Primary microglial cells were pretreated with indirubin-3 oxime (IMX) at 2 ␮M for 30 min before LPS was added. After stimulation with LPS for 30 min, whole cell lysates were analyzed by Western blotting. Representative blots from three independent experiments are shown. The band intensities were measured using a densitometer, and the phosphorylated protein levels were normalized against the total form of each kinase level. (B) BV2 cells were transfected, treated with the various experimental conditions assigned, and analyzed for NF␬B luciferase activity as described in the text. Data are expressed as means ± SEM from triplicate assays. ## p < 0.001 vs. control group; **p < 0.001, *p < 0.05 vs. LPS-only treated group.

products by indirubin-3 -oxime. In a report on the role of GSK-3 in NO and RANTES production, 6-bromo-indirubin-3 -monoxime also inhibited NO synthesis in microglia [10]. In contrast to our data, 6-bromo-indirubin-3 -monoxime increased LPS-elicited NF␬B activation in nuclear translocation [10]. These differences may be attributable to the differences in the nature of the cultured cells, and the differences in the chemical structures of the indirubin compounds used in each study. Nevertheless, our data together with those from the groups of Huang and Yuskaitis [10,34] consistently suggest an inhibitory role of indirubin-3 -oxime in microglial activation. In accordance with previous results [10,34], lithium chloride, a structurally distinct GSK-3 inhibitor, decreased LPSinduced NO production in microglial cells (Fig. 5). Altogether, these results may invite more detailed studies to confirm the role of GSK3␤ in LPS-induced microglial inflammation. In an effort to develop neuroprotective drugs, strategies to ameliorate the inflammatory microenvironment, which indirectly damage neurons via glial cell mediators, are promising [24]. Indirubin-3 -oxime represses diverse inflammatory mediators induced by LPS, including NO, TNF-␣, IL-1␤, PGE2 , intracellular

Fig. 4. Effect of indirubin-3 -oxime on LPS-induced hippocampal cell death. Organotypic hippocampal slice cultures were pretreated with indirubin-3 -oxime (IMX) at the indicated concentrations for 30 min before LPS was added to 10 ␮g/ml. After stimulation with LPS for 72 h, the culture medium was replaced with fresh serumfree medium containing PI. (A) PI fluorescence images. Scale bar = 500 ␮m. (B) Quantification of PI images. Data are expressed as the percentage of the LPS value (means ± SEM, n = 10–15 each). Means with the same letter are not significantly different. Differences were considered significant for p < 0.05.

Fig. 5. Effect of lithium chloride on nitrite production in microglial cells. Primary microglial cells were incubated in the absence (control) or presence of LPS. The cells were pretreated with the indicated amounts of lithium chloride for 30 min prior to the addition of LPS. Twenty-four hours after LPS was administered, the cultures were subjected to a nitrite assay Data are expressed as means ± SEM from triplicate assays. ## p < 0.001 vs. control group; **p < 0.001 vs. LPS-only treated group.

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