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Silibinin suppresses astroglial activation in a mouse model of acute Parkinson׳s disease by modulating the ERK and JNK signaling pathways
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Research Report
Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways Yujeong Lee, Hye Jeong Chun, Kyung Moon Lee, Young-Suk Jung, Jaewon Leen Department of Pharmacy, College of Pharmacy, Molecular Inflammation Research Center for Aging Intervention, Pusan National University, Busan 609-735, Republic of Korea
art i cle i nfo
ab st rac t
Article history:
Parkinson’s disease (PD) is the second-most common neurodegenerative disease after
Received 14 April 2015
Alzheimer’s disease, and is characterized by dopaminergic neuronal loss in midbrain. The
Received in revised form
MPTP-induced PD model has been well characterized by motor deficits and selective
24 July 2015
dopaminergic neuronal death accompanied by glial activation. Silibinin is a constituent of
Accepted 24 September 2015
silymarin, an extract of milk thistle seeds, and has been proposed to have hepatoprotective, anti-cancer, anti-oxidative, and neuroprotective effects. In the present study, the
Keywords: MPTP Astrocyte Parkinson's disease Silibinin Anti-inflammatory MAPK
authors studied the neuroprotective effects of silibinin in an acute MPTP model of PD. Silibinin was administered for 2 weeks, and then MPTP was administered to mice over 1 day (acute MPTP induced PD). Silibinin pretreatment effectively ameliorated motor dysfunction, dopaminergic neuronal loss, and glial activations caused by MPTP. In addition, an in vitro study demonstrated that silibinin suppressed astroglial activation and ERK and JNK phosphorylation in primary astrocytes in response to MPPþ treatment. These findings show silibinin protected dopaminergic neurons in an acute MPTP-induced mouse model of PD, and suggest its neuroprotective effects might be mediated by the suppression of astrocyte activation via the inhibition of ERK and JNK phosphorylation. In conclusion, the study indicates silibinin should be viewed as a potential treatment for PD and other neurodegenerative diseases associated with neuroinflammation. & 2015 Published by Elsevier B.V.
1.
that include tremors, rigidity, and bradykinesia (Olanow and
Introduction
Tatton, 1999). Although the etiology of PD is not understood, PD is a progressive neurodegenerative disorder characterized by
evidence suggests it involves oxidative stress, mitochondrial
selective dopaminergic neuronal loss and by clinical symptoms
dysfunction, environmental factors, susceptibility genes, and
Abbreviations: MPTP,
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD,
Parkinson's disease; MPPþ,
1-methyl-4-phenylpyridine;
TH, Tyrosine hydroxylase; GFAP, Glial fibrillary acidic protein; Iba-1, Ionized calcium binding adapter molecule-1 n Corresponding author. Fax: þ82 51 513 6754. E-mail address: [email protected] (J. Lee). http://dx.doi.org/10.1016/j.brainres.2015.09.029 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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aging (Dawson et al., 2010). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that induces Parkinson’s disease. MPTP can cross the blood-brain barrier and after doing so it acted upon by astroglial monoamine oxidase B (MAO-B) to form an active product 1-methyl-4-phenylpyridine (MPPþ). MPPþ is then selectively transported into dopaminergic neurons where it blocks mitochondrial complex Ι, and subsequently generates oxidative stress and neuroinflammation, which lead to dopaminergic neuronal death (Smeyne and Jackson-Lewis, 2005). It has been demonstrated astroglial homeostasis plays an important role in the regulation of dopaminergic neuronal degeneration and neuroprotection in PD (Rappold and Tieu, 2010). Glial cells like astrocytes are known to support and protect neurons under normal conditions (Ciesielska et al., 2009; Kurkowska-Jastrzebska et al., 1999), but under pathologic situations of neuronal loss/ damage, such as those, observed in PD and MPTP-induced neuronal loss (Deleidi and Gasser, 2013; Tansey and Goldberg, 2010), glial cells produce reactive oxygen and nitrogen species, inflammatory cytokines, and chemokines that cause neuroinflammation (Davalos et al., 2005; Glass et al., 2010; Hirsch and Hunot, 2009). Likewise, dopaminergic neuron degeneration is well associated with astrocyte and microglial activations in MPTP-induced PD models and other brain damage such as Alzheimer’s disease (Hunot and Hirsch, 2003). Therefore, the modulation of glial activation offers a possible target for treating PD-associated pathologies. Silibinin is a major active component of silymarin, which is obtained from the milk thistle (the chemical structure of silibinin is shown in Fig. 1A). Furthermore, silibinin and silymarin are known to hepatoprotective, anti-oxidant, anti-cancer, and neuroprotective effects, and several studies have shown silibinin has neuroprotective effects in models of ischemia, dementia, Alzheimer's disease, and PD (Baluchnejadmojarad et al., 2010; Neha et al., 2014; Raza et al., 2011; Yaghmaei et al., 2014). Previously, we demonstrated that co- and post-treatment of silibinin protected against dopaminergic neuronal loss in a subchronic
Fig. 1 – (A) The chemical structure of silibinin (IUPAC name (2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b] [1,4] dioxin-6-yl]chroman-4-one; C25H22O10) and (B) schematic of the in vivo experiment.
MPTP-induced mouse model of PD by stabilizing mitochondrial membrane potentials (Lee et al., 2015). In addition, it has been reported that silibinin protects brain against methamphetamine-induced cognitive deficits (Lu et al., 2010) and streptozotocin-induced memory loss (Lu et al., 2010). It has also been proposed that the neuroprotective effects of silibinin in a MPPþ induced rat model of PD involve its anti-apoptotic and anti-inflammatory properties (Geed et al., 2014). In a previous study, we showed that silibinin protects neurons in a subchronic MPTP-induced PD model, but we failed to observe microglial activation or the modulatory effects of silibinin on glial activations in this model. Therefore, in the present study, we focused on the modulatory effect of silibinin on neuroinflammation in an acute MPTPinduced mouse model of PD, in which neuroinflammatory responses are prominent.
2.
Results
2.1. Silibinin ameliorated motor disability in the MPTPinduced PD model To observe the effects of silibinin on motor dysfunction in our PD model, the rota-rod test was used to measure motor ability. Pre-training was performed such that all animals were able to maintain themselves on the rod for 180 s at 25 rpm. MPTP treated group exhibited motor dysfunctions, which included bradykinesia and tremor, at 6, 24, 48, and 72 h after last MPTP injection. However, silibinin 10 mg/kg group showed reduced levels of motor dysfunction as compared with the MPTP treated group at 48 and 72 h after MPTP injection (Fig. 2).
Fig. 2 – Silibinin prevented motor dysfunction in the acute MPTP-induced PD model. MPTP-induced motor disability was evaluated using the rota-rod test. Mice were pre-trained for 5 days to remain on the rod for at least 180 s. Overt motor dysfunction was observed in the MPTP treated group, but this was reduced in the silibinin treated group (10 mg/kg) as compared with the MPTP treated group at 48 h and 72 h after final MPTP treatment. Values shown are means7SEs (n ¼5–6 mice/group). **po0.01 vs. the naïve control group. #po0.05 vs. the MPTP group (the analysis was performed using ANOVA with Fisher’s PLSD procedure).
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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2.2. Silibinin protected dopaminergic neurons in the STR and SN in the MPTP-induced PD model To observe the neuroprotective effects of silibinin, we performed TH (Tyrosine hydroxylase, a marker of dopaminergic neurons) staining in STR (Striatum) and SN (Substantia nigra). TH levels were significantly lower in the STR (Fig. 3A) and SN (Fig. 3D) of the MPTP treated group than in the naïve control
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group. However, silibinin (1 or 10 mg/kg) pretreatment prevented these reductions in TH levels in STR (Fig. 3A) and SN (Fig. 3D). To confirm these results, we performed western blot for TH protein in STR. It was found that TH protein expression was significantly lower in the MPTP treated group than in the naïve control group, and that silibinin (1 or 10 mg/kg) pretreatment effectively blocked the TH losses observed in the MPTP treated group (Fig. 3B). Levels of TH expression were
Fig. 3 – Silibinin attenuated MPTP-induced dopaminergic neuronal loss in the acute PD model. TH immunostaining analyses were performed in STR (A) and SN (D). TH-positive immunostaining was significantly less in the MPTP treated group than in the naïve control group and more TH-positive neurons were observed in MPTPþsilibinin groups than in the MPTP treated group in STR and SN. (B) Western blot analysis was performed in STR homogenates, and the MPTP treated group showed lower TH levels than the naïve control group. However, silibinin pretreatment attenuated this TH level reduction. (C) Quantitative analysis was performed to confirm western blot results in the STR. (E) Quantitative analysis revealed that silibinin pretreatment effectively reduced the MPTP-induced loss of TH-positive dopaminergic neurons in the SN. Values shown are means7SEs (n¼ 5–6 mice/group). *po0.05 vs. the naïve control group, **po0.01 vs. the naïve control group, #po0.05 vs. the MPTP group, and ##po0.01 vs. the MPTP group (by ANOVA with Fisher's PLSD procedure). Scale bar¼100 μm. Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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quantified by densitometry, and TH immunostaining in SN was confirmed by counting numbers of dopaminergic neurons (Fig. 3E). The results obtained showed that silibinin pretreatment protected mice from MPTP-induced dopaminergic neuron loss.
2.3. Silibinin suppressed MPTP-induced glial activation in the STR and SN
associated with PD (Barcia, 2013). To observe glial activation in our PD model, we performed double immunohistochemistry using antibodies against glial fibrillary acidic protein (antiGFAP, an astrocyte marker) and ionized calcium binding adapter molecule-1 (anti-Iba-1, a microglial marker). Astrocyte and microglial activations were significantly elevated in the MPTP treated group vs. the naïve control group, but silibinin (10 mg/kg) pretreatment significantly inhibited astroglial and
Glia critically support neuron homeostasis, but conversely glial activation might promote the neurodegenerative processes
microglial activations by MPTP in STR (Fig. 4A and B) and SN (Fig. 4C and D).
Fig. 4 – Silibinin suppressed MPTP-induced glial activation in the STR and SN. (A) Brain sections of the STR were double immunostained with anti-GFAP and anti-Iba-1 antibodies (n ¼5–6 mice/group). The MPTP treated group showed more GFAP expression and Iba-1 expression in the STR than the naïve control group. Silibinin (10 mg/kg) pretreatment decreased glial activation induced by MPTP. (B) Fluorescence intensities of GFAP and Iba-1 in STR were analyzed by FV10-ASW program of FV10i FLOUVIEW confocal microscope. (C) Brain sections of the SN were double immunostained with anti-GFAP and anti-Iba-1 antibodies (n ¼5–6 mice/group). In the SN, MPTP administration elevated GFAP and Iba-1 expression vs. the naïve control group, and silibinin (10 mg/kg) pretreatment reduced MPTP-induced glial activation. (D) Fluorescence intensities of GFAP and Iba-1 in SN were analyzed by FV10-ASW program Values shown are means7SEs (n ¼5 mice/group). *po0.05 vs. the naïve control group, **po0.01 vs. the naïve control group, #po0.05 vs. the MPTP group, and ##po0.01 vs. the MPTP group (by ANOVA with Fisher’s PLSD procedure). Scale bar¼ 50 μm.
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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Fig. 4 – (continued)
2.4. Silibinin attenuated MPPþ-induced astrocyte activation in primary astrocytes by suppressing ERK and JNK signaling In order to confirm our in vivo results, we examined whether silibinin ameliorates astrocyte activation by MPPþ in primary astrocytes. MPPþ was found to significantly induce astrocytes activation, as indicated by elevated GFAP levels. However, silibinin (10 μM) pretreatment astrocytes attenuated GFAP elevation without affecting cell numbers (Fig. 5A). Phasecontrast images were used to confirm that cells were seeded at same densities. Western blot analysis was performed to evaluate GFAP protein expression levels in primary astrocytes (Fig. 5B). These results showed that the neuroprotective effects of silibinin were associated with the inhibition of astroglial activation. Furthermore, it has been reported that the activation of stress response kinases, such as, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), contribute to astroglial activation (Gadea et al., 2008; Lee et al., 2014; Son et al., 2009). To evaluated the effects of silibinin on ERK and JNK phosphorylation, primary astrocytes were pretreated with silibinin for 6 h and then with
MPPþ for 30 min. Western blot analysis demonstrated MPPþ induced ERK and JNK phosphorylation, and that silibinin (10 μM) pretreatment inhibited these phosphorylations of ERK and JNK in primary astrocytes (Fig. 5C). Quantifications of ERK and JNK expression by Western blotting confirmed that pretreatment with silibinin at 10 μM significantly attenuated MPPþ-induced ERK and JNK phosphorylation in primary astrocytes (Fig. 5C). In addition, to evaluate the effects of silibinin on neuroinflammation, primary astrocytes were pretreated with silibinin for 6 h and then with MPPþ for 24 h. We found that MPPþ caused elevated expression of cyclooxydatenase-2 (COX-2; a proinflammatory factor) and silibinin was effective to modulate COX-2 expression in MPPþ-treated astrocytes (Fig. 5D). Taken together with in vivo experiments, these findings suggest that silibinin ameliorates the neuroinflammation the current PD model.
3.
Discussion
Several previous studies have demonstrated that silymarin (a mixture of silibinin, isosilybinin, silydianin, and silychristin)
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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Fig. 5 – Silibinin attenuated MPPþ-induced GFAP expression and ERK and JNK phosphorylation in primary astrocytes. (A) MPPþ treated astrocytes showed greater GFAP expression than control astrocytes, but silibinin pretreatment suppressed this GFAP expression induced by MPPþ. (B) The western blot analysis confirmed that MPPþ increased GFAP level, and silibinin attenuated GFAP expression induced by MPPþ. (C) Western blotting revealed MPPþ significantly induced ERK and JNK activation, but that silibinin significantly inhibited phospho-ERK and JNK induction by MPPþ. A blot representative of three independent experiments that yielded similar results is shown. (D) Silibinin was effective to block elevated expression of COX-2 by MPPþ. Quantitative analysis was performed to confirm western blot analysis. Values shown are means7SEs (n ¼3). *po0.05 vs. the control cells and #po0.05 vs. MPPþ treated cells (by ANOVA with Fisher’s PLSD procedure). Scale bar¼ 50 μm.
ameliorates memory deficits by down-regulating glutathione levels in dementia mice model (Neha et al., 2014), reducing Aβ plaque accumulation, decreasing APP mRNA levels in an Alzheimer’s disease model (Yaghmaei et al., 2014), protecting neurons due to its anti-oxidant effects in a rat model of ischemia (Raza et al., 2011), and by protecting neurons against the effects of 6-hydroxydopamine (6-OHDA) in a PD model (Baluchnejadmojarad et al., 2010) and against MPTP in a PD model (Perez et al., 2014). Silibinin has also been reported to protect neurons by regulating Akt/mTor signaling in a SD (Sprague Dawley) rat model of ischemia (Wang et al., 2012), and to have beneficial effects in a streptozotocininduced memory impairment model by modulating energy metabolism and cholinergic function (Tota et al., 2011). Neuroinflammation and the activations of astro- and microglia are closely associated with neurodegeneration and neuropathologies (Kurkowska-Jastrzebska et al., 1999; McGeer and McGeer, 2008). In a previous study, it was demonstrated that a high dose of silibinin (200 mg/kg of oral administration) protected dopaminergic neurons and showed anti-inflammatory and anti-apoptotic activities in an MPPþ injected SD rat model (Geed et al., 2014). It is generally accepted that the neuroprotective effects of natural phytochemicals in neurodegenerative models, including MPTPinduced PD mouse models, are mediated via the amelioration of neuroinflammation (Choi et al., 2012; Lee et al., 2014; Moon et al., 2009). However, in a recent study, we found low doses
silibinin (1 mg/kg and 10 mg/kg) had neuroprotective effects in a MPTP-induced PD model and that these neuroprotective effects were mediated by the stabilization of mitochondrial membrane potentials in neurons and not by the modulation of neuroinflammation (Lee et al., 2015). Therefore, the present study was undertaken to investigate the modulatory roles of silibinin on neuroinflammation in an acute MPTP-induced mouse model of PD, in which neuroinflammatory responses predominate. In our previous study, MPTP-induced microglial activation was not observed in the STR or SN, which was attributed to a 5 day sub-chronic MPTP administration schedule, whereas in the present study, the administration of four injections in one day significantly elevated glial activations in the STR and SN. In addition, in the present study, we found that 2-weeks silibinin pretreatment (1 or 10 mg/kg per one day) effectively blocked MPTP-induced neuroinflammation, and this finding was supported by the observation that silibinin pretreatment attenuated GFAP activation induced by MPPþ in primary astrocytes. MPTP is a neurotoxic chemical that selectively induces dopaminergic neuron loss. MPTP can cross the blood brain barrier, and is metabolized to MPPþ by MAO-B in astrocytes. Furthermore, MPPþ inhibits mitochondrial complex Ι, generates oxidative stress, induces dopaminergic neuronal death, and causes reactive gliosis (Hirsch et al., 2003; Kopin, 1992; Liu and Hong, 2003; Liu et al., 2003). However, different MPTP administration schedules resulted in different mouse
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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behaviors and inflammation. In the present study, acute MPTP administration caused dramatic motor dysfunction, as determined by rota-rod analysis. In addition, more prominent glial activation was observed after acute MPTP administration, and interestingly, we observed that silibinin decreased MPTP-induced glial activations in the STR and SN. In the present study, studies of primary astrocyte cultures confirmed the toxic metabolite MPPþ induces astrocyte activation without affecting cell survival or proliferation, and that silibinin pretreatment effectively inhibited glial activation induced by MPPþ (Fig. 5). It has been reported that ERK and JNK are activated by MPPþ in SH-SY5Y cells, primary cultured astrocytes, and SH-EP1 cells (Gomez-Santos et al., 2002; Kim et al., 2013; Wang et al., 2010; Yang et al., 2010). Furthermore, it has been reported that the activations of stress response kinases contribute to astroglial activation. For example, in a previous study, it was demonstrated stress response kinases, including ERK and JNK, are phosphorylated by astrocyte activation, and that ERK signaling plays an important role in the proliferation of both immortalized and primary astrocytes (McLennan et al., 2008). Moreover, it was shown that endothelin-1 mediated GFAP expression was prevented by the combination of inhibitors of ERK and JNK, but not by the individual inhibitors (Gadea et al., 2008). This suggests that suppressive effects of silibinin on GFAP expression might be well-related with down-modulation of both ERK and JNK. ERK and JNK are mitogen-activated protein kinases (MAPKs), and are activated by many stimuli, such as, mitogenic signals, cytokines, cellular stress, and antigen receptor ligation (Ip and Davis, 1998; Sakata et al., 1999), and the ERK and JNK pathways are known to play major regulatory roles in cellular processes related to PD (Anderson et al., 2007; Peng and Andersen, 2003; Peterson and Flood, 2012). Taken together, our findings suggest that silibinin ameliorates the type of elevated neuroinflammation observed in PD. In the present study, silibinin ameliorates motor dysfunction and dopaminergic neuronal loss, and inhibited astroglial activation in our acute MPTP-induced PD model. Furthermore, the study shows MPPþ induced astrocyte activation and silibinin pretreatment attenuated astrocyte activation by inhibiting the ERK and JNK signaling pathway. In our previous study, we concluded that silibinin be considered a potential therapeutic for treating PD, because its neuroprotective effects were found to be due to the stabilization of neuronal MMPs (Lee et al., 2015). Overall, our findings indicate that silibinin has multi-therapeutic potential for the treatment and prevention of PD and other neurodegenerative disease.
4.
Experimental procedures
4.1.
Reagents
(St. Louis, MO). Alexa Flour 488 and 568 were purchased from Invitrogen (Eugene, OR). Western blot detection reagent (ECL solution) was obtained from Advansta (Menlo Park, CA).
4.2.
Animals and drug administration
Male C57B/6 mice (7 weeks old, weight 18–21 g) were obtained from Daehan Biolink Co. Ltd. (Chungbuk, South Korea). Animals were housed under temperature- and lightcontrolled conditions (20–23 1C under a 12 h light/dark cycle), provided food and water ad libitum, and randomly allocated to four groups of 10–12 animals; a naïve control group, a MPTP treated group (MPTPþvehicle), or one of two MPTP plus silibinin pretreated groups (MPTPþ1 mg/kg, MPTPþ10 mg/ kg). All animals were acclimatized for 1 week prior to drug administration. Silibinin (1 or 10 mg/kg of body weight, dissolved in phosphate-buffered saline (PBS) containing 5% ethanol and 2% Tween 20) was injected into mice (i.p.) for 14 days and then on the 15th day MPTP was administered intraperitoneally (i.p.) at 20 mg/kg of body weight four times in one day with 2 h intervals. Mice in the naïve control group and in the MPTP treated group were administered the same volume of PBS containing 5% ethanol and 2% Tween 20 (vehicle). A schematic of in vivo administrations is provided in Fig. 1B. The animal protocol used in this study was reviewed and approved by the Pusan National University Institutional Animal Care Committee (PNU-IACUC; Approval Number PNU-2013-0392).
4.3.
Motor performance testing
Motor performance was performed using a rota-rod apparatus as previously described (Borlongan et al., 1995). All mice were pre-trained for 5 days to ensure they could maintain themselves on the rod for 180 s. Training was performed using four consecutive runs at a rod speed of 25 rpm. All mice were tested 6, 24, 48, and 72 h after final MPTP administration.
4.4.
Tissue preparation
For histological studies, mice were anesthetized with ethyl ether and perfused intracardially with 0.1 M PBS (pH 7.4) containing 0.9% NaCl and then with 0.1 M PBS containing 4% paraformaldehyde. Brains were removed, placed in the same paraformaldehyde fixation solution at 4 1C overnight, and transferred to a 30% sucrose solution. Cryoprotected brains were sectioned serially at 40 μm in the coronal plane using a freezing microtome (MICROM, Walldorf, Germany), and sections were stored at 4 1C in Dulbecco’s phosphatebuffered saline (DPBS) solution containing 0.1% sodium azide.
4.5.
3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridine (MPPþ), and silibinin [(2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4] dioxin-6-yl] chroman-4-one; C25H22O10 ] were obtained from Sigma-Aldrich
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DAB immunostaining
Brain sections were treated with 0.6% H2O2 in Tris-buffered saline (TBS; pH 7.5) to quench endogenous peroxidase activity, blocked in TBS/0.1% Triton X-100/3% goat serum (TBS-TS) for 30 min, and incubated with primary antibody; anti-TH antibody (mouse monoclonal (1:1000), Chemicon, Temecula, CA) in TBS-TS at 4 1C. Sections were further processed using appropriate biotinylated secondary goat anti-mouse IgG
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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antibodies (1:400; Vector Laboratories, Burlingame, CA) at room temperature for 3 h, incubated in ABC solution (Vectastain Elite ABC Kit, Vector Laboratories) at room temperature for 1 h, and developed using diaminobenzidine (DAB) solution. Images were obtained using a Nikon ECLIPSE TE 2000-U microscope (Nikon, Tokyo, Japan).
4.6.
Double-label immunostaining
For double-label fluorescence immunostaining, brain sections were blocked with TBS-TS for 30 min at room temperature, and incubated with primary antibodies, that is, anti-GFAP (mouse polyclonal (1:500), Cell Signaling, MA) and anti-Iba-1 antibody (rabbit polyclonal (1:500), Wako, Tokyo, Japan) in TBS-TS at 4 1C overnight. Sections were then washed with TBS, incubated with anti-mouse IgG labeled with Alexa Fluor 488 and with anti-rabbit IgG labeled with Alexa Flour 568 for 3 h at room temperature. Images were obtained using a FV10i FLOUVIEW Confocal Microscope (Olympus, Tokyo, Japan). Zstacked images of 20 consecutive brain sections (of thickness about 2 mm taken from the brain section of 40 mm) were obtained using a 60 objective.
4.7.
Primary astrocyte cultures
Primary astrocyte cultures were established using a Sprague Dawley (SD) rat cortex obtained at postnatal day (PND) 1 or 2 (Daehan Biolink Co. Ltd., Chungbuk, South Korea). Briefly, cortices were dissected and diffused in ice-cold Hanks' balanced salt solution (HBSS; Welgene). Cells were treated with 0.25% trypsin for 30 min at room temperature, washed with HBSS, mechanically dissociated, and plated in Dulbecco's modified Eagle's medium (nutrient mixture F-12 (DMEM/ F12) medium containing 10% FBS and 1% penicillin–streptomycin) on a poly-L-lysine-coated plastic culture dishes. Experiments were performed using 14–21 day cultures.
4.8.
Immunocytochemistry
Primary astrocytes were seeded in 60 φ poly-L-lysine-coated plastic culture dishes, and pretreated the following day with silibinin (Vehicle, 0.1, 1, and 10 mM) for 6 h, and then treated with MPPþ 500 μM for 24 h, washed with PBS, and fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 min at 37 1C. They were then blocked with TBS-TS for 30 min, incubated with primary antibody overnight at 4 1C, washed, and incubated with anti-mouse IgG labeled with Alexa Fluor 488 for 3 h at room temperature. Images were obtained using a FV10i FLOUVIEW Confocal Microscope (Olympus).
4.9.
Western blot analysis
After experimental treatment, cell or tissue homogenates were solubilized in SDS-polyacrylamide gel electrophoresis sample buffer, and protein concentrations were determined using a Bio-Rad (Hercules, CA) protein assay kit with bovine serum albumin as the standard. Total protein equivalents in each sample (20 μg per lane) were then separated in 10% SDSpolyacrylamide gels and electrophoretically transferred to Immobilon-PSQ transfer membranes (Millipore, Bedford,
MA). Membranes were immediately placed into a blocking solution (5% nonfat milk) at room temperature for 30 min and then incubated with diluted primary antibodies: GFAP (rabbit; 1:500; Cell signaling), pJNK (mouse; 1:500; Santa Cruz Biotechnology), JNK (rabbit; 1:500, Cell signaling), pERK (mouse; 1:500, Cell signaling), ERK (rabbit; 1:500; Cell signaling), COX-2 (rabbit; 1:1000; Santa Cruz Biotechnology), and β-actin (mouse; 1:5000; Sigma) in TBS-T (Tris–HCl-based buffer with 0.2% Tween 20, pH 7.5) at 4 1C overnight. On the next day, membrane were washed for 10 min and incubated with secondary antibody, polyclonal anti-rabbit antibody or monoclonal anti-mouse antibody (1:10000; Santa Cruz Biotechnology), in TBS-T buffer at room temperature for 2 h. Horseradish peroxidase (HRP)-conjugated secondary antibody labeling was detected with an enhanced chemiluminescence (ECL) and cooled CCD camera system ATTO Ez-Capture (Atto Corp., Tokyo, Japan) and then quantified by densitometry. Prestained blue markers were used for molecular weight determinations.
4.10.
Statistical analysis
The significances of differences between groups were determined by analysis of variance (ANOVA) with Fisher’s protected least significant difference (PLSD) test. P values of o0.05 were considered statistically significant. Analyses were performed using Statview software (Version 5.0.1., SAS Institute Inc., Cary, NC).
Conflict of interest statement The authors have no potential conflict of interest to declare.
Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP) (Grant no. 2009-0083538). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (No. NRF-2013R1A2A2A01067388).
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Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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Research Report
Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways Yujeong Lee, Hye Jeong Chun, Kyung Moon Lee, Young-Suk Jung, Jaewon Leen Department of Pharmacy, College of Pharmacy, Molecular Inflammation Research Center for Aging Intervention, Pusan National University, Busan 609-735, Republic of Korea
art i cle i nfo
ab st rac t
Article history:
Parkinson’s disease (PD) is the second-most common neurodegenerative disease after
Received 14 April 2015
Alzheimer’s disease, and is characterized by dopaminergic neuronal loss in midbrain. The
Received in revised form
MPTP-induced PD model has been well characterized by motor deficits and selective
24 July 2015
dopaminergic neuronal death accompanied by glial activation. Silibinin is a constituent of
Accepted 24 September 2015
silymarin, an extract of milk thistle seeds, and has been proposed to have hepatoprotective, anti-cancer, anti-oxidative, and neuroprotective effects. In the present study, the
Keywords: MPTP Astrocyte Parkinson's disease Silibinin Anti-inflammatory MAPK
authors studied the neuroprotective effects of silibinin in an acute MPTP model of PD. Silibinin was administered for 2 weeks, and then MPTP was administered to mice over 1 day (acute MPTP induced PD). Silibinin pretreatment effectively ameliorated motor dysfunction, dopaminergic neuronal loss, and glial activations caused by MPTP. In addition, an in vitro study demonstrated that silibinin suppressed astroglial activation and ERK and JNK phosphorylation in primary astrocytes in response to MPPþ treatment. These findings show silibinin protected dopaminergic neurons in an acute MPTP-induced mouse model of PD, and suggest its neuroprotective effects might be mediated by the suppression of astrocyte activation via the inhibition of ERK and JNK phosphorylation. In conclusion, the study indicates silibinin should be viewed as a potential treatment for PD and other neurodegenerative diseases associated with neuroinflammation. & 2015 Published by Elsevier B.V.
1.
that include tremors, rigidity, and bradykinesia (Olanow and
Introduction
Tatton, 1999). Although the etiology of PD is not understood, PD is a progressive neurodegenerative disorder characterized by
evidence suggests it involves oxidative stress, mitochondrial
selective dopaminergic neuronal loss and by clinical symptoms
dysfunction, environmental factors, susceptibility genes, and
Abbreviations: MPTP,
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD,
Parkinson's disease; MPPþ,
1-methyl-4-phenylpyridine;
TH, Tyrosine hydroxylase; GFAP, Glial fibrillary acidic protein; Iba-1, Ionized calcium binding adapter molecule-1 n Corresponding author. Fax: þ82 51 513 6754. E-mail address: [email protected] (J. Lee). http://dx.doi.org/10.1016/j.brainres.2015.09.029 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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aging (Dawson et al., 2010). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that induces Parkinson’s disease. MPTP can cross the blood-brain barrier and after doing so it acted upon by astroglial monoamine oxidase B (MAO-B) to form an active product 1-methyl-4-phenylpyridine (MPPþ). MPPþ is then selectively transported into dopaminergic neurons where it blocks mitochondrial complex Ι, and subsequently generates oxidative stress and neuroinflammation, which lead to dopaminergic neuronal death (Smeyne and Jackson-Lewis, 2005). It has been demonstrated astroglial homeostasis plays an important role in the regulation of dopaminergic neuronal degeneration and neuroprotection in PD (Rappold and Tieu, 2010). Glial cells like astrocytes are known to support and protect neurons under normal conditions (Ciesielska et al., 2009; Kurkowska-Jastrzebska et al., 1999), but under pathologic situations of neuronal loss/ damage, such as those, observed in PD and MPTP-induced neuronal loss (Deleidi and Gasser, 2013; Tansey and Goldberg, 2010), glial cells produce reactive oxygen and nitrogen species, inflammatory cytokines, and chemokines that cause neuroinflammation (Davalos et al., 2005; Glass et al., 2010; Hirsch and Hunot, 2009). Likewise, dopaminergic neuron degeneration is well associated with astrocyte and microglial activations in MPTP-induced PD models and other brain damage such as Alzheimer’s disease (Hunot and Hirsch, 2003). Therefore, the modulation of glial activation offers a possible target for treating PD-associated pathologies. Silibinin is a major active component of silymarin, which is obtained from the milk thistle (the chemical structure of silibinin is shown in Fig. 1A). Furthermore, silibinin and silymarin are known to hepatoprotective, anti-oxidant, anti-cancer, and neuroprotective effects, and several studies have shown silibinin has neuroprotective effects in models of ischemia, dementia, Alzheimer's disease, and PD (Baluchnejadmojarad et al., 2010; Neha et al., 2014; Raza et al., 2011; Yaghmaei et al., 2014). Previously, we demonstrated that co- and post-treatment of silibinin protected against dopaminergic neuronal loss in a subchronic
Fig. 1 – (A) The chemical structure of silibinin (IUPAC name (2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b] [1,4] dioxin-6-yl]chroman-4-one; C25H22O10) and (B) schematic of the in vivo experiment.
MPTP-induced mouse model of PD by stabilizing mitochondrial membrane potentials (Lee et al., 2015). In addition, it has been reported that silibinin protects brain against methamphetamine-induced cognitive deficits (Lu et al., 2010) and streptozotocin-induced memory loss (Lu et al., 2010). It has also been proposed that the neuroprotective effects of silibinin in a MPPþ induced rat model of PD involve its anti-apoptotic and anti-inflammatory properties (Geed et al., 2014). In a previous study, we showed that silibinin protects neurons in a subchronic MPTP-induced PD model, but we failed to observe microglial activation or the modulatory effects of silibinin on glial activations in this model. Therefore, in the present study, we focused on the modulatory effect of silibinin on neuroinflammation in an acute MPTPinduced mouse model of PD, in which neuroinflammatory responses are prominent.
2.
Results
2.1. Silibinin ameliorated motor disability in the MPTPinduced PD model To observe the effects of silibinin on motor dysfunction in our PD model, the rota-rod test was used to measure motor ability. Pre-training was performed such that all animals were able to maintain themselves on the rod for 180 s at 25 rpm. MPTP treated group exhibited motor dysfunctions, which included bradykinesia and tremor, at 6, 24, 48, and 72 h after last MPTP injection. However, silibinin 10 mg/kg group showed reduced levels of motor dysfunction as compared with the MPTP treated group at 48 and 72 h after MPTP injection (Fig. 2).
Fig. 2 – Silibinin prevented motor dysfunction in the acute MPTP-induced PD model. MPTP-induced motor disability was evaluated using the rota-rod test. Mice were pre-trained for 5 days to remain on the rod for at least 180 s. Overt motor dysfunction was observed in the MPTP treated group, but this was reduced in the silibinin treated group (10 mg/kg) as compared with the MPTP treated group at 48 h and 72 h after final MPTP treatment. Values shown are means7SEs (n ¼5–6 mice/group). **po0.01 vs. the naïve control group. #po0.05 vs. the MPTP group (the analysis was performed using ANOVA with Fisher’s PLSD procedure).
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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2.2. Silibinin protected dopaminergic neurons in the STR and SN in the MPTP-induced PD model To observe the neuroprotective effects of silibinin, we performed TH (Tyrosine hydroxylase, a marker of dopaminergic neurons) staining in STR (Striatum) and SN (Substantia nigra). TH levels were significantly lower in the STR (Fig. 3A) and SN (Fig. 3D) of the MPTP treated group than in the naïve control
3
group. However, silibinin (1 or 10 mg/kg) pretreatment prevented these reductions in TH levels in STR (Fig. 3A) and SN (Fig. 3D). To confirm these results, we performed western blot for TH protein in STR. It was found that TH protein expression was significantly lower in the MPTP treated group than in the naïve control group, and that silibinin (1 or 10 mg/kg) pretreatment effectively blocked the TH losses observed in the MPTP treated group (Fig. 3B). Levels of TH expression were
Fig. 3 – Silibinin attenuated MPTP-induced dopaminergic neuronal loss in the acute PD model. TH immunostaining analyses were performed in STR (A) and SN (D). TH-positive immunostaining was significantly less in the MPTP treated group than in the naïve control group and more TH-positive neurons were observed in MPTPþsilibinin groups than in the MPTP treated group in STR and SN. (B) Western blot analysis was performed in STR homogenates, and the MPTP treated group showed lower TH levels than the naïve control group. However, silibinin pretreatment attenuated this TH level reduction. (C) Quantitative analysis was performed to confirm western blot results in the STR. (E) Quantitative analysis revealed that silibinin pretreatment effectively reduced the MPTP-induced loss of TH-positive dopaminergic neurons in the SN. Values shown are means7SEs (n¼ 5–6 mice/group). *po0.05 vs. the naïve control group, **po0.01 vs. the naïve control group, #po0.05 vs. the MPTP group, and ##po0.01 vs. the MPTP group (by ANOVA with Fisher's PLSD procedure). Scale bar¼100 μm. Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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quantified by densitometry, and TH immunostaining in SN was confirmed by counting numbers of dopaminergic neurons (Fig. 3E). The results obtained showed that silibinin pretreatment protected mice from MPTP-induced dopaminergic neuron loss.
2.3. Silibinin suppressed MPTP-induced glial activation in the STR and SN
associated with PD (Barcia, 2013). To observe glial activation in our PD model, we performed double immunohistochemistry using antibodies against glial fibrillary acidic protein (antiGFAP, an astrocyte marker) and ionized calcium binding adapter molecule-1 (anti-Iba-1, a microglial marker). Astrocyte and microglial activations were significantly elevated in the MPTP treated group vs. the naïve control group, but silibinin (10 mg/kg) pretreatment significantly inhibited astroglial and
Glia critically support neuron homeostasis, but conversely glial activation might promote the neurodegenerative processes
microglial activations by MPTP in STR (Fig. 4A and B) and SN (Fig. 4C and D).
Fig. 4 – Silibinin suppressed MPTP-induced glial activation in the STR and SN. (A) Brain sections of the STR were double immunostained with anti-GFAP and anti-Iba-1 antibodies (n ¼5–6 mice/group). The MPTP treated group showed more GFAP expression and Iba-1 expression in the STR than the naïve control group. Silibinin (10 mg/kg) pretreatment decreased glial activation induced by MPTP. (B) Fluorescence intensities of GFAP and Iba-1 in STR were analyzed by FV10-ASW program of FV10i FLOUVIEW confocal microscope. (C) Brain sections of the SN were double immunostained with anti-GFAP and anti-Iba-1 antibodies (n ¼5–6 mice/group). In the SN, MPTP administration elevated GFAP and Iba-1 expression vs. the naïve control group, and silibinin (10 mg/kg) pretreatment reduced MPTP-induced glial activation. (D) Fluorescence intensities of GFAP and Iba-1 in SN were analyzed by FV10-ASW program Values shown are means7SEs (n ¼5 mice/group). *po0.05 vs. the naïve control group, **po0.01 vs. the naïve control group, #po0.05 vs. the MPTP group, and ##po0.01 vs. the MPTP group (by ANOVA with Fisher’s PLSD procedure). Scale bar¼ 50 μm.
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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Fig. 4 – (continued)
2.4. Silibinin attenuated MPPþ-induced astrocyte activation in primary astrocytes by suppressing ERK and JNK signaling In order to confirm our in vivo results, we examined whether silibinin ameliorates astrocyte activation by MPPþ in primary astrocytes. MPPþ was found to significantly induce astrocytes activation, as indicated by elevated GFAP levels. However, silibinin (10 μM) pretreatment astrocytes attenuated GFAP elevation without affecting cell numbers (Fig. 5A). Phasecontrast images were used to confirm that cells were seeded at same densities. Western blot analysis was performed to evaluate GFAP protein expression levels in primary astrocytes (Fig. 5B). These results showed that the neuroprotective effects of silibinin were associated with the inhibition of astroglial activation. Furthermore, it has been reported that the activation of stress response kinases, such as, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), contribute to astroglial activation (Gadea et al., 2008; Lee et al., 2014; Son et al., 2009). To evaluated the effects of silibinin on ERK and JNK phosphorylation, primary astrocytes were pretreated with silibinin for 6 h and then with
MPPþ for 30 min. Western blot analysis demonstrated MPPþ induced ERK and JNK phosphorylation, and that silibinin (10 μM) pretreatment inhibited these phosphorylations of ERK and JNK in primary astrocytes (Fig. 5C). Quantifications of ERK and JNK expression by Western blotting confirmed that pretreatment with silibinin at 10 μM significantly attenuated MPPþ-induced ERK and JNK phosphorylation in primary astrocytes (Fig. 5C). In addition, to evaluate the effects of silibinin on neuroinflammation, primary astrocytes were pretreated with silibinin for 6 h and then with MPPþ for 24 h. We found that MPPþ caused elevated expression of cyclooxydatenase-2 (COX-2; a proinflammatory factor) and silibinin was effective to modulate COX-2 expression in MPPþ-treated astrocytes (Fig. 5D). Taken together with in vivo experiments, these findings suggest that silibinin ameliorates the neuroinflammation the current PD model.
3.
Discussion
Several previous studies have demonstrated that silymarin (a mixture of silibinin, isosilybinin, silydianin, and silychristin)
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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Fig. 5 – Silibinin attenuated MPPþ-induced GFAP expression and ERK and JNK phosphorylation in primary astrocytes. (A) MPPþ treated astrocytes showed greater GFAP expression than control astrocytes, but silibinin pretreatment suppressed this GFAP expression induced by MPPþ. (B) The western blot analysis confirmed that MPPþ increased GFAP level, and silibinin attenuated GFAP expression induced by MPPþ. (C) Western blotting revealed MPPþ significantly induced ERK and JNK activation, but that silibinin significantly inhibited phospho-ERK and JNK induction by MPPþ. A blot representative of three independent experiments that yielded similar results is shown. (D) Silibinin was effective to block elevated expression of COX-2 by MPPþ. Quantitative analysis was performed to confirm western blot analysis. Values shown are means7SEs (n ¼3). *po0.05 vs. the control cells and #po0.05 vs. MPPþ treated cells (by ANOVA with Fisher’s PLSD procedure). Scale bar¼ 50 μm.
ameliorates memory deficits by down-regulating glutathione levels in dementia mice model (Neha et al., 2014), reducing Aβ plaque accumulation, decreasing APP mRNA levels in an Alzheimer’s disease model (Yaghmaei et al., 2014), protecting neurons due to its anti-oxidant effects in a rat model of ischemia (Raza et al., 2011), and by protecting neurons against the effects of 6-hydroxydopamine (6-OHDA) in a PD model (Baluchnejadmojarad et al., 2010) and against MPTP in a PD model (Perez et al., 2014). Silibinin has also been reported to protect neurons by regulating Akt/mTor signaling in a SD (Sprague Dawley) rat model of ischemia (Wang et al., 2012), and to have beneficial effects in a streptozotocininduced memory impairment model by modulating energy metabolism and cholinergic function (Tota et al., 2011). Neuroinflammation and the activations of astro- and microglia are closely associated with neurodegeneration and neuropathologies (Kurkowska-Jastrzebska et al., 1999; McGeer and McGeer, 2008). In a previous study, it was demonstrated that a high dose of silibinin (200 mg/kg of oral administration) protected dopaminergic neurons and showed anti-inflammatory and anti-apoptotic activities in an MPPþ injected SD rat model (Geed et al., 2014). It is generally accepted that the neuroprotective effects of natural phytochemicals in neurodegenerative models, including MPTPinduced PD mouse models, are mediated via the amelioration of neuroinflammation (Choi et al., 2012; Lee et al., 2014; Moon et al., 2009). However, in a recent study, we found low doses
silibinin (1 mg/kg and 10 mg/kg) had neuroprotective effects in a MPTP-induced PD model and that these neuroprotective effects were mediated by the stabilization of mitochondrial membrane potentials in neurons and not by the modulation of neuroinflammation (Lee et al., 2015). Therefore, the present study was undertaken to investigate the modulatory roles of silibinin on neuroinflammation in an acute MPTP-induced mouse model of PD, in which neuroinflammatory responses predominate. In our previous study, MPTP-induced microglial activation was not observed in the STR or SN, which was attributed to a 5 day sub-chronic MPTP administration schedule, whereas in the present study, the administration of four injections in one day significantly elevated glial activations in the STR and SN. In addition, in the present study, we found that 2-weeks silibinin pretreatment (1 or 10 mg/kg per one day) effectively blocked MPTP-induced neuroinflammation, and this finding was supported by the observation that silibinin pretreatment attenuated GFAP activation induced by MPPþ in primary astrocytes. MPTP is a neurotoxic chemical that selectively induces dopaminergic neuron loss. MPTP can cross the blood brain barrier, and is metabolized to MPPþ by MAO-B in astrocytes. Furthermore, MPPþ inhibits mitochondrial complex Ι, generates oxidative stress, induces dopaminergic neuronal death, and causes reactive gliosis (Hirsch et al., 2003; Kopin, 1992; Liu and Hong, 2003; Liu et al., 2003). However, different MPTP administration schedules resulted in different mouse
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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behaviors and inflammation. In the present study, acute MPTP administration caused dramatic motor dysfunction, as determined by rota-rod analysis. In addition, more prominent glial activation was observed after acute MPTP administration, and interestingly, we observed that silibinin decreased MPTP-induced glial activations in the STR and SN. In the present study, studies of primary astrocyte cultures confirmed the toxic metabolite MPPþ induces astrocyte activation without affecting cell survival or proliferation, and that silibinin pretreatment effectively inhibited glial activation induced by MPPþ (Fig. 5). It has been reported that ERK and JNK are activated by MPPþ in SH-SY5Y cells, primary cultured astrocytes, and SH-EP1 cells (Gomez-Santos et al., 2002; Kim et al., 2013; Wang et al., 2010; Yang et al., 2010). Furthermore, it has been reported that the activations of stress response kinases contribute to astroglial activation. For example, in a previous study, it was demonstrated stress response kinases, including ERK and JNK, are phosphorylated by astrocyte activation, and that ERK signaling plays an important role in the proliferation of both immortalized and primary astrocytes (McLennan et al., 2008). Moreover, it was shown that endothelin-1 mediated GFAP expression was prevented by the combination of inhibitors of ERK and JNK, but not by the individual inhibitors (Gadea et al., 2008). This suggests that suppressive effects of silibinin on GFAP expression might be well-related with down-modulation of both ERK and JNK. ERK and JNK are mitogen-activated protein kinases (MAPKs), and are activated by many stimuli, such as, mitogenic signals, cytokines, cellular stress, and antigen receptor ligation (Ip and Davis, 1998; Sakata et al., 1999), and the ERK and JNK pathways are known to play major regulatory roles in cellular processes related to PD (Anderson et al., 2007; Peng and Andersen, 2003; Peterson and Flood, 2012). Taken together, our findings suggest that silibinin ameliorates the type of elevated neuroinflammation observed in PD. In the present study, silibinin ameliorates motor dysfunction and dopaminergic neuronal loss, and inhibited astroglial activation in our acute MPTP-induced PD model. Furthermore, the study shows MPPþ induced astrocyte activation and silibinin pretreatment attenuated astrocyte activation by inhibiting the ERK and JNK signaling pathway. In our previous study, we concluded that silibinin be considered a potential therapeutic for treating PD, because its neuroprotective effects were found to be due to the stabilization of neuronal MMPs (Lee et al., 2015). Overall, our findings indicate that silibinin has multi-therapeutic potential for the treatment and prevention of PD and other neurodegenerative disease.
4.
Experimental procedures
4.1.
Reagents
(St. Louis, MO). Alexa Flour 488 and 568 were purchased from Invitrogen (Eugene, OR). Western blot detection reagent (ECL solution) was obtained from Advansta (Menlo Park, CA).
4.2.
Animals and drug administration
Male C57B/6 mice (7 weeks old, weight 18–21 g) were obtained from Daehan Biolink Co. Ltd. (Chungbuk, South Korea). Animals were housed under temperature- and lightcontrolled conditions (20–23 1C under a 12 h light/dark cycle), provided food and water ad libitum, and randomly allocated to four groups of 10–12 animals; a naïve control group, a MPTP treated group (MPTPþvehicle), or one of two MPTP plus silibinin pretreated groups (MPTPþ1 mg/kg, MPTPþ10 mg/ kg). All animals were acclimatized for 1 week prior to drug administration. Silibinin (1 or 10 mg/kg of body weight, dissolved in phosphate-buffered saline (PBS) containing 5% ethanol and 2% Tween 20) was injected into mice (i.p.) for 14 days and then on the 15th day MPTP was administered intraperitoneally (i.p.) at 20 mg/kg of body weight four times in one day with 2 h intervals. Mice in the naïve control group and in the MPTP treated group were administered the same volume of PBS containing 5% ethanol and 2% Tween 20 (vehicle). A schematic of in vivo administrations is provided in Fig. 1B. The animal protocol used in this study was reviewed and approved by the Pusan National University Institutional Animal Care Committee (PNU-IACUC; Approval Number PNU-2013-0392).
4.3.
Motor performance testing
Motor performance was performed using a rota-rod apparatus as previously described (Borlongan et al., 1995). All mice were pre-trained for 5 days to ensure they could maintain themselves on the rod for 180 s. Training was performed using four consecutive runs at a rod speed of 25 rpm. All mice were tested 6, 24, 48, and 72 h after final MPTP administration.
4.4.
Tissue preparation
For histological studies, mice were anesthetized with ethyl ether and perfused intracardially with 0.1 M PBS (pH 7.4) containing 0.9% NaCl and then with 0.1 M PBS containing 4% paraformaldehyde. Brains were removed, placed in the same paraformaldehyde fixation solution at 4 1C overnight, and transferred to a 30% sucrose solution. Cryoprotected brains were sectioned serially at 40 μm in the coronal plane using a freezing microtome (MICROM, Walldorf, Germany), and sections were stored at 4 1C in Dulbecco’s phosphatebuffered saline (DPBS) solution containing 0.1% sodium azide.
4.5.
3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridine (MPPþ), and silibinin [(2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4] dioxin-6-yl] chroman-4-one; C25H22O10 ] were obtained from Sigma-Aldrich
7
DAB immunostaining
Brain sections were treated with 0.6% H2O2 in Tris-buffered saline (TBS; pH 7.5) to quench endogenous peroxidase activity, blocked in TBS/0.1% Triton X-100/3% goat serum (TBS-TS) for 30 min, and incubated with primary antibody; anti-TH antibody (mouse monoclonal (1:1000), Chemicon, Temecula, CA) in TBS-TS at 4 1C. Sections were further processed using appropriate biotinylated secondary goat anti-mouse IgG
Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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antibodies (1:400; Vector Laboratories, Burlingame, CA) at room temperature for 3 h, incubated in ABC solution (Vectastain Elite ABC Kit, Vector Laboratories) at room temperature for 1 h, and developed using diaminobenzidine (DAB) solution. Images were obtained using a Nikon ECLIPSE TE 2000-U microscope (Nikon, Tokyo, Japan).
4.6.
Double-label immunostaining
For double-label fluorescence immunostaining, brain sections were blocked with TBS-TS for 30 min at room temperature, and incubated with primary antibodies, that is, anti-GFAP (mouse polyclonal (1:500), Cell Signaling, MA) and anti-Iba-1 antibody (rabbit polyclonal (1:500), Wako, Tokyo, Japan) in TBS-TS at 4 1C overnight. Sections were then washed with TBS, incubated with anti-mouse IgG labeled with Alexa Fluor 488 and with anti-rabbit IgG labeled with Alexa Flour 568 for 3 h at room temperature. Images were obtained using a FV10i FLOUVIEW Confocal Microscope (Olympus, Tokyo, Japan). Zstacked images of 20 consecutive brain sections (of thickness about 2 mm taken from the brain section of 40 mm) were obtained using a 60 objective.
4.7.
Primary astrocyte cultures
Primary astrocyte cultures were established using a Sprague Dawley (SD) rat cortex obtained at postnatal day (PND) 1 or 2 (Daehan Biolink Co. Ltd., Chungbuk, South Korea). Briefly, cortices were dissected and diffused in ice-cold Hanks' balanced salt solution (HBSS; Welgene). Cells were treated with 0.25% trypsin for 30 min at room temperature, washed with HBSS, mechanically dissociated, and plated in Dulbecco's modified Eagle's medium (nutrient mixture F-12 (DMEM/ F12) medium containing 10% FBS and 1% penicillin–streptomycin) on a poly-L-lysine-coated plastic culture dishes. Experiments were performed using 14–21 day cultures.
4.8.
Immunocytochemistry
Primary astrocytes were seeded in 60 φ poly-L-lysine-coated plastic culture dishes, and pretreated the following day with silibinin (Vehicle, 0.1, 1, and 10 mM) for 6 h, and then treated with MPPþ 500 μM for 24 h, washed with PBS, and fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 min at 37 1C. They were then blocked with TBS-TS for 30 min, incubated with primary antibody overnight at 4 1C, washed, and incubated with anti-mouse IgG labeled with Alexa Fluor 488 for 3 h at room temperature. Images were obtained using a FV10i FLOUVIEW Confocal Microscope (Olympus).
4.9.
Western blot analysis
After experimental treatment, cell or tissue homogenates were solubilized in SDS-polyacrylamide gel electrophoresis sample buffer, and protein concentrations were determined using a Bio-Rad (Hercules, CA) protein assay kit with bovine serum albumin as the standard. Total protein equivalents in each sample (20 μg per lane) were then separated in 10% SDSpolyacrylamide gels and electrophoretically transferred to Immobilon-PSQ transfer membranes (Millipore, Bedford,
MA). Membranes were immediately placed into a blocking solution (5% nonfat milk) at room temperature for 30 min and then incubated with diluted primary antibodies: GFAP (rabbit; 1:500; Cell signaling), pJNK (mouse; 1:500; Santa Cruz Biotechnology), JNK (rabbit; 1:500, Cell signaling), pERK (mouse; 1:500, Cell signaling), ERK (rabbit; 1:500; Cell signaling), COX-2 (rabbit; 1:1000; Santa Cruz Biotechnology), and β-actin (mouse; 1:5000; Sigma) in TBS-T (Tris–HCl-based buffer with 0.2% Tween 20, pH 7.5) at 4 1C overnight. On the next day, membrane were washed for 10 min and incubated with secondary antibody, polyclonal anti-rabbit antibody or monoclonal anti-mouse antibody (1:10000; Santa Cruz Biotechnology), in TBS-T buffer at room temperature for 2 h. Horseradish peroxidase (HRP)-conjugated secondary antibody labeling was detected with an enhanced chemiluminescence (ECL) and cooled CCD camera system ATTO Ez-Capture (Atto Corp., Tokyo, Japan) and then quantified by densitometry. Prestained blue markers were used for molecular weight determinations.
4.10.
Statistical analysis
The significances of differences between groups were determined by analysis of variance (ANOVA) with Fisher’s protected least significant difference (PLSD) test. P values of o0.05 were considered statistically significant. Analyses were performed using Statview software (Version 5.0.1., SAS Institute Inc., Cary, NC).
Conflict of interest statement The authors have no potential conflict of interest to declare.
Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP) (Grant no. 2009-0083538). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (No. NRF-2013R1A2A2A01067388).
r e f e r e n c e s
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Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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Please cite this article as: Lee, Y., et al., Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.09.029
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