Baicalein attenuates α-synuclein aggregation, inflammasome activation and autophagy in the MPP+-treated nigrostriatal dopaminergic system in vivo

Author’s Accepted Manuscript Baicalein attenuates α-synuclein aggregation, inflammasome activation and autophagy in the MPP+-treated nigrostriatal dopaminergic system in vivo Kai-Chih Hung, Hui-Ju Huang, Yi-Ting Wang, Anya Maan-Yuh Lin www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)31277-6 http://dx.doi.org/10.1016/j.jep.2016.10.040 JEP10498

To appear in: Journal of Ethnopharmacology Received date: 28 July 2016 Revised date: 10 October 2016 Accepted date: 10 October 2016 Cite this article as: Kai-Chih Hung, Hui-Ju Huang, Yi-Ting Wang and Anya Maan-Yuh Lin, Baicalein attenuates α-synuclein aggregation, inflammasome activation and autophagy in the MPP+-treated nigrostriatal dopaminergic system in vivo, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.10.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Baicalein attenuates α-synuclein aggregation, inflammasome activation and autophagy in the MPP+-treated nigrostriatal dopaminergic system in vivo Kai-Chih Hunga , Hui-Ju Huangb1,Yi-Ting Wanga, Anya Maan-Yuh Linb,c* 1

a

Department of Physiology, National Yang-Ming University, Taipei, Taiwan

b

Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan

c

Department of Pharmacology, National Yang-Ming University, Taipei, Taiwan

[email protected] [email protected] [email protected] [email protected] *

Corresponding author. Tel.: +886-2-28712121x2688; fax: +886-2-28751562.

Keywords: baicalein, -synuclein aggregation, inflammasome activation, caspase 1, autophagy.

ABSTRACT Ethnopharmacological relevance Neuroinflammation, oxidative stress, and protein aggregation form a vicious cycle in the pathophysiology of Parkinson’s disease (PD); activated microglia is the main location of neuroinflammation. A Chinese medicine book, “Shanghan Lun”, known as the “Treatises on Cold damage Diseases” has suggested that Scutellaria baicalensis Georgi is effective in treating CNS diseases. The anti-inflammatory mechanisms of baicalein, a phenolic flavonoid in the dried root of Scutellaria baicalensis Georgi, remain to be explored. Aim of the study The neuroprotective mechanisms of baicalein involving α-synuclein aggregation, inflammasome activation, and programmed cell death were investigated in the nigrostriatal dopaminergic system of rat brain in vivo. Materials and methods Intranigral infusion of 1-methyl-4-phenylpyridinium (MPP+, a Parkinsonian neurotoxin) was performed on anesthetized Sprague-Dawley rats. Baicalein was daily administered via intraperitoneal injection. Striatal dopamine levels were measured using high performance liquid chromatography coupled with electrochemical detection. Cellular signalings were measured by 1

two authors share equal contribution to this work. 1

Western blot assay, immunofluorescent staining assay and enzyme-linked immunosorbent assay. Results: Systemic administration of baicalein attenuated MPP+-induced reductions in striatal dopamine content and tyrosine hydroxylase (a biomarker of dopaminergic neurons) in the infused substantia nigra (SN). Furthermore, MPP+-induced elevations in α-synuclein aggregates (a pathological hallmark of PD), ED-1 (a biomarker of activated microglia), activated caspase-1 (a proinflammatory caspase), IL-1β and cathepsin B (a cysteine lysosomal protease) in the infused SN were attenuated in the baicalein-treated rats. Moreover, intense immunoreactivities of caspase 1 and cathepsin B were co-localized with that of ED-1 in the MPP+-infused SN. At the same time, baicalein inhibited MPP+-induced increases in active caspases 9 and 12 (biomarkers of apoptosis) as well as LC3-II levels (a biomarker of autophagy) in the rat nigrostriatal dopaminergic system. Conclusion: Our in vivo study showed that baicalein possesses anti-inflammatory activities by inhibiting α-synuclein aggregation, inflammasome activation and cathepsin B production in the MPP+-infused SN. Moreover, baicalein is of therapeutic significance because it inhibits MPP+-induced apoptosis and autophagy in the nigrostriatal dopaminergic system of rat brain.

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1. Introduction Neuroinflammation, oxidative stress, and protein aggregation form a vicious cycle in the pathophysiology of neurodegenerative diseases in the central nervous system (CNS) (Hsieh & Yang 2013); activated glia (the major immune cells in the CNS) is the main location of neuroinflammation (Sanchez-Guajardo et al 2013). Clinical studies observed significant microglial activation in the affected brain regions of patients with CNS neurodegenerative diseases, including Parkinson’s disease (PD) and Alzheimer’s disease (McGeer et al 1988). Consequently, several pro-inflammatory features were detected in the nigrostriatal dopaminergic system of PD patients, such as elevated oxidative stress and accumulation of cytokines (Brown & Vilalta 2015, Chakraborty et al 2010, Nagatsu et al 2000, Nagatsu & Sawada 2007). In addition, activated microglia reportedly produce lysosomal cysteine proteases, such as cathepsin B (Brown & Vilalta 2015, Kingham & Pocock 2001, Nakanishi 2003) which is proposed to induce neuronal damages under various pathological conditions (Hook et al 2012, Kingham & Pocock 2001, Nakanishi 2003). ɑ-Synuclein aggregation clinically detected in the inclusion bodies of the postmortem brain tissues of PD patients (Spillantini et al 1998), has been suggested to activate microglia (Kim et al 2013, Roodveldt et al 2008, Sanchez-Guajardo et al 2015).Taken together, neuroinflammation in the activated microglia appears to be neurotoxic to neurons (Brown & Vilalta 2015, Gallegos et al 2015, Ghio et al 2016, Sanchez-Guajardo et al 2013). Currently, searching for herbal medicines with anti-inflammatory activities has become focus of interest in the field of neuroprotective research. A Chinese medicine book, “Shanghan Lun”, known as the “Treatises on Cold damage Diseases” has suggested that Scutellaria baicalensis Georgi (Huang-qin), one of the components of modified Xiao-Tsai-Hu decoction is effective in treating several CNS diseases, including insomnia, seizure and delirium. To support this notion, non-clinical studies have shown that extracted compounds of Scutellaria baicalensis Georgi are beneficial for insomnia (Shi et al 2014), seizure (Sugaya et al 1988) and brain ischemia (Shang et al 2006). Furthermore, our previous study has demonstrated a neuroprotective effect of an S/B remedy prepared from Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd, two components of modified Xiao-Tsai-Hu decoction (Lin et al 2011). Among all the flavonoids in the dried root of Scutellaria baicalensis Georgi, baicalein (5,6,7-trihydroxyflavone) reportedly possesses therapeutic activities against anxiety, seizure (de Carvalho et al 2011), stroke (Cui et al 2010, Liu et al 2010) and PD (Cheng et al 2008, Mu et al 2009). Moreover, clinical studies have 3

investigated the pharmacokinetics of baicalein (Li et al 2014) and the therapeutic effects of baicalein on PD patients (Yu et al 2012), indicating that baicalein is clinically useful for treating the CNS neurodegenerative diseases. A significant body of studies has focused on the anti-inflammatory mechanisms underlying baicalein-induced neuroprotection (Orhan et al 2015, Spencer et al 2012). Baicalein, a polyphenol with an o-trihydroxyl structure in the ring A (Spencer et al 2012), reportedly acts as a free radical scavenger to eliminate reactive oxygen/nitrogen species (Cheng et al 2008, Im et al 2005, Lee et al 2014, Wang et al 2013, Zhang et al 2010). Furthermore, baicalein may be anti-inflammatory by interacting with Nrf2 to regulate redox balance (Lee et al 2014). Moreover, baicalein reportedly protects mitochondrial functions by modulating mitochondrial membrane potentials, Bcl2/Bax ratio and reducing oxidative stress (de Oliveira et al 2015, Zhang et al 2010). In addition, baicalein reportedly exerts its anti-inflammatory action by inhibiting glial activation (Cheng et al 2008, Mu et al 2009) as well as reducing pro-inflammatory enzymes and cytokines, including Interleukin (IL)-1β (Chen et al 2008, Spencer et al 2012, Xue et al 2014). IL-1β is known to be activated by caspase 1, a pro-inflammatory caspase activated during the inflammasome activation (Chakraborty et al 2010, Sollberger et al 2014, Walsh et al 2014), the mechanism of baicalein-induced reduction in IL-1β levels involving inflammasome activation remains to be determined. Due to the pro-inflammatory role of ɑ-synuclein aggregates, biophysical studies and many in vitro studies have demonstrated that baicalein is capable of mitigating ɑ-synuclein aggregation and reducing cytotoxicity (Caruana et al 2012, Jiang et al 2010, Lu et al 2011, Zhu et al 2004). However, only one in vivo study has reported the baicalein-induced inhibition of ɑ-synuclein oligomers in the midbrain of rotenone-treated mice (Hu et al 2016). In the present study, the goal was twofold: 1) to study the anti-inflammatory mechanisms underlying baicalein-induced neuroprotection involving ɑ-synuclein aggregation, inflammasome activation and cathepsin B production and 2) to investigate the involvement of programmed cell death, including apoptosis and autophagy in the baicalein-induced neuroprotection using a PD animal model. 2. Materials and methods Adult, male Sprague-Dawley rats, weighing 300-350g, were supplied by the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, R.O.C.. Three rats were housed in a cage in an air-conditioned room (22±2°C) on a 12-h light/dark cycle (06:00–18:00 h 4

light) and had free access to food and water. Acclimation time for rats to a new environment was 24 hr. The use of animals has been approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C.. All experiments were performed in the accordance with the approved guidelines. The approval number is IACUC2014-089. 2.1. Surgery and intranigral infusion of MPP+ Intranigral infusion of MPP+ was performed on rats anesthetized with chloral hydrate (Hung et al 2014). Then, rats were immobilized in a stereotaxic instrument (David Kopf Instruments, Palo Alto, CA); the scalp was incised to expose the parietal bone; one hole was drilled above the cortical surface for a unilateral infusion of MPP+ (3 μg/μl/injection, Sigma, St. Louis, MO) in the substantia nigra (SN). The stereotaxic coordinates of rat SN were 2.3 mm above and 3.2 mm anterior to the interaural zero, 2.1 mm lateral to the midline and 3.5 mm below the incisor bar. The infusion rate of MPP+ was 0.3 l/min via a 30 gauge stainless steel needle. After MPP+ infusion, the injection needle was held in place for an additional 3 min. At the end of the surgery, rats recovered from anesthesia and were placed in home cages for the indicated times. 2.2. Baicalein treatment To investigate the effect of baicalein (product #465119; batch # 02031TO, Sigma-Aldrich, St. Louis, MO) on dopamine content of rat striatum, rats were randomly divided into 3 groups which received high baicalein (30 mg/kg/day), low baicalein (10 mg/kg/day) and vehicle (10% DMSO in saline) for 7 days. To study the tyrosine hydroxylase (TH) levels in the SN, rats were divided into 2 groups which received baicalein (30 mg/kg/day) and vehicle respectively for 7 days. For the studies on cellular signalings using Western blot assay, ELISA and immunostaining assay, rats were divided into 2 groups which received baicalein (30 mg/kg/day) and vehicle for 2 days. 2.3. High pressure liquid chromatography coupled with electrochemical detection (HPLC-ECD) for analysis of striatal dopamine content After decapitation, striata were dissected, immediately frozen in liquid nitrogen and stored at -80 C until analysis. Striatal dopamine contents were measured using an HPLC (CC5/PM80, Bioanalytical Systems Inc., West Lafayette, IN) with electrochemical detection (LC-4C, Bioanalytical Systems Inc., West Lafayette, IN) procedure (Hung et al 2014). Mobile phase (one liter) contained 2.1g heptanesulfonic acid, 3.5 ml triethylamine, 3ml phosphoric acid, 0.1 g NaEDTA and 170 ml acetonitrile. Applied potential was set at 0.75 volt vs Ag/AgCl as reference. 5

The retention time for dopamine was about 7.5 min 2.4. Western blot analysis of relevant proteins At the end of in vivo experiments, rats were sacrificed by decapitation. Forty microliters of ice-cold protease inhibitor cocktail (Calbiochem, San Diego, CA) were used to homogenize dissected SN. The lysates were centrifuged at 4 C at 12,000x g for 30 min; the supernatant was stored at -80C. PierceMT BCA protein assay (Thermo Scientific, Rockford, lL) using albumin as a protein standard was employed to measure protein concentrations. Protein samples (3 or 30 μg) were run on 10-15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) at 80 V for 120 minutes. Blots were probed with a monoclonal antibody against TH (1:3000; Chemicon, Temecula, CA), α-synuclein (1:3000; BD Transduction Lab., Lexington, KY), ED-1 (1:1000; AbD Serotec, Raleigh, NC), caspase 1 (1:1000; Santa Cruz Biotech, Santa Cruz, CA), cathepsin B (1:1000; Cell Signaling, Beverly, MA), active caspase 9 and 12 (1:1000; Cell Signaling Tech., Beverly, MA), and LC3 (1:1000; Novus, Littleton, CO) at room temperature for 2 h. Afterwards, the membrane was washed and incubated with horseradish peroxidase-conjugated secondary IgG (1:3000; Chemicon, Temecula, CA) for 1 h at room temperature. The immunoreaction was visualized by Amersham enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). After this detection, the bound primary and secondary antibodies were removed by incubating the membrane with the stripping buffer (100 mM 2-mercaptoethanol, 2% SDS) at 50 C for 45 min. The membrane was then reprobed with a mouse -actin antibody (1:5000; Millipore, Bedford, MA). The density of the blots was scanned and reported as relative optical density of the specific proteins and calculated using image J. 2.5. Enzyme-linked immunosorbent assay (ELISA) of IL-1β Dissected SN was homogenized in Mammalian cell Lysis/Extraction Reagent (CelLyticMT, Sigma) according to the manufacturer’s instructions. Protein concentrations were determined using PierceMT BCA protein assay (Thermo Scientific, Rockford, lL) as above-mentioned. The IL-1β levels of the SN were determined using an IL-1β kit (R&D Systems, Minneapolis, MN) and ELISA reader (Molecular Devices Corp, Silicon Valley, CA) according to the manufacturer’s instructions. 2.6. Immunofluorescence staining Rats were deeply anesthetized and transcardially perfused with chilled 0.9% saline followed by 6

4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Rat brains were carefully removed and placed in 30 % sucrose-PBS until sunken. Rat brains were sectioned coronally at 30 μm thickness using a cryostat (Leica CM 1950, Leica Biosystems, Germany). After permeabilization and blocking, sections were incubated overnight at 4°C with primary antibodies including ED-1 (1:100), caspase 1 (1:100) and cathepsin B (1:100), followed by incubation with secondary antibodies (1:500) conjugated with rhodamine and fluorescein isothiocyanate for 1 h at room temperature. Nuclei were labeled with 4', 6-diamidino-2-phenylindole (1mg/ml) for 10 min at room temperature. After final washes and mounting, fluorescence images were viewed with a fluorescence laser-scanning confocal microscope (Olympus FV10i, Center Valley, PA). 2.7. Statistics All data are expressed as the mean ± S.E.M. Statistical comparisons of time-dependent effects of MPP+ were made by one-way analysis of variance (one-way ANOVA) and followed by the LSD test as a post-hoc method. Statistical comparisons of baicalein effects were made using Kruskal-Wallis test and followed by the Mann-Whitney U test as a post-hoc method. A value of p<0.05 was considered significant.

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3. Results 3.1. Baicalein prevents MPP+-induced neurotoxicity and α-synuclein aggregation in the nigrostriatal dopaminergic system of rat brain To study the neuroprotective effect of baicalein, a PD animal model was employed by a local infusion of MPP+ (3 μg/1 μl/injection) in the SN of anesthetized rats. Seven days after intranigral infusion of MPP+, dopamine content was significantly reduced in the striatum ipsilateral to the MPP+-infused SN while compared with that of the control SN (Figure 1A, p<0.05). At the same time, Western blot assay showed that MPP+ reduced the levels of tyrosine hydroxylase (TH, the rate-limiting enzyme of dopamine biosynthesis) in the infused SN (Figure 1B). These data show MPP+-induced neurodegeneration of the nigrostriatal dopaminergic system of rat brain. Two doses of baicalein (10 and 30 mg/kg/day) were intraperitoneally administered for 7 days. Compared with vehicle-treated rats, low dose of baicalein (10 mg/kg/day) did not alter the MPP+-induced reduction in striatal dopamine content. In contrast, high dose of baicalein (30 mg/kg/day) significantly attenuated the MPP+-induced reductions in dopamine content in striatum (Figure 1A, p<0.05) and TH in the infused SN (Figure 1B, p<0.05). The effect of baicalein on α-synuclein aggregation was investigated after 2-d baicalein treatment. Western blot assay showed that baicalein attenuated MPP+-induced α-synuclein aggregation in the infused SN (Figure 1C). Our in vivo data demonstrate that baicalein reduced MPP+-induced α-synuclein aggregation and neurotoxicity in the nigrostriatal dopaminergic system of rat brain.

Figure 1. Effects of baicalein on MPP+-induced neurotoxicity and α-synuclein aggregation in the nigrostriatal dopaminergic system of rat brain. (A) Intranigral infusion of MPP+ (3 μg/injection) was performed in anesthetized rats. Baicalein (Bai., 10 or 30 mg/kg/day) was administered as described in the methods section. After 7-d baicalein treatment, striatal dopamine content was detected by HPLC-ECD. Values are the mean ± S.E.M. (n=7). (B) Intranigral infusion of MPP+ 8

(3 μg/injection) was performed in anesthetized rats. After 7-d baicalein treatment (30 mg/kg/day), tyrosine hydroxylase (TH) in the substantia nigra (SN) was measured using Western blot assay. Each lane contained 3 μg proteins for all experiments. (C) Intranigral infusion of MPP+ (3 μg/injection) was performed in anesthetized rats. After 2-d baicalein (30 mg/kg/day) treatment, α-synuclein in the SN was measured using Western blot assay. Each lane contained 30 μg proteins for all experiments. Graphs show statistical results from relative optical density of bands on the blots estimated by Image J. Values are the mean ± S.E.M. (n=3). *, P < 0.05 in the MPP+-infused SN compared with the control SN, # P< 0.05 in the MPP+-infused SN of Bai-treated rats compared with that of vehicle-treated rats by Kruskal-Wallis test and followed by the Mann-Whitney U test as a post-hoc method. 3.2. Baicalein prevents neuroinflammation in MPP+-activated microglia: involvement of inflammasome activation The anti-inflammatory effect of baicalein on MPP+-induced neurotoxicity was evaluated in several ways. First, intranigral infusion of MPP+ demonstrated a time-dependent elevation in ED-1 levels (a specific marker for activated microglia) in the infused SN (Figure 2A). Treatment with baicalein for 2 days significantly reduced MPP+-induced elevation in ED-1levels (Figure 2B), indicating that baicalein was capable of inhibiting microglial activation in the MPP+-infused SN.

Figure 2. Effects of baicalein on MPP+-induced microglia activation in the nigrostriatal dopaminergic system of rat brain. (A) Intranigral infusion of MPP+ (3 μg/injection) was performed in anesthetized rats. Time-dependent effects on ED-1 in the substantia nigra (SN) were measured using Western blot assay. Each lane contained 30 μg proteins for all experiments. Graphs show statistical results from relative optical density of bands on the blots estimated by 9

Image J. Values are the mean±S.E.M. (n = 3/group). *P < 0.05 in the MPP+-treated SN compared with the control SN by one-way analysis of variance (one-way ANOVA) and followed by the LSD test as a post-hoc method. (B) Intranigral infusion of MPP+ (3 μg/injection) was performed in anesthetized rats. Baicalein (Bai., 30 mg/kg) was administered as described in the methods section. After 2-d baicalein treatment, ED-1 in the SN was measured by Western blot assay. Values are the mean±S.E.M. (n = 3/group) *, P < 0.05 in the MPP+-infused SN compared with the control SN, # P< 0.05 in the MPP+-infused SN of Bai-treated rats compared with that of vehicle-treated rats by Kruskal-Wallis test and followed by the Mann-Whitney U test as a post-hoc method. The cellular mechanisms underlying baicalein-induced anti-inflammation involving inflammasomes were investigated by measuring caspase 1 activation and IL-1β levels. Intranigral infusion of MPP+ time-dependently induced caspase 1 activation in the infused SN; the activated caspase 1 levels peaked 2 d and maintained for 7 d after MPP+ infusion (Figure 3A). Furthermore, our immunostaining data showed that low immunoreactivity of caspase 1 was detected in the control SN while intense immunoreactivities of caspase 1 and ED-1 were co-localized in the MPP+-infused SN (Figure 3B), indicating that MPP+ induced caspase 1 activation in the activated microglia. Moreover, systemic administration of baicalein attenuated MPP+-induced inflammasome activation by attenuating MPP+-induced caspase 1 activation (Figure 3C) and elevation in IL-1β levels (Figure 3D) using Western blot assay and ELISA, respectively.

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Figure 3. Effects of baicalein on MPP+-induced inflammasome activation in the nigrostriatal dopaminergic system of rat brain. (A) Intranigral infusion of MPP+ (3 μg/injection) was performed in anesthetized rats. Time-dependent effects on procaspase 1 (pCaspase 1) and activated caspase 1 (aCaspase 1) in the substantia nigra (SN) were measured using Western blot assay. Each lane contained 30 μg proteins for all experiments. Graphs show statistical results from relative optical density of bands on the blots estimated by Image J. Values are the mean±S.E.M. (n = 3/group). *P < 0.05 in the MPP+-treated SN compared with the control SN by one-way analysis of variance (one-way ANOVA) and followed by the LSD test as a post-hoc method. (B) Representative immunostaining study showed co-localization of immunoreactivities of caspase 1 and ED-1 in the infused SN 4 days after MPP+ infusion. (C) Intranigral infusion of MPP+ (3 μg/injection) was performed in the anesthetized rats. Baicalein (Bai., 30 mg/kg) was administered as described in the methods section. After 2-d baicalein treatment, active caspase 1(aCaspase 1) in the SN were measured using Western blot assay. Each lane contained 30 μg proteins for all experiments. Graphs show statistical results from relative optical density of bands on the blots estimated by Image J. Values are the mean ± S.E.M. (n=3). (D) The levels of IL-1β in the SN were measured using ELISA. The IL-1β level in the MPP+-infused SN was normalized to that in the contralateral SN of the same rat. Values are the mean ± S.E.M. (n=5). *, P < 0.05 in the MPP+-infused SN compared with the control SN, # P< 0.05 in the MPP+-infused SN of Bai-treated rats compared with that of vehicle-treated rats by Kruskal-Wallis test and followed by the Mann-Whitney U test as a post-hoc method. In addition, intranigral infusion of MPP+ elevated cathepsin B levels in the infused SN in a time-dependent manner. We found that MPP+ mildly increased cathepsin B levels 2 days after MPP+ infusion and peaked 4 days after MPP+ infusion (Figure 4A). Co-localization of strong immunoreactivities of cathepsin B and ED-1 was demonstrated in the MPP+-infused SN (Figure 4B), indicating that MPP+ was capable of producing cathepsin B in the activated microglia. Compared with vehicle-treated rats, systemic administration of baicalein (30 mg/kg/day) for 2 days significantly attenuated MPP+-induced cathepsin B production in the infused SN of rat brain (Figure 4C).

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Figure 4. Effects of baicalein on MPP+-induced elevation in cathepsin B in the nigrostriatal dopaminergic system of rat brain. (A) Intranigral infusion of MPP+ (3 μg/injection) was performed in anesthetized rats. Time-dependent effects on Cathepsin B in the substantia nigra (SN) were measured using Western blot assay. Each lane contained 30 μg proteins for all experiments. Graphs show statistical results from relative optical density of bands on the blots estimated by Image J. Values are the mean±S.E.M. (n = 3/group). *P < 0.05 in the MPP+-treated SN compared with the control SN by one-way analysis of variance (one-way ANOVA) and followed by the LSD test as a post-hoc method. (B) Representative immunostaining data showed co-localization of immunoreactivities of cathepsin B and ED-1 in the infused SN 4 days after MPP+ infusion. (C) Intranigral infusion of MPP+ (3 μg/injection) was performed on anesthetized rats. Baicalein (Bai., 30 mg/kg) was administered as described in the methods section. After 2-d baicalein treatment, cathepsin B levels in the SN were measured using Western blot assay. Values are the mean ± S.E.M. (n=3). *, P < 0.05 in the MPP+-infused SN compared with the control SN, # P< 0.05 in the MPP+-infused SN of Bai-treated rats compared with that of vehicle-treated rats by Kruskal-Wallis test and followed by the Mann-Whitney U test as a post-hoc method. . 3.3. Baicalein prevents MPP+-induced apoptosis and autophagy 12

The anti-apoptotic effect of baicalein was studied by measuring levels of active caspase 9 and active caspase 12, two apoptotic proteins involved in the mitochondria and endoplasmic reticulum pathways, respectively. Western blot assay demonstrated that 2-d treatment of baicalein prevented MPP+-induced elevations in active caspase 9 and active caspase 12 in the infused SN (Figures 5A and 5B). Furthermore, the involvement of autophagy in the baicalein-induced neuroprotective effect was studied by measuring LC3-II levels, a biomarker of autophagy. Systemic administration of baicalein for 2 days significantly attenuated MPP+-induced elevation in LC3-II in the infused SN (Figure 5C). These data indicate that baicalein may exert its neuroprotection via inhibiting MPP+-induced apoptosis and autophagy.

Figure 5. Effects of baicalein on MPP+-induced apoptosis and autophagy in the nigrostriatal dopaminergic system of rat brain. Intranigral infusion of MPP+ (3 μg/injection) was performed on the anesthetized rats. Baicalein (Bai., 30 mg/kg) was administered as described in the methods section. After 2-d baicalein treatment, the levels of active caspase 9 (A), active caspase 12 (B) and LC3-II at 17 kD (C) in the MPP+-infused substantia nigra were measured using Western blot assay. Each lane contained 30 μg proteins for all experiments. Graphs show statistical results from relative optical density of bands on the blots estimated by Image J. Values are the mean ± S.E.M. (n=3). *, P < 0.05 in the MPP+-infused SN compared with the control SN, # P< 0.05 in the MPP+-infused SN of Bai-treated rats compared with that of vehicle-treated rats by Kruskal-Wallis test and followed by the Mann-Whitney U test as a post-hoc method. .

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4. Discussion The neuroprotective effects of baicalein in PD have been widely investigated in in vitro, in vivo and in on-going clinical studies (Cheng et al 2008, Im et al 2005, Jiang et al 2010, Lee et al 2014, Li et al 2014, Mu et al 2009, Wang et al 2013, Xue et al 2014, Yu et al 2012). Anti-inflammation has been proposed as one of the major mechanisms underlying baicalein-induced neuroprotection, such as baicalein-induced inhibition of NFκb activity via c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways as well as decreases in pro-inflammatory enzymes and cytokine levels (Cheng et al 2008, Lee et al 2014, Mu et al 2009, Xue et al 2014). In the present study, we demonstrated baicalein-induced anti-inflammation by mitigating ɑ-synuclein aggregation. Because α-synuclein aggregation has been proposed to correlate with the α-synuclein-induced neurotoxicity in PD pathology (Mu et al 2009, Zhang et al 2010), baicalein was neuroprotective by inhibiting α-synuclein oligomerization and stabilizing β-sheet-enriched oligomers to prevent the formation of α-synuclein fibrils (Liu et al 2010, Lu et al 2011, Zhu et al 2004). Furthermore, baicalein-reduced α-synuclein aggregation was found to attenuate neurotoxicity in PC12 pheochromocytoma cells (Jiang et al 2010). Consistent to the baicalein-induced inhibition of ɑ-synuclein oligomers in the midbrain of rotenone-treated mice (Hu et al 2016), the present study shows that baicalein is capable of attenuating α-synuclein aggregation in the MPP+-treated nigrostriatal dopaminergic system of rat brain. This finding is consistent with our previous study in which an S/B remedy was found to attenuate α-synuclein aggregation in the iron-infused SN (Lin et al 2011). Accordingly, our in vivo data imply two mechanisms of baicalein-induced reduction in α-synuclein aggregation, one is anti-inflammation since α-synuclein aggregation reportedly triggers inflammasome activation and IL-1β production (Lin et al 2011, Zhu et al 2004); the other mechanism is inhibition of MPP+-induced neurotoxicity due to a direct toxic activity of α-synuclein aggregation (Gallegos et al 2015, Ghio et al 2016). Activated microglia play a critical role in inflammation, and inhibition of microglial activation has been found to be a useful therapeutic strategy for CNS neurodegenerative diseases (Manthripragada et al 2011). Both the present study and others have shown that baicalein is capable of inhibiting microglial activation by decreasing ED-1 levels (Chen et al 2008, Lee et al 2014). Due to the co-localization of caspase 1 and ED-1 immunoreactivities, we suggest that MPP+-induced caspase 1 activation happens in the activated microglia and may be responsible for 14

the MPP+-induced IL-1β elevation. Furthermore, we demonstrated that baicalein reduced MPP+-induced caspase 1 activation and IL-1β elevation in the infused SN. These data are the first to suggest that baicalein exerts its anti-inflammatory action via inhibiting inflammasome activation in the MPP+-activated microglia. In addition, our finding helps resolving the cellular mechanisms underlying baicalein-induced reduction in proinflammatory cytokines in the animals with CNS neurodegeneration (Chen et al 2008, Xue et al 2014). Many studies have suggested neurotoxic mechanisms of cathepsin B, such as inducing Bid aggregation on mitochondria, a pro-apoptotic response (Lamparska-Przybysz et al 2005) as well as cleaving procaspase 1 and proIL-1β (Hentze et al 2003, Turk et al 2012). Therefore, MPP+-induced cathepsin B elevation in the ED-1 positive microglia may contribute to MPP+-induced neurotoxicity. From the time-dependent studies, the maximal MPP+-induced caspase 1 activation preceded the maximal MPP+-induced elevation in cathepsin B, suggesting that MPP+-induced caspase 1 activation depends on self-activation in the inflammasome complex as well as cathepsin B-mediated activation. Based on these findings, baicalein appears to be neuroprotective by reducing cathepsin B level and its subsequent caspase 1 activation, indicating that cathepsin B may be a novel therapeutic target of baicalein-induced neuroprotection. Baicalein has been suggested for cancer treatment via inducting apoptosis of cancer cells (Chen et al 2000, Lee et al 2008, Zhou et al 2009). Furthermore, baicalein-induced apoptosis was observed in retina ganglion cells in responsive to oxidative stress (Li et al 2009). However, a neuroprotective role of baicalein has been proposed by inhibiting apoptosis in hippocampal neurons and cortical neurons (Choi et al 2010, Lebeau et al 2001). To delineate the safety of baicalein, our data did not detect any significant alterations in TH levels, ɑ-synuclein aggregation, microglial activation, inflammasome activation in the control SN of baicalein-treated rats compared with those of vehicle-treated rats, suggesting that the dose of baicalein used in the present study is safe. In addition, our data show that baicalein-induced attenuation of MPP+-induced apoptosis is mediated via inhibiting mitochondria and ER stress pathways because baicalein inhibited MPP+-induced elevation in active caspases 9 and 12. Regarding the effect of baicalein on autophagy, baicalein was found to induce a protective autophagy in the HepG2 cancer cells (Wang et al 2015) but a prodeath autophagy in human breast and prostate cancer cells (Aryal et al 2014). In our previous study, a prodeath role of autophagy was defined in the MPP+-induced neurotoxicity because in vivo transfection of Atg7siRNA prevented autophagy 15

and MPP+-induced neurotoxicity in the nigrostriatal dopaminergic system of rat brain (Hung et al 2014). Based on these findings, baicalein may exert its neuroprotective action by reducing MPP+-induced autophagy in the PD animals. In conclusion, our study shows baicalein-induced inhibition of MPP+-induced α-synuclein aggregation in vivo. Furthermore, baicalein possesses anti-inflammatory activities by inhibiting MPP+-induced inflammasome activation and cathepsin B production in the activated microglia in the infused SN. Moreover, baicalein is of therapeutic significance since it also inhibited MPP+-induced apoptosis and autophagy in CNS neurodegenerative diseases. Competing interest The authors declare that they have no competing interests. Authors’ contributions KCH and YTW carried out animal experiments, HPLC-ECD and Western blot assay. HJH carried out Western blot assay and immunofluorescent staining study. AMYL conceived, designed and interpreted the experiments as well as prepared the manuscript. All authors approved the final version of the manuscript. Acknowledgements The authors express their special thanks to Dr. R.K. Freund at the University of Colorado-Denver, Anschutz Medical Campus, U.S.A. for editing this paper. This study was supported by NSC100-2320-B-010-005, VGH104-033, a grant from Ministry of Education, Aim for the Top University Plan, Taipei, Taiwan, R.O.C.. References Aryal P, Kim K, Park PH, Ham S, Cho J, Song K. 2014. Baicalein induces autophagic cell death through AMPK/ULK1 activation and downregulation of mTORC1 complex components in human cancer cells. The FEBS journal 281: 4644-58 Brown GC, Vilalta A. 2015. How microglia kill neurons. Brain research 1628: 288-97 Caruana M, Neuner J, Hogen T, Schmidt F, Kamp F, et al. 2012. Polyphenolic compounds are novel protective agents against lipid membrane damage by alpha-synuclein aggregates in vitro. Biochimica et biophysica acta 1818: 2502-10 Chakraborty S, Kaushik DK, Gupta M, Basu A. 2010. Inflammasome signaling at the heart of central nervous system pathology. Journal of neuroscience research 88: 1615-31 Chen CH, Huang LL, Huang CC, Lin CC, Lee Y, Lu FJ. 2000. Baicalein, a novel apoptotic agent 16

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