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Feb 12, 2017 - attenuated apomorphine-induced rotation (Figure 1). Moreover, the role of vanillin in the fourth week is more effective than the second week ...
International Journal of

Molecular Sciences Article

Vanillin Protects Dopaminergic Neurons against Inflammation-Mediated Cell Death by Inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway Xuan Yan 1 , Dian-Feng Liu 1 , Xiang-Yang Zhang 1 , Dong Liu 2 , Shi-Yao Xu 1 , Guang-Xin Chen 1 , Bing-Xu Huang 1 , Wen-Zhi Ren 1 , Wei Wang 1 , Shou-Peng Fu 1, * and Ju-Xiong Liu 1, * 1

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College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China; [email protected] (X.Y.); [email protected] (D.-F.L.); [email protected] (X.-Y.Z.); [email protected] (S.-Y.X.); [email protected] (G.-X.C.); [email protected] (B.-X.H.); [email protected] (W.-Z.R.); [email protected] (W.W.) Animal Husbandry and Veterinary Medicine, Cangzhou Technic College, Cangzhou 061001, China; [email protected] Correspondence: [email protected] (S.-P.F.); [email protected] (J.-X.L.); Tel./Fax: +86-431-8783-6163 (S.-P.F. & J.-X.L.)

Academic Editor: Maurizio Battino Received: 1 January 2017; Accepted: 6 February 2017; Published: 12 February 2017

Abstract: Neuroinflammation plays a very important role in the pathogenesis of Parkinson’s disease (PD). After activation, microglia produce pro-inflammatory mediators that damage surrounding neurons. Consequently, the inhibition of microglial activation might represent a new therapeutic approach of PD. Vanillin has been shown to protect dopaminergic neurons, but the mechanism is still unclear. Herein, we further study the underlying mechanisms in lipopolysaccharide (LPS)-induced PD models. In vivo, we firstly established rat models of PD by unilateral injection of LPS into substantia nigra (SN), and then examined the role of vanillin in motor dysfunction, microglial activation and degeneration of dopaminergic neurons. In vitro, murine microglial BV-2 cells were treated with vanillin prior to the incubation of LPS, and then the inflammatory responses and the related signaling pathways were analyzed. The in vivo results showed that vanillin markedly improved the motor dysfunction, suppressed degeneration of dopaminergic neurons and inhibited microglial over-activation induced by LPS intranigral injection. The in vitro studies demonstrated that vanillin reduces LPS-induced expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and IL-6 through regulating ERK1/2, p38 and NF-κB signaling. Collectively, these data indicated that vanillin has a role in protecting dopaminergic neurons via inhibiting inflammatory activation. Keywords: vanillin; inflammation; microglia; Parkinson’s disease; MAPK; NF-κB

1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder that affects millions of people in the world [1–3]. It is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in a substantial loss of dopamine afferents to the striatum and subsequent motor impairment. Although the precise pathophysiology that precipitates the development of PD is unknown, accumulating evidence suggested a potential role of neuroinflammation in the pathogenesis of PD [4–7]. Although microglia represent only 5%–20% of the Central Nervous System (CNS) cells, they act as the main effector cells on the procession of neuroinflammation [8,9]. Microglia are the main participants of neuroinflammation. Over-active microglia can produce a large amount of inflammatory mediators, which are involved in the Int. J. Mol. Sci. 2017, 18, 389; doi:10.3390/ijms18020389

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progression of neuronal in PD Therefore, the inhibition of microglia activation which are involved in degeneration the progression of [10,11]. neuronal degeneration in PD [10,11]. Therefore, the may be a potential therapeutic strategy for PD. inhibition of microglia activation may be a potential therapeutic strategy for PD. Vanillin (4-hydroxy-3-methoxybenzaldehyde) Vanillin (4-hydroxy-3-methoxybenzaldehyde) is is the the major major component component of of natural natural vanilla, vanilla, which which is one of the most widely used flavor components in food and personal products [12]. Besides is one of the most widely used flavor components in food and personal products [12]. Besides its its flavor qualities, vanillin exhibits exhibits the the anti-microbial, anti-microbial, anti-mutagenic, anti-mutagenic, anti-angiogenetic anti-angiogenetic effects effects [13,14]. [13,14]. flavor qualities, vanillin Dhanalakshmi et et al. al. have have found found that that vanillin vanillin plays plays an an important important role role in in the Dhanalakshmi the rotenone-induced rotenone-induced rat rat model of PD and protects against rotenone-induced neurotoxicity in SH-SY5Y cells, suggesting model of PD and protects against rotenone-induced neurotoxicity in SH-SY5Y cells, suggesting that that vanillin is a potential neuroprotective in PD models, themechanism exact mechanism vanillin is a potential neuroprotective agent agent in PD models, but thebut exact remainsremains unclear unclear [15,16]. Lots of studies have shown that vanillin has anti-inflammatory effects in peripheral [15,16]. Lots of studies have shown that vanillin has anti-inflammatory effects in peripheral tissues, tissues, liver,and colon and macrophages However, no reports whether such as such liver,as colon macrophages [17–19].[17–19]. However, no reports showedshowed whether vanillinvanillin has an has an anti-inflammatory effect the central nervous we hypothesize that vanillin anti-inflammatory effect on the on central nervous system.system. Herein,Herein, we hypothesize that vanillin might might have a potential role in inhibiting microglial over-activation and then alleviates further have a potential role in inhibiting microglial over-activation and then alleviates further PD PD development. In study, we aim investigate the neuroprotective and anti-inflammatory properties development. Inthis this study, we to aim to investigate the neuroprotective and anti-inflammatory of vanillin of in vanillin lipopolysaccharide (LPS)-induced in vivo andininvivo vitro PDinmodels so models as to reveal properties in lipopolysaccharide (LPS)-induced and vitro PD so asthe to mechanism of vanillin in inhibiting inflammatory response. reveal the mechanism of vanillin in inhibiting inflammatory response. 2. Results Results 2. Treatment Improves 2.1. Vanillin Treatment Improves Motor Motor Dysfunction Dysfunction Induced Induced by by LPS LPS Intranigral IntranigralInjection Injection

LPS, which is injected into unilateral SN, leading to microglial activation and the damage of dopaminergic neurons. of of dopaminergic neurons resulted in theincompensatory hyper dopaminergic neurons.Unilateral Unilateralinjury injury dopaminergic neurons resulted the compensatory function of dopamine receptors. Due to the different receptor activity on both sides, the receptor hyper function of dopamine receptors. Due to the different receptor activity on both sides, the agonist leads to the rotation behaviorbehavior towardstowards the injection side. The therapeutic effects effects of drugs receptor agonist leads to the rotation the injection side. The therapeutic of on lesion in PD animal models models are characterized by a rotational behaviorbehavior assay. Inassay. order In to drugs on degree lesion degree in PD animal are characterized by a rotational examine the effectthe of vanillin on motoron dysfunction, after LPS after injection, rats were order to examine effect ofadministration vanillin administration motor dysfunction, LPS the injection, the subjected rotationaltobehavior assay at two and fouratweeks. Notably, rats were tosubjected rotational behavior assay two and four vanillin weeks. dose-dependently Notably, vanillin attenuated apomorphine-induced rotation (Figure 1). Moreover, role of1). vanillin in the fourth week dose-dependently attenuated apomorphine-induced rotation the (Figure Moreover, the role of is more effective thanweek the second week (Figure 1). the second week (Figure 1). vanillin in the fourth is more effective than

Figure administration improves the motor dysfunction by lipopolysaccharide (LPS) intranigral Figure 1.1.Vanillin Vanillin administration improves the motor dysfunction by lipopolysaccharide (LPS) injection. Rats were treated with the vehicle or vanillin (5, 10, or 20 mg/kg/day) for three days priorfor to intranigral injection. Rats were treated with the vehicle or vanillin (5, 10, or 20 mg/kg/day) LPS and this lasted for 24 The number of turns two (A); and after four threeintranigral days priorinjection to LPS intranigral injection anddays. this lasted for 24 days. Theafter number of turns weeks (B)and was induced disease (PD) model rats. #(PD) p < 0.01 vs.rats. the two (A); four weeksby (B)apomorphine was inducedin byParkinson’s apomorphine in Parkinson’s disease model sham-operated control rats; and * p < 0.05 and ** p < 0.01 vs. the vehicle treated LPS-injected rats. # p < 0.01 vs. the sham-operated control rats; and * p < 0.05 and ** p < 0.01 vs. the vehicle treated LPS-injected rats.

2.2. Vanillin Adminastration Increases the Survival Rate of Dopaminergic Neurons in the SN 2.2. Vanillin Survival Rate ofdopaminergic Dopaminergic neurons, Neurons in thenumber SN In orderAdminastration to investigate Increases whether the vanillin protects the of tyrosine hydroxylase neurons andprotects the expression of neurons, TH were carriedof tyrosine out by In order to(TH)-positive investigate whether vanillin dopaminergic the number immunohistological and Western blot analysis respectively. As the result has showed, compared hydroxylase (TH)-positive neurons and the expression of TH were carried out by immunohistological with sham-operated rats, LPS-injected rats reducedcompared TH-positive neurons in the SN and Western blot analysis respectively. Ashave the significantly result has showed, with sham-operated section (p < 0.01). In contrast, vanillin (5, 10, or 20 mg/kg/day) administration markedly increased the number of TH-positive neurons in a dose-dependent manner (Figure 2A,B). The results of Western

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rats, LPS-injected rats have significantly reduced TH-positive neurons in the SN section (p < 0.01). In contrast, vanillin Int. J. Mol. Sci. 2017, 18, 389(5, 10, or 20 mg/kg/day) administration markedly increased the number 3 of of 12 TH-positive neurons in a dose-dependent manner (Figure 2A,B). The results of Western blot showed blot showed that therereduction is significant ininTH in injection. the SN after LPS treatment injection. that there is significant in THreduction expression theexpression SN after LPS Vanillin Vanillin treatment significantly increased theinexpression of TH inmanner a dose-dependent manner (Figure significantly increased the expression of TH a dose-dependent (Figure 2C). Thus, vanillin 2C). Thus, vanillin can protect dopaminergic neurons in the SN LPS-induced PD model rats. can protect dopaminergic neurons in the SN of LPS-induced PD of model rats.

Figure Vanillin administration increases the survival of dopaminergic neurons in the Figure 2.2.Vanillin administration increases the survival rate ofrate dopaminergic neurons in the substantia substantia (SN). The rats PD model rats were anaesthetized and decapitated to SN obtain the after nigra (SN).nigra The PD model were anaesthetized and decapitated to obtain the after 24SN days of 24 days of vanillin administration. (A) Immunohistochemical results of tyrosine hydroxylase vanillin administration. (A) Immunohistochemical results of tyrosine hydroxylase (TH)-positive cells, (TH)-positive cells, 1.0 scale bar(B) represents 1.0 mm; Thedopaminergic survival rationeurons; of the dopaminergic neurons; scale bar represents mm; The survival ratio(B) of the and (C) TH expression. and THvs. expression. # p < 0.01 vs. the sham-operated rats; * p