Tetramethylpyrazine Ameliorates Rotenone-Induced Parkinson’sDisease in Rats: Involvement of Its Anti-Inflammatoryand Anti-Apoptotic Actions

Haidy E. Michel1 & Mariane G. Tadros1 & Ahmed Esmat1 &

Amani E. Khalifa1 & Ahmed M. Abdel-Tawab2

Received: 5 March 2016 /Accepted: 1 August 2016# Springer Science+Business Media New York 2016

Abstract Parkinson’s disease (PD) is a slowly progressiveneurodegenerative movement disorder. Apoptosis, neuroin-flammation, and oxidative stress are the current hypothesizedmechanisms for PD pathogenesis. Tetramethylpyrazine(TMP), the major bioactive component of Ligusticumwallichii Franchat (ChuanXiong), Family Apiaceae, reported-ly has anti-apoptotic, anti-inflammatory and antioxidant ef-fects. This study investigated the role of ‘TMP’ in preventingrotenone-induced neurobiological and behavioral sequelae. Apreliminary dose–response study was conducted where ratsreceived TMP (10, 20, and 40mg/kg, i.p.) concomitantly withrotenone (2 mg/kg, s.c.) for 4 weeks. Catalepsy, locomotoractivity, striatal dopamine content, and tyrosine hydroxylaseBTH^ and α-synuclein immunoreactivity were evaluated. Theselected TMP dose (20 mg/kg) was used for western blotanalysis of Bax, Bcl2, and DJ-1, immunohistochemical detec-tion of nuclear factor kappa B (NF-кB), inducible nitric oxidesynthase (iNOS), cyclooxygenase-2 (COX2), and glial fibril-lary acidic protein (GFAP) expression, in addition to biochem-ical analysis of caspase-3 activity, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) levels.Results showed that TMP (20 mg/kg) significantly improvedmidbrain and striatal TH expression and striatal dopaminecontent as well as the motor deficits, compared to rotenone-treated group. These results were correlated with reduction incaspase-3 activity and α-synuclein expression, along with

improvement of midbrain and striatal Bax/Bcl2 ratio com-pared to rotenone-treated group. TMP also attenuatedrotenone-induced upregulation of Nrf2/HO-1 pathway.Furthermore, TMP downregulated rotenone-induced neuroin-flammation markers: NF-кB, iNOS, COX2, and GFAP ex-pression in both the midbrain and striatum. Taken together,the current study suggests that TMP is entitled to, at leastpartially, preventing PD neurobiological and behavioral defi-cits by virtue of its anti-apoptotic, anti-inflammatory, and an-tioxidant actions.

Keywords Parkinson’s disease . Rotenone .

Tetramethylpyrazine . Neuroinflammation . Apoptosis

Introduction

Parkinson’s disease is the most common neurodegenerativemovement disorder. It is clinically characterized by bradyki-nesia, resting tremor, rigidity, and postural instability [1, 2].Pathological features of PD include progressive loss of dopa-minergic neurons in the substantia nigra pars compacta(SNpc) and the presence of proteinaceous cytoplasmic inclu-sions known as Lewy bodies (LBs) in the surviving dopami-nergic neurons [3].α-synuclein is the main protein constituentof LBs and Lewy neurites. PD likely results from a combina-tion of genetic and environmental factors [4]. The main mech-anisms underlying PD neuropathology are attributed to mito-chondrial dysfunction, alteration in protein folding and degra-dation via ubiquitin proteasomal degradation pathway [5], ox-idative stress, ATP depletion, apoptosis, excitotoxicity, andneuroinflammation [6].

Developing clinically relevant animal models of PD is stillchallenging, as long as adequately effective disease-modifying therapies are not yet at hand. Rotenone, a pesticide

* Mariane G. Tadrosmarianeg@pharma.asu.edu.eg

1 Department of Pharmacology and Toxicology, Faculty of Pharmacy,Ain Shams University, Cairo, Egypt

2 Department of Pharmacology, Faculty of Medicine, Ain ShamsUniversity, Cairo, Egypt

Mol NeurobiolDOI 10.1007/s12035-016-0028-7

derived from the plant roots of Family Leguminosae, has beenused in different modalities in-search for the provision of amodel with high translational value [7]. Chronic rotenone ad-ministration has been demonstrated to produce selectivenigrostriatal dopaminergic neurodegeneration, as well as be-havioral, neurochemical, and neuropathological features ofPD in rodents including formation of ubiquitin and α-synuclein positive nigral inclusions and motor deficits[8–10]. Hence, rotenone-induced PD, is probably a model thatmeets many of the expectations of research studies on PD[11].

Traditional Chinese medicine is one of the most ancientand sti l l widely used tradit ional medicines [12].Tetramethylpyrazine (TMP) is a purified chemical that hasbeen identified as a component of Ligusticum wallichiiFranchat (ChuanXiong), a Chinese herb that is largely usedin the treatment of neurovascular and cardiovascular diseases[13–15]. TMP was shown to possess neuroprotective effectsin vitro and in vivo [16, 17]. Furthermore, TMP was found toprotect ischemic brain damage, and promote cell proliferationand differentiation stimulated by ischemia [18]. Moreover,TMP improved dopamine turnover in the striatum and re-duced oxidative damage in PD rats induced by levodopa[19]. TMP also had neuroprotective effects against MPTP-induced dopaminergic neurotoxicity in a mouse model ofPD [20].

To extend the scope of relevant studies, the presentstudy aimed to investigate the potential neuroprotectiveeffect of TMP in chronic rotenone-induced model of PDin rats as well as to characterize the neuroprotectivemechanisms of TMP with respect to neuroinflammation,apoptosis and its effects on the expression of the oxida-tive stress sensor DJ-1 and the pro-oxidant stressors’sensors Nrf2/HO-1.

Materials and Methods

Animals

Fifty-four male Spargue dawley rats weighing 200–250 gwere purchased from the Nile Company, El Amyria,Cairo, Egypt. They were housed in plastic cages at con-stant temperature (21 ± 2 °C), with alternating 12 h light/dark cycle. Animal chow and water were provided adlibitum. One week before the experiment, all animalswere acclimatized to laboratory conditions. All animaltreatments strictly adhered to institutional and interna-tional ethical guidelines of the care and use of laboratoryanimals. The experimental protocol was approved by AinShams Universi ty Faculty of Pharmacy ReviewCommittee for the use of animal subjects.

Chemicals

Rotenone and TMP were purchased from Sigma chemicals(St. Louis, MO, USA). The other reagents were of the highestpure grade commercially available.

Rotenone was suspended in sunflower oil at the concentra-tion of 2 mg/ml, and vortexed thoroughly just before injectionto ensure uniform suspension. It was administered subcutane-ously at a dose of 2 mg/kg daily for 4 weeks [21]. TMP wasdissolved in saline and was co-administered intraperitoneallywith rotenone in doses of 10, 20, and 40 mg/kg [22, 23].

Experimental Groups

A preliminary dose–response study was conducted in whichrats were randomly divided into six groups, six animals each.The first group, serving as a control group, received saline(i.p.) and sunflower oil (s.c.) for 4 weeks. The second groupreceived saline (i.p.) and rotenone (2 mg/kg/d, s.c.) for4 weeks. The third to fifth groups received TMP (10, 20 and40 mg/kg, i.p.) concomitantly with rotenone (2 mg/kg/d, s.c.)for 4 weeks. The sixth group received TMP (20 mg/kg, i.p.)and sunflower oil (s.c.) for 4 weeks. Rats were tested forcatalepsy (bar and grid tests) and locomotor activity 24 h afterthe last injection, then they were decapitated. Whole brainswere excised and one hemisphere was fixed in 10 % formalin(pH .4) for the preparation of paraffin blocks. Washing wasdone in tap water then serial dilutions of alcohol (methyl,ethyl, and absolute ethyl) were used for dehydration.Specimens were cleared in xylene then embedded in paraffinat 56 °C in hot air oven for 24 h. Paraffin sections (1.6 to2.8 mm posterior to the bregma and 0 to 1.2 mm anterior tothe bregma) were cut from each brain [24]. These sectionswere used for histological examination, toluidine blue stainingand immunohistochemical detection of tyrosine hydroxylase(TH) and α-synuclein. The striatum of the other hemispherewas dissected out, stored at −80 and used for determination ofdopamine content. The most effective TMP dose was selectedfor further investigations.

Afterwards, a mechanistic study was conducted in whichrats were randomly divided into four groups, six animals each.The first group received the respective vehicles. The secondgroup received saline (i.p.) and rotenone (2 mg/kg/d, s.c.) for4 weeks. The third group was intraperitoneally injected withthe selected TMP dose in addition to rotenone (2 mg/kg/d,s.c.) for 4 weeks. The fourth group received TMP (20 mg/kg,i.p.) and sunflower oil (s.c.) for 4 weeks. Rats were decapitat-ed, brains were excised, midbrains and striata of one hemi-sphere were dissected out, stored at −80 °C and used for neu-rochemical analyses. The other hemisphere was fixed in 10 %formalin (pH .4) for the preparation of paraffin blocks as be-fore. These sections were used for immunohistochemical de-tection of NF-кB (p65), iNOS, COX-2, and GFAP.

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Behavioral Tests

Catalepsy Test

The catalepsy set-up consisted of a vertical grid and a hori-zontal bar to ascertain inert or static behavior. The tests werecarried out between 9 am and 3 pm, under standard conditions[25]. All rats were tested for catalepsy 24 h after the lastinjection. (1) Grid test [26]: Gridiron of 30 cm wide and35 cm high with a space of 1.2 cm between each wire wasused. Each rat was hung by all four paws on the vertical gridand the time taken by each rat to descend the grid was noted asdescent latency. Maximum descent latency time was fixed at180 s. (2) Bar test [27]: rats were placed with both fore pawson a bar, 10 cm above the surface in half rearing position.Time taken by each rat to remove one paw from the bar wasrecorded. Maximum latency time was set at 180 s.

Locomotor Activity

Activity monitor (Opto-Varimex-Mini Model B, ColumbusInstruments, Columbus, OH, USA) was used to measure thelocomotor activity of animals. Such activity was measuredbased on the traditional infrared photocell principle whereinterruption of 15 infrared beams (wavelength = 875 nm, scanrate = 160 Hz, diameter = 0.32 cm, spacing = 2.65 cm)reflected total activity of the animal. Twenty-four hours afterthe last injection, rats were habituated in the recording cham-ber for 2 min and then activity was recorded for 5 min andexpressed as counts per 5 min [28].

Histological Examination

Hematoxylin and Eosin Staining

Paraffin beeswax tissue blocks were prepared for sectioning at4 μm thickness by slide microtome. The obtained tissue sec-tions were collected on glass slides, deparaffinized, stained byH&E, and examined using a light microscope [29].

Toluidine-Blue Staining and Evaluationof the Neurodegeneration

Paraffin sections of 5 μm thickness were prepared as previ-ously described [30]. Degenerated midbrain and striatal neu-rons were counted by capturing six non-overlapping fieldsfrom the levels of midbrain and whole striatum using a mag-nification of ×200 in three sections per group (n = 3rats/group). The number of midbrain and striatal degeneratedneurons was then calculated and expressed as a percentage oft h e t o t a l numbe r o f n eu r on s [% Degene r a t e dneurons = Number of degenerated neurons × 100 / Total num-ber of neurons]. Neurons with rounded nuclei and visible

nucleoli were considered undamaged, while deeply stainedshrunken neurons were considered damaged neurons [31].

Immunohistochemistry

The sections were prepared as previously described [32, 33].For the dose–response study, sections were incubated withpolyclonal rabbit anti-TH and anti-α-synuclein antibodies(1:50, Biorbyt LLC with catalog numbers orb127600 andorb11441, respectively). For the mechanistic study and immu-nohistochemical detection of NF-кB (p65), iNOS, COX-2,and GFAP, the sections were incubated with the correspond-ing polyclonal rabbit antibodies (1:100, Thermo FisherScientific, UK, with catalog numbers RB-9034-P0, RB-9242-R7, RB-9072-P, and 180,063 respectively). All primaryantibody incubations were carried out overnight at 4 °C,followed by incubation with biotinylated goat-anti-rabbitIgG at room temperature for 10 min. After three times of 3-min PBS rinses, sections were incubated for 10 min withstreptavidin horseradish peroxidase. The antibody bindingsites were visualized by incubation with diaminobenzidine–H2O2 solution. Sections incubated with PBS instead of theprimary antibody were used as negative controls. Brown gran-ules in cytoplasm or nuclei were recognized to be positivelystained. Computerized image analysis to calculate the areapercent (A%) was performed using imageJ software (version1.48).

Western Blot

Midbrain and striatal lysates were prepared according to stan-dard protocol using RIPA buffer (Abcam plc, Cambridge,MA). Protein (80 μg) was loaded per well of a 10 % SDS-PAGE gel. After electrophoresis, the gel was transferred ontoa PVDF membrane (Bio-Rad Laboratorie, Hercules, CA,USA). Membranes were blocked in TBST with 5 % BSAand incubated overnight with one of the following primaryantibodies (1:1,000); polyclonal rabbit anti-DJ-1, anti-Bax,anti-Bcl2, and anti-β-actin for loading correction (SantaCruz Biotechnology, Dallas, TX, USA, with catalog numbers:sc-32,874, sc-6236, sc-492, and sc-130657, respectively).Then, they were incubated with secondary goat anti-rabbitIgG HRP-linked antibody (1:5,000). Development was doneusing an Enhanced Chemiluminescence (ECL) Western blot-ting kit (sc-2048, Santa Cruz Biotechnology) on the X-rayfilm (GE Healthcare, Piscataway, NJ, USA).

Biochemical Analyses

Protein Estimation in Midbrain and Striatal Tissues

Protein levels were determined by BCA protein assay kit(Biovision Inc., Mountain View, CA, USA).

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Assessment of Striatal Dopamine Content

Striatal dopamine content was analyzed using high-performance liquid chromatography (HPLC) with electro-chemical detector. Frozen striatal tissue samples weresuspended in cold 0.1 M perchloric acid containing 0.4 mMsodium metabisulphite and homogenized with an ultrasoniccell disrupter (Vibracell 72,434, Bioblock, Illkrich-Cedex)The suspension was centrifuged at 10,000×g for 25 min at4 °C and the supernatants were filtered through a 0.22-μmfilter and frozen at −80 °C until analysis. The supernatantwas then injected into a Waters 2695 HPLC separation mod-ule (Waters Corp., Milford, MA, USA) using an autosamplerwithin the module and the sample temperature was maintainedat 4 °C. The mobile phase contained 0.06 M sodium phos-phate monobasic, 0.03M citric acid, 8%methanol, 1.075mM1-octanesulfonic acid, 0.1 mM ethylenediaminetetraacetic ac-id, and 2 mM sodium chloride, at pH .5. Chromatography wasisocratic with a flow rate of 1.0 mL/min. Dopamine was sep-arated on a Waters XBridge C18 4.6 × 150 mm column with a3.5-mm particle size and detected on a Waters 2465 electro-chemical detector with a glassy carbon electrode set at750 mV referenced to an ISAAC electrode. Striatal dopamineconcentration in each sample was calculated from the integrat-ed chromatographic peak area and expressed as nanogram permilligram protein. The method was adopted from Zagrodzkaet al. [34].

Assessment of Midbrain and Striatal Caspase-3 Activity

Caspase-3 activity was determined using a colorimetric assaykit purchased from (Sigma Aldrich Chemical Co., productcode: CASP-3-C), according to the manufacturer’s protocol.

Assessment of Midbrain and Striatal Nrf2 and HO-1 Levels

Nrf-2 and HO-1 quantification were carried out using ELISAkits (Cayman Chemical, Ann Arbor, MI, USA, item number:600590 and Enzo Life Sciences, Farmingdale, NY, USA, cat-alog number: ADI-EKS-810 A, respectively) according to themanufacturers’ protocols.

Statistical Analysis

Data were expressed as the mean ± SEM. Significant differ-ences between groups were calculated using one-wayANOVA followed by Tukey test as a post hoc test. All statis-tical analyses were performed using the GraphPad Prism soft-ware (version 5.01, Inc., 2007, San Diego, CA, USA).Probability values of less than 0.05 were considered statisti-cally significant.

Results

Dose–Response Study

Effect of TMP on Rotenone-Induced Catalepsy

Rotenone administration induced catalepsy in rats as indicatedby prolongation of descent latency in grid and bar tests by23.9- and 18.3-folds, respectively, as compared to the controlgroup (Fig. 1a). Co-administration of TMP (10 mg/kg) did notshow any significant difference from rotenone-treated group.However, TMP (20 mg/kg) significantly prevented rotenone-induced catalepsy by reducing descent latency nearly to base-line values. TMP (40 mg/kg) produced the same effects as the(20 mg/kg) dose.

Effect of TMP on Locomotor Activityin Rotenone-Treated Rats

Rotenone significantly reduced locomotor activity by 85.15%compared to control group (p < 0.001) (Fig. 1b). Co-administration of TMP (10 mg/kg) showed no significant dif-ference from rotenone-treated group. TMP (20 and 40 mg/kg)significantly attenuated rotenone-induced hypokinesia by834.24 and 820 % respectively (p < 0.001).

Histological Examination

Hematoxylin and Eosin Staining As shown in Fig. 2, histo-logical examination of the midbrains and striata of the controlgroup showed normal histological structure. Sections fromrats treated with rotenone (2 mg/kg, s.c.) for 4 weeks showedsevere hemorrhage in the midbrain, as well as hyalinosis as-sociated with congestion of the blood vessels in the midbrainand striatum. Co-administration of TMP (10 mg/kg, i.p.) alsoshowed focal hemorrhage in the midbrain and striatum, inaddition to hyalinosis and congestion in the midbrain. Co-treatment with TMP (20 and 40 mg/kg, i.p.) showed restora-tion of normal histological structure in the midbrain andstriatum.

Toluidine-Blue Staining for Quantitation of NeuronalDamage As shown in Fig. 3a, the midbrain and striatal neu-rons of the control group showed a conservation of thegeneral tissue histo-architecture, with neurons havinground body, clear cytoplasm, and defined nucleus. Inrotenone-treated rats, there is a complete loss of tissuehisto-architecture and neurons appear hyperchromaticand have lost the round form of normal ones and phan-tom nuclei appear scattered in the field. Sections fromrats co-treated with TMP (10 mg/kg, i.p.) showed severeneuronal damage in both the midbrain and striatum ascompared to the rotenone-treated group. However, the

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global appearance of the tissue in sections co-treatedwith TMP (20 and 40 mg/kg, i.p.) was closer to normalwith apparent increased number of normal neurons as com-pared to rotenone-treated rats. Quantitation of the percentageof degenerated neurons showed a significant increase in thenumber of degenerated neurons in the midbrain and striatumby 417 and 492 %, respectively, in the rotenone-treated groupas compared to the control. Co-administration of TMP(10 mg/kg, i.p.) showed no significant difference from therotenone-treated group. However, co-treatment with TMP(20 and 40 mg/kg, i.p.) significantly reduced damaged neu-rons in the midbrain by 60 and 71 %, respectively, and in thestriatum by 64 and 68 %, respectively as compared to therotenone-treated group (Fig. 3b).

Immunohistochemistry

Sections from rotenone-treated rats showed decreasedTH immunoreactivity, compared with control rats(Fig. 4a). Co-administration of TMP (10 mg/kg, i.p.)showed no significant difference from rotenone-treatedrats. However, co-treatment with TMP (20 and40 mg/kg, i.p.) significantly increased TH immunoreac-tivity, compared to the rotenone-treated group. On theother hand, patches of α-synuclein-positive cells werespread over the whole areas of rotenone and TMP(10 mg/kg, i.p.)-treated rats, whereas fewer positivecells were detected in sections from rats treated withTMP (20 and 40 mg/kg, i .p.) (Fig. 4a). These

Fig. 1 Effect of TMP (10, 20,and 40 mg/kg) on rotenone-induced behavioral changes. Dataare presented as means ± SEM(n = 6). a Catalepsy bar and gridtests. b Locomotor activity test.*,#p < 0.001 versus control androtenone-treated groups,respectively (one-way ANOVAfollowed by Tukey’s test formultiple comparisons betweengroups)

Fig. 2 Representative photomicrographs of H&E-stained rat midbrain and whole striatal sections. h, hemorrhage; v, hyalinosis; c, blood vesselcongestion (×200)

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Fig. 4 a Immunohistochemical staining of midbrain and striatal TH andα-synuclein expression (200×). b Quantitative image analysis for THimmunohistochemical staining expressed as mean area% (A%). cQuantitative image analysis for α-synuclein immunohistochemical

staining expressed as mean area% (A%). Data are presented asmeans ± SEM (n = 6). *,#p < 0.001 versus control and rotenone-treatedgroups, respectively (one-way ANOVA followed by Tukey test formultiple comparisons between groups)

Fig. 3 a Representative photomicrographs of toluidine-blue stained ratmidbrain and whole striatal sections (×200). b Quantitative analysis ofmidbrain and striatal neuronal degeneration calculated as the percentageof degenerated neurons in comparison to total neurons. Data are presented

as means ± SEM (n = 6). *,#p < 0.05, statistically significant compared tocontrol and rotenone-treated groups, respectively, using one-wayANOVA followed by Tukey’s test for multiple comparisons betweengroups

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photomicrographs greatly correlate with the values ofthe mean A% shown in (Fig. 4b, c).

Effect of TMP on Striatal Dopamine Contentin Rotenone-Treated Rats

As shown in Fig. 5, rotenone administration resulted in asubstantial depletion of striatal dopamine content by 60 %compared to the control group (p < 0.001). Co-administration of TMP (10 mg/kg) showed no significant dif-ference from rotenone-treated group. TMP (20 and 40 mg/kg)significantly increased striatal dopamine content by 201 and224 %, respectively in comparison to rotenone-treated group(p < 0.01).

Based on the previous results, the intermediate dose ofTMP (20 mg/kg, i.p.) was selected for further investigations.

Mechanistic Study

Effect of TMP on Rotenone-Induced Apoptosis

In order to evaluate apoptosis-related alterations, we detectedcaspase-3 activity, in addition to Bax and Bcl2 protein expres-sion. As shown in Fig. 6a, caspase-3 activity was significantlyincreased in the midbrain and striatum of rotenone-treated ratsby 9- and 6.4-folds respectively, as compared with control ratswhile TMP administration significantly ameliorated rotenone-stimulated increase in its activity both in the midbrain andstriatum by 62.23 and 51.34 % respectively. Furthermore,western blot analysis showed that rotenone injection increasedBax and decreased Bcl2 expression in the midbrain and stria-tum. These effects were prevented by TMP administration(Fig. 7).

Effect of TMP on Rotenone-Induced Neuroinflammation

Immunohistochemical staining and the values of the mean A% of midbrain and striatal NF-кB, COX2, iNOS, and GFAP-positive cells immunized with goat-anti-rabbit IgG is shown inFig. 8. Only faint immunoreactive staining was detected in the

brain areas of the control and TMP alone-treated groups.Sections from rotenone-treated rats showed patches ofNF-кB, COX2, iNOS, and GFAP-positive cells spread overthe whole areas whereas fewer positive cells were detected insections from rats treated with TMP (20 mg/kg, i.p.). Thesephotomicrographs greatly correlate with the values of themean A% shown in Table 1.

Effect of TMP on Rotenone-Induced Oxidative Stress

As shown in Fig. 9, rotenone increased DJ-1 expression in themidbrain and striatum. This effect was not prevented by TMPadministration. In addition, we measured Nrf2 and HO-1levels in the midbrain and striatum. As shown in Fig. 6b, c,we observed significant upregulation of Nrf2/HO-1 in themidbrain and striatum of rotenone-treated rats, compared withthat of control. However, TMP administration could signifi-cantly attenuate rotenone-induced increase of Nrf2 and HO-1levels.

Discussion

L. wallichii Franchat (Chuan Xiong) has long been used intraditional Chinese medicine for the treatment of pain, cardio-vascular diseases, angina pectoris, and cerebral ischemic syn-dromes [35, 36]. TMP is one of its major bioactive compo-nents, which can permeate the blood brain barrier [37].Experimental evidence has shown that TMP markedly re-duces cerebral and spinal cord ischemia/reperfusion injurythrough suppression of inflammatory cell activation and pro-inflammatory cytokine production [38, 39]. In addition, TMPhas been reported to inhibit neuronal apoptosis and preventneuronal loss [18]. In the present study, the neuroprotectiveeffect of TMP against rotenone-induced PD in rats was inves-tigated and the possible underlying mechanisms wereelucidated.

Since PD is both a chronic and progressive disease with itssymptoms growing worse over time, the effectiveness of long-term administration of TMP was studied in a chronically de-veloping rotenone model of PD which faithfully recapitulates

Fig. 5 Effect of TMP (10, 20, and40 mg/kg) on striatal dopaminecontent in rotenone-treated rats.*,#p < 0.01, significantly differentcompared to control and rotenone-treated groups, respectively (one-way ANOVA followed by Tukeytest for multiple comparisonsbetween groups)

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the progressive pathological and phenotypic features of PDwithin 4 weeks of its administration [40]. In this study, co-administration of TMP (20 and 40 mg/kg, i.p.) successfullyattenuated rotenone-induced cataleptic behavior and locomo-tor hypoactivity. Rotenone is known to produce these PD-likebehavioral features [41] by destroying the midbrain dopami-nergic neurons, thereby decreasing the striatal dopaminergicinput. In context, our results showed significant decline of TH(dopamine biosynthetic enzyme) immunoreactivity in themidbrain and striatal regions of rotenone treated rats, in addi-tion to reduced striatal dopamine content in rotenone-treatedanimals and this decrease was attenuated by co-treatment withTMP (20 and 40 mg/kg, i.p.).

It is reported that α-synuclein-positive nigral inclusionswere shown in rotenone-treated rats [42], thus reproducingthe pathological hallmark of PD. α-synuclein is a presynapticprotein whose function is not completely understood.However, increased levels of α-synuclein are observed inPD forming LBs [43]. Increased levels of such dysfunctionalprotein may facilitate neuronal death through either necrosisor apoptosis [44]. The mechanism by which α-synuclein ac-cumulates inside neurons involves, at least in part, oxidativestress [45]. It has been demonstrated that a vicious cycle ismaintained when dysfunctional α-synuclein aggregates andhalters proteasomal activity, which would be responsible forunfolded proteins degradation [46]. Interestingly, in the pres-ent study, TMP (20 and 40 mg/kg, i.p.) administrationprohibited rotenone-induced α-synuclein accumulation inthe midbrain and striatum.

Histological examination of midbrains and whole striatarevealed pathological changes following subcutaneous rote-none administration shown as neurodegeneration, hemor-rhage, hyalinosis, and congestion of blood vessels. Thesefindings are in accordance with previous studies which con-firmed that rotenone causes neuronal damage in the variousbrain regions via vascular changes [47, 48]. Histological ex-aminations of rat brain sections co-treated with TMP (20 and40 mg/kg, i.p.) revealed restoration of normal histologicalstructure.

Apoptosis has recently been regarded as an important modeof cell death in PD. This has been mainly discovered by theidentification of key markers including Bax/Bcl2 ratio andcaspase-3 activation [49]. Apoptosis-related alterations havebeen demonstrated in the SNpc and dopaminergic brain re-gions in the post-mortem brains of PD patients, includingincreased caspase-3 activity in the SN [50], as well as elevatedimmunoreactivity of the pro-apoptotic protein Bax [51]. Thus,it can be concluded that apoptosis may play a pivotal role inthe pathophysiology of PD, therefore targeting of the molec-ular pathways activated during apoptosis may lead to noveltreatment strategies for PD [52–54]. Indeed the present datashowed significant alteration in the Bax/Bcl2 ratio, in additionto increased caspase-3 activity in the midbrains and striata of

Fig. 6 Effect of TMP (20 mg/kg) on caspase-3 (a), HO-1 (b), and Nrf2(c) level/activity in the midbrain and striatum of rotenone-treated rats.*,#p < 0.01 significantly different compared to control and rotenone-treated groups, respectively (one-way ANOVA followed by Tukey testfor multiple comparisons between groups)

Fig. 7 aWestern blot analysis of midbrain and striatal Bax/Bcl2 ratio. (1)control, (2) rotenone-treated, (3) TMP (20 mg/kg) and rotenone-treated,(4) TMP (20 mg/kg) treated. Rotenone (2 mg/kg, s.c.) was administeredfor 4 weeks. TMP (20 mg/kg, i.p.) was co-administered with rotenone for4 weeks. bDensitometric quantitation of Bax/Bcl2. Data are presented asmeans ± SEM (n = 3). *,#p < 0.001, statistically significant compared tocontrol and rotenone-treated groups, respectively, using one-wayANOVA followed by Tukey’s test for multiple comparisons betweengroups

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rotenone-treated rats compared to control rats. Rotenone isknown universally to induce an increase in apoptotic markers,such as, caspase-3 and caspase-9 activities, Bax expression,and cytochrome c release [55–57]. Sufficient activation ofBax and Bak facilitates mitochondrial outer-membrane per-meabilization, which releases death-inducing factors resultingin apoptotic and nonapoptotic cell death [58]. Interestingly,the present study showed that TMP ameliorated rotenone-induced apoptosis via improving the Bax/Bcl2 ratio and de-creasing caspase-3 activity in the midbrains and striata of par-kinsonian rats. These findings are supported by previous stud-ies which revealed the anti-apoptotic effect of TMP in thespinal cord [59], neurons [60], renal tubular cells [61], andin MPTP-induced dopaminergic neurotoxicity [20].

Several studies support the hypothesis that NF-кB activa-tion plays an important role in PD pathogenesis. It was report-ed that NF-кB activation is induced in the SNpc of patientswith PD and MPTP-treated mice [62], as well as in rotenone-

treated rats [63]. When NF-кB is activated, it translocates tothe nucleus and stimulates the gene transcription of severalproinfammatory factors including iNOS, interleukin (IL)-1β,IL-6, COX-2, and tumor necrosis factor-α (TNF-α) by themicroglia [64, 65]. The current study showed an inhibitoryeffect of TMP on rotenone-induced increase of NF-кB expres-sion. In harmony, TMP inhibited NF- B activation in a ratmodel of spinal cord ischemia-reperfusion injury [38].Moreover, it was reported that TMP reduced NF-кB expres-sion, while enhancing the expression of inhibitor κappa B andthe anti-inflammatory cytokine IL-10 in a rat model of trau-matic spinal cord injury [66].

COX-2 is a crucial mediator of inflammation whose levelshave been observed to increase drastically in the PD-affectedbrain [67]. COX-2 mediates the potentiation of cytotoxic ef-fects not only through the ROS generated in conversion ofprostaglandins-G to prostaglandins-H [68] but also via pro-duction of pro-inflammatory prostaglandins that cause

Table 1 Quantitative imageanalysis of theimmunohistochemical staining ofCOX2, iNOS, NF-кB (p65) andGFAP expressed as A%

Brain area/group COX2 iNOS NF-кB (p65) GFAP

Control

Midbrain 12.16 ± 0.86 19.63 ± 0.99 24.88 ± 2.07 11.70 ± 1.47

Striatum 17.52 ± 1.75 16.26 ± 1.02 18.44 ± 1.18 6.23 ± 0.66

Rotenone

Midbrain 33.32 ± 2.90* 41.60 ± 1.68* 37.61 ± 2.29* 31.21 ± 1.08*

Striatum 39.34 ± 3.68* 38.77 ± 1.74* 51.30 ± 4.07* 22.78 ± 1.05*

Rotenone + TMP (20 mg/kg)

Midbrain 12.40 ± 0.93# 20.32 ± 1.34# 27.63 ± 1.69# 18.85 ± 0.49*, #

Striatum 18.76 ± 1.11# 23.60 ± 0.88# 22.36 ± 2.41# 11.50 ± 0.72*, #

TMP (20 mg/kg)

Midbrain 11.95 ± 1.01 22.70 ± 0.45 25.51 ± 1.52 11.50 ± 0.52

Striatum 20.87 ± 1.28 20.09 ± 0.63 19.44 ± 1.22 7.57 ± 0.36

Rotenone (2 mg/kg, s.c.) was administered for 4 weeks

TMP (20 mg/kg, i.p.) was co-administered with rotenone or vehicle for 4 weeks

Data are presented as means ± SEM (n = 6)

*,#p<0.001, statistically significant compared to control and rotenone-treated groups, respectively, using one-way ANOVA followed by Tukey’s test

for multiple comparisons between groups

Fig. 8 Immunohistochemical staining of midbrain and striatal COX-2, iNOS, NF-кB, and GFAP expression (200×)

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microglial activation [69]. Besides COX-2, NOS enzymeisoforms also participate in dopaminergic neurodegener-ation. Production of iNOS by activated microglia resultsin increased formation of NO, which in turn has a del-eterious effect on proteins and DNA [67]. The presentstudy figured out rotenone-induced increased COX-2and iNOS immunoreactivity in the midbrain and stria-tum. This finding is supported by previous studieswhich reported increased COX-2 and iNOS expression/activity in rotenone-treated rats [63, 40]. This effect wasruled out by TMP administration. Experimental evidencesupports the anti-inflammatory effects of TMP, where anin vitro study demonstrated that TMP inhibited produc-tion of nitric oxide and iNOS in LPS-induced N9microglial cells [70]. Moreover, it was reported thatTMP treatment not only reduced the up-regulation ofiNOS expression, but also decreased TNF-α, IL-1βand COX-2 mRNA in the spinal tissue at 4 h afterspinal cord compression injury in mice [71].

Astrocytes are the major glial cell population in thebrain. They have been implicated in neuronal homeosta-sis [72], development and/or maintenance of blood–brain barrier characteristics [73], clearance and releaseof extracellular glutamate [74], scavenging oxygen freeradicals [75], maintenance of extracellular ionic environ-ment and pH [76]. A great body of evidence shows thatastrocytes play pivotal roles in the neuroinflammatoryprocesses in PD. They respond to the inflammatory in-sults such as LPS, IL-1β, and TNF-α by producing pro-inflammatory cytokines both in vitro and in vivo [77,

78]. Reactive astrogliosis characterized by the increasedexpression levels of GFAP, hypertrophy of cell bodyand cell extensions have been reported in various PDanimal models [79, 9, 80–82]. In line, the current studyshowed increased GFAP expression in the midbrainsand striata of rotenone-treated rats, an effect that wasinhibited by TMP administration. This finding is sup-ported by in vivo and in vitro studies which reportedthat TMP is capable of inhibiting pro-inflammatory cy-tokines production and reducing astrocytes activation[83–86].

DJ-1 is a ubiquitous redox responsive, cytoprotective pro-tein with various functions. DJ-1 mutations can cause earlyonset of PD, leading to selective neurodegeneration of thenigrostriatal dopaminergic neurons, which accounts for par-kinsonian symptoms. DJ-1 may act as a sensor for oxidativestress, and it apparently protects neurons against oxidativestress and cell death [87]. Increased DJ-1 expression in themidbrains and striata of rotenone-treated rats was observed.This finding is in line with a previous study which reportedincreased DJ-1 expression due to chronic rotenone adminis-tration [88].

The current study demonstrated rotenone-induced upregu-lation of Nrf2 and HO-1, an effect that was attenuated by TMPadministration. Likewise, former studies reported upregula-tion of Nrf2 and HO-1 after rotenone administration[89–91]. Nrf2 and HO-1 act as pro-oxidant stressors sensorsthat are activated/ induced as a compensatory mechanism toreduce the damaging effects of free radicals [92]. Indeed, theinvolvement of the Nrf2 pathways in PD [93], as well as theincreased expression of HO-1 in post mortem brain tissuefrom PD patients have been well documented. HO-1 degradesheme to carbon monoxide (CO), free iron and biliverdin [94].Biliverdin is further converted to bilirubin, which is a verypotent antioxidant [95]. On the other hand, CO and free ironcan damage the mitochondrial membrane and induce oxida-tive stress under certain circumstances [96, 97]. One possibleinterpretation is that HO-1 upregulation can induce acytoprotective effect in neurodegenerative disorders, or, onthe contrary, it can accelerate neuronal cell damage. The pres-ent study showed that TMP could attenuate rotenone-inducedupregulation of HO-1 in rats. This result proposes that TMPcan suppress ROS production and subsequently attenuate HO-1 expression.

In conclusion, the present study investigated the neuropro-tective effect of TMP against rotenone-induced model of PDin rats. TMP effectively prevented rotenone-induced motordeficits, dopaminergic neurons’ damage and α-synuclein ac-cumulation. The anti-apoptotic and anti-inflammatory roles ofTMP are central to the protective effect of TMP. Regulation ofNrf2/HO-1 pathway by TMP contributes to its antioxidantrole. These data suggest that TMP is a promising candi datefor modifying the neuropathological course of PD.

Fig. 9 a Western blot analysis of midbrain and striatal DJ-1 expression.(1) control, (2) rotenone-treated, (3) TMP (20 mg/kg) and rotenone-treated, (4) TMP (20 mg/kg) treated. Rotenone (2 mg/kg, s.c.) wasadministered for 4 weeks. TMP (20 mg/kg, i.p.) was co-administeredwith rotenone for 4 weeks. b Densitometric quantitation of DJ-1expression. Data are expressed as mean ± S.E. M (n = 3). *p < 0.001versus control group. (one-way ANOVA followed by Tukey’s test formultiple comparisons between groups)

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Compliance with Ethical Standards

Conflict of Interest The authors declare that there are no conflicts ofinterest.

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