Ghrelin protects MES23.5 cells against rotenone via inhibiting mitochondrial dysfunction and apoptosis

    Ghrelin Protects MES23.5 Cells against Rotenone via Inhibiting Mitochondrial Dysfunction and Apoptosis. Jianhan Yu, Huamin Xu, Xiaoli Shen, Hong Jiang PII: DOI: Reference:

S0143-4179(15)00100-6 doi: 10.1016/j.npep.2015.09.011 YNPEP 1668

To appear in: Received date: Revised date: Accepted date:

26 March 2015 25 September 2015 29 September 2015

Please cite this article as: Yu, Jianhan, Xu, Huamin, Shen, Xiaoli, Jiang, Hong, Ghrelin Protects MES23.5 Cells against Rotenone via Inhibiting Mitochondrial Dysfunction and Apoptosis., (2015), doi: 10.1016/j.npep.2015.09.011

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ACCEPTED MANUSCRIPT Ghrelin Protects MES23.5 Cells against Rotenone via Inhibiting Mitochondrial Dysfunction and Apoptosis

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Jianhan Yu, Huamin Xu, Xiaoli Shen, Hong Jiang*

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Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Medical College

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of Qingdao University, Qingdao,266071, Chin

*Correspondence to: Prof. Hong Jiang, Department of Physiology, Shandong Provincial Key

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Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key

Qingdao, China, 266071

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Tel: +86 532 83780191;

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Disciplines: Physiology, Medical College of Qingdao University, No. 308 Ningxia Road,

Fax: +86 532 83780136;

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E-mail:[email protected]

ACCEPTED MANUSCRIPT Abstract Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor and has several important physiological functions. Recently, particular attention has been paid to

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its neuroprotective effect. Rotenone is used to investigate the pathogenesis of Parkinson’s disease (PD) for its ability to inhibit mitochondrial complex I. The current study was carried out

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to investigate the neuroprotective effects of ghrelin against rotenone in MES 23.5 dopaminergic cells and explored the possible mechanisms underlying this protection. Our results showed that rotenone induced significant decrease in cell viability which was

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counteracted by ghrelin treatment. In addition, rotenone challenge reduced mitochondrial membrane potential, inhibited the activity of mitochondrial complex I and depressed

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cytochrome C release from mitochondria. This mitochondrial dysfunction was reversed by the effect of ghrelin treatment. Furthermore, our results demonstrated that ghrelin protected

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MES23.5 cells against rotenone-induced apoptosis by inhibiting activation of caspase-3.

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Overall, our findings showed ghrelin provided protective effects on MES23.5 dopaminergic cells against rotenone via restoring mitochondrial dysfunction and inhibiting mitochondrial

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dependent apoptosis.

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Keywords: Parkinson’s disease; Ghrelin; rotenone; dopamine neuron; apoptosis

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ACCEPTED MANUSCRIPT 1. Introduction Parkinson’s disease (PD) is a neurodegenerative disease characterized by resting tremor, rigidity and bradykinesia. The primary pathological changes of PD is the loss of dopaminergic

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neurons of the substangia nigra pars compacta (SNpc) (Fahn, 2003). Although genetic mutations contribute to the development of PD, mitochondrial dysfunction, protein

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mishandling, inflammatory response and environment factors were reported to play an important role in the pathogenesis of sporadic PD for most of the cases (Modgil et al., 2014). Among the risk factors, accumulating evidence showed that exposure to the environmental

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neurotoxin rotenone increased the probability of developing PD (Sanders and Greenamyre, 2013). Rotenone is a kind of fat-solubility environmental toxin which is used as the insecticide.

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Epidemiological study showed that a long time rotenone exposure was associated with the one set of PD (Tsui et al., 1999). Rotenone can pass through the cell membrane and the

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mitochondrial outer membrane. And then it inhibits the activity of mitochondrial complex I, an

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enzyme essential for the oxidation respiratory chain (Friedrich et al., 1994; Hollingworth et al., 1994). And this leads to the release of ROS and cytochrome C from mitochondria to

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cytoplasm which induce oxidative stress and apoptotic cascade of cells (Bedner et al., 1999; Zamzami et al., 1996). Braak and colleagues have proposed a hypothesis for PD progression that rotenone might initiate PD pathogenesis by inducing a-synuclein pathology from enteric

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nervous system and olfactory bulb to the midbrain (Braak et al., 2003). Due to these properties, it was widely used to induce both cell and animal models of PD (Alam and Schmidt, 2002; Betarbet et al., 2000; Hoglinger et al., 2003; Sherer et al., 2003). Ghrelin is a 28-amino acid peptide found in 1999 (Kojima et al., 1999). It’s the only endogenous ligand for the growth hormone secretagogue receptor (GHS-R) (Bednarek et al., 2000; Kojima and Kangawa, 2005). GHS-R has two types: GHS-R1a and GHS-R1b. It was reported that ghrelin exerted its bioactivities by binding to GHS-R1a (Howard et al., 1996). Ghrelin is mainly secreted by stomach, and it could also be secreted by pancreas, hypothalamus and pituitary etc (Cowley et al., 2003; Kojima et al., 1999). The main function of ghrelin is promoting appetite and adiposity and regulating the energy metabolism (Korbonits et al., 2002; Lin et al., 2004; Lindeman et al., 2002; Theander-Carrillo et al., 2006; Tritos et al., 2003). Recently, accumulating evidence supported that ghrelin exerted the function of 3

ACCEPTED MANUSCRIPT neuroprotection and anti-apoptosis (Baldanzi et al., 2002; Chung et al., 2007; Kim et al., 2004; Kim et al., 2005; Nanzer et al., 2004). It is reported that ghrelin protected hypothalamus neuron from oxygen and glucose deprivation (Chung et al., 2007; Kim et al., 2005). Our

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previous studies also demonstrated that ghrelin antagonized 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-induced neurotoxicity (Dong et al., 2009; Jiang et al., 2008). The

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mechanisms underlying this protection were mediated by its anti-oxidant effect by inhibiting the production of ROS and anti-apoptosis effect by decreasing the Bcl-2/Bax ratio. However, whether ghrelin could exert the protective effect against environmental neurotoxin rotenone-

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induced neurotoxicity is not clear. The aim of this study was to investigate the cytoprotective potential of ghrelin against rotenone-induced toxicity in MES23.5 dopaminergic cells and

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elucidated the possible mechanisms underlying this neuroprotection.

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2. Materials and methods

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2.1 Materials

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis.

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MO, USA). Dulbecco’s modified Eagle’s medium/Nutrient Mixture-F12 (Ham; DMEM/F12) was from Gibco (Gibco, Grand Island, NY, USA). The mitochondria isolation kit was from Clontech (Clontech, USA). Mitochondrial respiratory chain complexes I activity colorimetric

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assay kit were from Nanjing Jiancheng Bioengineering Institute. The phycoerythrin (PE)conjugated monoclonal active caspase-3 antibody apoptosis kit was from BD Bioscience Company (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA). Hoechst 33258 was from Beyotime (Beyotime, Jiangsu, China).

2.2 Cell culture MES23.5 cells were offered by Dr. Wei-Dong Le (Baylor Collegeof Medicine, TX, USA). It is a dopaminergic cell line hybridized from murine neuroblastoma-glioma N18TG2 cells with rat mesencephalic neurons, which exhibits several properties similar to the primary neurons originated in the substantia nigra (Crawford et al., 1992). Cells were cultured in DMEM/F12 containing Sato's components growth medium supplemented with 5% FBS, 100 units/mL of penicillin and 100 mg/mL of streptomycin at 37 °C, in a humid 5% CO2, 95% air environment. 4

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For experiments, cells were seeded at a density of 1×10 /cm in the plastic flasks. 96-well plates were used for MTT assay and 6-well plates were used for other experiments. Rotenone was prepared with DMSO, the final concentration of DMSO in cells was 0.1% and the same

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were pretreated with ghrelin (10

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concentration was used as a vehicle control. To study the protective effects of ghrelin, cells mol/L) for 20 min, and then ghrelin was washed away after

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20 min preincubation and new ghrelin was added to incubate with rotenone (500 nmol/L) for

2.3 Methyl thiazolyl tetrazolium (MTT) assay

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24 hours.

To assess the neuroprotective effects of ghrelin, MES23.5 cells were seeded in 96-well 4

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plates at 2×10 cells/well in 150 μL culture medium. After 24 h, cells were pre-incubated with -9

ghrelin (10 mol/L) dissolved in DMEM/F12 without serum supplement for 20 min, and then

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treated with rotenone (500 nmol/L) for another 24 h. MTT was added (5 mg/mL) to culture

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medium for 4 h at 37°C, and cell viability was assessed at 494 nm and 630 nm with a

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spectrophotometer (Tecan, Grodig, Austria).

2.4 Mitochondria extraction

ApoAlert® Cell Fractionation Kit (Clontech) was used to extract mitochondria. After

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centrifuging at 600 g for 5 min at 4°C, cells were resuspended in 1 ml ice-cold wash buffer. After that, cells were centrifuged at 600 g for 5 min at 4°C. Then remove supernatant and resuspend cells in 0.8 ml ice-cold fractionation buffer mix. After incubating on ice for 10 min, cells were homogenized in an ice-cold dounce tissue grinder. Next, the homogenate was transferred to a 1.5 ml microcentrifuge tube, and centrifuged at 700 g for 10 min at 4°C. Then, the supernatant was transferred to a fresh, 1.5 ml tube, and centrifuged at 10000 g for 25 min at 4°C. Finally the supernatant was collected as the cytosolic fraction, and the pellet was resuspended in 0.1 ml fractionation buffer mix as the mitochondrial fraction. COXIV expression in mitochondria and β-actin expression for cytoplasm were used as control.

2.5 Mitochondrial respiratory chain complexes I (NADH dehydrogenase) activity assay Mitochondrion was isolated as described above and was normalized for protein. NADH 5

ACCEPTED MANUSCRIPT dehydrogenase which is located in the inner mitochondrial membrane could catalyze the transfer of electrons from NADH to coenzyme Q (CoQ). NADH dehydrogenase activity was determined at 340 nm using spectrophotometer by following the decrease in NADH +

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absorbance that occurred when NADH was converted to oxidize NAD . The reaction was started by the incubation of the reaction mixture at 30℃ for 3 minutes according to the

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manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute). Then samples were added to the reaction mixture to detect NADH dehydrogenase activity. Results were

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expressed as changes in NADH level in micromoles per minute per milligram of protein.

2.6 Measurement of mitochondrial transmembrane potential (ΔΨm) -9

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After pretreatment with ghrelin (10 mol/L) for 20 min, cells were treated with rotenone (500 nmol/L) without serum for a further 24 h, and then incubated with rhodamine123 at a

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final concentration of 5 μmol/L for 10 min at 37°C. Fluorescence intensity was recorded at 488

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2.7 Western blot analysis

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nm excitation and 525 nm emissions by flow cytometry.

Mitochondria fraction and cytosolic fraction were separated by 8% sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. Blots were

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probed with anti-cytochrome C monoclonal antibody (Clontech, 1:200). Blots were also probed with anti-β-actin monoclonal antibody (Sigma, 1:5000) and anti-COXIV monoclonal antibody (Clontech, 1:500) as a loading control.

2.8 Active caspase-3 assay Caspase-3 activity was measured by flow cytometry using a PE-conjugated monoclonal active caspase-3 antibody apoptosis kit (BD Bioscience). After washing twice with cold phosphate-buffered saline, cells were resuspended in Citofix/Cytoperm™ (0.5 mL) at a 6

density of 1×10 cells/0.5 mL and incubated on ice for 20 min. Cells were then washed twice with Perm/Wash buffer and incubated with antibody (100 μL Perm/Wash buffer plus 20 μL antibody per sample) for 30 min. After one wash with Perm/Wash buffer, cells were resuspended in 0.5 mL Perm/Wash buffer and analyzed by flow cytometry at 523 nm 6

ACCEPTED MANUSCRIPT excitation and 658 nm emissions. Apoptosis was evaluated as the percentage of caspase-3positive cells in the total number of cells using cellquest software (BD Bioscience).

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2.9 Hoechst 33258 staining

MES23.5 cells were seeded on sterile cover glasses placed in a six-well plate at a density of 4

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1.0×10 cells per square centimeter. Cells in different groups were fixed and washed twice with phosphate buffer solution (PBS) and then stained with Hoechst 33258 staining solution according to the manufacturer’s instructions (Beyotime, Jiangsu, China). Cells were then

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examined immediately and photographed under a fluorescence microscope (Olympus, Japan) with an excitation wave length of 330–380 nm. Apoptotic cells were defined on the basis of

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nuclear morphological changes, such as chromatin condensation and fragmentation. The total number of condensed cells was counted manually by researchers blinded to the treatment

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schedule using unbiased stereology and a fluorescence microscope (Olympus, Japan). For 2

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each well, we delineated a 400 μm frame and counted all condensed and normal nuclei with at least ten different fields in one well. Average sum of condensed and normal nuclei was

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calculated per well. The data were expressed as a percentage of condensed nuclear number to the total number.

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2.10 Statistical analysis

Results were presented as mean±SEM. One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test was used to compare differences between means in more than two groups. A probability value of P<0.05 was considered to be statistically significant.

3. Results 3.1 Ghrelin Protected MES23.5 Cells against Rotenone-Induced Cytotoxicity Cell viability was measured in MES23.5 cells after 0-500 nmol/L rotenone treatment for 24 hours. A significant reduction of cell viability was observed when treated with 100-500 nmol/L rotenone as shown in Fig.1A (P<0.05). 500 nmol/L rotenone was chosen to do the following experiments. To investigate whether ghrelin could antagonize rotenone-induced cell injury in vitro in 7

ACCEPTED MANUSCRIPT MES23.5 cells, MTT assay was used to detect the effect of ghrelin on rotenone-induced decrease in cell viability. Results showed that ghrelin pretreatment (10

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mol/L) significantly

increased the cell viability compared to the cells with solely rotenone treatment. The

mol/L) pretreatment as shown

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inFig.1B (P<0.05).

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(P<0.05); however, it increased to 95.27% after ghrelin (10

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percentage of viable cells in rotenone group decreased to 60.95% compared to the control

3.2 Ghrelin Restored Rotenone-Induced Inhibition of Mitochondrial Respiratory Chain

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Complex I Activity and Prevented Rotenone-Induced Decrease in ΔΨM. It was reported that rotenone exerted its neurotoxicity mainly by inhibiting the activity of

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mitochondrial respiratory chain complex I. So we analyzed the activity of mitochondrial respiratory chain complex I to investigate whether the neuroprotection of ghrelin against

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rotenone insult was related to its ability to restore the activity of mitochondrial respiratory

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chain complex I. Results showed that there’s a significant inhibition of the activity of mitochondrial respiratory chain complex I in rotenone-treated group (P<0.05), and this effect

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was completely reversed by ghrelin pretreatment as shown in Fig. 2A (P<0.05). In addition, changes of mitochondrial membrane potential are markers of mitochondria function. To further confirm the protective effect of ghrelin on mitochondrial dysfunction in

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MES23.5 cells, ΔΨM was measured using rhodamine 123 by flow cytometry. Results showed ΔΨM decreased by 31.38% in the cells incubated with rotenone compared to the control (P<0.05). However, ghrelin pretreatment significantly restored the ΔΨM from rotenone treatment to a decrease of 14.55% compared to the control (P<0.05) (Fig. 2B). This indicated ghrelin could protect MES 23.5 dopaminergic neurons against rotenone by inhibiting the mitochondrial dysfunction.

3.3 Ghrelin Antagonized Rotenone-Induced Cytochrome C Release The impairment of mitochondria leads to cytochrome C release from mitochondria to cytosolic compartment. Therefore, to further investigate the mechanisms underlying the protective effect of ghrelin, we detected the protein expressions of cytochrome C in mitochondria and cytosolic compartment by western blots. Results showed a decreased 8

ACCEPTED MANUSCRIPT protein expression of cytochrome C in mitochondria was observed in rotenone-treated cells compared to the control (P<0.05). Ghrelin pretreatment could increase mitochondrial protein levels of cytochrome C compared to rotenone-treated group (P<0.05) (Fig.3 A, C). Cytosolic

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protein expression of cytochrome C significantly increased in rotenone-treated cells compared to the control (P<0.05). And this increase was inhibited by ghrelin pretreatmentcompared to

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rotenone-treated group (P<0.05) (Fig.3B, D). These results suggested that ghrelin could counteract rotenone-induced release of cytochrome C from mitochondria to cytosolic

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compartment.

3.4 Ghrelin Inhibited Rotenone-Induced Caspase-3 Activation and Nuclear Fragmentation

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The release of cytochrome C could promote oxidative stress and finally lead to caspase3 activation. To further confirm the effect of ghrelin on rotenone-induced apoptosis, we

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measured the activation of caspase-3 using a PE-conjugated monoclonal active caspase-3

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antibody apoptosis kit by flow cytometry. Results demonstrated that there was an increased activation of caspase-3 after incubation with rotenone compared to the control (P<0.05), and

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this effect could be significantly reversed by ghrelin pretreatment (P<0.05, compared to rotenone-treated group) (Fig.4). To further confirm the cell apoptosis, the nuclear morphology was analyzed by Hoechst staining. The nuclei of MES23.5 cells appeared hyper-condensed

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and fragmentation of chromatin in rotenone-treated group. % of condensed nuclei increased in rotenone-treated group compared to the control (P<0.05). Pretreatment with ghrelin could significantly abolish these nuclear morphological changes (P<0.05) (Fig.5). These results suggested that ghrelin protected MES23.5 cells against rotenone-induced apoptosis by inhibiting activation of caspase-3.

4. Discussion In the present study, we provided data showing that ghrelin could significantly antagonize rotenone-induced neurotoxicity in MES23.5 cells. This protection is mediated by restoration of mitochondria transmembrane potential, inhibition of the activity of mitochondrial complex Ⅰand cytochrome C release from mitochondria. We also demonstrated that ghrelin could exert its anti-apoptotic effect by inhibiting rotenone-induced activation of caspase-3. 9

ACCEPTED MANUSCRIPT Ghrelin is the only endogenous ligand for the GHS-R. It has been reported that ghrelin exerted its bioactivities by binding to GHS-R1a (Howard et al., 1996).The main function of ghrelin is promoting appetite and adiposity and regulating the energy metabolism (Korbonits

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et al., 2002; Lin et al., 2004; Lindeman et al., 2002; Theander-Carrillo et al., 2006; Tritos et al., 2003). Recent studies showed that ghrelin could stimulate proliferation, migration and

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differentiation of neural progenitors, protect hippocampus from ischemia reperfusion and mitigate neuroinflammation by inhibiting the release of IL-6 (Beynon et al., 2013; Li et al., 2014; Liu et al., 2009). Ghrelin also could protect dopaminergic cells by blocking microglial

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activation and influencing the electrophysiological activity of dopaminergic neurons (Andrews et al., 2009; Moon et al., 2009). Our previous study showed that GHS-R1a was expressed on

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dopaminergic cells and could protect against MPTP-induced neurotoxicity to the dopaminergic neurons in vivo and in vitro (Chung et al., 2007; Kim et al., 2005). The

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mechanisms underlying this protection were mediated by its anti-oxidant effect by inhibiting

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the production of ROS and anti-apoptosis effect by decreasing the Bcl-2/Bax ratio (Dong et al., 2009; Jiang et al., 2008). Our previous data also demonstrated that ghrelin could enhance

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firing of nigral dopaminergic neurons by inhibiting voltage-gated potassium Kv7/KCNQ/Mchannels through its receptor GHS-R1a and activation of the PLC-PKC pathway (Shi et al., 2013). This provides evidence that ghrelin has the potential to become a neuroprotective

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agent for PD.

Accumulated evidence has proved the important role of mitochondrial dysfunction in the pathogenesis of PD (Dexter and Jenner, 2013; Esteves et al., 2011; Hauser and Hastings, 2012). Therefore, protection against mitochondrial dysfunction has a great potential as a target for PD therapy. In this study, we investigated whether ghrelin could exert the protective effect on environmental neurotoxin rotenone-induced mitochondrial dysfunction and elucidated the possible mechanisms. Study showed that the activity of mitochondria respiratory chain complex I in SNpc of PD patients was significantly lower than healthy person (Schapira et al., 1990). As complex I is the direct target of rotenone, it is reasonable to think that the mechanism of ghrelin protection might be related to impairing rotenone action on complex I, as this is the first defect induced by rotenone treatment. Our results showed that ghrelin restored rotenone-induced inhibition of mitochondrial respiratory chain complex I activity. Further studies showed that ghrelin also had significant effect on stability the mitochondrial transmembrane potential, which is one of the important events reflecting 10

ACCEPTED MANUSCRIPT mitochondrial functions. What is the mechanism underlying the effect of ghrelin on rotenoneinduced mitochondrial dysfunction? It has been proved that ghrelin could promote SNpc dopamine function by increasing mitochondrial biogenesis (Andrews et al., 2009). They

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showed that ghrelin promoted mitochondrial respiration and proliferation via activation of the

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AMPK-UCP2 pathway (Andrews et al., 2009). However, there was no significant difference in complex I activity between ghrelin-treated cells and control in MES23.5 dopaminergic cells

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(Data not shown). In addition, our previous in vivo study also demonstrated that ghrelin solelytreated had no effect on the number of TH-ir neurons in substantial nigra, DA content in the striatum and caspase-3 activation in C57BL/6 mice (Jiang et al., 2008). In line with this,

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results of Andrews also showed that there was no difference in total number of TH cells in the entire SNpc in mice treated with ghrelin and saline (Andrews et al., 2009). The observation that ghrelin promotes mitochondrial proliferation raises the possibility that ghrelin could

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strengthen the nigrostriatal dopaminergic system during increased cellular stress such as rotenone to resistant the toxicity of rotenone on mitochondrion. This might be one of the mechanisms underlying the protective effect of ghrelin on rotenone-induced mitochondrial

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dysfunction of MES23.5 dopaminergic cells.

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The mitochondrial transmembrane potential collapse induced the formation of ROS, and further ROS formation damages the mitochondrial membrane. This also could lead to

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cytochrome C in mitochondria releases to cytoplasm (Sanders et al., 2013). The released cytochrome C could combine with apoptotic protease activating factor 1 (Apaf-1) when ATP is existed, then caspase-9 is recruited and activated. Finally caspase-3 activation is caused by

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the activated caspase-9 and then initiates the cell apoptosis (Bouchier-Hayes et al., 2005). Our results confirmed that ghrelin could inhibit rotenone-induced release of cytochrome C from mitochondria to cytoplasm. Accordingly, in this study, the rotenone-induced apoptosis and caspase-3 activation were significantly alleviated by ghrelin pretreatment. This indicated that mitochondrial dysfunctions and the subsequent classic apoptotic pathway activation were both inhibited by ghrelin pretreatment. In summary, we demonstrated that ghrelin could significantly antagonize rotenoneinduced neurotoxicity in MES23.5 cells via ameliorating the mitochondrial dysfunction including inhibition of mitochondrial respiratory chain complex I activity and stabilizing the mitochondrial transmembrane potential, thus decreased the release of cytochrome C from mitochondria. This further inhibited caspase-3 activation and apoptosis. These results

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ACCEPTED MANUSCRIPT provided experimental basis for the development of drugs for PD, suggesting that ghrelin may be a novel therapeutic strategy against environmental toxin for the preventive and/or

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complementary therapies of PD.

Acknowledgements

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This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2011CB504102), the National Foundation of Natural Science of China (81430024, 31471114, 81171207, 31271131).

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Taishan Scholars Construction Project, Shandong.

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ACCEPTED MANUSCRIPT Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M., Kroemer, G., 1996. Mitochondrial control of nuclear apoptosis. The Journal of experimental medicine 183, 1533-

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Figure legends -9

Fig. 1 Changes in cell viability with rotenone treatment and ghrelin (10 mol/L) pretreatment.

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A. MTT assay was used to determine the viability of MES23.5 cells treated with different doses of rotenone. A significant reduction of cell viability was observed when treated with 100-

*P<0.05, compared to the control. B. Changes in cell viability with ghrelin (10

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500 nmol/L rotenone. Data were presented as mean±SEM of 6 individual experiments.

mol/L) pretreatment were measured by MTT

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assay. Ghrelin significantly inhibited the rotenone (500 nmol/L)-induced reduction in cell viability. Data were presented as mean±SEM of 6 individual experiments. *P<0.05, compared #

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to the control; P<0.05, compared to rotenone-treated group.

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Fig.2 Ghrelin inhibited rotenone-induced mitochondrial dysfunction.

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A. Changes of NADH level in MES23.5 cells with ghrelin pretreatment. The rotenone-induced inhibition of mitochondrial respiratory chain complexes I activity was completely blocked by

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ghrelin pretreatment. NADH dehydrogenase activity was expressed in micromoles per minute per milligram of protein (μmol NADH/min per mg protein). Data were presented as mean±SEM of 6 individual experiments. *P<0.05, compared to the control;

#

P<0.05,

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compared to rotenone-treated group. B. Flow cytometry was applied to assess the mitochondrial transmembrane potential (ΔΨm). Data were presented as means±SEM of 6 individual experiments. Fluorescence values of the #

control were set to 100%. *P<0.05 compared to the control; P<0.05 compared to rotenonetreated group.

Fig.3 Expression of cytochrome C was detected in MES23.5 cells. The expression of cytochrome C in mitochondria (A, C) and cytoplasm (B, D) were shown. Results showed that rotenone-induced release of cytochrome C could be blocked by ghrelin pretreatment. Data were presented as mean±SEM of 7 individual experiments. *P<0.05 compared to the control; #

P<0.05 compared to rotenone-treated group.

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ACCEPTED MANUSCRIPT Fig.4 Flow cytometry was applied to assess the caspase-3 activation in rotenone-treated MES23.5 cells with ghrelin pretreatment. A. Representatives of the fluorometric assay of active caspase-3. B. Statistical analysis. This assay confirmed that ghrelin could inhibit the

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activation of caspase-3 induced by rotenone treatment. Data were presented as means±SEM of 6 individual experiments. Fluorescence values of the control were set to 100%. *P<0.05, #

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compared to the control; P< 0.05, compared to rotenone-treated group.

Fig.5 Morphological changes in rotenone-treated MES23.5 cells with ghrelin pretreatment. A.

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Representative photographs of Hoechst 33258 staining. B. Statistical analysis. Rotenone treatment resulted in nuclear condensation; however, ghrelin pretreatment could significantly

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attenuate this effect. Data were presented as means±SEM of 6 individual experiments. #

*P<0.05, compared to the control; P< 0.05, compared to rotenone-treated group. Scale

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bar=25 µm.

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Figure 5

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Highlights 1. Ghrelin provided protective effects on MES23.5 dopaminergic cells against

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rotenone

2. Rotenone-induced mitochondrial dysfunction was reversed by ghrelin.

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3. Ghrelin protected against rotenone-induced apoptosis by inhibiting

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caspase-3 activation.

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