Neurochem Res DOI 10.1007/s11064-014-1249-7
ORIGINAL PAPER
Mangiferin Antagonizes Rotenone: Induced Apoptosis Through Attenuating Mitochondrial Dysfunction and Oxidative Stress in SK-N-SH Neuroblastoma Cells Mani Kavitha • Thamilarasan Manivasagam • Musthafa Mohamed Essa • Kuppusamy Tamilselvam • Govindasamy Pushpavathy Selvakumar • Subran Karthikeyan • Justin Arokiasamy Thenmozhi • Selvaraju Subash
Received: 14 July 2013 / Revised: 18 January 2014 / Accepted: 21 January 2014 Ó Springer Science+Business Media New York 2014
Abstract In the present study, using a human neuroblastoma SK-N-SH cells, we explored antioxidant, mitochondrial protective and antiapoptotic properties of mangiferin against rotenone-mediated cytotoxicity. SK-NSH cells are divided into four experimental groups based on 3-(4,5-dimethyl2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay—untreated cells, cells incubated with rotenone (100 nM), cells treated with mangiferin (20 lg) (pretreatment 4 h before) ? rotenone (100 nM) and mangiferin alone treated. 24 h after treatment with rotenone and 28 h after treatment with mangiferin, levels of ATP thiobarbituricacid reactive substances and reduced glutathione and activities of enzymatic antioxidants including superoxide dismutase, catalase and glutathione peroxidise were measured. Finally mitochondrial transmembrane potential and expressions of apoptotic protein were also analysed. Pre-treatment with mangiferin significantly enhanced cell viability, ameliorated decrease in mitochondrial membrane potential and decreased rotenoneinduced apoptosis in the cellular model of Parkinson’s disease. Moreover oxidative imbalance induced by
M. Kavitha T. Manivasagam (&) K. Tamilselvam G. P. Selvakumar S. Karthikeyan J. A. Thenmozhi Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India e-mail:
[email protected] M. M. Essa (&) S. Subash Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, P.O. 34, Al-Khoud, Muscat 123, Oman e-mail:
[email protected] M. M. Essa S. Subash Ageing and dementia research Group, Sultan Qaboos University, P.O. 34, Al-Khoud, Muscat 123, Oman
rotenone was partially rectified by mangiferin. Our results indicated that anti-apoptotic properties of this natural compound due to its antioxidant and mitochondrial protective function protect rotenone induced cytotoxicity. Keywords Rotenone Mangiferin Mitochondrial dysfunction Oxidative stress Apoptosis
Introduction Parkinson’s disease (PD) is the most common neurodegenerative disorder of movement, mainly affecting aged persons. The pathological hallmark of PD is the selective loss of dopaminergic neurons in the substantia nigra (SN). These dopaminergic neurons are required for proper motor function, and their loss is associated with tremors, rigidity, bradykinesia, and postural instability. Though the cause of PD is not known exactly, environmental factors including exposure to chemical pollutants such as agricultural pesticides have a strong correlation with an increased incidence of idiopathic PD [1]. Rotenone, a natural pesticide and insecticide, is a specific inhibitor of complex I of the mitochondrial respiratory chain. Due to its extremely hydrophobic nature, it crosses biological membranes easily and does not depend on the dopamine transporter for acquiring access to the cytoplasm of dopaminergic neurons. Inside the nerve cells, rotenone inhibits mitochondrial respiration, and thus elicits ATP deficiency, produces reactive oxidant species, and disrupts Ca2? homeostasis. Further downstream mitochondrial damage leads to the release of cytochrome C and caspase-3 activation and finally leads to apoptotic death [2]. Severity and prevalence of this disease are not yet under control despite the availability of various treatment strategies. Hence, alternative and complementary medicines
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including herbal extracts containing phytochemicals are being used in the management of neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease [3– 6]. Mangiferin is the primary polyphenol component of mangifera, its antioxidant properties have been extensively evaluated in various cells, including neurons [7]. In vitro activities of mangiferin show that it protects oligodendrocytes, brain neurons and cortical neurons [8] from excitotoxicity, protects neuronal loss in the hippocampus due to forebrain ischemia and hypoxia in experimental animals [9]. Due to its antioxidant property, mangiferin protected N2A cells against MPP?-induced cytotoxicity, by quenching the reactive oxygen intermediates, restoring glutathione (GSH) content and down-regulating both sod1 and cat mRNA expression. However in this study, we investigated the neuropreventive effect of mangiferin on rotenone induced cellular model of PD by analysing its antioxidantal, mitochondrial protective and antiapoptotic properties.
Based on the MTT assay, we found that the effective dose of rotenone was 100 nM (*50 % cell viability). Cells were pre-treated with different concentrations of mangiferin (2.5, 5, 10, 20 and 40 lg/ml) for 4 h, and then incubated with rotenone (100 nM) for 24 h. Based on the cell viability assay, the effective dose of mangiferin was used for further study (Estimation of MMP, ATP, apoptosis, thiobarbituricacid reactive substances (TBARS) and antioxidants; protein expression studies of apoptotic markers were carried out 24 h after the treatment of rotenone). Experimental Design Group Group Group Group
I: II: III: IV:
Untreated control cells Rotenone (100 nM) Mangiferin (20 lg) ? Rotenone (100 nM) Mangiferin (20 lg)
Cell Viability Assay Materials and Methods Chemicals Rotenone, mangiferin, thiobarbituric acid (TBA), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), 5,5dithiobis(2-nitrobenzoic acid) (DTNB), MTT, 2-7-diacetyl dichlorofluorescein (DCFH-DA), Rhodamine 123 (Rh-123), heat-inactivated fetal calf serum (FCS), Dulbecco’s modified Eagle’s medium (DMEM), glutamine, penicillin–streptomycin, EDTA, and trypsin were acquired from Sigma Chemicals Co., (St. Louis, USA). Anti-Bcl-2, anti-Bax, Caspase-3, caspase-9 and cytochrome-C antibodies were obtained from Cell Signalling (USA) and b-actin antibodies were purchased from Santa Cruz Biotechnology, Inc, (USA). ATP Bioluminescence Assay Kit HS II was obtained from (Roche Molecular Biochemicals). Anti-mouse and anti-rabbit secondary antibodies were purchased from Genei, (Bangalore, India).
Cell viability was measured by the MTT method [10]. SK-NSH (1 9 103cells/well) was pre-treated with mangiferin (2.5, 5, 10, 20 and 40 lg/100 ll of medium). After 4 h incubation, 100 nM of rotenone was added to respective groups. 0.5 ml of MTT reagent was added to each well after 24 h. Then the cells were centrifuged for 10 min and the supernatant was removed, 200 ll of DMSO were added into each well to dissolve the formazan crystals and absorbance was measured in a microplate reader at 560 nm. Cell viability was expressed as a percentage of the control culture value. Determination of Intracellular ATP Levels Cells were collected by centrifugation and intracellular ATP was measured using an ATP Bioluminescence Assay Kit HS II (Roche Molecular Biochemicals) according to the manufacturer’s instructions.
Cell Culture and Media Preparation
Estimation of Oxidant and Antioxidant Indices
SK-N-SH neuroblastoma cell line obtained from National Center for Cell Science (NCCS) Pune, India were cultured with Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum (FBS) (Himedia), 2 mM glutamine and penicillin/streptomycin (100 U/ml) in an incubator humidified with 95 % air and 5 % CO2 and the medium was changed for every 2 days. Cells were maintained at 37 °C in CO2 incubator in a saturated humidity atmosphere containing 95 % air and 5 % CO2. Rotenone and mangiferin were made fresh in DMSO (0.05 %) prior to each experiment. Mangiferin was added 4 h prior to rotenone treatment.
Following the incubation with mangiferin and/rotenone, SK-N-SH neuroblastoma cells (5 9 103 cells/well in a six well plate) were suspended in 130 mM KCl and 50 mM PBS containing 0.1 ml of 0.1 M dithiothreitol and centrifuged at 20,000g for 15 min (4 °C). The supernatant was taken for further biochemical analysis.
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Measurement of TBARS The level of lipid peroxidation was determined by analyzing TBARS according to the protocol of [11]. The pink coloured chromogen formed by the reaction of 2-TBA with
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breakdown products of lipid peroxidation was measured. 0.2 mL of sample was diluted with double distilled water (0.2 mL) and mixed well, and then 2.0 mL of TBA-TCAHCl reagent was added. The mixture was kept in a boiling water bath for 15 min, after cooling, the tubes were centrifuged at 1,000g for 10 min and the supernatant was estimated at 535 nm. Assay of SOD Total superoxide dismutase (SOD) activity was examined in the supernatants according to the method described by Del Maestro and McDonald [12]. This assay is based on the ability of SOD to scavenge superoxide anion radical (O2), which decreases the overall rate of pyrogallol autoxidation. In brief, 1 mL of 0.05 mol/L Tris–HCl buffer (pH 8.2) containing 1 mmol/L DTPA was added to 200 ll of sample. The reaction was initiated by the addition of 0.2 mmol/ L pyrogallol, and the change in optical density at 420 nm was recorded for 3 min. In a separate reaction, specific MnSOD activity was measured by the addition of 1 mmol/ L sodium cyanide to the sample to inhibit CuZnSOD. SOD activity was calculated as units per milligram of protein, with 1 U of SOD defined as the amount that inhibited the rate of pyrogallol autoxidation by 50 %. We calculated CuZnSOD activity by subtracting the value using cyanide from the total SOD value. Assay of Catalase Catalase activity was assayed by the decomposition of hydrogen peroxide in the previous method [10], a decrease in absorbance due to H2O2 degradation was monitored at 240 nm for 1 min. The reaction mixture contained 50 mM phosphate buffer (7.0), 40 mM of H2O2 and 200 ll of cell lysate [13]. One unit of catalase is defined as the amount of enzyme that transforms 1 lmol of hydrogen peroxide per minute at 25 °C. Assay of GPx Glutathione peroxidase activity was measured by the method described by Rotruck et al. [14]. The reaction mixture contained 2.0 ml of 0.4 M Tris–HCl buffer, pH 7.0, 0.01 ml of 10 mM sodium azide, 200 ll of cell lysate, 0.2 ml of 10 mM glutathione and 0.5 ml of 0.2 mM. H2O2. The contents were incubated at 37 °C for 10 min followed by the termination of the reaction by the addition of 0.4 ml 10 % (v/v) TCA, centrifuged at 5,000 rpm for 5 min. The absorbance of the product was read at 430 nm and calculated as nmol of NADPH consumed/min/mg protein and expressed as mU/mg protein.
Determination of GSH GSH was determined by the method of Ellman’s [15]. In brief, to 200 ll of cell lysate, 0.5 ml of Ellmans reagent (19.8 mg of 5, 50 -dithiobisnitro benzoic acid (DTNB) in 100 ml of 0.1 % sodium nitrate), 3.0 ml of phosphate buffer (0.2 M, pH 8.0) and 0.4 ml of distilled water was added. The mixture was thoroughly mixed and the absorbance was read at 412 nm, expressed as nmol/mg protein. Changes in Mitochondrial Transmembrane Potential (Dwm) The change in Dwm in different treatment groups was observed microscopically and determined fluorimetrically using fluorescent dye Rh-123. To the control and experimental neuroblastoma cells, 1 ll of fluorescent dye Rh-123 (5 mmol/L) was added and kept in incubator for 15 min [10]. Then the cells were washed with PBS and observed under fluorescence microscope and estimated by using blue filter (450–490 nm) (Shimadzu RF-5301 PC spectrofluorimeter). Polarized mitochondria emit orange-red fluorescence and depolarized mitochondria emit green fluorescence. The fluorescence intensity was measured at 535 nm. Western Blot Analysis Briefly, cells in 6 well plates were harvested and washed with PBS. Cells were lysed in 100 ll lysis buffer (20 mM Tris–Hcl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 30 lg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) followed by centrifugation at 1,000g for 5 min at 4 °C. The supernatants (cytosolic fractions) were saved and the pellets solubilized in the same volume of mitochondrial lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2 % Triton X-100, 0.3 % NP-40, 100 lM PMSF, 10 lg/ml leupeptin, 2 lg/ml aprotinin), kept on ice and vortex for 20 min followed by pelleting at 10,000g for 10 min at 4 °C and subjected to 12.5 % poly acrylamide gel electrophoresis lane [16]. A total volume of 40 lg of protein was loaded per lane. The separated proteins were blotted onto a PVDF membrane by semi-dry transfer (BIORAD). After blocking with 5 % non fat milk in TBS, the membranes were then incubated with various antibodies: caspases-3 and 9, cytochrome c and b-actin. The following dilutions were used for cytochrome c, caspases-3 and 9 (1:1,000), and b-actin (1:2,000). After primary antibody incubation, the membranes were incubated with secondary antibody at a concentration of 1:2,000. Then the membranes were washed with Tris-buffered saline and 0.05 % Tween-20 thrice for 10 min interval, after extensive washes in TBST, the bands was visualized by treating the membranes with 3, 30 -diaminobenzidine tetrahydrochloride
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Statistical analysis was performed by one-way analysis of variance followed by Duncan’s multiple range test (DMRT) using Statistical Package for the Social Science (SPSS) software package version 12.0. Results were expressed as mean ± SD for four experiments in each group. p values (p \ 0.05) were considered significant.
Results Effect of Mangiferin on Rotenone-Induced Changes in Cell Viability and Oxidative Stress Rotenone treatment (2.5, 5, 50, 100 and 200 nM for 24 h) of SK-N-SH cells induced a dose-dependent cytotoxicity, with approximately half-maximal inhibition of cell viability obtained at 100 nM rotenone concentration (Fig. 1a). Mangiferin dose-dependently (0.5, 5, 10, 20 and 40 lg/ml) protected against cell death induced by 100 nM rotenone (Fig. 1b), with about 85 % protection at 20 lg in mangiferin. Table 1 shows the levels of TBARS and GSH and activities of Mn-SOD, CuZn-SOD catalase and glutathione peroxidise (GPx) in rotenone-treated SK-N-SH cells incubated with or without mangiferin. Compared with untreated cells, rotenone treatment (100 nM) increased the levels of TBARS and decreased the levels of GSH significantly in SK-N-SH cells. Pre-treatment with mangiferin (20 lg) to rotenone decreased levels of TBARS and enhanced GSH significantly, compared to rotenone alone treated group Compared with untreated cells, rotenone (100 nM) treatment decreased Mn-SOD, catalase and GPx activities in SK-N-SH cells. Pre-treatment with mangiferin to rotenone treated cells enhanced the activities of Mn-SOD, catalase and GPx significantly, compared to rotenone alone treated group. There is no significance difference between the activities of Cu–Zn SOD in control and experimental groups. Rotenone treatment depleted cellular ATP levels (*65 % at 100 nM) (Fig. 2). Pre-treatment with mangiferin enhanced the ATP levels significantly as compared to rotenone alone treated group. No significant changes in ATP levels were detected in SK-N-SH cells treated only with mangiferin and the activities of CuZn-SOD. Effect of Mangiferin on Rotenone-Induced Mitochondrial Dysfunction and Apoptosis Figure 3a, b shows the mitochondrial membrane potential (DWm) measured by determining the red/green fluorescence ratio after the treatment with rhodamine-123.
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Cell viability (% of control)
Data Analysis
(A) 120 100
a
a b
80
c
60
d
40 20 0 0.5
5.0
50
100
200
Rotenone (nM)
(B) Cell viability ( % of control)
(Western blot detection reagent, Sigma, USA). Densitometry was done using ‘Image J’ analysis software.
Mangiferin
120
Mangiferin+ Rotenone
c
100 80 60
ab
d
b
a
40 20 0 2.5
5
10
20
40
Mangiferin
Fig. 1 a and b Effect of mangiferin on rotenone induced cell death (MTT assay) in SK-N-SH neuroblastama cells. a shows the dose dependent effect of rotenone (0.5,5,50,100 and 200 nM) induced cell viability after 24 h. Values are presented as mean ± SD of four experiments in each groups. An approximately half-maximal inhibition of cell viability was obtained at 100 nM rotenone concentration. b illustrates the dose dependent effect of mangiferin (2.5, 5, 10, 20 and 40 lg) alone and against rotenone induced cell death. Values are presented as mean ± SD of four experiments in each group. Values not sharing a common superscript letter differ significantly (p \ 0.05). Mangiferin alone treatment (blue column) (2.5, 5, 10 and 20 lg) did not induced any toxicity (100 % cell viability), whereas 40 lg decreased cell viability significantly (p \ 0.05). Mangiferin (2.5, 5, 10 and 20 lg) pre-treatment dose dependently enhanced the cell viability against rotenone toxicity, whereas 40 lg of mangiferin reduced cell viability significantly (brown column) (Color figure online)
Rotenone treated cells are depolarised and show significant dissipation of DWm with highly green colour fluorescence as compared with control. Mangiferin pre-treatment to rotenone treated cells displayed red/green fluorescence indicating a polarized state of mitochondrial membrane as compared to rotenone treated cells. Pretreated mangiferin suppressed the rotenone (100 nM) induced apoptosis as shown in Fig. 4a, b. Administration of 100 nM rotenone for 24 h induced morphological changes typical of apoptosis (nuclei fragmentation with bright staining) observed by fluorescent microscopy. Mangiferin pretreatment (20 lg) prevented the morphological changes observed in neuroblastoma cells treated with rotenone indicating that mangiferin prevented the apoptosis induced by rotenone in SK-N-SH cells.
Neurochem Res Table 1 Effect of mangiferin (20 lg) on rotenone (100 nM) induced lipid peroxidation and anti-oxidative stress TBARS nmol/mg protein Control Rotenone Mangiferin ? rotenone Mangiferin
GSH mg/dl
Mn-SOD U1/mg of protein
Cu–Zn-SOD U1/mg of protein
CAT U2/mg of protein
GPx U3/mg of protein
12.56 ± 1.2a
51.14 ± 5.1a
1.32 ± 0.11a
14.7 ± 1.15a
735.30 ± 32.5a
936.80 ± 41.4a
4.11 ± 0.49
b
6.74 ± 0.42
b
329.10 ± 18.2
b
894.30 ± 49.4
a
3.41 ± 0.21
c
11.40 ± 0.81
c
566.40 ± 37.6
c
916.70 ± 71.0
a
1.30 ± 0.14
a
a
758.20 ± 33.5
a
949.50 ± 31.4
a
a
14.74 ± 1.6
5.09 ± 0.48
b
21.27 ± 1.6b
9.01 ± 0.71
c
38.41 ± 3.2c
12.62 ± 1.4
51.19 ± 5.3a
Rotenone treatment significantly increased the levels of TBARS and decreased GSH and activities of Mn-SOD, catalase and GPx respectively as compared to control cells, while mangiferin pretreatment significantly, decreased the levels of TBARS and increased the GSH and activities of Mn-SOD, catalase and GPx significantly as compared to rotenone alone treated cells. There is no signifance difference between the activities of Cu–Zn SOD in control and experimental groups. Values are given as mean ± SD of four experiments in each group. Values not sharing a common alphabet differ significantly at p \ 0.05 (Duncan’s multiple range test-DMRT) a
Enzyme concentration required for 50 % inhibition of nitroblue tetrazolium reduction in 1 min
b
Micromoles of hydrogen peroxide consumed per minute
c
Micrograms of glutathione consumed per minute
ATP (% of control)
120
a
a
100 80
c
60
b
40 20 0
Control
Rotenone
Mangiferin+Rotenone
Mangiferin
Fig. 2 Measurement of ATP levels. Rotenone (100 nM) treatment significantly reduced the levels of ATP as compared to control cells, while mangiferin (20 lg) pretreatment significantly enhanced the levels of ATP as compared to rotenone alone treated cells. Values are given as mean ± SD of four experiments in each group. bp \ 0.05 compared to control and cp \ 0.05 compared to rotenone group (Duncan’s multiple range test-DMRT)
To further characterize the mechanism by which mangiferin prevents rotenone-induced apoptosis, the expression of cyt c and caspase 3 and 9 were studied by Western blot analysis (Fig. 5a, b). The protein expression of cytosol cyt c and caspase 3 and 9 were increased significantly and mitochondrial cyto c was decreased significantly in the rotenone-treated cells compared to the control cells. Mangiferin, which had no effects in control cells receiving vehicle, prevented the increase in the expression of cytosol cyt c and caspase 3 and 9 and the decrease in mitochondrial cyto c observed in the rotenone treated cells.
Discussion It was observed that rotenone destroyed SK-N-SH cells in a dose-dependent manner and approximately half-maximal
inhibition of cell viability was obtained at 100 nM rotenone concentration. This is corroborating with the results of rotenone treatment in other in vitro models (PC12 and SK-N-MC cells) of PD. The direct effect of rotenone toxicity was thought to be mainly attributable to energy metabolic impairment and production of free radicals. Addition of mangiferin significantly enhanced cell viability in a dose-dependent manner in the present study. Our results showed that rotenone treatment induced ATP deficiency and reactive oxidant species production which were attenuated by low doses of mangiferin. Mangiferin is reported to have non-toxic effect in animal experiments, but a high dose decreased cell viability in in vitro experiment, which may be due to change in microenvironment of cells. For example, administration of Epigallocatechin gallate (EGCG), a potent neuroproprotective polyphenol of green tea, at high concentrations (25 and 50 lM), diminished cell viability in SH-SY-5Y cells. In higher dose, EGCG promoted, rather than prevents, neuronal cell damage induced by 6-hydroxydopamine [17] and rotenone [18]. Due to the complex I inhibition, formation of superoxide ion (O2-) is also expected to be enhanced significantly via inhibition of NADH dehydrogenase [19]. It is followed by an increase in NADH. This may thereby result in activation of NAD(P)H oxidase and subsequently, increased O2- generation. This appears to be the primary mechanism of rotenoneinduced formation of O2-, which undergoes spontaneous or SOD-catalyzed dismutation to form H2O2. Catalase and glutathione peroxidase catalyzes the decomposition of H2O2 to H2O and O2. Increased production of ROS during neurodegenerative diseases is an indication of the oxidative stress and leads to a rapid consumption and depletion of endogenous scavenging antioxidants.Decreased activities of enzymatic antioxidants such as SOD, catalase and GPx were probably due to a response towards increased concentration
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(A)
Rotenone(100nM)
Control
(B)
Mangiferin(20µg)+ Rotenone (100nM)
Mangiferin(20µg)
% of fluorescence intensity
MMP 100
a
a
80
c
60 40 b
20 0 Control
Rotenone
Mangiferin+Rotenone
Mangiferin
Fig. 3 The alteration in mitochondria membrane potential of control, mangiferin and rotenone treated SK-N-SH cells. Rotenone (100 nM) significantly decreased mitochondria membrane potential, while mangiferin (20 lg) pretreatment significantly increased MMP
in rotenone treated SK-N-SH cells (a, b). Values are given as mean ± SD of four experiments in each group. bp \ 0.05 compared to control; cp \ 0.05 compared to rotenone groups (Duncan’s multiple range test-DMRT)
of reactive oxygen species and lipid peroxidation in rotenone treated cells. Moreover, GSH depletion, the first indicator of oxidative stress during PD progression, suggests a concomitant increase in reactive oxygen species. Mangiferin seems to balance the endogenous antioxidant by enhancing the activities of SOD, GPx and catalase in therapeutic group. It was reported that the complex I inhibition by rotenone induces increase in the MnSOD activity without altering CuZnSOD in the brain tissue [20, 21]. Mangiferin is a potent antioxidant, offers more protection against TPA-induced oxidative damage as compared to vitamins C and E [7]. But in this study, antioxidant potential of mangiferin is not compared with other well known antioxidants such as vitamins C and E. The impact of rotenone on mitochondria was normally investigated by taking the loss of mitochondrial membrane potential. Disruption of the mitochondrial membrane potential is one of the earliest intracellular events that occur following induction of apoptosis. Previous findings have demonstrated that rotenone induces mitochondrial swelling which is a result of an increase in inner membrane permeability and opening of the mitochondrial permeability transition pores [22]. The opening of the mitochondrial permeability transition pore (PTP) has been considered as the main mechanism in apoptosis activated by different
signalling pathways [23]. It results in the release of mitochondrial cytochrome c and the latter binds to apoptotic protease-activating factor-1 (Apaf-1) leading to the caspase cascade activation, which is a central effector mechanism promoting apoptosis in response to death inducing signals from cell surface receptors or from mitochondria [24]. The release of cyt c from mitochondria leads to a decrease in its content in mitochondria fraction and a simultaneous increase in cytosol. High levels of mitochondrial membrane potential are necessary to maintain closure for the multiprotein pore and the mitochondrial permeability transition pore. In our study, mangiferin significantly prevented rotenone-induced crumple of mitochondrial membrane potential, which inhibited the opening of the mitochondrial permeability transition pore (PTP) causing diminishing release of apoptogenic substances such as cytochrome c from mitochondria into cytosol and subsequently inactivated apoptosis processes. Mitochondria are the site of ATP biosynthesis by oxidative phosphorylation providing energy to power cellular activities. Oxidative phosphorylation requires the coordinated action of five enzyme complexes (I–V), which together composes different structural proteins. A reduction/ inhibition of any one of the above could lead to disrupts the balance between ATP production and consumption and
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(A)
Control
Rotenone (100nM)
Mangiferin(20µg)+ Rotenone (100nM)
(B)
Mangiferin(20µg)
Dual staining % of apoptotic cell
100
b
80 60
c
40 20
a
a
0 Control
Rotenone
Mangiferin+Rotenone
Mangiferin
Fig. 4 The effect of mangiferin on rotenone induced dual staining in SK-N-SH cells. a Photo micrograph illustrates apoptotic morphological changes in SK-N-SH cells treated with mangiferin and rotenone. b depicts apoptotic morphological changes in control, rotenone and
mangiferin treated SK-N-SH cells. Values are given as mean ± SD of four experiments in each group. bp \ 0.05 compared to non-treated cells; cp \ 0.05 compared to rotenone-treated cells
results the reduced ATP levels. Rotenone, even in a low concentration decreased ATP levels to a greater extent in dopaminergic cells than non-dopaminergic cells by specifically inhibiting the mitochondrial NADH dehydrogenase (complex I), one of the five enzymes complexes of the inner mitochondrial membrane involved in oxidative phosphorylation [25]. Prabhu et al. [26] reported that the anti-lipid peroxidative and antioxidant property of mangiferin could have reduced mitochondrial lipid peroxidation to maintain the ETC and ATP concentration for the normal functioning of heart mitochondria. To understand the mechanism behind antiapoptotic effect of mangiferin against rotenone-induced neuronal cell death, we assessed the expression of apoptotic markers caspase 9 (initiator) and caspase 3 (executioner). Caspase-3 is believed to be the final executor of apoptotic DNA damage, as a marker of apoptosis and studies have associated neuronal death in PD has been associated with activation of caspase-3. In the cell-intrinsic pathway, apoptotic signals converge on mitochondria to trigger the release of cytochrome c into the cytosol, causing caspase-9 and -3 activation and cell death. Our results showed that
rotenone (100 nM) activate caspase-3 and 9 and lead to DNA fragmentation, without affecting plasma membrane integrity, which is in agreement with the occurrence of an apoptotic process. Rotenone showed a biphasic pattern of neurotoxicity, causes apoptosis at low doses (1 lM) and necrosis when applied at high doses (10 and 100 lM) [27]. Moreover rotenone in low doses, promoted cell membrane rupture after several hours (above 48 h) and lesser LDH leakage when compared to high doses. Since the present experiment is designed with low dose of rotenone (100 nM), plasma membrane integrity is not affected. However if the activity of LDH was measured then it will offer clear idea. Moreover treatment with rotenone significantly increased the number of cells showing apoptotic features (fragmentation of nuclear chromatin). Mangiferin (20 lg) conferred protection to the cells against oxidative damage thereby preventing much of DNA damage. There was a considerable increase in the percentage of live cells when stained with AO/EtBr with a significant decrease in the percent of apoptotic cell population in cells treated with optimal dose of mangiferin (20 lg).
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(A)
(B)
2.5 Relative intensity (Fold of control)
Fig. 5 The effect of mangiferin on rotenone induced cyt c and caspases 3 and 9 expressions in SK-N-SH cells. a Lane 1 Control, Lane 2 Rotenone, Lane 3 Mangiferin ? Rotenone, Lane 4 Mangiferin. b Rotenone (100 nM) significantly enhanced the expressions of caspases 3, 9 and cytosol cyt c and diminished the expression of mitochondrial cyto C, while mangiferin (20 lg) pretreatment significantly diminished the expressions of caspases 3, 9 and cytosol cyt c and enhanced mitochondrial cyto c, in rotenone treated SK-N-SH cells (a, b). Immunoblot data are quantified by using b-actin as an internal control and the values are expressed as arbitrary units and given as mean ± SD of four in each group. bp \ 0.05 compared to control; cp \ 0.05 compared to rotenone groups
b
2
c 1.5
b
b a
a
a
a
c
b
1
a
a
0.5 0 Cas-3
Cas-9 C
Control Mangiferin+Rotenone
Conclusion As to conclude, the cytotoxicity of rotenone has been reported to be arbitrated by oxidative stress, mitochondria inhibition and ultimately apoptotic cell death, which were attenuated by mangiferin. Conflict of interest
None.
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7.
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