http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1005749
ORIGINAL ARTICLE
Digitoflavone provokes mitochondrial biogenesis in PC12 cells: A protective approach to oxidative stress Asal Yans1,2, Shima Zareh Shahamati1,2, Amir Hossein Maghsoudi1,2, and Nader Maghsoudi1,2 NeuroBiology Research Center and 2Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Abstract
Keywords
Context: Reactive oxygen species (ROS) are known to be one of the main causes of neurodegenerative disorders, and flavonoids play characteristic roles in a variety of biological activities, and specially are known to be antioxidant reagents. Objective: In this study, we investigated neuroprotective effects of digitoflavone to suppress H2O2 -induced cell death in neuron-like PC12 cells. Material and methods: PC12 cells were pre-treated with digitoflavone for 2 h and then cells were exposed to H2O2 for 18 h. The cells’ viability was evaluated by MTT assay. Rhodamine 123 staining was used for the determination of mitochondrial membrane potential (DCm). The intracellular ROS aggregation was determined by using 20 ,70 -dichlorofluorescein diacetate. Also, the level of mitochondrial biogenesis factors was measured by western blot. The antioxidant capacity of digitoflavone was also determined by measuring reduced glutathione (GSH) level and catalase (CAT) activity quantification. Results: Digitoflavone significantly elevated cells’ viability at concentrations of 10 and 20 mM. Also, digitoflavone attenuated intracellular level of ROS, and stabilized DCm. Moreover, digitoflavone increased phosphorylation of AMP-activated protein kinase (AMPK) and, consequently, elevated mitochondrial biogenesis factors which were reduced after H2O2 exposure. We emphasized on the protective effect of digitoflavone through increasing mitochondrial biogenesis by specifically inhibiting AMPK. Antioxidant ability of digitoflavone was indicated by the elevation of GSH level and CAT activity. Conclusion: As a result, digitoflavone stabilize DCm, enhanced cell viability through inducing mitochondrial biogenesis pathway, and increased antioxidant capacity of the cells which lead to better combating the oxidative stress.
Antioxidant, flavonoids, mitochondrial membrane potential, neuroprotection, reactive oxygen species
Introduction Mitochondria provide an integrated functional network to produce adenosine triphosphates (ATP), beside which it generates a large amount of reactive oxygen species (ROS) mainly through the electron transfer chain (Gibson & Shi, 2010). In pathological conditions, the generation of ROS exceeds the antioxidant capacity of the cells which then results in oxidative damage. Since the neuronal cells possess high oxygen consumption, they are prone to ROS-induced oxidative insult. Besides, neurons represent low antioxidant activity, in which the mitochondria are one of the most susceptible organelles to be affected by ROS, and this damage to mitochondria may lead to cell death (Estaquier et al., 2012). Increasing mitochondrial biogenesis is one of the defense systems of cells against ROS elevation; mitochondrial biogenesis is a complex process that requires the synthesis, import, and incorporation of proteins and lipids to the existing
Correspondence: Dr. Nader Maghsoudi, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel: +98 21 22429768. Fax: +98 21 22432047. E-mail: [email protected]
History Received 21 July 2014 Revised 21 November 2014 Accepted 5 January 2015 Published online 9 April 2015
mitochondrial reticulum, as well as replication of the mitochondrial DNA (mtDNA). Mitochondrial biogenesis is concomitant with the upregulation of the peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a) and its downstream effectors, mitochondrial transcription factor A (TFAM), and nuclear respiratory factors (NRFs) (Dai & Rabinovitch, 2009). PGC-1a has emerged as a master regulator of mitochondrial biogenesis and respiration (Kelly & Scarpulla, 2004; Lehman et al., 2000; Puigserver et al., 1998; Wu et al., 1999), which itself is directly phosphorylated by AMP-activated protein kinase (AMPK) (Lo´pez-Lluch et al., 2008). It has been shown that AMPK phosphorylation promotes mitochondrial biogenesis via some downstream proteins such as PGC-1a and TFAM in cardiac cells (Irrcher et al., 2003). In vivo studies have reported that following oxidative stress, PGC-1a protein expression increases trying to regulate or compensate the components of the ROS defense system (St-Pierre et al., 2006). However, evidence have shown that following the critical limits of ROS, PGC-1a expression started to decline significantly and subsequently resulted in downregulation of its downstream targets, i.e., NRF-1, NRF-2 and TFAM (Shaerzadeh et al., 2014; Sharma et al., 2013).
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Maintenance of a proper mitochondrial membrane function drives the synthesis of ATP and maintains oxidative phosphorylation. As it seems, major alterations in mitochondrial membrane function have been shown to be significantly involved in the programmed cell death (Khodorov et al., 1996). Excessive ROS production and mitochondrial Ca2+ overload have been suggested to be two dependent processes, where ROS can stimulate Ca2+ release and Ca2+ increases mitochondrial ROS production, both of which can disrupt mitochondrial membrane potential (DCm) (Sedlic et al., 2010). Interestingly, mitochondrial depolarization is accompanied by the production of ROS contributing to excessive oxidative damage (Kromer et al., 1997). Flavonoids represent a large class of phenolic phytochemicals that constitute important components of the human diet, contained mostly in fruits, vegetables, and spices as well as in some medicinal herbs. It has been demonstrated that flavonoids prevent cancer, oppose osteoporosis, act as antiinflammatory agents, and subsides atherosclerosis (Sawa et al., 1999). In this regard, recent studies have indicated that anti-oxidative and anti-inflammatory properties of flavonoids can contribute to their neuroprotective effects in neurodegeneration (Lee et al., 2003). Beneficial effects of flavonoids are discussed to be related to direct scavenging of free radicals, increasing antioxidant capacity, and interacting with specific proteins central to the intracellular signaling cascades (Schroeter et al., 2002). In particular, accumulating evidence has indicated that flavonoids increase the PGC-1a level and subsequently enhance mitochondrial biogenesis (Davis et al., 2009). Previously, it has been shown that digitoflavone has neuroproective effects mostly related to its antioxidant feature (Kang et al., 2004). In the current study, we aimed to find out the impact of a proper dose of digitoflavone on H2O2-induced alterations of mitochondrial antioxidant enzyme activities and the level of factors involved in mitochondrial biogenesis in differentiated PC12 cells. In this regard, we also detected mitochondrial depolarization in accordance with cellular survival following digitoflavone administration.
were preserved in dump atmosphere at 37 C in a 5% CO2. The medium of the cells was substituted three times a week. Cells were differentiated using 50 ng/ml nerve growth factor (NGF) treatment every other day for 6 d. The cells were pretreated with 10 and 20 mM digitoflavone, subsequently, after 2 h, PC12 cells were exposed to 200 mM H2O2 for 18 h to measure digitflavone’s protection quality against H2O2. Moreover, in the other group, PC12 cells were pretreated with digitoflavone (10 and 20 mM) and then (6-[4-(2piperidin1-yl-ethoxy)-phenyl)]3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidine (known as Compound C) (10 mM) was added to inhibit AMPK, and after that were exposed to H2O2.
Materials and methods
Measurement of intracellular ROS
Materials Primary antibody against PGC-1a was acquired from ABCAM (Cambridge, UK). Other antibodies against AMPK, p-AMPK, and b-actin were obtained from Cell Signaling Technology (Beverly, MA), the antibody against NRF-1 was acquired from Santa Cruz Biotechnology (Santa Cruz, CA), and TFAM was obtained from BioVision (Palo Alto, CA). Electro chemiluminescenc (ECL) kit was prepared from Amersham Bioscience, Piscataway, NJ. The other reagents used in enzymatic assays and cell staining were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture, differentiation, and treatment PC12 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) – F12 supplemented with 5% fetal bovine serum, 10% horse serum, and 1% antibiotic mixture comprising penicillin–streptomycin and
Measurement of cell viability By MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction assay, PC12 cells’ viability was quantified. For this purpose, the dark blue formazan crystals were resolved in dimethyl sulfoxide and the absorbance was measured at 630 nm with the ELISA reader. Rhodamine 123 (Rh123) staining Rh123 was added to the cultured cells with the concentration of 3 mg/ml. Cells were incubated for 30 min at 37 C, then the medium was removed and cells were washed twice with phosphate-buffered saline (PBS). Finally, cells were observed by florescent microscopy acquired from Olympus, Tokyo, Japan (IX71). Measurement of Rh123 density Rh123 florescent dye was used as an indicator of DCm. Initially, PBS containing 5 mL Rh123 was added to PC12 cells and incubated for 30 min at 37 C. Subsequently, the cells were leached with PBS and trypsinized at room temperature and suspended in PBS. Then, the fluorescence intensities were quantified by Varian Cary Eclipse spectrofluorometer (Varian Inc., Santa Clara, CA) with excitation and emission wavelengths of 485 nm and 530 nm, respectively.
For monitoring intracellular reposition of ROS, 20 ,70 dichlorofluorescein diacetate (DCF-DA) was used. To this end, to cell suspension (1 106 cells/mL), 10 mM of DCF-DA was added and incubated at 37 C for 1 h. Afterward, the mixture was leached by PBS and quantified by Varian Cary Eclipse spectrofluorometer (Varian Inc., Santa Clara, CA) with stimulation and emission wavelengths of 485 nm and 530 nm, respectively. Western blot analysis For Western blot analysis, total protein concentrations of cells were determined by the Bradford (1976) method. By using bovine serum albumin, a standard plot was generated. Proteins were electrophoresed on 12.5% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Then, the membranes get blocked with blocking solutions and were incubated with proprietary primary antibodies (1:1000 dilutions). Secondary antibody (1:3000 dilutions) was used after
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Reduced glutathione level measurement
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removing unbound primary antibody by rinsing the membrane. By using the ECL reagent and autoradiography, the immunoreactive band of proteins on membranes was detected. The quantitative value of band over the film was then detected via Image J software (National Institutes of Health, Bethesda, MD).
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The contents of intrinsic glutathione (GSH) of the cells were measured by the Ellman (1959) method, briefly by adding 5–50 -dithiobis(2-nitrobenzoic acid) (DTNB) to cell lysate and measuring the absorbance at 412 nm.
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Catalase activity measurement By the Aebi (1984) method, the decomposition of H2O2 at 204 nm was measured with a spectrophotometer to determine the catalase (CAT) activity. For this purpose, 50 mM phosphate buffer (pH 7.0) and cell lysate were mixed in a cuvette. By adding 0.01 mM H2O2 that prepared freshly, the reaction was initiated. Data analysis All data are illustrated as the mean ± S.E.M. (standard error mean). Evaluation of the groups was done using one-way analysis of variance (ANOVA) and subsequently by the Tukey post hoc test (compare all pairs of columns), a proper post hoc test for analyzing the diversity. The importance of statistics had been reached when p50.05 (* or #p50.05, ** or ##p50.01 and *** or ### or gggp50.001).
Results Digitoflavone protected PC12 cells against H2O2induced cell death We used MTT assay to detect neuroprotective effect of digitoflavone (10, 20 and 50 mM) against cell death in H2O2exposed PC12 cells. We observed that H2O2 reduced cell viability to 47% compared with control cells. Although 10 mL and 20 mL concentrations of digitoflavone protected cells against H2O2-induced cell death and enhanced cell viability by 1.29- and 1.46-folds compared with H2O2exposed cells, 50 mM concentration of digitoflavone showed a toxic impact, so we discontinued using this concentration (Figure 1).
Figure 1. Effect of digitoflavone on viability of H2O2-exposed cells. PC12 cells were pretreated with 10, 20, and 50 mM concentrations of digitoflavone for 2 h and then exposed to H2O2 (200 mM) for 18 h. MTT assay was used to determine cells’ viability. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. #p50.05 and ###p50.001 Significantly different from H2O2-treated cells.
DCm is necessary for cellular viability and proper functions of respiratory chain enzymes to produce ATP (Joshi & Bakowska, 2011). Reduction in DCm has been shown in early stages of cell apoptosis (Kromer et al., 1997). ROS also causes depolarization and decreases DCm (Gottlieb et al., 2000), in which Rh123 gets permeable into mitochondrial matrix with a bright radiation (Poot et al., 1996). As shown in Figure 2(B) and (C), on one hand, H2O2 insult increased Rh123 density in PC12 cells which indicated a decrease in DCm after ROS accumulation in cells. On the other hand, treating H2O2-insulted cells with digitoflavone in concentrations of 10 and 20 mM did not alter Rh123 density. Also, our data indicated that the concentration of 20 mM is more suitable for protection against H2O2-induced mitochondrial depolarization that saves mitochondria as unscathed. Digitoflavone enhanced AMPK phosphorylation and upregulated mitochondrial biogenesis factors’ level
Flavonoids are known to have wide antioxidant properties and digitoflavone were examined for this purpose. In this investigation, we found that the level of ROS in H2O2-treated cells raised 1.79-fold compared with control cells. But, 10 mM and 20 mM concentrations of digitoflavone decrease this amount 20.95% and 32.1% relative to H2O2 exposure cells, respectively (Figure 2A).
Increasing the number of mitochondria within the cells is controlled by the activation of several signaling pathways with specific transcription factors and regulators. AMPK is a main regulator of mitochondrial biogenesis and, PGC-1a, NRF-1, and TFAM are the major markers of mitochondrial biogenesis that control the expression of mitochondrial proteins and response to the cell energy demand in oxidative conditions. Our data showed that while the level of AMPK declined significantly in H2O2-insulted cells, both 10 and 20 mM of digitoflavone increased its level near to the control, the same result was seen for PGC-1a, NRF-1, and TFAM levels. Also, here, 20 mM concentration of digitoflavone displayed better protective effect than 10 mM on rising PGC-1a, NRF-1, and TFAM levels in H2O2-exposed PC12 cells (Figure 3).
Digitoflavone stabilized DCm that was depolarized by H2O2
Digitoflavone incremented mitochondrial GSH level and CAT activity
Rh123 is a lipophilic cation accumulated by mitochondria in proportion to DCm (Russell & Lee, 1999). Maintenance of
GSH is one of the abundant molecules in the nervous system that as an intracellular antioxidant is responsible for ROS
Digitoflavone reduced level of intracellular ROS
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Figure 2. Effect of digitoflavone on H2O2induced intracellular ROS and altered Rhodamine density as an indicator of DCm in PC12 cells. Cells were pretreated with 10 and 20 mM concentrations of digitoflavone for 2 h and then exposed to 200 mM H2O2 for additional 18 h. (A) The level of ROS formation was measured using DCF-DA fluorescent probe. (B) and (C) Rhodamine density. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. #p50.05 and ###p50.001 Significantly different from H2O2-treated cells.
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Intera Cellular ROS Levels (DCFDA fluorescence)
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detoxification. As shown in Figure 4(A), GSH level declined in H2O2-exposed cells to 70.83% compared with the control group. While 10 and 20 mM concentrations of digitoflavone increased mitochondrial GSH level, declined by H2O2 by 1.16- and 1.22-folds. GSH level in 20 mM concentration of digitoflavone pretreated cells was higher than 10 mM. CAT is one of the cytoplasmic antioxidant enzymes that act as ROS scavenger in the cell. As shown in Figure 4(B), H2O2 decreased the CAT activity to 52.8% compared with the control cells. While 10 and 20 mM concentrations of
Digitoflavone (10 μM)
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digitoflavone compensated this decrease by 1.42- and 1.54folds compared with the H2O2-exposed group, respectively. Cell viability declined by AMPK inhibition in digitoflavone pretreated, H2O2-insulted cells Compound C specifically inhibits AMPK and decreases mitochondrial biogenesis consequently. In our experiment, adding compound C (10 mM) to the H2O2-exposed, digitoflavone pretreated cells decreased cell viability to a
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DOI: 10.3109/13880209.2015.1005749
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Figure 3. Western blot analysis to measure the effect of digitoflavone on pAMPK/AMPK ratio and mitochondrial biogenesis factors PGC-1a, NRF-1, and TFAM in PC12 cells. (A) PC12 cells pretreated for 2 h with digitoflavone (10 and 20 mM) and then exposed to H2O2 (200 mM) for 18 h. About 20 mg of proteins were separated on SDS-PAGE, Western blotted, probed with anti-pAMPK, PGC-1a, -NRF-1, and -TFAM and reprobed with anti-AMPK and b-actin antibodies. (One representative Western blot was shown; n ¼ 3). (B) The density of p-AMPK bands was measured and the ratio to AMPK was calculated. The mean of three independent experiments is shown. (C) The density of PGC-1a bands was measured and the ratio to b-actin was calculated. The mean of three independent experiments is shown. (D) The density of NRF-1 bands to the b-actin ratio. (E) The density of TFAM bands to the b-actin ratio. ***p50.001 Significantly different from control cells. #p50.05, ##p50.01 and ###p50.001 Significantly different from H2O2treated cells.
significant extent compared with the H2O2-exposed digitoflavone-treated cells (p50.01) which indicates that increasing mitochondrial biogenesis is one of the main pathways that digitoflavone can exert its protective effects, also in here 20 mM of digitoflavone had more prominent protective effect (Figure 5).
Discussion Since the neuronal tissue consists of high oxygen consuming cells, excessive ROS formation in mitochondria is a common finding in the brain, this fact with the lack of a powerful antioxidant network are important underlying causes of
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Figure 5. Effect of AMPK inhibition on viability of digitoflavone pretreated, H2O2-insulted cells. PC12 cells were pretreated with 10 and 20 mM concentrations of digitoflavone and compound C, then exposed to H2O2 (200 mM) for 18 h. MTT assay was used to determine cell viability. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. ###p50.001 Significantly different from digioflavone (10 mM and/or 20 mM) pretreated, H2O2insulted cells.
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Figure 4. Effect of digitoflavone on CAT activity and GSH level in PC12 cells exposed to H2O2. PC12 cells were pretreated with 10 and 20 mM concentrations of digitoflavone for 2 h and then were exposed to H2O2 (200 mM) for 18 h. (A) GSH level was determined by adding DTNB and measuring the absorbance at 412 nm. (B) Activity of CAT was measured based on the decomposition of H2O2. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. #p50.05 and ###p50.001 Significantly different from H2O2-treated cells.
neuronal cell death. Our data suggested that administration of digitoflavone increased cell viability by preventing H2O2induced ROS accumulation in the cells and increasing mitochondrial biogenesis factors in oxidative stress condition, besides the over activation of mitochondrial antioxidants defense system. These results were more prominent while administrating 20 mL of digitoflavone. H2O2 increases ROS level in the cell and its over production in neurons is featured in aging (Auerbach & Segal, 1997; Sohal & Dubey, 1994). Generation of H2O2 has been proposed as the leading component in neuronal damage observed in Parkinson’s and Alzheimer’s diseases (Schapira, 1994). In this manner, early phase of H2O2-induced oxidative stress has been shown to result in an impaired mitochondrial function, evidenced by a decline in ATP/adenosine diphosphate ratio in nerve terminals (Tretter et al., 1997). An emerging number of studies have evidenced an intimate link between an excessive generation of ROS and the development of neuronal death, mostly apoptosis in neurological disorders (Cadenas & Davies, 2000; Hoogeboom & Burgering, 2009). Our results support this idea that the cells undergoing
oxidative stress by H2O2 showed a considerable increase in ROS levels and decreased overall cell survival. However, 20 mM of digitoflavone lowered the ROS level even near to the intact cells. Flavonoids such as digitoflavone are structurally heterogeneous, polyphenolic compounds that are mostly present in vegetables and fruits and have been reported to provide both short and long-term protection against oxidative insult. Many flavonoids act directly as antioxidants, by donating hydrogen ions, and it has also been indicated recently that they can modulate intracellular signaling pathways (Williams et al., 2004). Digitoflavone, a natural polyphenolic flavone, is present in a wide variety of food plants, including carrots, olive oil, peppermint, rosemary, and more (Lo´pez-La´zaro, 2009). In vivo studies have implied that supplementation of digitoflavone decreased the lipid peroxidation and enhanced the levels of enzymatic antioxidants (Samy et al., 2006). Since it has been proposed that radical scavenging activity of flavonoids is directly related to the number of hydroxyl groups, digitoflavone has a strong antioxidative potential that contains four hydroxyl groups at the 30 , 40 , 50 , and 70 positions (Ueda et al., 2002). For many years, polyphenols were thought to protect cells against oxidative damage through scavenging of free radicals. However, this concept now appears to be an oversimplified view of their action (Azzi et al., 2004). In this regard, it has been indicated recently that cells respond to polyphenols mainly through direct interactions with receptors or enzymes involved in signal transduction, which may result in the modification of the redox status of the cell and may trigger a series of redox-dependent reactions (Forman et al., 2002; Halliwll et al., 2005). Previous studies have indicated that specific flavonoids like naringenin and digitoflavone increased CAT activity and GSH level in H2O2-insulted cells (Ramprasath et al., 2014; Zhao et al., 2012). This is in consistent with our data that increase of CAT activity and GSH level in the H2O2-exposed group was displayed in response to digitoflavone pretreatment.
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DOI: 10.3109/13880209.2015.1005749
Mitochondria play a crucial role in the aging process not only due to their role as the main intracellular generators of ROS but also they are targets of ROS themselves. Although there is a wide spectrum of protective mechanisms against ROS, including enzymatic and small molecule antioxidants, an excess of ROS content may lead to a progressive damage in the cell and decrease of mitochondrial physiologic function (Mariani et al., 2005). AMPK is shown to directly phosphorylate PGC-1a and trigger mitochondrial biogenesis pathway, it also acts as a PGC-1a regulator in post-translational level and has been showed to be increased by flavonoids in different conditions (Ahn et al., 2008; Lu et al., 2010). PGC-1a has been accepted as the main modulator of mitochondrial biogenesis and respiration (Kelly & Scarpulla, 2004; Lin et al., 2002; Puigserver et al., 1998). Activation of mitochondrial biogenesis by PGC-1a results from the co-activation of estrogenrelated receptor a, NRF-1, and NRF-2 (Mootha et al., 2004; Wu et al., 1999). In this manner, Sharma et al. (2013) have reported that increasing ROS level by aluminum directly downregulated PGC-1a expression in neurons at both the mRNA and the protein levels. The reduction of the PGC-1a mRNA level suggests that PGC-1a is transcriptionally regulated in neurons and, as we mentioned, AMPK is one of its regulators acting at the post-translational level. Cellular researches on Hela and drosophila S2 cells have shown the stimulating effect of polyphenols on sirtuin 1 that deacetylates PGC-1a at multiple lysine sites and increases its activity (Howitz et al., 2003; Wood et al., 2004). Davis et al. (2009) have reported that quercetin, a flavonoid, enhanced the expression of genes involved in mitochondrial biogenesis. Consistently, in our current study, we observed an increase in the PGC-1a level by adding digitoflavone to the medium of the cells, along with its downstream factors including TFAM and NRF-1 after H2O2 exposure. We used compound C as the AMPK-specific inhibitor to see if the protective effect of digitoflavone after H2O2 insult was only because of its antioxidant property or caused by digitoflavone-induced increase in mitochondrial biogenesis factors either. Our results supported the idea that digitoflavone exerts a central role in protecting cells via increasing mitochondrial biogenesis factors besides its antioxidant activity. The other indicator of mitochondrial dysfunction is the DCm. Chang et al. (2001) reported that ROS accumulation in the epithelial cells depolarized the mitochondrial membrane. Impaired DCm results in mitochondrial dysfunction and decreased ATP production, AMPK senses the decrease of ATP and phosphorylates in response, then the phosphorylated AMPK activates its related proteins (Mukherjee et al., 2008). There are also evidences that proposed changes in the DCm are an early, irreversible event in the cell death signaling pathway such as cytokine-induced apoptosis (Barbu et al., 2002). However, there are conflicting ideas in this regard (Oubrahim et al., 2001). In our experiment, H2O2 depolarized the mitochondrial membrane and this dissipation of DCm was then modulated by digitoflavone administration.
Conclusion Overall, our data indicated that digitoflavone not only exert its protective effect by direct radical scavenging but also
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interacting with cellular survival signaling pathways, such as antioxidant overexpression and activation of mitochondrial biogenesis, which led to a strong cellular defense against ROS-induced oxidative insult. Further studies are progressing in our laboratory to unveil detailed underlying mechanisms in the way of digitoflavone cellular protection.
Declaration of interest The authors report that they have no conflicts of interest. This work was supported financially by Iran National Science Foundation (Grant no. 91004247).
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ORIGINAL ARTICLE
Digitoflavone provokes mitochondrial biogenesis in PC12 cells: A protective approach to oxidative stress Asal Yans1,2, Shima Zareh Shahamati1,2, Amir Hossein Maghsoudi1,2, and Nader Maghsoudi1,2 NeuroBiology Research Center and 2Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Abstract
Keywords
Context: Reactive oxygen species (ROS) are known to be one of the main causes of neurodegenerative disorders, and flavonoids play characteristic roles in a variety of biological activities, and specially are known to be antioxidant reagents. Objective: In this study, we investigated neuroprotective effects of digitoflavone to suppress H2O2 -induced cell death in neuron-like PC12 cells. Material and methods: PC12 cells were pre-treated with digitoflavone for 2 h and then cells were exposed to H2O2 for 18 h. The cells’ viability was evaluated by MTT assay. Rhodamine 123 staining was used for the determination of mitochondrial membrane potential (DCm). The intracellular ROS aggregation was determined by using 20 ,70 -dichlorofluorescein diacetate. Also, the level of mitochondrial biogenesis factors was measured by western blot. The antioxidant capacity of digitoflavone was also determined by measuring reduced glutathione (GSH) level and catalase (CAT) activity quantification. Results: Digitoflavone significantly elevated cells’ viability at concentrations of 10 and 20 mM. Also, digitoflavone attenuated intracellular level of ROS, and stabilized DCm. Moreover, digitoflavone increased phosphorylation of AMP-activated protein kinase (AMPK) and, consequently, elevated mitochondrial biogenesis factors which were reduced after H2O2 exposure. We emphasized on the protective effect of digitoflavone through increasing mitochondrial biogenesis by specifically inhibiting AMPK. Antioxidant ability of digitoflavone was indicated by the elevation of GSH level and CAT activity. Conclusion: As a result, digitoflavone stabilize DCm, enhanced cell viability through inducing mitochondrial biogenesis pathway, and increased antioxidant capacity of the cells which lead to better combating the oxidative stress.
Antioxidant, flavonoids, mitochondrial membrane potential, neuroprotection, reactive oxygen species
Introduction Mitochondria provide an integrated functional network to produce adenosine triphosphates (ATP), beside which it generates a large amount of reactive oxygen species (ROS) mainly through the electron transfer chain (Gibson & Shi, 2010). In pathological conditions, the generation of ROS exceeds the antioxidant capacity of the cells which then results in oxidative damage. Since the neuronal cells possess high oxygen consumption, they are prone to ROS-induced oxidative insult. Besides, neurons represent low antioxidant activity, in which the mitochondria are one of the most susceptible organelles to be affected by ROS, and this damage to mitochondria may lead to cell death (Estaquier et al., 2012). Increasing mitochondrial biogenesis is one of the defense systems of cells against ROS elevation; mitochondrial biogenesis is a complex process that requires the synthesis, import, and incorporation of proteins and lipids to the existing
Correspondence: Dr. Nader Maghsoudi, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel: +98 21 22429768. Fax: +98 21 22432047. E-mail: [email protected]
History Received 21 July 2014 Revised 21 November 2014 Accepted 5 January 2015 Published online 9 April 2015
mitochondrial reticulum, as well as replication of the mitochondrial DNA (mtDNA). Mitochondrial biogenesis is concomitant with the upregulation of the peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a) and its downstream effectors, mitochondrial transcription factor A (TFAM), and nuclear respiratory factors (NRFs) (Dai & Rabinovitch, 2009). PGC-1a has emerged as a master regulator of mitochondrial biogenesis and respiration (Kelly & Scarpulla, 2004; Lehman et al., 2000; Puigserver et al., 1998; Wu et al., 1999), which itself is directly phosphorylated by AMP-activated protein kinase (AMPK) (Lo´pez-Lluch et al., 2008). It has been shown that AMPK phosphorylation promotes mitochondrial biogenesis via some downstream proteins such as PGC-1a and TFAM in cardiac cells (Irrcher et al., 2003). In vivo studies have reported that following oxidative stress, PGC-1a protein expression increases trying to regulate or compensate the components of the ROS defense system (St-Pierre et al., 2006). However, evidence have shown that following the critical limits of ROS, PGC-1a expression started to decline significantly and subsequently resulted in downregulation of its downstream targets, i.e., NRF-1, NRF-2 and TFAM (Shaerzadeh et al., 2014; Sharma et al., 2013).
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Maintenance of a proper mitochondrial membrane function drives the synthesis of ATP and maintains oxidative phosphorylation. As it seems, major alterations in mitochondrial membrane function have been shown to be significantly involved in the programmed cell death (Khodorov et al., 1996). Excessive ROS production and mitochondrial Ca2+ overload have been suggested to be two dependent processes, where ROS can stimulate Ca2+ release and Ca2+ increases mitochondrial ROS production, both of which can disrupt mitochondrial membrane potential (DCm) (Sedlic et al., 2010). Interestingly, mitochondrial depolarization is accompanied by the production of ROS contributing to excessive oxidative damage (Kromer et al., 1997). Flavonoids represent a large class of phenolic phytochemicals that constitute important components of the human diet, contained mostly in fruits, vegetables, and spices as well as in some medicinal herbs. It has been demonstrated that flavonoids prevent cancer, oppose osteoporosis, act as antiinflammatory agents, and subsides atherosclerosis (Sawa et al., 1999). In this regard, recent studies have indicated that anti-oxidative and anti-inflammatory properties of flavonoids can contribute to their neuroprotective effects in neurodegeneration (Lee et al., 2003). Beneficial effects of flavonoids are discussed to be related to direct scavenging of free radicals, increasing antioxidant capacity, and interacting with specific proteins central to the intracellular signaling cascades (Schroeter et al., 2002). In particular, accumulating evidence has indicated that flavonoids increase the PGC-1a level and subsequently enhance mitochondrial biogenesis (Davis et al., 2009). Previously, it has been shown that digitoflavone has neuroproective effects mostly related to its antioxidant feature (Kang et al., 2004). In the current study, we aimed to find out the impact of a proper dose of digitoflavone on H2O2-induced alterations of mitochondrial antioxidant enzyme activities and the level of factors involved in mitochondrial biogenesis in differentiated PC12 cells. In this regard, we also detected mitochondrial depolarization in accordance with cellular survival following digitoflavone administration.
were preserved in dump atmosphere at 37 C in a 5% CO2. The medium of the cells was substituted three times a week. Cells were differentiated using 50 ng/ml nerve growth factor (NGF) treatment every other day for 6 d. The cells were pretreated with 10 and 20 mM digitoflavone, subsequently, after 2 h, PC12 cells were exposed to 200 mM H2O2 for 18 h to measure digitflavone’s protection quality against H2O2. Moreover, in the other group, PC12 cells were pretreated with digitoflavone (10 and 20 mM) and then (6-[4-(2piperidin1-yl-ethoxy)-phenyl)]3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidine (known as Compound C) (10 mM) was added to inhibit AMPK, and after that were exposed to H2O2.
Materials and methods
Measurement of intracellular ROS
Materials Primary antibody against PGC-1a was acquired from ABCAM (Cambridge, UK). Other antibodies against AMPK, p-AMPK, and b-actin were obtained from Cell Signaling Technology (Beverly, MA), the antibody against NRF-1 was acquired from Santa Cruz Biotechnology (Santa Cruz, CA), and TFAM was obtained from BioVision (Palo Alto, CA). Electro chemiluminescenc (ECL) kit was prepared from Amersham Bioscience, Piscataway, NJ. The other reagents used in enzymatic assays and cell staining were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture, differentiation, and treatment PC12 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) – F12 supplemented with 5% fetal bovine serum, 10% horse serum, and 1% antibiotic mixture comprising penicillin–streptomycin and
Measurement of cell viability By MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction assay, PC12 cells’ viability was quantified. For this purpose, the dark blue formazan crystals were resolved in dimethyl sulfoxide and the absorbance was measured at 630 nm with the ELISA reader. Rhodamine 123 (Rh123) staining Rh123 was added to the cultured cells with the concentration of 3 mg/ml. Cells were incubated for 30 min at 37 C, then the medium was removed and cells were washed twice with phosphate-buffered saline (PBS). Finally, cells were observed by florescent microscopy acquired from Olympus, Tokyo, Japan (IX71). Measurement of Rh123 density Rh123 florescent dye was used as an indicator of DCm. Initially, PBS containing 5 mL Rh123 was added to PC12 cells and incubated for 30 min at 37 C. Subsequently, the cells were leached with PBS and trypsinized at room temperature and suspended in PBS. Then, the fluorescence intensities were quantified by Varian Cary Eclipse spectrofluorometer (Varian Inc., Santa Clara, CA) with excitation and emission wavelengths of 485 nm and 530 nm, respectively.
For monitoring intracellular reposition of ROS, 20 ,70 dichlorofluorescein diacetate (DCF-DA) was used. To this end, to cell suspension (1 106 cells/mL), 10 mM of DCF-DA was added and incubated at 37 C for 1 h. Afterward, the mixture was leached by PBS and quantified by Varian Cary Eclipse spectrofluorometer (Varian Inc., Santa Clara, CA) with stimulation and emission wavelengths of 485 nm and 530 nm, respectively. Western blot analysis For Western blot analysis, total protein concentrations of cells were determined by the Bradford (1976) method. By using bovine serum albumin, a standard plot was generated. Proteins were electrophoresed on 12.5% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Then, the membranes get blocked with blocking solutions and were incubated with proprietary primary antibodies (1:1000 dilutions). Secondary antibody (1:3000 dilutions) was used after
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Reduced glutathione level measurement
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removing unbound primary antibody by rinsing the membrane. By using the ECL reagent and autoradiography, the immunoreactive band of proteins on membranes was detected. The quantitative value of band over the film was then detected via Image J software (National Institutes of Health, Bethesda, MD).
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The contents of intrinsic glutathione (GSH) of the cells were measured by the Ellman (1959) method, briefly by adding 5–50 -dithiobis(2-nitrobenzoic acid) (DTNB) to cell lysate and measuring the absorbance at 412 nm.
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Catalase activity measurement By the Aebi (1984) method, the decomposition of H2O2 at 204 nm was measured with a spectrophotometer to determine the catalase (CAT) activity. For this purpose, 50 mM phosphate buffer (pH 7.0) and cell lysate were mixed in a cuvette. By adding 0.01 mM H2O2 that prepared freshly, the reaction was initiated. Data analysis All data are illustrated as the mean ± S.E.M. (standard error mean). Evaluation of the groups was done using one-way analysis of variance (ANOVA) and subsequently by the Tukey post hoc test (compare all pairs of columns), a proper post hoc test for analyzing the diversity. The importance of statistics had been reached when p50.05 (* or #p50.05, ** or ##p50.01 and *** or ### or gggp50.001).
Results Digitoflavone protected PC12 cells against H2O2induced cell death We used MTT assay to detect neuroprotective effect of digitoflavone (10, 20 and 50 mM) against cell death in H2O2exposed PC12 cells. We observed that H2O2 reduced cell viability to 47% compared with control cells. Although 10 mL and 20 mL concentrations of digitoflavone protected cells against H2O2-induced cell death and enhanced cell viability by 1.29- and 1.46-folds compared with H2O2exposed cells, 50 mM concentration of digitoflavone showed a toxic impact, so we discontinued using this concentration (Figure 1).
Figure 1. Effect of digitoflavone on viability of H2O2-exposed cells. PC12 cells were pretreated with 10, 20, and 50 mM concentrations of digitoflavone for 2 h and then exposed to H2O2 (200 mM) for 18 h. MTT assay was used to determine cells’ viability. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. #p50.05 and ###p50.001 Significantly different from H2O2-treated cells.
DCm is necessary for cellular viability and proper functions of respiratory chain enzymes to produce ATP (Joshi & Bakowska, 2011). Reduction in DCm has been shown in early stages of cell apoptosis (Kromer et al., 1997). ROS also causes depolarization and decreases DCm (Gottlieb et al., 2000), in which Rh123 gets permeable into mitochondrial matrix with a bright radiation (Poot et al., 1996). As shown in Figure 2(B) and (C), on one hand, H2O2 insult increased Rh123 density in PC12 cells which indicated a decrease in DCm after ROS accumulation in cells. On the other hand, treating H2O2-insulted cells with digitoflavone in concentrations of 10 and 20 mM did not alter Rh123 density. Also, our data indicated that the concentration of 20 mM is more suitable for protection against H2O2-induced mitochondrial depolarization that saves mitochondria as unscathed. Digitoflavone enhanced AMPK phosphorylation and upregulated mitochondrial biogenesis factors’ level
Flavonoids are known to have wide antioxidant properties and digitoflavone were examined for this purpose. In this investigation, we found that the level of ROS in H2O2-treated cells raised 1.79-fold compared with control cells. But, 10 mM and 20 mM concentrations of digitoflavone decrease this amount 20.95% and 32.1% relative to H2O2 exposure cells, respectively (Figure 2A).
Increasing the number of mitochondria within the cells is controlled by the activation of several signaling pathways with specific transcription factors and regulators. AMPK is a main regulator of mitochondrial biogenesis and, PGC-1a, NRF-1, and TFAM are the major markers of mitochondrial biogenesis that control the expression of mitochondrial proteins and response to the cell energy demand in oxidative conditions. Our data showed that while the level of AMPK declined significantly in H2O2-insulted cells, both 10 and 20 mM of digitoflavone increased its level near to the control, the same result was seen for PGC-1a, NRF-1, and TFAM levels. Also, here, 20 mM concentration of digitoflavone displayed better protective effect than 10 mM on rising PGC-1a, NRF-1, and TFAM levels in H2O2-exposed PC12 cells (Figure 3).
Digitoflavone stabilized DCm that was depolarized by H2O2
Digitoflavone incremented mitochondrial GSH level and CAT activity
Rh123 is a lipophilic cation accumulated by mitochondria in proportion to DCm (Russell & Lee, 1999). Maintenance of
GSH is one of the abundant molecules in the nervous system that as an intracellular antioxidant is responsible for ROS
Digitoflavone reduced level of intracellular ROS
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Figure 2. Effect of digitoflavone on H2O2induced intracellular ROS and altered Rhodamine density as an indicator of DCm in PC12 cells. Cells were pretreated with 10 and 20 mM concentrations of digitoflavone for 2 h and then exposed to 200 mM H2O2 for additional 18 h. (A) The level of ROS formation was measured using DCF-DA fluorescent probe. (B) and (C) Rhodamine density. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. #p50.05 and ###p50.001 Significantly different from H2O2-treated cells.
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detoxification. As shown in Figure 4(A), GSH level declined in H2O2-exposed cells to 70.83% compared with the control group. While 10 and 20 mM concentrations of digitoflavone increased mitochondrial GSH level, declined by H2O2 by 1.16- and 1.22-folds. GSH level in 20 mM concentration of digitoflavone pretreated cells was higher than 10 mM. CAT is one of the cytoplasmic antioxidant enzymes that act as ROS scavenger in the cell. As shown in Figure 4(B), H2O2 decreased the CAT activity to 52.8% compared with the control cells. While 10 and 20 mM concentrations of
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digitoflavone compensated this decrease by 1.42- and 1.54folds compared with the H2O2-exposed group, respectively. Cell viability declined by AMPK inhibition in digitoflavone pretreated, H2O2-insulted cells Compound C specifically inhibits AMPK and decreases mitochondrial biogenesis consequently. In our experiment, adding compound C (10 mM) to the H2O2-exposed, digitoflavone pretreated cells decreased cell viability to a
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Figure 3. Western blot analysis to measure the effect of digitoflavone on pAMPK/AMPK ratio and mitochondrial biogenesis factors PGC-1a, NRF-1, and TFAM in PC12 cells. (A) PC12 cells pretreated for 2 h with digitoflavone (10 and 20 mM) and then exposed to H2O2 (200 mM) for 18 h. About 20 mg of proteins were separated on SDS-PAGE, Western blotted, probed with anti-pAMPK, PGC-1a, -NRF-1, and -TFAM and reprobed with anti-AMPK and b-actin antibodies. (One representative Western blot was shown; n ¼ 3). (B) The density of p-AMPK bands was measured and the ratio to AMPK was calculated. The mean of three independent experiments is shown. (C) The density of PGC-1a bands was measured and the ratio to b-actin was calculated. The mean of three independent experiments is shown. (D) The density of NRF-1 bands to the b-actin ratio. (E) The density of TFAM bands to the b-actin ratio. ***p50.001 Significantly different from control cells. #p50.05, ##p50.01 and ###p50.001 Significantly different from H2O2treated cells.
significant extent compared with the H2O2-exposed digitoflavone-treated cells (p50.01) which indicates that increasing mitochondrial biogenesis is one of the main pathways that digitoflavone can exert its protective effects, also in here 20 mM of digitoflavone had more prominent protective effect (Figure 5).
Discussion Since the neuronal tissue consists of high oxygen consuming cells, excessive ROS formation in mitochondria is a common finding in the brain, this fact with the lack of a powerful antioxidant network are important underlying causes of
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Figure 5. Effect of AMPK inhibition on viability of digitoflavone pretreated, H2O2-insulted cells. PC12 cells were pretreated with 10 and 20 mM concentrations of digitoflavone and compound C, then exposed to H2O2 (200 mM) for 18 h. MTT assay was used to determine cell viability. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. ###p50.001 Significantly different from digioflavone (10 mM and/or 20 mM) pretreated, H2O2insulted cells.
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Figure 4. Effect of digitoflavone on CAT activity and GSH level in PC12 cells exposed to H2O2. PC12 cells were pretreated with 10 and 20 mM concentrations of digitoflavone for 2 h and then were exposed to H2O2 (200 mM) for 18 h. (A) GSH level was determined by adding DTNB and measuring the absorbance at 412 nm. (B) Activity of CAT was measured based on the decomposition of H2O2. The mean of three independent experiments is shown. ***p50.001 Significantly different from control cells. #p50.05 and ###p50.001 Significantly different from H2O2-treated cells.
neuronal cell death. Our data suggested that administration of digitoflavone increased cell viability by preventing H2O2induced ROS accumulation in the cells and increasing mitochondrial biogenesis factors in oxidative stress condition, besides the over activation of mitochondrial antioxidants defense system. These results were more prominent while administrating 20 mL of digitoflavone. H2O2 increases ROS level in the cell and its over production in neurons is featured in aging (Auerbach & Segal, 1997; Sohal & Dubey, 1994). Generation of H2O2 has been proposed as the leading component in neuronal damage observed in Parkinson’s and Alzheimer’s diseases (Schapira, 1994). In this manner, early phase of H2O2-induced oxidative stress has been shown to result in an impaired mitochondrial function, evidenced by a decline in ATP/adenosine diphosphate ratio in nerve terminals (Tretter et al., 1997). An emerging number of studies have evidenced an intimate link between an excessive generation of ROS and the development of neuronal death, mostly apoptosis in neurological disorders (Cadenas & Davies, 2000; Hoogeboom & Burgering, 2009). Our results support this idea that the cells undergoing
oxidative stress by H2O2 showed a considerable increase in ROS levels and decreased overall cell survival. However, 20 mM of digitoflavone lowered the ROS level even near to the intact cells. Flavonoids such as digitoflavone are structurally heterogeneous, polyphenolic compounds that are mostly present in vegetables and fruits and have been reported to provide both short and long-term protection against oxidative insult. Many flavonoids act directly as antioxidants, by donating hydrogen ions, and it has also been indicated recently that they can modulate intracellular signaling pathways (Williams et al., 2004). Digitoflavone, a natural polyphenolic flavone, is present in a wide variety of food plants, including carrots, olive oil, peppermint, rosemary, and more (Lo´pez-La´zaro, 2009). In vivo studies have implied that supplementation of digitoflavone decreased the lipid peroxidation and enhanced the levels of enzymatic antioxidants (Samy et al., 2006). Since it has been proposed that radical scavenging activity of flavonoids is directly related to the number of hydroxyl groups, digitoflavone has a strong antioxidative potential that contains four hydroxyl groups at the 30 , 40 , 50 , and 70 positions (Ueda et al., 2002). For many years, polyphenols were thought to protect cells against oxidative damage through scavenging of free radicals. However, this concept now appears to be an oversimplified view of their action (Azzi et al., 2004). In this regard, it has been indicated recently that cells respond to polyphenols mainly through direct interactions with receptors or enzymes involved in signal transduction, which may result in the modification of the redox status of the cell and may trigger a series of redox-dependent reactions (Forman et al., 2002; Halliwll et al., 2005). Previous studies have indicated that specific flavonoids like naringenin and digitoflavone increased CAT activity and GSH level in H2O2-insulted cells (Ramprasath et al., 2014; Zhao et al., 2012). This is in consistent with our data that increase of CAT activity and GSH level in the H2O2-exposed group was displayed in response to digitoflavone pretreatment.
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DOI: 10.3109/13880209.2015.1005749
Mitochondria play a crucial role in the aging process not only due to their role as the main intracellular generators of ROS but also they are targets of ROS themselves. Although there is a wide spectrum of protective mechanisms against ROS, including enzymatic and small molecule antioxidants, an excess of ROS content may lead to a progressive damage in the cell and decrease of mitochondrial physiologic function (Mariani et al., 2005). AMPK is shown to directly phosphorylate PGC-1a and trigger mitochondrial biogenesis pathway, it also acts as a PGC-1a regulator in post-translational level and has been showed to be increased by flavonoids in different conditions (Ahn et al., 2008; Lu et al., 2010). PGC-1a has been accepted as the main modulator of mitochondrial biogenesis and respiration (Kelly & Scarpulla, 2004; Lin et al., 2002; Puigserver et al., 1998). Activation of mitochondrial biogenesis by PGC-1a results from the co-activation of estrogenrelated receptor a, NRF-1, and NRF-2 (Mootha et al., 2004; Wu et al., 1999). In this manner, Sharma et al. (2013) have reported that increasing ROS level by aluminum directly downregulated PGC-1a expression in neurons at both the mRNA and the protein levels. The reduction of the PGC-1a mRNA level suggests that PGC-1a is transcriptionally regulated in neurons and, as we mentioned, AMPK is one of its regulators acting at the post-translational level. Cellular researches on Hela and drosophila S2 cells have shown the stimulating effect of polyphenols on sirtuin 1 that deacetylates PGC-1a at multiple lysine sites and increases its activity (Howitz et al., 2003; Wood et al., 2004). Davis et al. (2009) have reported that quercetin, a flavonoid, enhanced the expression of genes involved in mitochondrial biogenesis. Consistently, in our current study, we observed an increase in the PGC-1a level by adding digitoflavone to the medium of the cells, along with its downstream factors including TFAM and NRF-1 after H2O2 exposure. We used compound C as the AMPK-specific inhibitor to see if the protective effect of digitoflavone after H2O2 insult was only because of its antioxidant property or caused by digitoflavone-induced increase in mitochondrial biogenesis factors either. Our results supported the idea that digitoflavone exerts a central role in protecting cells via increasing mitochondrial biogenesis factors besides its antioxidant activity. The other indicator of mitochondrial dysfunction is the DCm. Chang et al. (2001) reported that ROS accumulation in the epithelial cells depolarized the mitochondrial membrane. Impaired DCm results in mitochondrial dysfunction and decreased ATP production, AMPK senses the decrease of ATP and phosphorylates in response, then the phosphorylated AMPK activates its related proteins (Mukherjee et al., 2008). There are also evidences that proposed changes in the DCm are an early, irreversible event in the cell death signaling pathway such as cytokine-induced apoptosis (Barbu et al., 2002). However, there are conflicting ideas in this regard (Oubrahim et al., 2001). In our experiment, H2O2 depolarized the mitochondrial membrane and this dissipation of DCm was then modulated by digitoflavone administration.
Conclusion Overall, our data indicated that digitoflavone not only exert its protective effect by direct radical scavenging but also
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interacting with cellular survival signaling pathways, such as antioxidant overexpression and activation of mitochondrial biogenesis, which led to a strong cellular defense against ROS-induced oxidative insult. Further studies are progressing in our laboratory to unveil detailed underlying mechanisms in the way of digitoflavone cellular protection.
Declaration of interest The authors report that they have no conflicts of interest. This work was supported financially by Iran National Science Foundation (Grant no. 91004247).
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