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• W2274935216 abstract "Introduction: Neurodegenerative diseases are progressive disorders that could impair neuronal functions and structures. Oxidative stress and mitochondrial dysfunction are involved in the etiology of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and etc. Gemfibrozil is used as a therapeutic drug for hyperlipidemia. It has been shown that gemfibrozil is neuroprotective via modulation of mitochondrial biogenesis pathway under oxidative stress condition and in a sex-dependent manner. Materials and Methods: In this study, neuronal-like PC12 cells with were pretreated with different concentrations of gemfibrozil and H2O2, concomitantly. Results: In gemfibrozil pretreated groups, reduced level of caspase-3 and raised mitochondrial transcription factor A (TFAM) levels were detected. In contrast, adding fulvestrant, an Estradiol receptor antagonist, prevents the impact of gemfibrozil on oxidative stress condition, reducing its efficacy to protect the neurons against stress. Conclusion: Our results indicated the involvement of estradiol receptors in gemfibrozil neuroprotective mechanism, in diminishing oxidative stress-induced damage via reducing caspase-3 and inducing the level of TFAM that plays a crucial role in the mitochondrial biogenesis. 254 | Physiol Pharmacol 19 (2016) 253-262 Ashabi et al. oxidative stress (Wei et al., 2001; Onyango et al., 2010), as well as metabolic disease (Knutti and Kralli, 2001; Finck and Kelly, 2006), brain stroke (Dong et al., 2007) and mitochondrial diseases (Wenz, 2009). Mitochondrial biogenesis is regulated by mitochondrial and nuclear genomes causing e mitochondrial proliferation and differentiation, which is altered during oxidative stress (Ostronoff et al., 1996; Fernandez-Silva et al., 2003). Mitochondrial transcription factor A (TFAM) is considered as a key player in initiating mitochondrial biogenesis (Miranda et al., 1999). According to some studies, induction of H2O2 produces reactive oxygen species (ROS) and ROS subsequently triggers the procaspase-3 cleavage and consequent release of cytochrome c from mitochondria, leading to apoptosis (Tang et al., 2005). Gemfibrozil is a lipid lowering agent that belongs to a group of drugs known as fibrates(Xu et al., 2001a). It has been demonstrated that fibrates as peroxisome proliferator-activated receptor (PPAR)-α agonists, that protect against oxidative stress via anti-oxidant and anti-inflammatory mechanisms (Deplanque et al., 2003; Bordet et al., 2006; Xu et al., 2007). Also, it has been claimed that benzafibrate and fenofibrate also in the fibrate category could induce mitochondrial biogenesis in the skeletal muscle and liver (Nagai et al., 2002). Moreover, researches have proved that fibrates affect metabolism of steroid hormones and have direct estrogenic activity through binding to estrogen receptors as well (Xu et al., 2001a; Fan et al., 2004; Isidori et al., 2009). Estrogen receptor (ER) antagonist such as fulvestrant is mainly considered for treatment of hormone sensitive metastatic breast cancer(Osborne et al., 2004). Gemfibrozil has been able to significantly attenuate superoxide production resulting in inhibition of apoptosis (Calkin et al., 2006). Our recent studies have showed that gemfibrozil pretreatment resulted in a sexually-dimorphic Outcome (Mohagheghi et al., 2013a; Mohagheghi et al., 2013b). Gemfibrozil activated Nuclear respiratory factor 1 (NRF-1) and Mitochondrial transcription factor A (TFAM) in mitochondrial biogenesis signaling pathway and subsequently inhibited the caspase-dependent apoptosis, resulting in protection of female rats; while in male rats, provoked both caspase-dependent and caspase–independent apoptotic pathways and resulted in suppression of mitochondrial biogenesis signaling factors, meaning gemfibrozil acted reversibly and led to neurodegeneration (Mohagheghi et al., 2013a; Mohagheghi et al., 2013b). On the basis of above results, we aimed to test the effect of three different dosages of (5, 10 and 20 μM) of gemfibrozil on TFAM and apoptotic factor (caspase-3) neuronal level in the differentiated rat pheochromocytoma cells being exposed to oxidative stress in two different time points (4 hours and 9 hours after induction), and then examined the extent of involvement of estrogen receptors when administrating fulvestrant (an antagonist of ER). Materials and methods Antibodies directed against caspase-3, TFAM and βactin were obtained from Cell Signaling Technology (Beverly, MA, USA). Electrochemiluminescence (ECL) kit was purchased from Amersham Bioscience (Piscataway, NJ, USA). Fetal Bovine Serum (FBS) was provided from Gibco (Big Cabin, Oklahoma, USA), and polyvinylidene fluoride membrane was obtained from Chemicon Millipor (Temecula, CA, USA). All the other reagents were from Sigma Aldrich (St. Louis, MO, USA). Cell Culture and treatment conditions Rat pheochromocytoma (PC12) cells were obtained from Pasteur Institute (Tehran, Iran) which were grown in Dulbecco’s modified Eagle’s medium (DMEM), enriched with 5% fetal bovine serum, 10% horse serum, and 1% antibiotic (penicillinstreptomycin), Cultures were maintained according to standard protocols at 37 °C in a 95% humidified incubator with 5% CO2 (Greene and Tischler, 1976). Growth medium was changed three times a week. The cells were differentiated by incubating with nerve growth factor (NGF; 50 ng/mL) for 6 days (Figure.1). PC12 cells were treated with different concentrations (5, 10 and 20 μM) of gemfibrozil and 500 nM of fulvestrant. To induce oxidative stress 150 μMH2O2 was added to culture plates. MTT cell viability assay Cell viability was determined using a 3(4,5dimethylthiazol-2yl) 2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion assay(Mosmann, 1983) s. Gemfibrozil protects neurons via estradiol receptors Physiol Pharmacol 19 (2016) 253-262 | 255 The dark blue formazan crystals formed in intact cells were solubilized in dimethyl sulfoxide, and the optical density (O.D.) of each well was measured with a spectrophotometer equipped with a 550 nm filter. Results were expressed as percentage of cell viability = (O.D. treated/O.D. control) × 100. Western blot technique For Western blot analysis, total proteins were electrophoresed in 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Then the membrane was incubated with specific antibodies. Immuno-reactivity was detected Fig.1. A schematic representation of experimental procedure. Neuronal like PC12 cells were pretreated with gemfibrozil (5, 10 and 20 μM of gemfibrozil) and H2O2. In 4 hours and 9 hours after induction of H2O2 and three doses of gemfibrozil in separate groups, the MTT assay was done and neurons were collected to measure caspase-3 and TFAM. Fig.2. Effect of gemfibrozil and fulvestrant on cell viability against oxidative stress. The cell viability was determined by the MTT reduction assay and the surviving cell values were expressed as the percentage of control cells. Experiments were replicated 3 times independently. &&& p<0.001 versus control. ### p<0.001 versus H2O2 (9 hours), * p<0.05 versus Gemfibrozil 10 μM + H2O2 (9 hours). 256 | Physiol Pharmacol 19 (2016) 253-262 Ashabi et al. 35 KDa Procaspase-3 45 KDa Cleaved caspase-3 17 KDa C o n tr o l Fu lv e st ra n t (9 h ) Fu lv e st ra n t+ H 2 O 2 (9 h )" @default.
• W2274935216 created "2016-06-24" @default.
• W2274935216 creator A5037995106 @default.
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• W2274935216 date "2015-12-10" @default.
• W2274935216 modified "2023-09-23" @default.
• W2274935216 title "Gemfibrozil protect PC12 cells through modulation of Estradiol receptors against oxidative stress" @default.
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