3- Bromopyruvate, a caloric restriction mimetic, exerts a mitohormetic effect to provide neuroprotection through activation of autophagy in rats during aging
In the present study, attempts have been made to evaluate the potential role of 3 Bromopyruvate (3-BP) a glycolytic inhibitor and a caloric restriction mimetic (CRM), to exert neuroprotection in rats during aging through modulation of autophagy. Young male rats (4 months), and naturally aged (22 months) male rats were supplemented with 3-BP (30 mg/kg b.w., orally) for 28 Daysdays. Concomitantly, oOur dataResults demonstrated decreased levela significant increase in the antioxidant biomarkers (ferric reducing antioxidant potential level, total thiol, superoxide dismutase and catalase activities) and decrease in the level of pro-oxidant biomarkers such as protein carbonyl after 3-BP supplementation in brain tissues. A significant increase in reactive oxygen species (ROS) was observed due to the mitohormetic effect of 3-BP supplementation in the treated rats. Furthermore, the 3-BP treatment also enhanced the activities of electron transport chain complexes I and IV in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy, neuroprotective and aging marker genes. RT-PCR data revealed that 3-BP up-regulated the expression of autophagy markers genes (Beclin-1 and LC3 β), sirtuin-1, and neuronal marker gene (NSE), respectively in the aging brain. The results suggest that 3-BP induces a mitohormetic effect through the elevation of ROS which reinforces defensive mechanism(s) targeted at regulating autophagy. These findings suggest that consistently low-dose 3-BP may be beneficial for neuroprotection during aging and age-related disorders.
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Preparation of brain tissue homogenate The whole brain was dissected from the experimental rats, weighed, and homogenized according to the procedure of Liapi et al (2013). Isolation of mitochondria To acquire mitochondrial preparations, the pellet was washed in almost the same manner (Navarro and Boveris 2004). The quality of the mitochondrial preparation was determined using succinate dehydrogenase. Complex I (NADH-CoQ oxidoreductase) The activity was evaluated using Hatefi and Rieske's (1967) procedure with minor changes. Complex II (succinate-CoQ oxidoreductase) The activity was assessed using Hatefi and Stiggall's (1978) technique with minor changes. Complex III (CoQ-cytochrome c oxidoreductase) The activity was measured using Shimomura's method (1984) with minor changes. Complex IV (cytochrome c oxidase) With minor changes, the activity was assessed using the technique of Taber & Morrison, 1964. Determination of pro-oxidant biomarkers associated with aging The generation of reactive oxygen species (ROS) in brain was evaluated as described before (Garg et al. 2017). The protein carbonyl (PCO) activity was calculated using Evans et al. 1999 method. Determination of Non-enzymatic and Enzymatic Antioxidants Ferric reducing antioxidant potential was evaluated according to Benzie and Strain's procedure (1996) T-SH (total thiol) was determined using the standard procedure (Sedlak and Lindsay 1968). Superoxide dismutase (SOD) activity was evaluated according to the published procedure (Kakkar et al. 1984). The H2O2 degradation test was used to evaluate catalase activity in brain homogenates of experimental groups (Hong et al. 2014) Gene expression analyses by a reverse transcriptase-polymerase chain reaction Gene expression profiling using reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out substantially as previously reported (Kumar et al. 2020). The Spectro-star nano spectrophotometer was used to measure RNA at 260 nm, and its quality was determined using agarose gel electrophoresis under denaturing conditions. cDNA was prepared using the New England Biolabs cDNA Synthesis Kit. Beclin-1, LC3β, Neuron-specific enolase (NSE), Sirtuin 1, and β-actin were amplified using gene-specific primers in 30 cycles of RT PCR using the PCR Protocol for Taq DNA Polymerase kit (M0273) (New England Biolabs). The BIORAD Gel-Doc system was used to visualize PCR results on 1.5 percent agarose containing ethidium bromide. The densitometry system (BIORAD Image analysis software) was used to examine the bands, with beta-actin serving as an endogenous control. Statistical analyses Graph-Pad Prism version 5.01 software was used for the statistical analysis. The mean and ± standard deviation is used to express the values. A one-way analysis of variance (ANOVA) was used in the analysis, followed by a post hoc Bonferroni's test. Statistical significance was defined by the P value.