Neuroprotective Effects of Methyl 3,4-Dihydroxybenzoate against TBHP-Induced Oxidative Damage in SH-SY5Y Cells

Liang Cai, Li-Fang Wang, Jun-Ping Pan, Xiang-Nan Mi, Zheng Zhang, Hai-Ju Geng, Jia-Hui Wang, Song-Hui Hu, Wei Zhang, Qin Gao, Wu-Tian Wu, Huan-Min Luo
2016 Molecules  
This study investigated the neuroprotective effects of methyl 3,4-dihydroxybenzoate (MDHB) against t-butyl hydroperoxide (TBHP) induced oxidative damage in SH-SY5Y (human neuroblastoma cells) and the underlying mechanisms. SH-SY5Y were cultured in DMEM + 10% FBS for 24 hours and pretreated with different concentrations of MDHB or N-acetyl-L-cysteine (NAC) for 4 hours prior to the addition of 40 μM TBHP for 24 hours. Cell viability was analyzed using the methylthiazolyltetrazolium (MTT) and
more » ... te dehydrogenase (LDH) assays. An annexin V-FITC assay was used to detect cell apoptosis rates. The 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay was used to determine intracellular ROS levels. The activities of antioxidative enzymes (GSH-Px and SOD) were measured using commercially available kits. The oxidative DNA damage marker 8-OHdG was detected using ELISA. Western blotting was used to determine the expression of Bcl-2, Bax, caspase 3, p-Akt and Akt proteins in treated SH-SY5Y cells. Our results showed that MDHB is an effective neuroprotective compound that can mitigate oxidative stress and inhibit apoptosis in SH-SY5Y cells. susceptible to oxidative stress injury because the level of consumed oxygen is higher in the brain while the anti-oxidation ability is lower. Maintenance of the redox balance is crucial to normal brain functions. Oxidative stress likely plays a role in the pathogenesis of neurodegenerative diseases including Alzheimer′s disease (AD) [4], Parkinson's disease (PD) [5] , and Huntington′s disease (HD). Oxidation markers, such as lipid, protein, and nucleic acid oxidation products, are found in patients with these diseases [6] . Therefore, antioxidant supplements may prevent or at least delay the advancement of neurodegenerative diseases and reduce ROS-induced neuronal damage. Cell apoptosis involves the characteristic cell shrinkage, nuclear pyknosis, DNA fragmentation and membrane vacuoles of autonomous, which activates a cysteine protease cascade of the caspase family and results in cell death [7] . ROS induces oxidative damage to DNA, which leads to apoptosis through an endogenous apoptotic pathway in mitochondria [8] . CytC, dATP and apoptotic proteaseactivating factor-1 (Apaf-1) compose the apoptosome [9], which activates caspase-9 and initiates the caspase cascade. Caspase-9 activates caspase-3, which is a key executor of apoptosis [10] to produce apoptosis-related morphological and biochemical change [11] The balance between the apoptoticpromoting Bax family proteins (e.g., Bad, Bax, and Bak) and the anti-apoptotic Bcl-2 family proteins (Bcl-XL and the Bcl-2) is a key factor. The ratio of Bcl-2/Bax is a "switch" that adjusts the mitochondrial permeability transition pore to activate or inhibit the cell death pathways of mitochondria [12] . The effect of bioactive small molecules extracted from plants on cell survival, apoptosis and the oxidative stress pathway has been widely studied in recent years. Methyl 3,4-dihydroxybenzoate (MDHB) is a small molecule compound that is extracted from East Asian Tang Materia, Malan and other natural plants [13] . Previous studies by our laboratory have demonstrated that MDHB has neurotrophic effects [14] , anti-apoptotic effects induced by and H2O2 [16] as well as the lifespan extension effects on C. elegans [17] . The present study used t-butyl hydroperoxide (TBHP) to induce cellular oxidative stress in cultured SH-SY5Y cells in vitro and examined the protective effect of MDHB on oxidative damaged in nerve cells and its associated mechanism. The results provide experimental evidence for oxidative stress-related neurodegenerative disease treatments for use in aging research. Results The Effect of MDHB on SH-SY5Y Cell Viability Cell viability was measured by MTT assay. First,The safety margin of MDHB, SH-SY5Y cells were cultured with MDHB at 2~128 μM and 0.1% DMOS for 24 h. The result showed that 0.1% DMSO and the concentration of 2~128 μM MDHB had no effect on the cell vitality ( Figure 1A ). SH-SY5Y cells were pretreated with 2, 4, or 8 μM of MDHB and 100 μM NAC for 4 h, followed by treatment with 40 μM TBHP for 24 h. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15
doi:10.3390/molecules21081071 pmid:27556437 fatcat:gjd32anosbegbo4bcxdq42te5y