Taken together, these investigations implied that an agonist of 7-nAChRs could be developed as a therapeutic agent against neurodegeneration. Multiple mechanisms were involved in the neuroprotective effects of nicotine, such as inhibition of astrocyte activation, PI3K signaling pathway, apoptotic signaling pathway, Wnt/-catenin signaling pathway, and Erk1/2 signaling pathway (Fodero et al., 2004; Yu et al., 2011; Liu et al., 2012; Lombardo dmDNA31 and Maskos, 2015). found that pretreatment with nicotine at low concentrations markedly recovered the cell cycle that was arrested at dmDNA31 the G2/M phase in the presence of H2O2 through reduced intracellular ROS generation. Moreover, nicotine attenuated H2O2-induced mitochondrial dysfunctions. Mechanistically, the application of nicotine significantly upregulated the levels of phosphorylated Erk1/2. The neuroprotective dmDNA31 effects of nicotine, in turn, were abolished by PD0325901, a selective Erk1/2 inhibitor. Further obtained investigation showed that nicotine exerted its neuroprotective effects specifically activating 7 nicotinic acetylcholine receptors (7-nAChRs). A selective inhibitor of 7-nAChRs, methyllycaconitine citrate (MLA), not only completely prevented nicotine-mediated antioxidation but also abolished expression of p-Erk1/2. Taken together, our findings suggest that nicotine suppresses H2O2-induced HT-22 cell injury through activating the 7-nAChR/Erk1/2 signaling pathway, which indicates that nicotine may be a novel strategy for the treatment of neurodegenerative disorders. oxidative stress in many different cell types. For instance, H2O2 caused intracellular ROS generation and repressed mitochondrial membrane potential, which then underwent apoptosis dmDNA31 in PC12 cells (Gao J. et al., 2018) and in SK-N-MC cells (Lee and Kim, 2019). Similarly, mitochondrial dysfunctions induced by H2O2 occurred in HT-22 cells as well (Dai et al., 2014). Evidence further exhibited that H2O2-induced mitochondrial membrane depolarization, swelling, and fragmentation could be due to the motility of mitochondria accompanied with mitochondrial elongation (Debattisti et al., 2017). Moreover, evidence showed that H2O2 could induce autophagic death in dopaminergic SY5Y cells through ROS-dependent endoplasmic reticulum stress and AMPK activation (Gao Z. et al., 2018). Therefore, it is of importance to identify a mechanism that exerts neuroprotective effects against oxidative injury. Nicotine has been recognized as the principal additive compound of tobacco that causes devastating health problems and even premature death for tobacco users (Hoffmann et al., 1990; Benowitz, 2009; Hatsukami et al., 2008). Nicotine abuse induces oxidative stress, apoptosis, and inflammation in brain cells (Oliveira-da-Silva et al., 2009; Benowitz, 2010; Cardinale et al., 2012; Motaghinejad et al., 2016) and also exacerbates behavioral impairments in mice (Shim et al., 2008). Chronic nicotine administration exacerbates tau pathology in a mouse model of AD (Oddo et al., 2005). Interestingly, frequency of dietary nicotine however has been reported to be inversely associated with Parkinsons disease (PD) risk (Nielsen et al., 2013). These studies suggest that nicotine might exert reverse functions with respect to neurodegeneration and neuroprotection. Indeed, an experimental study showed that nicotine prevents dopaminergic neuron loss in a rodent PD model (Liu Y. et al., 2017). Evidence has also accumulated that nicotine has been linked with decreased risk for AD (Oddo et al., 2005; Echeverria and Zeitlin, 2012; Moreno-Gonzalez et al., 2013; Lombardo and Maskos, 2015). Nicotine could attenuate A peptide-induced neurotoxicity in hippocampal neurons of rats (Liu and Zhao, 2004). Moreover, an study showed that nicotine is usually neuroprotective against NMDA-induced excitotoxicity (Dajas-Bailador et al., 2000). The actual results indicate the opposite effects of nicotine in the CNS, neuroprotective effects, and neurotoxic effects. Importantly, nicotine has been reported to encourage oxidative impairments in rats brain (Barr et al., 2007; Benowitz, 2010; Saad et al., 2020); Gipc1 nevertheless, increasing studies and showed the functions of nicotine on oxidative stress (Guan et al., 2003; Liu and Zhao, 2004; Hritcu et al., 2017). For instance, nicotine could neuroprotect against oxidative stress in primary cultures (Liu et al., 2015), in PC12 cells (Slotkin et al., 2015). Moreover, antioxidative functions of nicotine have been indicated in SY5Y cells (Parada et al., 2010). However, the contribution of nicotine on oxidative injury and its underlying mechanisms in mouse hippocampal HT-22 cell remain largely unknown. In the present study, we investigated whether nicotine could mitigate H2O2-induced oxidative damage in HT-22 cells and explored the potential molecular mechanisms. Thereby, a thorough understanding of the potential functions of nicotine on oxidative stress will be revealed, and this could promote the development of effective brokers in the treatment of these conditions. Materials and Methods Reagents and Antibodies The FITC-labeled Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (Canada). The ROS assay kit (DCFH-DA) was purchased from Meilun (China). The cell culture medium was obtained from HyClone (Utah, USA), and cell-cultured grade fetal bovine serum (FBS), penicillin/streptomycin, and trypsin were purchased from Gibco (Thornton, Australia). The antibodies of p-Erk1/2, Erk1/2, p-Akt, Akt, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9, -actin, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-nicotinic acetylcholine receptor 7 antibody was purchased from Abcam (ab216485, Abcam). The drugs were obtained from the following sources: nicotine, methyllycaconitine citrate (MLA), dihydro–erythroidine hydrobromide (DHE), and PD0325901 from MedChemExpress (MCE, USA) and H2O2 and EdU cell proliferation Kit with Alexa.
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