The H E staining and Nissl staining results showed that
The H&E staining and Nissl staining results showed that damage to the pyramidal neurons in the CA1 and CA3 regions of the hippocampus were observed in the 50 mg/kg/day DBP group, accompanied by an absence of Nissl bodies, suggesting that DBP exposure can lead to pathological changes in the neurons of the hippocampus, characterized by loss of synapses and loose and disordered arrangement. This observation is consistent with our previous study results (Ma et al., 2015). One of the mechanisms of cell apoptosis activated by the ERK 1/2 pathway is the release of cytochrome C (cyt C) from the mitochondria into cytosol, with caspase-3 being a key executive molecule in the process of mediated apoptosis (Porter and Jänicke, 1999; Goldstein et al., 2005). Cyt C is involved in the initiation of apoptosis, and also has an intermediate role in apoptosis (Goldstein et al., 2005). Caspase-3 has been found to be necessary for normal brain development as well as for its typical role in apoptosis (Porter and Jänicke, 1999). Both Cyt C and Caspase-3 levels can reflect the degree of early apoptosis. TNF-α, one of the cytokines that make up the acute-phase reaction, can be produced by neurons (Beg and Baltimore, 1996). Although the primary role of TNF-α is in the regulation of immune cells, its dysregulation has been implicated in a variety of human diseases including Alzheimer's disease and major depression, and this dysregulation is able to induce apoptotic cell death (Reichenberg et al., 2001). The results of this experiment showed that the levels of cyt C, caspase-3 and TNF-α in the hippocampi of the mice in the 50 mg/kg/day DBP group were significantly higher than those of the control group. The results of the Hoechst 33258 fluorescence test also showed that the number of apoptotic cells in the CA1 area of the hippocampus increased significantly after exposure to DBP. These results suggest that DBP exposure may lead to injury and apoptosis of neurons in the brain tissue of mice by activating the caspase-3 apoptotic pathway. Vitamin E (VE) is a fat-soluble organic antioxidant, which can directly eliminate O2– and other ROS (Traber and Atkinson, 2007). It is a strong antioxidant, and protects the cell membrane and nucleic torin synthesis from attack by free radicals to protect the neurons. Further data show that, VE as a non-enzymatic antioxidant, can reduce the production of ROS and MDA in cells, effectively blocking oxidative stress caused by environmental chemicals, and reducing damage to cells (Espinosadiez et al., 2015). The results from our experiment suggest that VE may attenuate to a certain extent, oxidative stress of neuronal cells in the hippocampal CA1 region that is induced by DBP, thereby exerting neuroprotective effects. It has also been reported that, nimodipine (NMDP), an L-type calcium channel antagonist, has great clinical potential for the treatment of brain related diseases, since NMDP directly affects neurons, and has neuropharmacological properties that can protect and promote memory and the recovery of intelligence (Shen et al., 2016; Hu et al., 2017). Recent studies have shown that NMDP can reduce the pathological state caused by excessive Ca2+ influx, and can decrease the spatial learning and memory impairment of test animals by inhibiting the pathological changes to the hippocampal CA1 neurons (Oh et al., 2016; Bittner et al., 2017). Other animal experiments have shown that pretreatment with NMDP, having the ability to lessen memory impairment, reduces the incidence of postoperative cognitive dysfunction (POCD) by decreasing the calcineurin mediated hippocampal neuroapoptosis in older rats (Zhang et al., 2018). The results from our experiment suggest that NMDP may play a neuroprotective role by blocking the Ca2+ influx to a certain extent, to alleviate the ERK 1/2 pathway activation of neurons in the CA1 region induced by DBP. In summary, the mechanism underlying the DBP-induced hippocampal neuron apoptosis in KM mice may be: Upstream molecular events: DBP can act on the hippocampus of the brain, resulting in ROS accumulation and excessive Ca2+ in the hippocampal neurons, thus activating the ERK 1/2 pathway; Midstream molecular events: p-ERK1/2 translocates into the nucleus to mediate nuclear transcription factor activity; Downstream molecular events: nuclear transcription factor-dependent gene expression changes, such as BDNF release and CREB phosphorylation, at the same time the equilibrium state of cell differentiation, growth and apoptosis is broken, and a caspase-3 cascade is activated; End effects: hippocampal neurons exhibit oxidative damage and apoptosis (Fig. 12 Graphical abstract).