Additionally both in vitro and in vivo
Additionally, both in vitro and in vivo studies showed that a Ca2+ influx is closely related to apoptosis (Rizzuto et al., 2003). Calmodulin (CaM) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells, which senses intracellular calcium levels by binding one Ca2+ ion. (Chin and Means, 2000). It is an intracellular target of the secondary messenger Ca2+, and the binding of Ca2+ is required for the activation of CaM. When the intracellular Ca2+ concentration exceeds 10−6 mol/L, the inactive CaM can bind to four Ca2+ per molecule (Waxham et al., 1998). Once bound to Ca2+, CaM acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatases, and is thus involved in mediating cell apoptosis, short-term and long-term memory and other activities (Soderling, 2000). Calcium/calmodulin-dependent protein kinase II (CaMK II) is widely distributed in nerve tissue, and accounts for between 1 and 2% of the total protein in the hippocampus. More importantly, CaMKII is also necessary for Ca2+ homeostasis and reuptake involved in many signaling cascades, including apoptosis (Hund, 2008), and is also thought to be an important mediator of learning and memory. Strong evidence suggests that its main function is to sense changes in post-synaptic Ca2+ levels, maintain synaptic plasticity and exert learning and memory functions (Incontro et al., 2018). Protein kinase C (PKC) is an NAD+ synthesis in the G protein coupled receptor system, and takes part in cellular responses to various agonists, including neurotransmitters (Nishizuka, 1992). In unstimulated cells, PKC is mainly distributed in the cytoplasm, showing inactive conformation. Once the second messenger, Ca2+ is present, PKC become membrane-bound enzymes (Keenan and Kelleher, 1998). These multifunctional enzymes can participate in the regulation of biochemical reactions, and also play prominent roles in Ca2+ signal transduction cascades. Our results showed that the levels of CaM and CaMK II in the hippocampi of the mice in the 50 mg/kg/day DBP group decreased significantly, while the level of PKC increased, suggesting that DBP could increase the intracellular concentration of Ca2+, which is consistent with the results presented by Palleschi et al. (2009). Currently, there is no direct evidence of an association between DBP exposure and the ERK 1/2 pathway. However, numerous different stimuli, including ROS accumulation and excessive Ca2+, can activate the ERKs pathway (Mccubrey et al., 2006; Kemmerling et al., 2007). The ERK family comprises five subgroups, including ERK 1–5. Among them, ERK 1 and ERK 2 are the most widely studied protein kinases belonging to the serine/threonine residues in the ERK family, and are principal pathways of cell signal transduction (Chang and Karin, 2001). More and more studies have confirmed that the ERK 1/2 pathway plays a key role in neurobehavioral responses, learning and memory (Thiels and Klann, 2001; Kamei et al., 2006; Peng et al., 2010). As a key member of the MAPK family, ERK 1/2 activity is necessary for long-term spatial memory (Atkins et al., 1998). English and Sweatt (1997) found that ERK 1/2 is involved in the formation of hippocampal synaptic plasticity. Another study reports that p-ERK 1/2 plays a regulatory role in long-term potentiation (LTP) of the hippocampus by affecting transcription factors and protein translation in the process of synaptic plasticity (Ying et al., 2002). Our results showed that the ERK 1/2 and p-ERK 1/2 levels in the 50 mg/kg/day DBP group were significantly higher than that in the control group, suggesting that DBP can activate the ERK 1/2 pathway in the hippocampus. Phosphorylated ERK1/2 (p-ERK1/2), translocating from the cytoplasm to the nucleus, mediates the activation of transcription factors such as CREB, and then regulates many biological functions including cell apoptosis, proliferation and differentiation (Ying et al., 2002). Roberson and Cobb (2002) confirmed that activation of the ERK 1/2 signaling pathway could lead to an increase of CREB phosphorylation (p-CREB) in the hippocampus, and that the ERK 1/2 inhibitor PD09895, could block the activation of ERK 1/2, thereby decreasing the phosphorylation of CREB. Hu et al. (2004) also found that the p-ERK 1/2 activated CREB in the hippocampus plays a well-documented role in a mouse model of traumatic brain injury. Another role of ERK 1/2 activation is to affect the expression of BDNF in the central nervous system (Ying et al., 2002). BDNF is one of the most active neurotrophins, helping to stimulate and control neurogenesis, and plays an important role in normal neural development for long-term memory storage (Bekinschtein et al., 2008). BDNF and p-CREB are two important proteins involved in neuronal protection mediated by transcription factors in the nucleus. Their expression levels can reflect the ability of neurons to resist cell damage and apoptosis. Our results showed that the levels of BDNF and p-CREB in the hippocampi of the mice from the 50 mg/kg/day DBP group were significantly lower than those seen in the control group, suggesting that DBP could affect the phosphorylation of CREB in the nucleus and promote the release of BDNF in neurons through activation of the ERK 1/2 pathway. As a result, with the activation of the ERK 1/2 pathway, the hippocampal neurons exposed to DBP will tend to damage or apoptosis.