**3. Discussion**

The concept of neuroprotection under brain insults, such as prolonged seizures (status epilepticus), should consist of not only the conservation of structural neuronal cell death, but also preservation of neuronal networks and functions [38,39]. In research on prevention of the deleterious effects of status epilepticus, the primary focus is on the development of chronic epilepsy and cognitive decline [38,39]. Thus, a more precise definition of neuroprotection in status epilepticus needs to include protection not just against neuronal death but also against neuronal and network dysfunction at the cellular and molecular levels [39,40]. However, the studies of neuroprotective mechanisms elicited by recurrent seizure activities, especially under status epilepticus, are limited. Considering the detrimental reaction in brain cells under status epilepticus, acute response proteins to counteract these detrimental effects elicited by sustained seizures can be a mechanism of endogenous neuroprotection

against seizure-induced neuronal cell death [39,41,42]. Following status epilepticus, the mechanism of endogenous neuronal survival signals is evolutionarily conserved and may actuate extensive signaling pathways to offer the neuroprotective effect which therefore may be strong candidates for therapeutic strategies [39,42]. In our previous studies, both in human epilepsy [43] and animal studies following status epilepticus [5,26,41,44–46], several endogenous neuroprotective mechanisms to lessen neuronal damage were proposed, including PPARγ [41], mitochondrial uncoupling protein 2 (UCP2) [41], heat shock protein 70 [43,44], brain-derived neurotrophic factor [45], mitochondrial dynamin-related protein 1 [46], PGC1-α [5,26], and sirtuin 1 [5,26].

The present study demonstrated that activation of PGC-1α activity regulated VEGF/VEGFR2 (Flk-1) signaling and showed the neuroprotective effect against neuronal cell death in the hippocampus following experimental status epilepticus. Particularly, we noted that PGC-1α expression was enhanced in the rat hippocampus following status epilepticus and upregulated VEGF and VEGFR2 (Flk-1) expression. Administration of the activator of PGC-1α, resveratrol, was accompanied by increased VEGF and VEGFR2 expression, and promotion of PI3K/AKT and MEK/ERK pathways and decreased neuronal cell death in the hippocampus following status epilepticus. Downregulation of PGC-1α by pretreatment of siRNA against *pgc-1*α reduced VEGF and VEGFR2 expression and inhibited PI3K/AKT and MEK/ERK pathways, also accompanied by heightened caspase-3 activity, and augmented neuronal damage in the hippocampal CA3 subfield. We also noted that the mRNA levels for *vegf* and *vegfr2* were reduced after pretreatment of siRNA against *pgc-1*α compared with the levels of control siRNA groups. This suggests an additional mechanism for seizure-induced activation following status epilepticus. Therefore, our results indicated that seizure activities triggered the upregulation of the VEGF/VEGFR2 (Flk-1) pathway, activation of PI3K/AKT and MEK/ERK signaling, and worked against the hippocampal neuronal apoptotic cell death following status epilepticus. Therefore, our results showed VEGF/VEGFR2 and related downstream PI3K/AKT and MEK/ERK signaling can exert endogenous neuroprotection in status epilepticus.

Recently, our research revealed that PGC-1α is an important transcriptional regulator that acts through regulating the expression of reactive oxygen species (ROS), mitochondrial UCP2, superoxide dismutase 2, and mitochondrial biogenesis, which plays a beneficial part in neuroprotection in the CNS following global ischemia and status epilepticus [5,26,28,41,47]. In addition, recent studies have demonstrated that the PGC-1α signaling pathway exerts potential neuroprotective properties in many neurological diseases [26,48,49], such as Alzheimer's disease [50], Parkinson's disease [51], acute stroke [28,52], epilepsy [53], and status epilepticus [26,53–55]. Whether activation of PGC-1α following sustained seizures promotes the endogenous activation of VEGF/VEGFR2 signaling is unclear. However, recent evidence showed activation of PGC-1α induces the expression of VEGF/VEGFR2 signaling, leading to protection from apoptotic cell damage in the neuronal cells and providing neuroprotective effects [15,29–31]. Thus, the activation of PGC-1α may regulate the VEGF/VEGFR2 signaling pathway in the neuronal cells and might be a crucial mechanism in neuroprotection following status epilepticus.

VEGFs are strong endothelial cell mitogens and major regulators of neurogenesis and angiogenesis [8,56]. In addition to the well-established effects of VEGF, recent research has demonstrated pivotal roles for VEGF/VEGFR2 in a broad range of neurotrophic and neuroprotective effects in the CNS that may relate to neurogenesis and angiogenesis [11–13]. The neurotrophic and neuroprotective effects of VEGF are predominantly mediated by VEGFR2, also called fetal Flk-1 [14,57–59], or kinase insert-domain containing receptor (KDR) [60]. VEGF/VEGFR2 (Flk-1) signaling may have neuroprotective effects in many neurological diseases, such as hemorrhagic or ischemic stroke [16–18], traumatic brain injury [61], amyotrophic lateral sclerosis [20], Huntington's disease [62], Alzheimer's disease [12,21], and Parkinson's disease [12,22]. Recent evidence showed that during status epilepticus, VEGF is upregulated and it protects against seizure-induced neuronal cell death in the hippocampus [14,19,23]. In vitro, VEGF administration suppresses ictal and interictal epileptiform activity via the VEGFR2 (Flk-1) receptor. Thus, upregulated VEGF signaling through

VEGFR2 (Flk-1) overexpression may regulate epileptogenesis and ictogenesis in mice and counteract the focal epileptic seizure [14].

VEGF can directly stimulate the proliferation of neuronal progenitors; however, the possible mechanism of seizure-induced activation of endogenous PGC-1α that promotes the following VEGF signaling that contributes to neuroprotection in the hippocampus following status epilepticus is not clear. Thus, we investigated two downstream pathways of the VEGF/VEGFR2 (Flk-1) signaling pathways that included PI3K/AKT and MEK/ERK signaling [13,63–65]. Activation of VEGF has been suggested to regulate PI3K/AKT and MEK/ERK cascades. Furthermore, VEGF is known to be involved in the trophic and neuroprotective effects of growth factors via binding to its tyrosine kinase receptors, particularly of which VEGFR2 (Flk-1) is proposed to mediate most of the neuron-specific effects of VEGF via mediating PI3K/AKT and MEK/ERK signaling [10–13,33].

Mitogen-activated protein kinases (MAPK) are considered to be a family of Ser/Thr protein kinases and conserved in eukaryotes and involved in many cellular programs. The MEK/ERK pathway is one of the MAPK cascades involved in transducing cell survival signals via growth factor receptors [66,67]. Interestingly, growing evidence revealed that improving mitochondrial function with activation of PI3K/AKT and MEK/ERK pathways might tend to inhibit phosphorylation of c-Jun-NH2-terminal kinase (JNKs) and p38, the second and third major signaling cassettes in the MAPK pathway, which majorly respond to inflammatory and cellular stress to promote inflammation and cell death, in neuronal cells under many neurological conditions, both in vitro and in vivo studies [66–68]. Overall, MEK/ERK pathways have been reported to play a crucial role in protecting neuronal cells from death under hypoxia, global ischemia, epilepsy, status epilepticus, and Parkinson's disease [33,63,68,69]. Therefore, following status epilepticus, endogenous activation of PGC-1α may regulate the VEGF/VEGFR2 (Flk-1) signaling pathway that triggers PI3K/AKT and MEK/ERK cascades and also promotes the cAMP-CREB signaling axis [45,69], which regulates the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family, the expression of autophagy [63] and lysosomal genes, and further contributes to neuron survival mechanisms in hippocampal neuronal cells [70–72].

Several studies showed that the PI3K/AKT signaling pathway can modulate cellular activities, such as neuronal proliferation, differentiation, cell survival, and synaptic plasticity, and this signaling cascade activation promotes an important cytoprotective mechanism that promotes neural survival associated with neurodegenerative disorders and epileptic seizures [13,73,74]. Binding of VEGF may further trigger VEGFR2 (Flk-1) to activate PI3K and then phosphorylate phosphatidylinositol 4,5-bisphosphate or PtdIns (4,5)*P*<sup>2</sup> (PIP2) to the second messenger, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)*P*<sup>3</sup> (PIP3), on the plasma membrane. PIP3 directly or indirectly binds to AKT to induce structural changes and facilitate the phosphorylation of AKT amino acid residues under neurodegenerative diseases [13,73,74]. Therefore, under different acute or chronic neurological conditions, activated AKT can promote neuronal cell survival in several ways, including subsequently regulating numerous downstream molecules, such as cyclin D, glycogen synthase kinase-3β, and mechanistic targets of rapamycin complex 1, thereby regulating cell functions and improving cell survival advantage [75]. Moreover, phosphorylated AKT regulates cell survival and anti-apoptosis by targeting the pro- or anti-apoptotic mediators, such as Bcl-2, Bax, and caspases [76–78].

In addition to effects of the PI3K/AKT signaling pathway on cellular proliferation and survival, the activation of the MEK/ERK pathway encourages cell survival and prevents cell apoptosis [63,65]. Therefore, the role in neuroprotection of the MEK/ERK pathway is emphasized in many acute or chronic neurological diseases [13,63,79]. Growing evidence has shown that the MEK/ERK pathway plays a central role as a potential therapeutic target in neurological disorder diseases including epilepsy and status epilepticus [13,80,81]. The MEK/ERK pathway is also a vital message transmission pathway in embryonic and adult neurogenesis and plays a role in the regulation of brain physiological function [80]. When the cell suffers external stress, such as prolonged seizures, VEGF may stimulate cellular responses by binding to a special receptor, VEGFR2 (Flk-1), and this activates receptors in the form of the dimer

which start signaling output and then triggers the activation of a series of signal transduction molecules, such as MEK/ERK signaling.

Evidence has shown that mitochondrial related autophagic pathways, such as the Jun N-terminal kinases-associated B-cell lymphoma-2 (JNK/Bcl-2) pathway, MEK/ERK signaling, and the sirtuin 1/forkhead box protein O1 pathway, may provide neuroprotective effects from neurotoxicity against dopamine neurons [82]. Activation of the MEK/ERK pathway by phosphorylation of threonine and tyrosine residues can quickly initiate ERK1/2-dependent biological processes in neurological diseases, including epilepsy and status epilepticus [39]. Recently, we noted exogenous resveratrol may provide neuroprotective effects through upregulation of PGC-1α, modulation of mitochondrial dynamics, and MEK/ERK regulated autophagy in rotenone-induced oxidative stress SH-SY5Y cell lines [63]. These results suggest activation PGC-1α regulated the VEGF/VEGFR2 pathway and further promoted MEK/ERK signaling which provided neuroprotective effects against epilepsy, status epilepticus, and neurodegenerative diseases.
