EGCG Attenuates CA1 Neuronal Death by Regulating GPx1, NF-κB S536 Phosphorylation and Mitochondrial Dynamics in the Rat Hippocampus following Status Epilepticus

Status epilepticus (SE) is a life-threatening neurological emergency that leads to neuronal death, cognitive impairments, and aberrant networks. Excessive Ca2+ influx through NMDAR contributes to excitotoxic, programmed necrotic death. Because mitochondrial dynamics (fission and fusion), bioenergetics, and ROS homeostasis are critical for neuronal survival, dysregulation of the fission mediator DRP1 contributes to SE-induced damage. Prior work shows SE downregulates DRP1 and causes mitochondrial hyperfusion in CA1 neurons. EGCG is an antioxidant that scavenges ROS and inhibits pro-oxidant enzymes; it also upregulates GPx1 and can inhibit NF-κB signaling. However, whether EGCG mitigates SE-induced mitochondrial dynamic impairment and related signaling in CA1 neurons was unclear. The study aimed to determine if EGCG protects CA1 neurons after SE by restoring mitochondrial fission via ERK1/2-DRP1 and by modulating GPx1 and NF-κB S536 phosphorylation.

- Mitochondrial dynamics: DRP1 S616 phosphorylation promotes fission; S637 inhibits it. Human DNM1L mutations impair fission and are associated with refractory SE and poor outcomes. In animal SE models, DRP1 downregulation leads to aberrant mitochondrial elongation and programmed necrosis in CA1 neurons. - EGCG actions: Antioxidant, ROS scavenger, and inhibitor of NADPH oxidase and xanthine oxidase. In SAH models, EGCG reduced DRP1 upregulation and excessive mitochondrial fragmentation. In SE models, EGCG previously protected pyramidal neurons via NF-κB inhibition, but effects on mitochondrial dynamics were not defined. - Oxidative stress and GPx1: GPx1 reduces H2O2 and its downregulation contributes to SE-induced CA1 death. EGCG can enhance GPx1, and oxidative stress disrupts mitochondrial dynamics, suggesting a link between EGCG, GPx1, mitochondrial function, and NF-κB signaling. - Signaling to DRP1: ERK1/2 and JNK regulate DRP1 S616 phosphorylation. SE reduces ERK1/2 and JNK phosphorylation. Other antioxidants (e.g., CDDO-Me) protect CA1 via ERK1/2 and JNK activation. - NF-κB: NF-κB S536 phosphorylation correlates with SE vulnerability; mitochondrial fission and NF-κB can reciprocally regulate each other; GPx1 inhibits NF-κB S536 phosphorylation.

- Animals: 70 male Sprague-Dawley rats (200–220 g). Controls n=28; experimental n=42. Standard housing. Approved protocol (Hallym 2021-3). - Intracerebroventricular infusion: Under isoflurane anesthesia, an infusion needle to right lateral ventricle was connected to an osmotic pump (model 1007D) containing: (1) vehicle (n=28), (2) EGCG 50 µM (n=28), or (3) EGCG 50 µM + U0126 25 µM (n=14). Pilot data showed no adverse neurological effects. - SE induction: Two days post-surgery, LiCl (127 mg/kg, i.p.). Next day, atropine methylbromide (5 mg/kg i.p.) 20 min prior to pilocarpine (30 mg/kg i.p.; n=42) to induce SE. Diazepam (10 mg/kg i.p.) administered 2 h after SE onset and as needed to terminate seizures. Controls received saline instead of pilocarpine. - Tissue collection: Three days after SE, animals were anesthetized. For Western blot (n=7/group): hippocampi homogenized in lysis buffer with protease and phosphatase inhibitors; protein quantification; SDS-PAGE; PVDF transfer; immunoblotting (primary antibodies listed in Table 1); β-actin as loading control; ECL detection; phosphorylation ratio computed as phospho:total. - Immunohistochemistry and mitochondrial morphometry: Perfusion fixation, cryoprotection, sectioning at 30 µm. Sections from dorsal hippocampus (−3.0 to −3.6 mm from bregma). Primary and fluorescent secondary antibodies used for DRP1, p-ERK1/2, NF-κB S536, GPx1, mitochondrial marker (MTCO1), NeuN. For morphometry, five CA1 pyramidal neurons per section (five sections per animal; n=7 rats/group) analyzed. Indices: (a) mitochondrial elongation index (area-weighted form factor = perimeter^2/4π) and (b) mitochondrial network aggregation (Σarea/Σperimeter). - Neuronal degeneration: Fluoro-Jade B staining on 4–5 sections/animal; quantification of FJB-positive neurons in CA1. - Statistics: Mann–Whitney test or Kruskal–Wallis test with Dunn–Bonferroni post hoc. Significance p<0.05. - Experimental comparisons: Vehicle vs EGCG in control and post-SE conditions; effect of EGCG on GPx1, DRP1 (total and S616), ERK1/2 and JNK phosphorylation, NF-κB S536 phosphorylation, mitochondrial morphology; effect of ERK1/2 inhibition (U0126) co-treatment on EGCG-mediated outcomes.

- Neuroprotection: EGCG reduced SE-induced CA1 neuronal degeneration (FJB-positive cells) vs vehicle (z=2.619; p=0.009; n=7 rats). - GPx1: SE decreased hippocampal GPx1 to 0.46-fold of control (p<0.001). EGCG increased it to 0.61-fold (p=0.041; χ2(3)=22.03; p<0.001; n=7). In CA1 neurons, SE reduced GPx1 fluorescence to 0.39-fold of control (p<0.001); EGCG significantly mitigated the decrease (χ2(3)=111.685; p<0.001; n=35 cells in 7 rats). - Mitochondrial dynamics: SE increased mitochondrial elongation (p<0.001) and aggregation (p<0.001) in CA1 neurons. EGCG reduced elongation (p=0.001; χ2(3)=42.741; p=0.001) and aggregation (p=0.022; χ2(3)=41.646; p<0.001; n=35 cells in 7 rats). Interpretation: EGCG inhibits SE-induced mitochondrial hyperfusion. - DRP1 total and S616 phosphorylation: SE reduced total DRP1 to 0.56-fold of control (p<0.001) and DRP1 S616 to 0.51-fold (p<0.001). EGCG increased total DRP1 to 0.8-fold (p=0.01; χ2(3)=21.275; p<0.001) and DRP1 S616 to 0.66-fold (p=0.016), and restored DRP1 expression in CA1 neurons (χ2(3)=105.805; p<0.001; n=35 cells in 7 rats). - ERK1/2 and JNK: SE decreased p-ERK1/2 to 0.54-fold (p<0.001) and p-JNK to 0.55-fold (p<0.001), without changing total protein levels. EGCG increased p-ERK1/2 to 0.78-fold (p=0.009; χ2(3)=22.627; p<0.001; n=7) and restored p-ERK1/2 in CA1 neurons (χ2(3)=109.938; p<0.001; n=35 cells), but did not affect decreased p-JNK. - NF-κB S536 phosphorylation: Baseline very low in CA1 neurons; SE significantly increased NF-κB S536 phosphorylation (p<0.001); EGCG markedly reduced this increase (χ2(3)=111.461; p<0.001; n=35 cells). - ERK1/2 inhibition (U0126 co-treatment with EGCG): Diminished EGCG neuroprotection (z=2.43; p=0.015; n=7). Increased mitochondrial elongation (z=3.172; p=0.002) and aggregation (z=4.939; p<0.001) compared to vehicle co-treatment, indicating loss of EGCG’s benefit on fission. U0126 reduced DRP1 S616 phosphorylation despite EGCG (z=2.492; p=0.013; n=7) but did not alter total DRP1, hippocampal GPx1 levels, or NF-κB S536 phosphorylation in CA1 neurons under EGCG treatment post-SE. - Summary: EGCG protects CA1 neurons post-SE by GPx1 upregulation, restoration of ERK1/2-DRP1 S616-mediated mitochondrial fission (independent of JNK), and reduction of NF-κB S536 phosphorylation.

The study addresses how EGCG confers neuroprotection after SE by examining mitochondrial dynamics and redox/inflammatory signaling in vulnerable CA1 neurons. SE leads to oxidative stress, DRP1 downregulation, ERK1/2/JNK inactivation, mitochondrial hyperfusion, and neuronal death. EGCG upregulated GPx1, attenuating ROS burden, which in turn restored ERK1/2 activation and DRP1 S616 phosphorylation, normalizing fission dynamics and preventing hyperfusion. This ERK1/2-DRP1 mechanism was necessary for neuroprotection, as ERK1/2 inhibition (U0126) abrogated EGCG’s effects on mitochondrial morphology and survival without affecting GPx1 or NF-κB status, indicating pathway specificity. EGCG also reduced NF-κB S536 phosphorylation in CA1 neurons, likely via GPx1-mediated redox control, suggesting an additional, ERK1/2-DRP1–independent anti-inflammatory/anti-stress pathway. EGCG did not rescue JNK phosphorylation, implying its mitochondrial benefits are primarily ERK1/2-driven, unlike other antioxidants (e.g., CDDO-Me) that engage both ERK1/2 and JNK. Collectively, findings demonstrate that EGCG counters SE pathology through dual GPx1-linked routes: (1) GPx1–ERK1/2–DRP1 S616 restoring mitochondrial fission and (2) GPx1-mediated inhibition of NF-κB S536 phosphorylation, contributing to CA1 neuron survival.

EGCG protects CA1 hippocampal neurons from SE-induced degeneration by inducing GPx1 and preserving ERK1/2-dependent DRP1 S616 phosphorylation, thereby restoring mitochondrial fission and preventing hyperfusion. EGCG also suppresses NF-κB S536 phosphorylation in CA1 neurons, indicating parallel GPx1–NF-κB modulation. These results highlight EGCG’s potential as a therapeutic strategy targeting ROS–GPx1–ERK1/2–DRP1 and ROS–GPx1–NF-κB pathways beyond its general antioxidant properties. Future work should address EGCG brain delivery/BBB permeability under pathological conditions, dissect hypoxia-related contributions during SE, and explore interactions between neuronal mitochondrial dynamics and neuroinflammatory mediators (e.g., IL-1β).

- The study did not investigate molecular mechanisms of hypoxia-related hippocampal damage during SE; potential contributions of hypoxia cannot be excluded. - EGCG blood–brain barrier permeability remains a limitation; intracerebroventricular administration was used because EGCG is poorly BBB-permeable in rats. Translation to systemic dosing requires further pharmacokinetic and BBB permeability studies. - The work focused on CA1 neurons at a single time point (3 days post-SE); broader regional and temporal dynamics were not assessed.