Digoxin and sodium valproate, but not celecoxib, render a pronounced effect on the cerebral cyclooxygenase pathway and neuron-specific enolase under the pentylenetetrazole-induced kindling in mice
Authors: Tsyvunin Vadim , Shtrygol’ Sergiy , Mishchenko Mariia , Havrylov Ihnat , Taran Andriy , Shtrygol' Diana , Horbach Tatiana 
Published: August 2022

Abstract
Epilepsy is a chronic neurological disease characterized by a persistent tendency to seizures. The development of new approaches to the treatment of epilepsy is an urgent issue in modern pharmacology. Such studies are especially necessary in view of the growing number of cases of drug-resistant epilepsy, which reaches 30 %. To improve the results of treatment, adjuvant non-antiepileptic drugs are offered. Promising adjuvant agents include, in particular, the lipophilic cardiac glycoside digoxin in subcardiotonic doses as well as non-steroidal anti-inflammatory drugs. Taking into account the important role of neuroinflammation in the pathogenesis of epilepsy, it is necessary to clarify the influence of standard antiepileptic drugs and adjuvant agents on individual links of the inflammatory process and neurons’ damage under the conditions of chronic epileptogenesis. The aim of the work is to determine the effect of digoxin, sodium valproate and celecoxib per se as well as the combination of digoxin with sodium valproate on the content of cyclooxygenase 1 and 2 types (COX-1, COX-2), prostaglandins (PG) E2, F2α, I2, thromboxane (TX) B2, 8-isoprostane and neuron-specific enolase (NSE) in the brain of mice in the pentylenetetrazole (PTZ)-induced kindling model. It was found that during 16 days of kindling (PTZ 30 mg/kg daily) sodium valproate in a sub-effective dose of 150 mg/kg and digoxin 0.8 mg/kg (1/10 LD50) per se moderately effectively affected the course of the seizure syndrome in mice. Sodium valproate significantly (p<0.05) increased the seizure latency and decreased the number of days with convulsions, when digoxin significantly (p<0.05) decreased the number of animals with seizures. Digoxin clearly potentiated the effect of sodium valproate – against the background of their combination, seizures were absent. Celecoxib 4 mg/kg only tended to reduce the number of animals with convulsions. In the brains of untreated animals with the PTZ-kindling model, a clear shift of the cyclooxygenase pathway of the arachidonic acid cascade was revealed, indicating neuroinflammation: the level of COX-1 and especially COX-2, PGF2α, and TXB2 increased, while the content of PGE2 and PGI2 decreased compared to the vehicle control group (p<0.001). These violations occurred against the background of strong oxidative stress, as evidenced by an almost 18-fold increase in the content of cerebral 8-isoprostane (p<0.001), and were accompanied by massive destruction of neurons, the marker of which is a 2.7-fold increase in the content of NSE (p<0.001). The anti-inflammatory and antioxidant properties of digoxin, sodium valproate and especially their combinations have been shown. They are manifested in a significant decrease in the level of COX-1 and COX-2 (p<0.001), an increase in the content of PGE2 and PGI2 (p<0.001), a decrease in the level of PGF2α and TXB2, 8-isoprostane and NSE (p<0.001) compared to the positive control (PC). Despite its selectivity for COX-2, celecoxib had almost no effect on the level of this cerebral COX isoform, mainly inhibiting COX-1. Compared with PC, celecoxib did not have a significant effect on COX-2, PGI2, and NSE, significantly inferior to valproate (except for the effect on PGF2α), digoxin and its combination (except for the effect on COX-1), which corresponds to the absence of a pronounced impact on the course of kindling and indicates the limitation of its anti-inflammatory and antioxidant properties in the CNS without a neurocytoprotective effect. Predominant suppression of COX-2 indicates a moderate selectivity of the combination of valproate and digoxin to this enzyme, which is a predictor of a reduction in the risk of side effects against the background of long-term use. Complete normalization of the level of 8-isoprostane under the combination of valproate + digoxin indicates the most favorable effect on the course of cerebral oxidative stress, as well as the maximum decrease in the content of NSE (p<0.001) indicates the neurocytoprotective properties of this combination in the model of chronic epileptogenesis. The set of obtained results experimentally substantiates the expediency of using a combination of digoxin with classical anticonvulsants, in particular with sodium valproate, to increase the effectiveness of pharmacotherapy of epilepsy.