Dingxian pill alleviates hippocampal neuronal apoptosis in epileptic mice through TNF-α/TNFR1 signaling pathway inhibition

Ethnopharmacological relevance: Dingxian Pill (DXP), a famous traditional Chinese medicine prescription, and has been widely proven to have positive therapeutic effects on "Xianzheng" (the name of epilepsy in ancient China). However, the anti-epileptic molecular mechanisms of DXP are not yet fully understood and remain to be further investigated. Aim of the study: To elucidate the molecular mechanism of DXP's improvement in epileptic neuronal loss, damage and apoptosis by regulating TNF-alpha/TNFR1 signaling pathway. Materials and methods: Sixty Kunming mice were randomly divided in 6 groups: control group (equal volume of normal saline), model group (180 mg kg-1 pilocarpine hydrochloride - used to establish the epilepsy animal model), carbamazepine group (30 mg kg- 1), and low, medium, and high-dose Dingxian Pill groups (4.08, 8.16, and 16.32 g kg-1, respectively - oral administration once daily for 2 weeks). Successful establishment of the epileptic mouse model was monitored with electroencephalography. Pathological changes in hippocampal tissue were analyzed with hematoxylin-eosin staining. Hippocampal neuronal apoptosis was analyzed with TUNEL staining. TNF-alpha, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein expression levels in hippocampal tissue were analyzed with real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot, respectively. Cleaved caspase-8 protein levels in hippocampal tissue were measured with immunohistochemistry and Western blot. Results: Compared to control, the model group showed an increase in continuous epileptic discharge waves on EEG, a damaged hippocampal neuron morphological structure, increased hippocampal neuronal apoptosis, and significantly increased TNF-alpha, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels, and increased caspase-8 cleavage (P < 0.05). Compared to the model group, the carbamazepine group as well as the low-, medium-, and high-dose Dingxian Pill groups showed decreased epileptic discharges on EEG, an obvious hippocampal neuron morphological structure restoration, varying degrees of attenuated hippocampal neuronal apoptosis, and significantly decreased TNF-alpha, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels as well as decreased caspase-8 cleavage (P < 0.05). Conclusions: Dingxian Pill exerts an anti-epileptic effect through inhibition of TNF-alpha/TNFR1 signaling pathwaymediated apoptosis in hippocampal neurons.