Effects of Tasimelteon Treatment on Traumatic Brain Injury Through NRF-2/HO-1 and RIPK1/RIPK3/MLKL Pathways in Rats

Abstract

Secondary brain damageafter traumatic brain injury (TBI) involves oxidative stress, neuroinflammation, apoptosis, and necroptosis and can be reversed by understanding these molecular pathways. The objective of this study was to examine the impact of tasimelteon (Tasi) administration on brain injury through the nuclear factor erythroid 2-related factor 2 (NRF-2)/heme oxygenase-1 (HO-1) and receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like (MLKL) pathways in rats with TBI. Thirty-two male Wistar albino rats weighing 300-350 g were randomly divided into four groups: the control group, trauma group, Tasi-1 group (trauma + 1 mg/kg Tasi intraperitoneally), and Tasi-10 group (trauma + 10 mg/kg Tasi intraperitoneally). At the end of the experimental phase, after sacrifice, blood samples and brain tissue were collected for biochemical, histopathological, immunohistochemical, and genetic analyses. Tasi increased the total antioxidant status and decreased the total oxidant status and oxidative stress index. In addition, Tasi caused histopathological changes characterized by a markedly reduced hemorrhage area in the Tasi-1 group. Normal brain and meningeal structure was observed in rats in the Tasi-10 group. Immunohistochemical analysis indicated that Tasi also decreased the expression of interferon-gamma, caspase-3, and tumor necrosis factor-alpha in the brain tissue. Although NRF-2 and HO-1 expression decreased, RIPK1/RIPK3/MLKL gene expression increased due to trauma. However, Tasi treatment reversed all these findings. Tasi protected against brain injury through the NRF-2/HO-1 and RIPK1/RIPK3/MLKL pathways in rats with TBI.

Keywords: NRF-2/HO-1; Neuroprotection; RIPK1/RIPK3/MLKL; Tasimelteon; Traumatic brain injury.

Fig. 1

Oxidative stress parameters of brain tissues. Tasi-1, Trauma + Tasi (1 mg/kg); Tasi-10, Trauma + Tasi (10 mg/kg); TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index. Values are presented as means ± standard deviation. A one-way ANOVA test was used. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig. 2

Histopathological appearance and statistical analysis of brains between the groups. A Mild hyperemia (arrowhead) and slight hemorrhage (arrow) in meningeal vessels in the control group. B Marked hyperemia (arrowhead) and hemorrhage foci (arrow) in the brain of a rat in the trauma group. C Markedly reduced hemorrhage area (arrowhead) in the Tasi-1 group. D Normal brain and meningeal structure in a rat in the Tasi-10 group, HE, scale bars = 50 µm. Values are presented as means ± standard deviation. A one-way ANOVA test was used. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig. 3

Expression and statistical analysis of IFN-ẟ (top row), Cas-3 (middle row), and TNF-α (bottom row) in the brain. (A) Negative IFN-ẟ, Cas-3, and TNF-α expression in the control group. (B) Significant increase in IFN-ẟ, Cas-3, and TNF-α expressions (arrows) in the trauma group. (C) Marked decrease in IFN-ẟ, Cas-3, and TNF-α expressions in the trauma and Tasi groups. (D) Negative IFN-ẟ, Cas-3, and TNF-α expressions in the Tasi groups, streptavidin–biotin peroxidase method, scale bars = 20 µm. Values are presented as means ± standard deviation. A one-way ANOVA test was used. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig. 4

The expression levels of RIPK1, RIPK3, MLKL, NRF-2, and HO-1 in brain tissues. NRF-2, nuclear factor E2-related factor 2; HO-1, heme oxygenase 1; RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3; MLKL, mixed lineage kinase domain-like pseudokinase. Values are presented as means ± standard deviation. A one-way ANOVA test was used. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001