Roxadustat attenuates brain injury in mice with heat stroke by regulating mitochondrial fission and fusion

Abstract

Objective To explore the protective effect and underlying mechanism of roxadustat (FG-4592), hypoxia-inducible factor-α (HIF-α) prolyl hydroxylase inhibitor, on brain injury caused by heat stroke (HS). Methods A total of 140 male C57BL/6J mice (6~8 weeks old, weighing 18~22 g) were subjected, and 40 of them were randomly divided into HS group, and low-, medium- and high-dose roxadustat groups (LD, MD and HD groups, 5, 10 and 20 mg/kg), with 10 mice in each group. The 24-hour survival rate was observed to determine the optimal dosage of roxadustat after modeling. Additionally, the remaining 100 mice were randomly allocated to normal control (Control) group, roxadustat (FG-4592) group, HS group, and roxadustat+HS (FG-4592+HS) group, with 25 mice in each. Heat shock was inflicted to establish mouse model of HS. Modified neurological severity score (mNSS) was used to assess neurological function. HE staining of brain sections was performed to examine pathological damage, and Fluoro-Jade C staining was applied to observe neuronal degeneration. The activity of total superoxide dismutase (SOD) and content of malondialdehyde (MDA) in brain tissue were measured to assess oxidative stress. Transmission electron microscopy was employed to visualize mitochondrial damage. Western blotting was performed to assess the protein levels of Caspase-3, Cleaved Caspase-3, Mfn1, Mfn2, Opa1, Fis1, HIF-1α, HO-1 and p-Drp1(Ser616)/Drp1 ratio in the cerebral cortex. Results Compared to the HS group, FG-4592 significantly improved the survival rate of HS mice within 24 h, with the MD group showing the highest survival rate. Compared to the Control group, the HS group showed an increase in mNSS score (P < 0.05), an elevation in the MDA content in the cerebral cortex (P < 0.05), and a decrease in total SOD activity in the cerebral cortex (P < 0.05); HE staining revealed pathological damage in the cerebral cortex, and Fluoro-Jade C staining displayed obvious neuronal degeneration in the cerebral cortex; Electron microscopy revealed obvious mitochondrial structural damage in the cerebral cortex tissue; The protein expression of Caspase-3, Cleaved Caspase-3, Fis1, HIF-1α, HO-1 and p-Drp1(Ser616)/Drp1 ratio was increased (P < 0.05), while that of Mfn1, Mfn2, and Opa1 was decreased (P < 0.05). Pretreatment with FG-4592 significantly reduced the mNSS score in HS mice (P < 0.05), decreased MDA content (P < 0.05), and enhanced total SOD activity (P < 0.05). Additionally, FG-4592 pretreatment improved pathological damage in the cerebral cortex, reduced neuronal degeneration, and mitigated mitochondrial structural damage. Furthermore, it decreased the protein levels of Caspase-3, Cleaved Caspase-3, Fis1 and p-Drp1(Ser616)/Drp1 ratio (P < 0.05), while increased the levels of Mfn1, Mfn2, Opa1, HIF-1α, and HO-1 (P < 0.05). Conclusion Roxadustat regulates the balance between mitochondrial fission and fusion, reduces mitochondrial structural damage, oxidative stress and apoptosis, and alleviates heat stroke-induced brain injury.

Keywords

• roxadustat
• heat stroke
• oxidative stress
• mitochondria
• hypoxia-inducible factor-1α