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).

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.

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).

Roxadustat regulates the balance between mitochondrial fission and fusion, reduces mitochondrial structural damage, oxidative stress and apoptosis, and alleviates heat stroke-induced brain injury.

To investigate the effect of microglial derived extracellular vesicles on neuronal damage in the context of heat stress.

After BV2 microglial cells were exposed to heat stress, the supernatant was collected and subjected to ultracentrifugation at different speeds to obtain large and small vesicles, respectively. Nano Particle Tracking and Zeta Potential Distribution Analyzer was used to measure and analyze the size distribution of the large vesicles and small vesicles. Western blotting was used to detect the expression of specific vesicle surface markers, TSG101, CD63 and flotillin-1. Microglial extracellular vesicles were labeled with PKH67 dye and then co-cultured with N2a cells to examine the uptake by capacity the neurons. After large and small vesicles derived from microglia after heat stress stimulation were co-cultured with N2a cells, respectively, CCK-8 assay, lactate dehydrogenase (LDH) assay, Trypan blue staining and TUNEL assay were employed to evaluate heat stress induced neuronal damage.

The small vesicles were in a particle size of 30~120 nm, and highly expressed TSG101 and CD63, whereas the large vesicles, in a size of 90~1 000 nm, highly expressed flotillin-1. The BV2-derived extracellular vesicles could be taken up by N2a cells and were proved to be involved in the modulation of N2a cell injury caused by heat stress. CCK-8 assay showed that both large and small vesicles of microglial cells inhibited the viability of N2a cells after heat exposure (P<0.05). The results of LDH assay, Trypan blue staining and TUNEL assay showed that both large (P<0.05) and small vesicles (P<0.01) significantly enhanced the LDH release, blue stain intensity and apoptosis of N2a cells after heat exposure, and the release, intensity and apoptosis were stronger in the cells treated with small vesicles than those group of large vesicles.

Microglia aggravate heat stress-induced neuronal damage through releasing extracellular vesicles.

To compare the differences in myocardial injury in rat models of exertional heat stroke（EHS）and classic heat stroke（CHS）in order to provide possible laboratory basis for clinical treatment of heat stroke.

A total of 80 SD rats were randomly divided into control group（n=10）, exercise group（EXC, n=10）, classic heat stroke group（CHS, n=30）and exertional heat stroke group（EHS, n=30）. The rats in the control and EXC groups were exposed to a condition of ambient temperature of（25±0.5）℃ and relative humidity of（50±5）%, and those of the EHS and CHS groups were placed in a simulated hot climate chamber with an ambient temperature of（39.0±0.5）℃ and a relative humidity of（50±5）%. The rats in the EHS and EXC groups were administered to treadmill exercise treatment under a running speed of 12 m/min and a slope of 0°, and rested for 2 min in every 8 min of exercise to monitor rectal temperature. When the core body temperature reached 42.3 ℃ or collapsed（tail pinching reflex disappeared）, the rats were taken to normal temperature and rewarmed. Echocardiography was used to detect the cardiac function, and serum myocardial enzymes（AST, LDH, CK-MB and CK）were detected by biochemical analyzer. HE staining was used to detect the pathological changes of rat myocardial tissue. The apoptosis of cardiomyocytes was detected by TUNEL staining. The expression levels of quinone oxidoreductase 1（Nrf2）, cleaved Caspase-3, anti-apoptotic protein（Bcl-2）, pro-apoptotic protein（Bax）and heme oxygenase-1（HO-1）were detected by Western blotting.

Compared with the CHS group, the EHS group had significantly shortened latent period（P<0.01）and reduced heat load in heat stroke（P<0.01）. The CHS group obtained obviously higher dehydration rate（P<0.01）but lower dehydration velocity（P<0.01）than the CHS group. There was no significant difference in rectal temperature between the 2 heat stroke groups（P>0.05）. Echocardiography indicated that statistically lower left ventricular ejection fraction（LVEF）（P<0.05）and higher AST and LDH levels（P<0.01）were observed in the EHS group than the CHS group. The pathological changes of acute myocardial injury including myocardial cell edema, disordered and broken muscle fibers, and inflammatory infiltration, were observed in both groups, but the pathological score of myocardial injury was notably higher in the EHS group than the CHS group（P<0.05）, so was the apoptotic rate of cardiomyocytes（P<0.05）. The protein levels of oxidative stress-related signaling pathway-related proteins were significantly higher in the EHS group than the CHS group.

High ambient temperature combined with exercise has a higher susceptibility to heat stroke. EHS-mediated abnormal cardiac dysfunction and myocardial apoptosis were more severe than CHS inducement, which may be related to the activation of oxidative stress-related signaling pathways.