World Journal of Emergency Medicine ›› 2026, Vol. 17 ›› Issue (2): 113-125.doi: 10.5847/wjem.j.1920-8642.2026.026
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Bingling Yin1, Haiyang Guo1, Yu Shao2, Chongxiao Xu3, Yueli Zhao4, Ting Chen5, Xuan He4, Shan Sun6, Caoyuan Wu6, Guodong Lin4,5(
), Zhiguo Pan1,5,6()
Received:2025-05-02
Accepted:2025-09-29
Online:2026-03-17
Published:2026-03-01
Contact:
Zhiguo Pan, Email: pzgsubject@126.comBingling Yin, Haiyang Guo, Yu Shao, Chongxiao Xu, Yueli Zhao, Ting Chen, Xuan He, Shan Sun, Caoyuan Wu, Guodong Lin, Zhiguo Pan. Role of NRF2 in regulating oxidative stress and alleviating mitochondrial and endoplasmic reticulum structural damage in heatstroke-induced brain injury[J]. World Journal of Emergency Medicine, 2026, 17(2): 113-125.
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URL: http://wjem.com.cn/EN/10.5847/wjem.j.1920-8642.2026.026
Figure 1.
The experimental flowchart of this study. Con: control; R0, R3, and R6: 0, 3, and 6 h groups of rewarming following heat stress, respectively; HS: heat stress; TBHQ: tert-butylhydroquinone; KO: knockout. H&E: hematoxylin & eosin; CCK8: cell counting kit-8; ER: endoplasmic reticulum; MAM: mitochondrial-associated endoplasmic reticulum membranes; RT-qPCR: real-time quantitative polymerase chain reaction (PCR); MDA: malondialdehyde; SOD: superoxide dismutase; ROS: reactive oxygen species.
Figure 2
Changes in the NRF2 pathway and the effects of TBHQ pretreatment on the survival rate and pathological damage to hippocampal neurons in heatstroke mice. (A-B) RT-qPCR and western blot analyses of NRF2 pathway-related mRNA expression in the hippocampus of mice following TBHQ pretreatment; (C) Survival rate of heatstroke mice after TBHQ pretreatment; (D) Results of H&E staining in the CA3 region of the hippocampus in heatstroke mice after TBHQ pretreatment. The black arrows indicate the shrunken and deeply stained neurons; (E) Results of Nissl staining in the CA3 region of the hippocampus in heatstroke mice after TBHQ pretreatment. The black arrows indicate neurons lacking Nissl bodies. Data are expressed as means ± standard deviations. Data analysis was performed using ordinary one-way ANOVA, and multiple comparisons were conducted with Tukey’s multiple comparisons test. n = 8. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
Figure 3.
Changes in the NRF2 pathway in the hippocampus of NRF2 KO mice after heatstroke and their effects on the survival rate and pathological damage to hippocampal neurons. (A-B) RT-qPCR and western blot analyses of NRF2 pathway-related protein expression in the hippocampus of NRF2 KO mice; (C) Survival rate of heatstroke model mice after NRF2 KO; (D) H&E staining of the CA3 region of the hippocampus in heatstroke mice after NRF2 KO. The black arrows indicate the shrunken and deeply stained neurons; (E) Nissl staining of the CA3 region of the hippocampus in heatstroke model mice after NRF2 KO. The black arrows indicate neurons lacking Nissl bodies. Data are expressed as means ± standard deviations. Data analysis was performed using ordinary one-way ANOVA, and multiple comparisons were conducted with Tukey’s multiple comparisons test. n = 8. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
Figure 4.
The changes in neuronal MAMs in the in vivo and in vitro models of heatstroke brain injury subjected to TBHQ pretreatment. (A) Transmission electron microscopy image showing MAMs in hippocampal neurons of TBHQ pretreated mice; (B) Changes in contact distance and contact platform length of MAMs in hippocampal neurons of heatstroke mice after TBHQ pretreatment (contact platform length denotes the outer mitochondrial membrane segment within 30 nm of the endoplasmic reticulum); (C) Western blot and RT-qPCR analyses of MFN2 protein and mRNA expression, respectively, in the hippocampus of heatstroke mice after TBHQ pretreatment; (D) Transmission electron microscopy showing changes in the MAM structure of SH-SY5Y cells after TBHQ pretreatment, followed by heat stress; (E) The impact of heat stress on the contact distance and platform length of MAMs (platforms with contact distances less than 30 nm) in SH-SY5Y cells was investigated after TBHQ pretreatment; (F) Western blot and RT-qPCR analyses of MFN2 protein and mRNA expression changes in SH-SY5Y cells after TBHQ pretreatment followed by heat stress. Data are expressed as means ± standard deviations. Data analysis was performed using ordinary one-way ANOVA, and multiple comparisons were conducted with Tukey’s multiple comparisons test. n = 8 (in vivo) or 3 (in vitro). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Figure 5.
The changes in neuronal MAMs in the in vivo and in vitro models of heatstroke brain injury following NRF2 gene knockout (KO). (A) Transmission electron microscopy observation of MAMs in hippocampal neurons of NRF2 KO mice; (B) Changes in contact distance and contact platform length of MAMs in hippocampal neurons of heatstroke NRF2 KO mice (contact length where the distance between mitochondria and the endoplasmic reticulum is < 30 nm); (C) Western blot and RT-qPCR analyses of MFN2 protein and mRNA expression, respectively, in the hippocampus of heatstroke NRF2 KO mice; (D) Transmission electron microscopy findings of changes in the MAM structure of SH-SY5Y cells after NRF2 siRNA transfection, followed by heat stress; (E) The effects of a 3 h recovery following heat stress on the contact distance and platform length of MAMs in SH-SY5Y cells after NRF2 siRNA transfection. (F) Western blot and RT-qPCR analyses elucidating the changes in MFN2 expression in SH-SY5Y cells after heat stress following NRF2 siRNA transfection. Data are presented as the means ± standard deviations. Data analysis was performed using ordinary one-way ANOVA, and multiple comparisons were conducted with Tukey’s multiple comparisons test. n = 8 (in vivo) or 3 (in vitro). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
Figure 6.
Schematic of the molecular mechanism. Following heatstroke, neuronal cells experience reactive oxygen species (ROS) overload, leading to structural damage to the mitochondria and the ER, with MAMs likely playing a significant role. This destruction activates the NRF2 antioxidant signaling pathway. However, the activation of NRF2 signaling pathway is inadequate to mitigate the damage caused by heatstroke. Thus, TBHQ was administered to activate the NRF2 signaling pathway, providing antioxidant effect, restoring the MAM structure, and protecting neuronal cells against heat stress.
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