Nrf2缺失影响甲基苯丙胺致前额叶皮质ASIC表达及神经元损伤OA

OBJECTIVE To investigate the effect of nuclear factor erythroid 2-related factor 2(Nrf2)deficiency on methamphetamine(METH)-induced changes in expressions of acid-sensing ion channel 1α(ASIC1α)and ASIC2 and on neuronal injury in the prefrontal cortex.METHODS ① Immunofluores-cence was used to detect Nrf2 and ASIC1α expressions in primary cortical neurons after 24 hours of treatment with METH at 1 mmol·L-1.An Nrf2 knockdown model was established in the mouse hippo-campal neuronal cell line HT22 by using short hairpin RNA targeting Nrf2(sh-Nrf2).After 24 hours of treatment with METH at 1 mmol·L-1,ASIC1α and ASIC2 protein expressions were detected by Western blotting.② The genotypes of mice were identified using a genotyping kit before male C57BL/6J wild-type(WT)and Nrf2-knockout(Nrf2-KO)mice were randomly assigned to a control group and a METH group.A repeated METH exposure model was established using an escalating-dose regimen in which METH and physiological saline were administered on alternating days.Scores of conditioned place preference(CPP)and rates of spontaneous alternation were determined in mice using the CPP test and Y-maze test,respectively.Double immunofluorescence staining was used to examine the co-expres-sion of ASIC1α+/vesicular glutamate transporter 1(VGLUT-1)+and ASIC2+/VGLUT-1+in VGLUT-1+neurons.HE staining and Nissl staining were performed to assess morphological injury to neurons.In WT mice,Western blotting was used to determine the protein expressions of total Nrf2(T-Nrf2),nuclear Nrf2(N-Nrf2),ASIC1α and ASIC2 in the prefrontal cortex while Western blotting and immunohistochemistry were adopted to determine ASIC1α and ASIC2 expressions in the prefrontal cortex of WT and Nrf2-KO mice.RESULTS ① After 24 hours of treatment with METH,Nrf2 and ASIC1α expressions were signifi-cantly increased in primary cortical neurons.Compared with the sh-NC-METH group,ASIC1α and ASIC2 protein expressions were significantly decreased in HT22 cells in the sh-Nrf2-METH group.② Behavioral results showed that CPP scores were increased in the post-test after METH exposure,accompanied by a decrease in spontaneous alternation in the Y-maze test.Compared with the WT-METH group,spontaneous alternation kept decreasing in the Nrf2-KO-METH group.Compared with the WT-control group,the proportions of ASIC1α+/VGLUT-1+and ASIC2+/VGLUT-1+neurons were significantly increased in the WT-METH group.HE staining and Nissl staining suggested that METH expo-sure induced neuronal morphological injury in the prefrontal cortex,characterized by neuronal shrinkage,nuclear hyperchromasia and an increased percentage of shrunken neurons.Neuronal injury was aggra-vated in the Nrf2-KO-METH group compared with the WT-METH group.Compared with the WT-control group,protein expressions of T-Nrf2,N-Nrf2,ASIC1α and ASIC2 in the prefrontal cortex were signif-icantly increased in the WT-METH group.Compared with the WT-METH group,ASIC1α and ASIC2 protein expressions and percentages of positive staining areas in the prefrontal cortex were signif-icantly decreased in the Nrf2-KO-METH group.CONCLUSION METH can activate Nrf2,upregulate ASIC1α and ASIC2,and cause neuronal injury in the prefrontal cortex.Nrf2 deficiency attenuates the methamphetamine-induced increase in ASIC1α and ASIC2,but aggravates neuronal injury and cognitive dysfunction in the prefrontal cortex.