灵芝酸A通过激活Nrf2/GPX4信号通路减轻七氟烷诱导的HT22细胞铁死亡

Ganoderic acid A reduces sevoflurane-induced ferroptosis of HT22 cells through activating the Nrf2/GPX4 signaling pathway

目的研究灵芝酸A(ganoderic acid A,GAA)对七氟烷诱导的HT22细胞铁死亡的影响和机制。方法采用CCK-8法检测不同浓度GAA对HT22细胞的增殖活性,筛选出合适的GAA处理浓度。将HT22细胞分成对照组、七氟烷诱导组(Sev组)、GAA-25μmol/L组、GAA-50μmol/L组、GAA-100μmol/L组。对照组细胞按照常规方法处理;其他组均利用七氟烷进行诱导处理,同时不同浓度GAA组给予相应浓度的GAA干预。采用Western blot检测氧化应激相关因子(Nrf2、GPX4)与铁死亡相关因子(ACSL4、SLC7A11)的蛋白表达。利用相应的试剂盒检测Fe^(2+)、ROS、MDA、GSH、4-HNE等因子的含量。结果根据CCK-8实验结果,选择对HT22细胞增殖活性无影响的25、50、100μmol/L GAA进行后续研究。与对照组比较,Sev组的HT22细胞存活率降低(P<0.05),Nrf2、GPX4、ACSL4、SLC7A11蛋白表达减少(P<0.05),Fe^(2+)、ROS、MDA、4-HNE含量升高(P<0.05),GSH含量降低(P<0.05)。与Sev组比较,随着CAA处理浓度的升高,HT22细胞存活率显著升高(P<0.05),Nrf2、GPX4、ACSL4、SLC7A11蛋白表达增多(P<0.05),Fe^(2+)、ROS、MDA、4-HNE含量降低(P<0.05),GSH含量升高(P<0.05)。结论GAA通过激活Nrf2/GPX4信号通路减轻七氟烷诱导的HT22细胞铁死亡,从而发挥神经保护作用。GAA可作为治疗七氟烷麻醉神经损伤的潜在药物。

Objective To study the effect and mechanism of ganoderic acid A(GAA)on the ferroptosis of HT22 cells induced by sevoflurane.Methods The proliferation activity of HT22 cells was detected by CCK-8 assay to selecte the appropriate concentration.HT22 cells were divided into control group,Sev group,and CAA-25μmol/L group,CAA-50μmol/L group and CAA-100μmol/L group.Cells in the control group were treated with conventional methods.The other groups were treated with sevoflurane induction,and the GAA groups with different concentrations were given corresponding concentrations of GAA intervention.Western blot was used to detect the protein expression of oxidative stress related factors(Nrf2,GPX4)and ferroptosis related factors(ACSL4,SLC7A11).The content of Fe^(2+),ROS,MDA,GSH,4-HNE was detected by using the corresponding concentration detection kits.Results According to the results of CCK-8 experiment,25,50,100μmol/L GAA which had no effect on the proliferation activity of HT22 cells were selected for follow-up study.Compared with control group,HT22 cells survival rate decreased(P<0.05),Nrf2,GPX4,ACSL4,SLC7A11 protein expression decreased(P<0.05),Fe^(2+) level and ROS,MDA,4-HNE content increased(P<0.05),and GSH content decreased in Sev group(P<0.05).Compared with Sev group,with the increase of CAA concentration,the survival rate of HT22 cells was increased(P<0.05),the protein expression of Nrf2,Gpx4,ACSL4,SLC7A11 increased(P<0.05),Fe^(2+) level and ROS,MDA,4-HNE content decreased(P<0.05),and the content of GSH increased(P<0.05).Conclusion GAA plays a neuroprotective role by activating Nrf2/GPX4 signaling pathway to reduce sevoflurane-induced ferroptosis in HT22 cells.GAA has great potential as a therapeutic agent for sevoflurane anesthetic nerve injury.