该研究基于线粒体铁蛋白(ferritin mitochondrial,FtMt)抑制铁死亡探讨藁本内酯(ligustilide,LIG)减轻氧糖剥夺/复糖复氧(oxygen and glucose-deprivation/reoxygenation,OGD/R)诱导小鼠海马神经元细胞(HT22)损伤的作用及机制.体外建立OGD/R诱导HT22细胞损伤的模型,HT22细胞随机分为正常组,模型组,LIG 5、10、20 μmol·L-1组,铁死亡抑制剂(ferrostatin-1,Fer-1,2 μmol·L-1)组.CCK-8法检测细胞活力,LDH试剂盒检测乳酸脱氢酶释放量,倒置显微镜观察HT22细胞形态,透射电镜观察线粒体的超微结构,化学发光法检测细胞内Fe2+含量.为进一步探究FtMt抑制铁死亡的机制,沉默HT22细胞的FtMt,并随机分为正常组、模型组、20 μmol·L-1LIG 组、si-NC 组、si-FtMt 组、si-FtMt+20 μmol·L-1LIG 组.免疫荧光和 Western blot 法检测FtMt的表达,化学发光法检测HT22细胞内NADPH/NADP+、GSH、MDA、ATP的含量,激光共聚焦观察mtROS荧光强度,流式细胞术检测细胞内Fe2+含量,Western blot法检测铁死亡相关蛋白Ferrtin、GPX4、ASCL4的表达.研究结果表明,与模型组相比,LIG可以显著提高HT22细胞存活率,改善损伤的HT22细胞形态及线粒体超微结构,降低细胞内Fe2+含量和降低促铁死亡蛋白ACSL4的表达,增加抗铁死亡蛋白Ferrtin、GPX4的表达.沉默FtMt后,LIG促进FtMt的表达;与si-FtMt组相比,LIG可以显著提高NADPH/NADP+、GSH含量,降低mtROS的荧光强度、MDA含量,提高ATP活性,降低HT22细胞内Fe2+含量,抑制促铁死亡蛋白ACSL4的表达,提高抗铁死亡蛋白Ferrtin、GPX4的表达.综上,LIG通过上调FtMt的表达改善线粒体功能抑制铁死亡,从而减轻OGD/R诱导HT22细胞损伤.
Mechanism of ligustilide in attenuating OGD/R-induced HT22 cell injury based on FtMt inhibition of ferroptosis
This study investigated the role and mechanism of ligustilide(LIG)in attenuating oxygen-glucose deprivation/reoxyge-nation(OGD/R)-induced damage to mouse hippocampal neuron cells(HT22)by inhibiting ferroptosis through mitochondrial ferritin(FtMt).An in vitro model of OGD/R-induced HT22 cell damage was established.HT22 cells were randomly divided into normal group,model group,LIG groups(5,10,and 20 μmol·L-1),and ferrostatin-1(Fer-1,2 μmol·L-1)group.Cell viability was mea-sured using the CCK-8 method,and lactate dehydrogenase(LDH)release was measured using an LDH assay kit.Cell morphology was observed under an inverted microscope,and mitochondrial ultrastructure was observed using transmission electron microscopy.Intracellular Fe2+content was detected using a chemiluminescence method.To further investigate the mechanism of FtMt inhibition of ferroptosis,FtMt in HT22 cells was silenced and divided into normal group,model group,LIG group(20 μmol·L-1),si-NC group,si-FtMt group,and si-FtMt+20 μmol·L-1 LIG group.Immunofluorescence and Western blot were used to detect FtMt expression.Chemiluminescence was used to measure the content of NADPH/NADP+,GSH,MDA,and ATP in HT22 cells.The mtROS fluorescence intensity was observed by laser confocal microscopy,and intracellular Fe2+content was measured by flow cytometry.The expression of ferroptosis-related proteins Ferrtin,GPX4,and ACSL4 was detected by Western blot.The results showed that compared with the model group,LIG significantly increased the survival rate of HT22 cells,improved the morphology of damaged HT22 cells and mitochondrial ultrastructure,decreased intracellular Fe2+content,and reduced the expression of the pro-ferroptosis protein ACSL4 while increasing the expression of anti-ferroptosis proteins Ferrtin and GPX4.After silencing FtMt,LIG promoted FtMt expression.Compared with the si-FtMt group,LIG significantly increased the content of NADPH/NADP+and GSH,reduced mtROS fluorescence intensity and MDA content,increased ATP activity,decreased intracellular Fe2+content,inhibited the expression of pro-ferroptosis protein ACSL4,and increased the expression of anti-ferroptosis proteins Ferrtin and GPX4.In summary,LIG improved mitochondrial function by upregula-ting FtMt expression to inhibit ferroptosis,thereby alleviating OGD/R-induced damage to HT22 cells.
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