FAM134B介导的内质网自噬对小鼠脑缺血再灌注损伤的影响及其机制

Effect and mechanism of FAM134B-mediated ER-phagy on cerebral ischemia-reperfusion injury in mice

目的观察序列相似性家族134B(FAM134B)介导的内质网自噬对小鼠脑缺血再灌注损伤的影响并探讨其机制。方法雄性C57BL/6小鼠随机分为假手术组、模型组、FAM134B沉默组,每组6只。FAM134B沉默组脑定位注射FAM134B敲低慢病毒,假手术组、模型组正常饲养不转染慢病毒。转染7 d后,模型组、FAM134B沉默组采用线栓法建立大脑中动脉阻塞模型,假手术组仅分离大脑中动脉但不插线栓。再灌注24 h后,采用HE及Nissl染色观察各组脑组织损伤情况,免疫荧光法观察脑组织细胞凋亡情况,ELISA法检测脑组织氧化应激指标丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT),Western blotting法检测脑组织内质网自噬相关蛋白FAM134B、微管相关蛋白1轻链3B(LC3B)、Beclin-1、p62。结果假手术组脑组织细胞未见明显病理改变及凋亡细胞;模型组脑组织病理学形态异常,神经元形态不规则,细胞核固缩或消失,可见大量凋亡细胞;FAM134B沉默组较模型组脑组织病理损伤减轻,凋亡细胞减少。与假手术组比较,模型组脑组织MDA含量升高,SOD、CAT活性降低;与模型组比较,FAM134B沉默组MDA含量降低,SOD、CAT活性升高(P均<0.05)。与假手术组比较,模型组脑组织p62蛋白表达降低,FAM134B、LC3B、Beclin-1蛋白表达升高;与模型组比较,FAM134B沉默组脑组织p62蛋白表达升高,FAM134B、LC3B、Beclin-1蛋白表达降低(P均<0.05)。结论抑制FAM134B介导的内质网自噬可改善小鼠脑缺血再灌注损伤,其机制可能与减轻脑组织氧化应激以及抑制细胞凋亡有关。

Objective To observe the effects of ER-phagy mediated by family with sequence similarity 134 member B(FAM134B)on cerebral ischemia-reperfusion(IR)injury in mice and to explore its mechanism.Methods Male C57BL/6 mice were randomly divided into the sham operation group(Sham group),cerebral IR group(IR group),and cerebral IR group with silenced FAM134B group(IR+siFAM134B group),with 6 mice in each group.Mice in the IR+si‑FAM134B group received stereotactic injection in the brain of FAM134B silencing lentivirus,while mice in the Sham group and IR group were normally fed without lentivirus transfection.Seven days after transfection,mice in the IR group and IR+siFAM134B group were subjected to middle cerebral artery occlusion using thread occlusion method,while in the Sham group,we only isolated the middle cerebral artery without inserting the thread.After 24 h of reperfusion,HE and Nissl staining were used to observe brain damage in each group.Immunofluorescence was employed to observe apoptosis rate in the brain tissues,ELISA was used to detect the levels of oxidative stress indices[malondialdehyde(MDA),superoxide dismutase(SOD),and catalase(CAT)],and Western blotting was utilized to detect the ER-phagy-related proteins(FAM134B,LC3B,Beclin-1,and p62).Results In the Sham group,no obvious pathological changes and apoptotic cells were seen in neuronal cells.In the IR group,brain tissues exhibited abnormal pathology,with irregular neuronal morphology,shrunken or absent nuclei,and a large number of apoptotic cells.While the IR+siFAM134B group showed reduced pathological damage and fewer apoptotic cells in comparison with those of the IR group.Compared with the Shamgroup,the activity of SOD and CAT decreased,while MDA content increased in the IR group(all P<0.05).Compared with the IR group,the levels of SOD,CAT increased,whereas MDA content decreased in IR+siFAM134B group(all P<0.05).Compared with the Sham group,IR group had decreased level of P62 and increased levels of FAM134B,LC3B,and Beclin-1(all P<0.05).Compared with the IR group,the level of P62 increased,whereas FAM134B,LC3B and Beclin-1 decreased in the IR+siFAM134B group(all P<0.05).Conclusion Inhibiting ER-phagy mediated by FAM134B can alleviate the cerebral IR injury in mice,and the mechanism may be related to reducing oxidative stress and inhibiting apoptosis in the brain tissues.