EFhd2和ATG16L1在二乙酰吗啡致神经元自噬中的作用研究

Study on the role of EFhd2 and ATG16L1 in neuronal autophagy induced by diacetylmorphine

摘要:

目的 探讨EF-Hand结构域家族成员2（EFhd2）和自噬相关蛋白16L1（ATG16L1）在二乙酰吗啡致神经元自噬中的作用。方法 12只雄性SD大鼠随机分为对照组和模型组，每组6只。模型组腹腔注射二乙酰吗啡，连续20 d，复制二乙酰吗啡成瘾模型；对照组注射生理盐水。取大鼠脑组织，蛋白质组学测序筛选差异蛋白，苏木精-伊红（HE）染色观察脑组织形态学改变，以及自噬蛋白表达水平变化。采用CCK-8法检测二乙酰吗啡干预细胞的最适浓度及细胞吸光度值；Western blotting检测EFhd2、ATG16L1及自噬相关蛋白P62、LC3蛋白水平；构建EFhd2敲低原代大鼠皮质神经元，Western blotting检测验证转染效率；GFP-RFP-LC3双荧光慢病毒检测神经元自噬通量；蛋白质作用位点预测，以及免疫共沉淀（CO-IP）验证EFhd2和ATG16L1相互作用。使用Peretinoin激活ATG16L1后检测细胞存活率及自噬水平。结果 蛋白质组学筛选出差异蛋白，在差异显著的蛋白中通过与自噬数据库交互确定研究主因子为EFhd2。HE染色结果显示，模型组相对于对照组大鼠脑组织染色较浅，细胞核数量减少。Western blotting检测结果显示，对照组P62蛋白相对表达量高于模型组（P <0.05），LC3Ⅱ/Ⅰ、EFhd2和ATG16L1蛋白相对表达量均低于模型组（P <0.05）。体外细胞实验显示，二乙酰吗啡干预神经元细胞后，与空载病毒对照组比较，DA组细胞吸光度值降低（P <0.05），自噬相关蛋白相对表达量升高（P <0.05）；而敲低EFhd2后使用二乙酰吗啡干预后，与DA组比较，shEFhd2+DA组细胞吸光度值升高（P <0.05），自噬蛋白相对表达量下降（P <0.05），自噬水平降低（P <0.05）。蛋白质相互作用预测及CO-IP结果显示，EFhd2和ATG16L1存在相互作用。激活ATG16L1后，与shEFhd2+DA组比较，shEFhd2+Peretinoin+DA组细胞吸光度值降低（P <0.05），自噬水平升高（P <0.05）。结论 EFhd2、ATG16L1参与了二乙酰吗啡致神经元自噬过程，干预EFhd2表达可降低二乙酰吗啡致神经元自噬水平，为神经系统疾病防治提供新靶点。

Abstract:

Objective To explore the roles of EF-Hand domain family member 2 (EFhd2) and Autophagy-related 16-like 1 (ATG16L1) in diacetylmorphine-induced neuronal autophagy.Methods Twelve male SD rats were randomly divided into a control group and a model group, with six rats in each group. The model group was intraperitoneally injected with diacetylmorphine for 20 consecutive days to establish a diacetylmorphine addiction model, while the control group was injected with saline. Rat brain tissues were collected for proteomic sequencing to screen differential proteins; HE staining was used to observe morphological changes in brain tissue; and changes in autophagy protein expression levels were assessed. CCK-8 was used to determine the optimal concentration and changes in cell viability following diacetylmorphine intervention; Western blotting was used to detect changes in the levels of EFhd2, ATG16L1, and autophagy-related proteicontrolP62 and LC3; primary rat cortical neurocontrolwith EFhd2 knockdown were prepared and transfection efficiency was verified by Western blot; GFP-RFP-LC3 dual-fluorescence lentivirus was used to detect neuronal autophagic flux; the protein interaction sites were predicted and EFhd2-ATG16L1 interaction was validated by CO-IP. After activating ATG16L1 with Peretinoin, cell survival rate and autophagy levels were measured.Results Proteomics identified differential proteins, and among the significantly different proteins, EFhd2 was determined to be the primary research factor through interaction with the autophagy database. HE staining results showed that the brain tissue of the model group showed weaker staining compared with the control group, with a reduced number of nuclei. Western blot results indicated significant differences in the levels of P62, LC3Ⅱ/Ⅰ, EFhd2, and ATG16L1 between the controlgroup and model group (P < 0.05); the control group had higher P62 levels than the model group (P < 0.05), while LC3Ⅱ/Ⅰ, EFhd2, and ATG16L1 levels were lower than those in the model group. In vitro cell experiments demonstrated that after diacetylmorphine intervention on neuronal cells, variance analysis showed statistically significant differences in cell viability and autophagy-related protein expression, with cell viability markedly decreased compared with the NC group (P < 0.05) and autophagy-related protein expression increased compared with the NC group (P < 0.05). Following EFhd2 knockdown with the use of diacetylmorphine intervention (shEFhd2 DA group), variance analysis indicated significant differences in cell viability and autophagy protein levels, with cell viability significantly higher than the diacetylmorphine treatment group (DA group) (P < 0.05) and autophagy protein expression lower compared with the DA group (P < 0.05), indicating reduced autophagy levels relative to the DA group. Protein interaction prediction and CO-IP results showed an interaction between EFhd2 and ATG16L1. After activation of ATG16L1, variance analysis of cell viability showed statistically significant differences, with cell viability reduced and autophagy levels increased relative to the shEFhd2 DA group (P < 0.05).Conclusion EFhd2 and ATG16L1 are involved in the neuronal autophagy process induced by diacetylmorphine. Modulating EFhd2 expression can reduce the level of diacetylmorphine-induced neuronal autophagy, providing a new target for the prevention and treatment of neurological diseases.