目的 探讨微小RNA（microRNA, miR）-30b通过调控泛素特异性肽酶14（ubiquitin specific peptidase 14, USP14）影响非PTEN诱导激酶1（PTEN-induced kinase 1, PINK1）/Parkin通路依赖性线粒体自噬在神经元氧糖剥夺/复氧（oxygen-glucose deprivation/reoxygenation, OGD/R）损伤中的作用机制，为新生儿缺氧缺血性脑病的治疗提供新思路。 方法 分离胎鼠皮层神经元并建立OGD/R模型。实验分为两部分：（1）将细胞随机分为对照组、OGD/R组、OGD/R+miR阴性对照（miR-NC）组、OGD/R+miR-30b模拟物（mimic）组、OGD/R+miR-30b mimic+过表达阴性对照组（oe-NC）组、OGD/R+miR-30b mimic+USP14过表达（oe-USP14）组；（2）另取细胞随机分为对照组、OGD/R组、OGD/R+miR-NC组、OGD/R+miR-30b mimic组、OGD/R+miR-30b mimic+siRNA阴性对照（si-NC）组、OGD/R+miR-30b mimic+siRNA敲降Parkin（si-Parkin）组、OGD/R+miR-30b mimic+siRNA敲降动力相关蛋白1（dynamin-related protein 1, Drp1）（si-Drp1）组。通过实时荧光定量PCR检测miR-30b、USP14 mRNA表达水平，CCK-8检测细胞活力，碘化丙啶（propidium iodide, PI）染色检测细胞死亡情况，Western blot法检测USP14、微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3, LC3）Ⅱ/LC3 Ⅰ、线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20, TOMM20）、PINK1、Parkin、Drp1蛋白表达水平，MitoTracker Green染色检测线粒体形态变化，MitoSOX Red荧光探针检测线粒体活性氧（reactive oxygen species, ROS）水平，AGO₂-RNA免疫共沉淀实验和双萤光素酶报告基因实验验证miR-30b与USP14的靶向结合关系。 结果 与OGD/R组相比，OGD/R组+miR-30b mimic组细胞活力、miR-30b表达、LC3 Ⅱ/LC3 Ⅰ比例、Drp1蛋白表达升高（P<0.05），PI阳性率、线粒体ROS水平、USP14 mRNA和蛋白表达、TOMM20蛋白表达、线粒体碎片化指数、线粒体体积降低（P<0.05）；两组PINK1、Parkin蛋白表达水平比较差异无统计学意义（P>0.05）。与OGD/R+miR-30b mimic+oe-NC组相比，OGD/R+miR-30b mimic+oe-USP14组细胞活力、miR-30b表达、LC3 Ⅱ/LC3 Ⅰ比例降低（P<0.05），PI阳性率、线粒体ROS水平、USP14 mRNA和蛋白表达、TOMM20蛋白表达、线粒体碎片化指数、线粒体体积升高（P<0.05）。miR-30b可靶向调节USP14的表达。与OGD/R+miR-30b mimic+si-NC与OGD/R+miR-30b mimic+si-Parkin组LC3 Ⅱ/LC3 Ⅰ比例、碎片化指数、线粒体平均体积比较差异无统计学意义（P>0.05）；与OGD/R+miR-30b mimic+si-NC组比较，OGD/R+ miR-30b mimic+si-Drp1组LC3 Ⅱ/LC3 Ⅰ比例降低，碎片化指数、线粒体平均体积升高（P<0.05）。 结论 miR-30b通过靶向抑制USP14，独立于PINK1/Parkin通路调控线粒体自噬，对氧糖剥夺/复氧神经元损伤发挥保护作用。

Objective To investigate how miR-30b regulates mitophagy independently of the PINK1/Parkin pathway by targeting ubiquitin specific peptidase 14 (USP14) in neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and to provide new insights for the treatment of neonatal hypoxic-ischemic encephalopathy. Methods Fetal rat cortical neurons were isolated and an OGD/R model was established. Experiments were conducted in two parts. In part 1, cells were randomized into control, OGD/R, OGD/R+microRNA (miR)-negative control (NC), OGD/R+miR-30b mimic, OGD/R+miR-30b mimic+oe-NC, and OGD/R+miR-30b mimic+oe-USP14 groups. In part 2, cells were randomized into control, OGD/R, OGD/R+miR-NC, OGD/R+miR-30b mimic, OGD/R+miR-30b mimic+si-NC, OGD/R+miR-30b mimic+si-Parkin, and OGD/R+miR-30b mimic+si-Drp1 (dynamin-related protein 1) groups. miR-30b and USP14 mRNA levels were measured by real-time quantitative PCR. Cell viability was assessed using the CCK-8 assay and cell death by propidium iodide staining. Protein levels of USP14, microtubule-associated protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, translocase of outer mitochondrial membrane 20 (TOMM20), PINK1, Parkin, and Drp1 were determined by Western blot. MitoTracker Green staining was used to evaluate mitochondrial morphology, and mitochondrial reactive oxygen species (ROS) were measured using the MitoSOX Red probe. AGO₂-RNA immunoprecipitation and dual-luciferase reporter assays were performed to validate the targeting relationship between miR-30b and USP14. Results Compared with the OGD/R group, the OGD/R+miR-30b mimic group showed higher cell viability, miR-30b expression, LC3 Ⅱ/LC3 Ⅰ ratio, and Drp1 protein expression (P<0.05), and lower PI positivity, mitochondrial ROS, USP14 mRNA and protein expression, TOMM20 protein expression, mitochondrial fragmentation index, and mitochondrial volume (P<0.05). PINK1 and Parkin protein levels did not differ significantly between these two groups (P>0.05). Compared with the OGD/R+miR-30b mimic+oe-NC group, the OGD/R+miR-30b mimic+oe-USP14 group exhibited reduced cell viability, miR-30b expression, and LC3 Ⅱ/LC3 Ⅰ ratio (P<0.05), and increased PI positivity, mitochondrial ROS, USP14 mRNA and protein expression, TOMM20 protein expression, mitochondrial fragmentation index, and mitochondrial volume (P<0.05). miR-30b was confirmed to target USP14. In addition, no significant differences were observed between the OGD/R+miR-30b mimic+si-NC and OGD/R+miR-30b mimic+si-Parkin groups in LC3 Ⅱ/LC3 Ⅰ ratio, fragmentation index, or average mitochondrial volume (P>0.05). Compared with the OGD/R+miR-30b mimic+si-NC group, the OGD/R+miR-30b mimic+si-Drp1 group showed a decreased LC3 Ⅱ/LC3 Ⅰ ratio and increased fragmentation index and average mitochondrial volume (P<0.05). Conclusions miR-30b regulates mitophagy by targeting USP14 independently of the PINK1/Parkin pathway, and confers protection against neuronal OGD/R injury.