Abstract:Objective To investigate the potential mechanism by which small ubiquitin - like modifier 2/3 protein（SUMO2/3）modification exerts brain protection by reducing excessive mitochondrial autophagy. Methods Eighty clean-grade healthy male SD rats, aged6-8weeks, withabodyweightof240-260g, wererandomlydividedinto4groups（n=20）：sham operation group（C group）, cerebral ischemia-reperfusion group（I/R group）, SUMO-specific protease 3（SENP3）inhibitor group（shSENP3 group）, and SENP3 inhibition control group（shNC group）. Except for group C, the other three groups were all prepared using the filament method to create a rat cerebral ischemia-reperfusion injury（CIRI）model. Rats in shSENP3 group and shNC group were injected into the lateral ventricle of recombinant adeno-associated virus（AAV）-sh-SENP3（AAV-sh-SENP3）and AAV-sh-NC respectively 3 weeks before modeling, while only the cervical vessels were exposed in C group. At 24 hours of reperfusion, the modified Neurological Deficit Severity Score（mNSS）was used to assess the severity of neurological deficits. Subsequently, the rats were anesthetized and decapitated to obtain the brain, and the brain slices were stained with 2, 3, 5-triphenyltetrazolium chloride（TTC）and the percentage of cerebral infarction volume was calculated. The ischemic penumbra tissue of the cerebral cortex was taken, and the relative positions and co-localization of mitochondria and lysosomes were observed by trans mission electron microscopy and confocal microscopy；the expression levels of mitochondrial autophagy proteins［microtubule-associated protein light chain 3（LC3）Ⅰ, LC3 Ⅱ , ubiquitin - binding protein P62（P62）, mitochondrial outer membrane translocation enzyme 20 homolog（TOM20）］, SENP3, total cytochrome C（Cyt C）, and cytoplasmic Cyt Cand the binding level of SUMO2/3 were detected by Western blot. Results Compared with C group and shNC group, the expression of SENP3 in the shSENP3 group was significantly decreased（P＜0.01）, and there was no statistically significant difference between C group and shNC group（P＞0.05）. Compared with C group, the other three groups showed increased mNSS scores and percentage of cerebral infarction volume, decreased expression of P62 and TOM20, upregulated expression of LC3Ⅱ/Ⅰ, total Cyt C, and cytoplasmic-Cyt C, along with increased SUMO2/3 conjugates levels（P＜0.01）；mitochondrial vacuolation was evident, the distance between mitochondria and lysosomes decreased, and colocalization of mitochondria and lysosomes increased. Compared with I/R group, shSENP3 group showed decreased mNSS and percentage of cerebral infarction volume, downregulated expression of LC3Ⅱ/Ⅰ, T-Cyt C, and cytoplasmic Cyt C, upregulated expression of P62 and TOM20, with further increased levels of SUMO2/3 conjugates（P＜0.01）；mitochondrial vacuolation weakened, the distance between mitochondria and lysosomes increased, and the co-localization of mitochondria and lysosomes decreased. There was no statistically significant difference in all indicators between the shNC group and the I/R group（P＞0.05）. Conclusion Inhibiting SENP3 can further increaseSUMO2/3 modification, reduce excessive mitochondrial autophagy during CIRI, and exert brain protection.