关键词: FBXL4, 线粒体动力学, 心肺复苏, 脑缺血再灌注损伤, 氧化应激, 凋亡

Abstract:

Objective To explore the molecular mechanism by which F-box/leucine-rich repeat protein 4 （FBXL4） inhibits oxidative stress and apoptosis， and prevents cerebral ischemia-reperfusion injury （CIRI） induced by cardiopulmonary resuscitation （CPR） after cardiac arrest （CA） through regulating dynamin-related protein 1 （Drp1）-mediated mitochondrial dynamics. Methods A total of 48 male SD rats were randomly divided into 4 groups， with 6 rats in each group （n = 6）： Control group， Control + FBXL4 overexpression （OE-FBXL4） group， CA/CPR group， and CA/CPR + OE-FBXL4 group. Survival analysis was also conducted， with 6 cases in each group. The rats were sacrificed at 24 h after the return of spontaneous circulation （ROSC） for sampling. The neurological deficit score （NDS） and the 7-day survival rate were detected. The expressions of FBXL4 and Drp1 mRNA and proteins were determined by RT-qPCR and Western blot. The expressions of mitochondrial dynamics proteins were measured by Western blot. The interaction between FBXL4 and Drp1 was verified by the co-immunoprecipitation method. The pathology of brain tissues was examined by HE staining and Nissl staining. The ultrastructure of mitochondria was observed using a transmission electron microscope. The levels of malondialdehyde （MDA）， superoxide dismutase （SOD）， glutathione （GSH）， and catalase （CAT） were measured by a biochemical method to evaluate the oxidative stress level. The expressions of apoptosis-related proteins ［B cell lymphoma-2 associated X protein （Bax）， B cell lymphoma-2 （Bcl-2）， cysteine aspartase peptidases （Caspase）-3/8］ were measured by Western blot. Results （1） When compared to the Control group， the Neurological Deficit Score （NDS） in the CA/CPR group was significantly lower （P < 0.01）， and the 7-day survival rate was also reduced. In the brain tissues， the expressions of FBXL4 mRNA and protein were significantly down-regulated （all P < 0.01）， while the expressions of Drp1， Fis1， and MFF were significantly up-regulated （all P < 0.01）， and the expressions of OPA1， MFN1， and MFN2 were significantly down-regulated （all P < 0.01）. HE and Nissl staining results indicated that in the cortex and the CA1 and CA3 regions of the hippocampus， neurons were arranged in a disordered manner， with cell body atrophy， dissolution of Nissl bodies， and a decreased number of surviving neurons. Transmission electron microscopy （TEM） revealed mitochondrial swelling， vacuolization， cristae fracture， and fragmentation. The content of MDA was increased （P < 0.01）， whereas the activities of SOD， GSH， and CAT were decreased （all P < 0.01）. The Bax/Bcl-2 ratio was elevated （P < 0.01）， and the expressions of Caspase-3 and Caspase-8 were increased （both P < 0.01）. （2） In comparison with the CA/CPR group， the Neurological Deficit Score （NDS） was significantly elevated in the CA/CPR + OE-FBXL4 group （P < 0.01）， and the 7-day survival rate was also significantly increased. The expression of the FBXL4 protein was at a high level. The levels of Drp1， Fis1， and MFF proteins decreased （all P < 0.01）， while the levels of OPA1， MFN1， and MFN2 proteins increased （all P < 0.01）. HE and Nissl staining indicated an improvement in neuronal morphology， a reduction in the number of degenerated and necrotic cells， and an increase in the number of survived neurons. Transmission electron microscopy （TEM） revealed the recovery of the mitochondrial pipe-network structure and a clear and complete cristae structure. The content of MDA decreased （P < 0.01）， and the activities of SOD， GSH， and catalase （CAT） increased （all P < 0.01）. The Bax/Bcl-2 ratio decreased （P < 0.01）， and the expressions of Caspase-3 and Caspase-8 decreased （both P < 0.01）. （3） A Co-IP experiment confirmed a protein-protein interaction between FBXL4 and Drp1. The formation amount of the FBXL4-Drp1 complex in the Control + OE-FBXL4 group was significantly higher than that in the Control group （P < 0.01）. The formation amount of the FBXL4-Drp1 complex decreased in the CA/CPR group， and the binding amount was significantly restored in the CA/CPR + OE-FBXL4 group. Conclusions FBXL4 serves as a crucial protective regulator against brain injury following CA/CPR. The overexpression of FBXL4 can restore mitochondrial dynamic balance by targeting Drp1， thus suppressing oxidative stress and neuronal apoptosis. Moreover， the further up-regulation of the Nrf2/HO-1 pathways may be synergistically involved in the antioxidant protection provided by FBXL4.

Key words: FBXL4, mitochondrial dynamics, cardiopulmonary resuscitation, cerebral ischemia-reperfusion injury, oxidative stress, apoptosis