RÉSUMÉ
BACKGROUND:After peripheral facial nerve injury,glial cell-derived neurotrophic factor(GDNF)can play a protective role in facial neurons.It has been found that GDNF can regulate the level of autophagy through mammalian target of rapamycin(mTOR),but it is unclear whether it can regulate facial neurons through the adenylate-activated protein kinase/Unc-51-like kinase 1(AMPK/ULK1)signaling pathway after facial nerve injury. OBJECTIVE:To establish a facial nerve injury model in Sprague-Dawley rats and explore the role of autophagy in facial nerve regeneration and the mechanism by which the GDNF/AMPK/ULK1 signaling pathway promotes facial nerve repair after injury. METHODS:Seventy-two Sprague-Dawley rats were randomly divided into sham group,model group and autophagy inhibitor 3-methyladenine(3-MA)group,with 24 rats in each group.Only the main trunk of the facial nerve was exposed in the sham group,while the remaining two groups were modeled for the compression injury of the facial nerve trunk.After successful modeling,the model group was given intraperitoneal injection of normal saline(15 mg/kg),and the 3-MA group was given intraperitoneal injection of 3-MA(15 mg/kg),both once daily for 7 days.The rats in each group were scored on the Simone 10-point behavioral scale at 1,4,7,14,21 and 28 days after surgery.Nissl staining was performed to observe the morphology and number of facial neuron cells at 7,14,21,and 28 days.The expression levels of p-AMPK,p-ULK1,Beclin1 and GDNF in the facial neuron tissues of rats were detected by western blot assay. RESULTS AND CONCLUSION:Behavioral scoring showed that the improvement of facial paralysis symptoms in the 3-MA group was worse and later than that in the model group(P<0.05).Nissl staining showed that the morphology and number of Nissl bodies in facial neurons in the 3-MA group recovered poorly and the number was less than that in the model group(P<0.05).Western blot detection results showed that the expression of p-AMPK and Beclin1 in the model group was higher than that in the 3-MA group and the sham group(P<0.05).The protein expression of p-ULK1 in the model group was lower than that in the 3-MA group and the sham group(P<0.05).To conclude,autophagy inhibitor delays nerve repair after facial nerve injury,which may be related to down-regulation of GDNF expression,inactivation of AMPK,and phosphorylation of ULK1,thereby inhibiting neuronal autophagy levels.
RÉSUMÉ
BACKGROUND: There are increasing reports about autophagy, but the relationship between the level of autophagy in neurons and the neuroprotection mechanism is not clear. OBJECTIVE: To investigate whether rapamycin, an mammalian target of rapacmycin (mTOR) autophagy pathway inhibitor, could activate autophagy by mediating the P70s6k and mTOR protein levels to protect spinal cord neurons in experimental autoimmune encephalomyelitis mice. METHODS: Fifty-four healthy female C57BL/6 mice were divided into three groups: control group, model group and treatment group, with 18 mice in each group. Mice in the model group and treatment group were injected with complete Freund’s adjuvant containing MOG35-55 and pertussis diluent for establishing models of experimental autoimmune encephalomyelitis. At the same time, the mice in the treatment group were given rapamycin (1 mg/kg per day), and those in the model and control groups were given the same amount of normal saline. The mice in the model and treatment were sacrificed at the peak of the onset, and the non-morbid mice, including those in the control group, were sacrificed after 4 weeks of feeding. The spinal cord tissue from each animal was taken to isolate the intumescentia lumbalis of the spinal cord. Nissl staining was used for pathological observation of the spinal cord tissue. Immunofluorescence double staining was used to observe the expression and co-localization of autophagy markers LC3 and NeuN in spinal cord tissue. Western blot was used to detect mTOR, P70S6K proteins and their phosphorylation levels in spinal cord tissue. RESULTS AND CONCLUSION: No mice in the control group had an attack, but those in the other groups developed experimental autoimmune encephalomyelitis to different extents. Compared with the model group, the treatment group had prolonged incubation time (P < 0.01), shortened progressive stage (P < 0.01), and decreased neurologic dysfunction score (P < 0.05). Compared with the control group, the model group had the significantly less number of Nissl bodies (P < 0.05), while the number of Nissl bodies in the treatment group was significantly higher than that in the model group, but still lower than that in the control group (P < 0.05). In the model group, LC3 was scattered in the spinal cord neurons and had no obvious dot-like aggregation, whereas in the treatment group, LC3 showed obvious dot-like aggregation, and its distribution was basically consistent with that of NeuN. The phosphorylation levels of mTOR and P70S6K proteins were highest in the model group, followed by the treatment group and control group in turn. To conclude, rapamycin might through inhibiting the phosphorylation levels of mTOR and P70S6K proteins activate the activity of autophagy to protect the spinal cord neurons in experimental autoimmune encephalomyelitis mice.
RÉSUMÉ
To explore the protective effect of nerve function of Buyang Huanwu Decoction on cerebral ischemia/reperfusion rats after the transplantation of neural stem cells (NSCs) . Methods Thread bolt method was used to establish middle cerebral artery occlusion model. Drug groups were given Buyang Huanwu Decoction (14. 8 g kg"1 d " 1) by gavage after the rats being sober. NSCs were transplanted to rat brain after making the model 24 hours later. Zea Longa neurobehavioral behavioral score was used to observe neural function defect, and TTC staining to detect the volume of cerebral infarction, and Nissl staining to detect Nissl body integrated optical density (IOD), and Immunohistochemical staining to detect expression of Bcl-2 and Bax. Results Compared with sham operation group, the nerve function defect appeared, and the volume of cerebral infarction increased significantly, the integral optical density of Nissl body was reduced and the ratio of Bcl-2 to Bax was reduced in model group (P < 0. 05) . Compared with model group, the nerve function defect was reduced, the volume of cerebral infarction was reduced, the integral optical density of Nissl body increased, and the ratio of Bcl-2 to Bax increased in BYHWD group, Transplant group and BYHWD + Transplant group (P < 0. 05). Compared with transplant group, the nerve function defect was reduced, the volume of cerebral infarction was reduced , the integrated optical density of Nissl body increased , and the ratio of Bcl-2 to Bax increased in BYHWD + Transplant group (P < 0. 05). Conclusions Buyang Huanwu Decoction can enhance the neuroprotective effect after NSCs transplantation in cerebral ischemia/reperfusion rats.