Intranasal Insulin Ameliorates Neurological Impairment Through AKT/GSK3β Signaling Pathway After Intracerebral Hemorrhage In Mice

Background:Intracerebral hemorrhage is a devastating subtype of stroke with high morbidity,mortality,disability rate and recurrence rate,which can cause severe neurological damage.At present,the effective treatments to reduce mortality and improve prognosis are still lacking.Therefore,it is imperative to explore effective methods to reduce neurological impairment after intracerebral hemorrhage.The available evidences suggest that peripheral insulin can enter the central nervous system and bind to insulin receptors which are widely distributed in the central nervous system and activate the corresponding insulin signaling pathways to exert a variety of peripheral and central biological effects.The AKT/GSK3β is an important signaling pathway which plays a key role in controlling cell survival and degeneration and apoptosis.The insulin that administrated through nose can rapidly reach a wide range of brain regions through the nasal mucosa,olfactory nerve and trigeminal nerve space to increase insulin content.Studies showed that intranasal insulin exert neuroprotective effects through the AKT/GSK3P signaling pathway in Alzheimer’s disease and ischemic stroke,however its role in intracerebral hemorrhage has not been reported.Objective:1)To investigate whether intranasal insulin has a neuroprotective effect on intracerebral hemorrhage in mice;2)To probe into whether its neuroprotective effect is related to AKT/GSK3β signaling pathway.Methods:1)Healthy male wild-type C57 mice at 6-8 weeks were randomly divided into 5 groups(n=6 per group).2)An autologous blood intracerebral hemorrhage model was established by injecting autologous blood from the mouse tail into the striatum,and the sham group was injected with an equal volume of normal saline.3)Experimental intracerebral hemorrhage mice models were subdivided into control and experimental groups,in which the control group was intranasally instilled with normal saline twice a day,the experimental group was intranasally instilled with insulin in dose of 0.5 IU,1 IU or 2 IU twice a day.The survival rate and neurological deficit score of mice in each group at 24 hours or 72 hours after operation were evaluated to assess the effect of intranasal insulin on the prognosis of intracerebral hemorrhage;5)The brain water content,hematoma volume and blood-brain barrier permeability of each group were detected at 24 hours or 72 hours after operation to assess the effect of intranasal insulin on neuropathological injury after intracerebral hemorrhage.6),The effect of intranasal insulin on neuronal degeneration damage after intracerebral hemorrhage was detected by FJB staining.7)The expression levels of AKT,GSK3β,p-AKT(ser473),and p-GSK3β(ser9)in perihematomal tissues were further measured by western blotting.Results:1)Compared with the saline-treatment group,1 IU intranasal insulin treatment significantly decreased the mortality and neurological deficit scores at 24 hours and 72 hours after surgery(P<0.05).2)Compared with the saline-treatment group,the brain edema,hematoma volume and blood-brain barrier permeability in the 1 IU intranasal insulin treatment group were significantly lower(P<0.05).3)In the intranasal insulin treatment group,the degenerated neurons in the perihematomal tissue after intracerebral hemorrhage were significantly reduced(P<0.01).4)Western blot assay suggested that the expression of p-AKT(ser473)and p-GSK3β(ser9)protein in the perihematomal tissue were significantly increased in the intranasal insulin treatment group(P<0.01).While,there was no significant difference in mice treated with 0.5IU and 2IU intranasal insulin compared with the control group in all indicators.Conclusion:1)Intranasal insulin can reduce the mortality and neurological impairment induced by intracerebral hemorrhage in mice.2)Intranasal insulin can reduce the hematoma volume,brain edema and the blood-brain barrier permeability of experimental intracerebral hemorrhage mice.3)Intranasal insulin can reduce the degeneration damage of neurons induced by intracerebral hemorrhage.4)The neuroprotective effect of intranasal insulin on intracerebral hemorrhage may be related to the regulation of AKT/GSK3β signaling pathway inhibition of GSK3β.