The Role Of ERK Signaling Pathway/Autophagy In SAH Rats Neurons And The Effects Of Mild Hypothermia Intervention

Objectives 1 To explore the relationship between ERK signaling pathway and autophagy in hippocampal neurons of rats with subarachnoid hemorrhage(Subarachnoid Hemorrhage,SAH)and the role of the latter in brain(early Injury,EBI)2.To explore the protective effect of hypothermia therapy on SAH and the possible mechanism.Methods 100 male SD rats(350g)were randomly assigned to five groups: sham operation group(group Sham),subarachnoid hemorrhage model group(group SAH),ERK signaling pathway inhibitor group(SAH+U0126 group),mild hypothermia treatment group(SAH+MH group)and mild hypothermia therapy combined with U0126 treatment group(SAH+ group U0126+MH)a total of 5 groups,20 rats in each group,each group Re number,random number and method according to the postoperative 24 h,72h divided into two sub groups,each sub group 10.The rat model of SAH was established by the method of intravascular puncture.The Sham group only received vascular surgery,but the blood vessels were not pierced.SAH+U0126 group was formed 30 min before modeling,and injected with U0126 solution(0.05mg/kg)through caudal vein.the rats of SAH+MH group use electric blanket with ice pack,alcohol bath 6h for the treatment of mild hypother mia,maintaining the anus temperature at 30+0.5?;SAH+U0126+MH group was formed 30 min before modeling,and injected with U0126 solution(0.05mg/k g)through caudal vein.After modeling success,give Mild hypothermia intervention immediately,method ibid.24 h ?72h after modeling success,rats were killed.The rats were sacrificed at the corresponding time point and the brain was taken.The brain edema index was detected by dry wet weig MH method,HE staining was used to observe the morphology of neurons in the hippocampal CA1 area and the quantitative analysis of the neurons.Immunohistochemical staining and Western-Blot were used to detect the expression of p-ERK1/2 protein and Beclin-1 and LC3-?in hip pocampus of rats.Results 1 Detection of water content in brain tissue: Compared with Sham group,SAH group were significantly higher in 24 h and 72 h after injury in brain edema index(P<0.05).Compared with SAH group,The cerebral edema index of U0126+SAH groupwas significantly increased(P<0.05),but decreased significantlyin SAH+MH group(P<0.05).Compared with the SAH+MH group,the brain edema index of SAH+MH+U0126 group was significantly increased(P<0.05);While compared with SAH group,SAH+MH+U0126 group have no significant differences in brain edema index(P>0.05).2 The result of morphological changes(HE staining):The cell of Sham groupvisible aligned neurons and nucleus positive round,clear nucleoli,patina indifferent,even visible axons in hippocampal CA1 region;The cell of SAH group neurons arranged in a relatively loose,hierarchical structure disorder and has been severely damaged,more nuclear pyknosis,nucleolus disappeared,and the surrounding the organization dissolved,showed vacuolization in hippocampal CA1 region;Compared with sham group,the nerve cell survival rate in hippocampus was decreased in SAH group at 24 h and 72h(P<0.05).Compared with SAH group,the nerve cell survival rate in hippocampus was further decreased in SAH+U0126 group(P<0.05).Compared with SAH group,the nerve cell survival rate was increased in SAH+MH group(P<0.05).3 Results of Beclin-1 and LC3 by immunohistochemistry and Western blott: Immunohistochemistry: immunohistochemistry: The immunohistochemical staining,were brown,mainly expressed in neuronal cel s in Sham group were rare ones;light staining in Sham group;Compared with the Sham group.,the SAH group had significant staining was enhanced,the positive cell number were increased(P<0.05);Compared with SAH group,but the staining weakenedin the SAH+U0126 group,the number of immunoreactive cells reducing(P<0.05);Comparison with the SAH group and SAH+MH group,the staining positive cel s deepermore in SAH+MH group(P<0.05);But Compared with SAH group,The immune positive cel s did not differ significantly in the SAH+U0126+MH group(P>0.05),While Compared with the SAH+MH group,the number of positive cel s decreased significantly(P<0.05).Western blotting: The expression level of Beclin-1 and LC3 is very less in the Sham group;Compared with Sham group,Its expression increased significa nt ly in SAH group at 24 h and 72h(P<0.05);Compared with SAH group,Its expression of SAH+U0126 group was significantly decreased(P<0.05);Compared with SAH group,Its expression of SAH+MH group was significantly up-regulated(P <0.05);Its protein expession in SAH + MH + U0126 group was significantly lower than that of SAH + MH group(P <0.05).4 p-ERK1/2 immunohistochemistry and Western blotting analysis results: Immunohistochemistry: The p-ERK1/2 protein expressed very weak in the Sham group;Compared with Sham group,the staining was increased and the number of immunoreac t ive cel s increased in SAH group at 24 h and 72 h,(P <0.05);Compared with SAH group,SAH + U0126 group cel s staining decreased and the number of immunoreactive cel s reduced(P <0.05),but the staining in SAH + MH group more deeper and more immunoreactive cel s(P <0.05);While the number of positive cel s in the immune not significantly in the SAH + U0126 + MH group have no different than SAH group,(P> 0.05).Western blotting: The expression level of p-ERK1/2 is very less in the Sham group;Compared with Sham group,Its expression increased significantly in SAH group at 24 h and 72h(P<0.05);Compared with SAH group,Its expression of SAH+U0126 group was significantly decreased(P<0.05);Compared with SAH group,Its expression of SAH+MH group was significantly upregulated(P <0.05);Its protein expession in SAH + MH + U0126 group was significa nt ly lower than that of SAH + MH group(P <0.05).Conclusion 1 After SAH the level of autophagy increased in EBI,which has neur oprotective effects.The activation of autophagy may be dependent on the ERK sign aling pathway.Autophagy down-regulated and the nerve injury aggravated when inhi bit ERK signaling pathway.2.Mild hypothermia therapy has neuroprotective effects,a nd the mechanism may relate to activating the ERK signaling pathway can increase the level of autophagy.