Effect Of ROCK2 Signaling Pathway On Autophagy In Hippocampus Of Rats After Subarachnoid Hemorrhage

Objectives The study of effect of ROCK2 signaling pathway on autophagy in the hippocampus of rats after subarachnoid hemorrhage.Methods 108 male Sprague-Dawley rats(350-450 g)of Specific Pathogens Free,after coding using random number table method assigned to three groups: Sham group,Subarachnoid hemorrhage group(SAH group),and SAH + ROCK2 inhibitor group(Drug group),every group was divided into three subgroups(6h,24 h and 72h).The Sham group only received intracranial vascular surgery,but the blood vessels were not pierced.Rat SAH model was made by carotid artery puncture method,The Drug group received inhibitor treatment after successful modeling,and intraperitoneal interjection was implemented with the 10 mg / kg dose.Shuttle box test was carried out to measure the time of escape response(ERT)and the number of escape response(ERN)in each group at all the time points,and to compare the changes of learning and memory of rats in each group.The shape and number of Rats nerve cells in hippocampus each group were studied by HE staining.The expression of ROCK2 protein and autophagy-related protein Beclin-1 and LC3-Ⅱin rat hippocampus were analyzed by the immunohistochemical staining method and Western Blot method.Results 1.Shuttle box test: The results of ERN in the SAH group at each time point all reduce and ERT largely extend compared with the Sham group(P<0.05);The ERN values increase but the ERT significantly is shortened in the Drug group at each time point compared with the SAH group(P<0.05).2.HE staining result: Rats in the Sham group more normal morphology of nerve cells,most of the normal morphology of nerve cells,arranged in neat,clear nucleus,and cytoplasmic uniforme in the hippocampal area.Compared with the Sham group,the hippocampal neurons of SAH group arranged disorder,deep purple nucleus and their shapes are spindle shrinkage and polygons,6h su bgroup can be seen nuclear pyknosis,nuclear fragmentation,the number of normal nerve cells decreased,24 h subgroup of nuclear pyknosis and nuclear fragmentation more,72 h subgroup nuclear pyknosis,nuclear fragmentation more obvious,the smallest number of normal nerve cells(P<0.05);Compared with the SAH group,nerve cells near normal shapes in Drug group normal nerve cells at each time point increased significantly,nuclear pyknosis and nuclear fragmentation decreased obviously(P<0.05).3.Results of Immunohistochemistry and Western Blot Analysis of Beclin-1,LC3-Ⅱ and ROCK2 Proteins.Immunohistochemistry: The expression of ROCK2,Beclin-1 and LC3-Ⅱprotein in the hippocampus of Sham group was positive and only in few cytoplasm at each subgroup.The SAH group: The expression of Beclin-1 and LC3-Ⅱat each time point is significantly up-regulated,the peak of positive expression increasing at 6h,the peak of positive expression at 24 h and decreasing at 72h(P<0.05);The expression of ROCK2 at each time point is significantly up-regulated,the peak of positive expression increasing at 6h,the peak of positive expression at 24 h and keeping peak at 72h(P<0.05);Compared with SAH group,the expression of Beclin-1 and LC3-Ⅱin Drug group at each time significantly increases,increasing at 6h,the expression reached the peak at 24 h,the expression began to decrease at 72 h and more than 6h time point,however,thenumber of ROCK2 protein positive cells at each time point was lower(P<0.05).Western Blot results: the expression level of Beclin-1,LC3-Ⅱand ROCK2 is extremely low in Sham group.Compared with Sham group,the expression of Beclin-1 and LC3-Ⅱ in Drug group at each time point significantly increases,increasing at 6h,the expression reached the peak at 24 h,the expression began to decrease at 72 h and more than 6h time point(P<0.05).The expression level of Beclin-1 and LC3-Ⅱ in Drug group at each time point also increases,however,the expression of ROCK2 protein at each time point was lower(P<0.05).Conclusion In the early stage of subarachnoid hemorrhage,ROCK2 signaling pathway is activated,which inhibits autophagy and decreases the expression of Beclin-1 and LC3-Ⅱ,damages neurons in the hippocampus,and impairs Learning and memory in rats.Therefore,proper inhibition of ROCK2 signaling pathway can protect neurons in hippocampus after SAH and improve learning and memory in rats.