Effect Of Edaravone On SAH Rat Hippocampus JNK-Autophagy Signaling Pathway

Objectives: 1. To explore the effect of edaravone on Subarachnoid Hemorrhage(SAH);2. To explore the effect of edaravone on rat hippocampus SAH JNK- autophagy signaling pathway.Method: 192 clean healthy male SD rats(350~450g) were randomly assigned to four groups: the Sham group, SAH group, SAH JNK group, and Edaravone group. The method of intracranial arterial puncture was adopted to prepare the SAH model rats(the Sham group only received intracranial vascular surgery, but the blood vessels were not pierced);SP600125 group was formed 30 min before modeling, and stereotactic instrument was used to inject SP600125 solution to intracerebroventricular; the Edaravone group received edaravone treatment after successful modeling, and intraperitoneal interjection was implemented with the 5mg / kg dose, and then repeated administration was conducted once 12 hours later, until the rats died. Each large group was divided into four subgroups by the time point of 6h, 24 h, 72 h and 144 h. forelimb tension of rats in the 4 groups was compared; and shuttle box test was carried out to measure the passive avoidance latency(PAL) of rats in each group at all the time points and active avoidance reaction rate(AARR) and evaluate the changes the situation of the behavior of rats in each group on the whole. The changes in shape and volume of rats neuron in hippocampus in each group after HE staining were observed; the immunohistochemical staining method and WesternBlot method were used to detect the expression of p-JNK protein and autophagy markers Beclin-1 and LC3-? in the hippocampus of rats.Result: 1. Test on holding power: compared with the Sham group, the values of rats forelimb tension in the SAH group at each time point significantly decrease, in which the difference has statistical significance(P < 0.05); compared with the SAH group, the values of rats forelimb tension in the SAH group at each time point increase; and the values of rats forelimb tension in the Edaravone group at each time point increase, in which the difference has statistical significance(P<0.05). 2. Shuttle box test: compared with the Sham group, the results of AARR in the SAH group at each time point all decrease, and PAL largely increase; compared with the SAH group, the avoidance capacity of rats in SP600125 group and the times of avoiding electric shocks increase,and AARR is improved, while the PAL values largely decrease, in which the difference has statistical significance(P<0.05); AARR values in the Edaravone group at each time point increase, while the PAL time significantly is shortened. 3. Result of HE staining:rats in the Sham group have complete brain tissue structure, and neuronal cells in hippocampus have a large number, while there are no changes in shape. Compared with the Sham group, hippocampus neurons in SAH group at each time point arrange disorderly, the number distinctly decreases, and their shapes are mainly polygons, in which the difference has statistical significance(P < 0.05); Compared with the SAH group, cells near normal shapes in SP600125 group distinctly increase, in which the difference has statistical significance(P < 0.05); and neuron necrosis rate distinctly decreases, and the number of cells with normal shape begins to increase, in which the difference has statistical significance(P < 0.05). 4. Results of Beclin-1, LC3-?immunohistochemistry(IHC) and Western Blot: IHC: in Sham group, Beclin-1and LC3-?in hippocampus neuron cytoplasm have low expression, without changing over time, positive expression in SAH group is mainly located in the cytoplasm of neurons.Compared with Sham group, expression in SAH group at each time point is significantly up-regulated, increasing at 6h, peaking at 24 h and decreasing at 72 h and 144 h with time extension, in which the difference has statistical significance(P<0.05); Compared with SAH group, the expression in SP600125 group at each time point significantly decreases,increasing at 6h, peaking at 24 h and decreasing at 72 h, in which the difference has statistical significance(P < 0.05); in Edaravone group, the expression hippocampus neuron cytoplasm distinctly reduces, increasing at 6h, peaking at 24 h and decreasing at72 h, in which the difference has statistical significance(P<0.05). Result of Western-Blot:in Sham group, the expression level of Beclin-1 and LC3-?is extremely low, and does not change with time. Compared with Sham group, expression in SAH group has high level, increasing at 6h, peaking at 24 h and decreasing at 72 h, in which the difference has statistical significance(P<0.05); the expression level in SP600125 group at each time point also decreases, in which the difference has statistical significance(P < 0.05).Results of p-JNK immunohistochemistry(IHC) and Western Blot: IHC result: p-JNK positive cells mainly exist in nucleus, only with a few expressing in cytoplasm. The expression in Sham group is weak, while the expression level in SAH group significantly increases, increasing at 6h, peaking at 24 h and decreasing at 72 h. Compared with SAH group, the expression in SP600125 group at each time point all significantly decreases(P<0.05), increasing at 6h, peaking at 24 h and decreasing at 72 h, in which the difference has statistical significance(P < 0.05); the expression level in Edaravone group decreases(P < 0.05), increasing at 6h, peaking at 24 h and steadily reducing at 72 h, in which the difference has statistical significance(P<0.05). Result of Western-Blot: there is nearly no expression in Sham group; the expression level in SAH group distinctly increases; compared with SAH group, expression level in SP600125 group significantly decreases, in which the difference has statistical significance(P<0.05); and expression level in Edaravone group also significantly reduces, in which the difference has statistical significance(P<0.05).Conclusion: 1. Edaravone can restrain the expression of Beclin-1 and LC3-?in hippocampus neuronal cells after SAH, thus reducing the early brain injury after SAH. 2.Edaravone can restrain the excessive activation of hippocampus JNK signal after SAH,reduce the expression of Beclin-1 and LC3-?, thus properly reducing the protection of autophagy activation degree on hippocampus neuronal cells after SAH.