| Objective: To observe the effects of recombinant human erythropoietin(rh EPO) on dysfunction of behavior and neuronal apoptosis in rats induced by chronic cerebral ischemia(CCI), to explore the mechanism of rh EPO in rats after CCI.Methods: Sixty healthy male SD rats were randomly divided into sham-operated group, 2VO group after being ligated the bilateral common carotid arteries,2VO+rh EPO group, 2VO+rh EPO+AG490(the specific antagonist of JAK2) group, 2VO+AG490 group,(n=12). It was adopted to establish the chronic permanent cerebral ischemia models by the bilateral occlusion of common carotid arteries in these rats. Each group undergone via intraperitoneal injection AG490(5mg/kg) or DMSO(the solvent of AG490) was given normal saline or rh EPO(150U/μl) by intranasal delivery after 30 minutes. Weekly all the rats were accepted drugs according to the above method 3 days after operation. Morris water maze test was performed 24 hours after the last accepted drug to assess the change of spatial learning and memory ability. It was observed the morphology change of nerve cells by HE staining, detected the protein level of JAK2, p-JAK2,STAT3, p-STAT3, Bcl-2, Bax by immunnohistochemistry and the neuronal apoptosis by TUNEL staining and measured m RNA expression of Bcl-2, Bax by real-time quantitative RT-PCR.Results: 1.The results of behavior test, evaluating the learning capacity of rats to acquire space information and spatial memory ability.(1) spatial navigation trials demonstrated that the mean swimming speed of the rats had not changed with the increasing days and were not different among every group on the same day, that the mean escape latencies were significantly decreased with the increase of training days in each group( p<0.0001). Compared with sham-operated group, rats of 2VO group had longer escape latency(p<0.05). Rh EPO improved the spatial learning capacity and shortened searching theplatform time under the water in 2VO+ rh EPO group. However, AG490 can attenuate the effect on rats in 2VO+ rh EPO+AG490 group induced by rh EPO.(2) Results of spatial probe test, compared with sham-operated group, dwell time shortened in the target quardrant and frequency of crossing the platform reduced in 2VO group(p<0.05). Compared with 2VO group,there were longer time and higher frequency in 2VO+rh EPO group(p<0.05). While, there were shorter time of staying at the region and lower freguency of crossing platform in 2VO+rh EPO+AG490 group than in 2VO+rh EPO group(p<0.05).2.HE staining showed there were least neurons, most serious morphology change, and more irregular arrangement in rats after 2VO. In Sham-operated group, neurons were arranged orderly and intactly. But morphology change in 2VO+rh EPO group were slighter than in 2VO group. The morphology change among 2VO group, 2VO+rh EPO+AG490 group and 2VO+AG490 group was similar.3.The results of immunnohistochemistry, real-time quantitative RT-PCR.(1). Compared with the sham-operated group, the expression of P-JAK2 and P-STAT3 enhanced and the expression of Bcl-2 and Bax was up-regulation in the 2VO group(p<0.05).(2). Compared with the 2VO group, the expression of P-JAK2 and P-STAT3 increased and the expression of Bcl-2 was up-regulation, Bax was down-regulation in the 2VO+rh EPO group(p<0.05).(3).Compared with the 2VO+rh EPO group, the expression of P-JAK2 and P-STAT3 was attenuated and the expression of Bcl-2 was down-regulation, Bax was up-regulation in the 2VO+rh EPO+AG490 group(p<0.05).(4) The 2VO+ AG490 group had no difference with the 2VO group and the 2VO+rh EPO+AG490group in the expression of P-JAK2, P-STAT3, Bcl-2 and Bax(p>0.05).4.Compared with sham-operated group, the index of neuronal apoptosis increased in groups after 2VO, and there were no significant differences between all groups.Conclusions: Rh EPO can attenuate neuronal apoptosis in rats induced by CCI. It can activate JAK2/STAT3 pathway, with the Bcl-2 up-regulation and Bax down-regulation, which is the possible molecular mechanism of improving the cognitive function. |
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