Effects of acupuncture on Beclin-1 and Caspase-3 expression in hippocampal tissue of rats with cerebral ischemia-reperfusion injury: Observations at different time points ☆基于不同时间点观察针刺对脑缺血再灌注损伤大鼠海马组织Beclin-1及Caspase-3表达的影响

Objective

To observe the changes in the neurological score, infarct size, and autophagy- and apoptosis-related indicators following the acupuncture treatment of rats with cerebral ischemia-reperfusion injury (CIRI) in order to investigate the effects of acupuncture on autophagy and apoptosis in the hippocampal tissue of CIRI rats

Methods

In this study, 170 Sprague-Dawley rats were randomly assigned to the normal group (n=34), sham group (n=34), and model establishment group (n=102). A CIRI rat model was established in the model establishment group. The rats of the model establishment group were then randomly and equally allocated to the middle cerebral artery occlusion/reperfusion group (MCAO/R group), acupuncture group (AC group), and edaravone group (ED group) (n=34 each). Each group was further subdivided into the 24-h and 72-h subgroups (n=17 each). The rats of the MCAO/R group were immobilized for 30 min and administered with an intraperitoneal injection of 0.9% sodium chloride solution. The rats of the AC group were immobilized, and they received acupuncture at the“Dà zhuī (大椎 GV 14) ”,“Shuĭ gōu (水沟 GV 26 ) ”, and “Băi huì (百会 GV 20) ” and an intraperitoneal injection of 0.9% sodium chloride solution, with the acupuncture needles left in place for 30 min. The rats of the ED group were immobilized for 30 min and administered with an intraperitoneal injection of edaravone (5 mg/kg). All interventions were performed once every 12 h. Investigations of the neurological score, infarct size using triphenyl tetrazolium chloride staining, hippocampal Caspase-3 protein expression by immunohistochemical assay, hippocampal Beclin-1 expression by Western blotting, autophagosomes in the hippocampus by transmission electron microscopy, and apoptotic rate in the brain tissue of the ischemic side using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay were performed both before and after the interventions.

Results

Before intervention, the neurological score of the model establishment group was significantly lower than that of the normal and sham groups (P<0.01). After 24 h and 72 h, the neurological scores of the ED and AC groups were significantly increased (P<0.01), whereas the infarct size ratio were smaller (P<0.05 or P<0.01) and the apoptotic rates and Caspase-3 expression were lower (P<0.01) when compared with the MCAO/R group. The hippocampal Beclin-1 expression in the ED and AC groups was downregulated after 24 h of intervention (P<0.05) but upregulated after 72 h of intervention (P<0.05 and P<0.01, respectively). In the ED and AC groups, 72 h of intervention led to significant increases in the neurological score (P<0.01) and significant reductions in the apoptotic rate and Caspase-3 expression (P<0.05 or P<0.01), but no significant changes in the Beclin-1 expression (P>0.05) when compared with 24 h of intervention. Moreover, the AC group showed a significant decrease in the infarct size ratio (P<0.01). Electron microscopy revealed that after 24 h of intervention, only individual lysosome structures could be observed in the tissues of the AC and ED groups. After 72 h of intervention, co-existence of autophagosomes and lysosomes was observed in the ED group, whereas co-existence of lysosomes and autolysosomes was observed in the AC group.

Conclusion

Acupuncture significantly improved the neurological score and reduced the infarct size in the CIRI rats. The mechanisms of these effects could be attributed to the regulation of autophagy and inhibition of apoptosis in the hippocampal tissue as well as the duration of the intervention to a certain extent.