Abstract:

Objective To study the effect of different concentrations of cystatin C (Cys C) pretreatment on apoptosis related proteins, the expression of heat shock protein 70(HSP70) and autophagy related proteins, the expression of microtubule-associated protein light chain-3(LC3) in rats following cerebral ischemia reperfusion injury. Methods The healthy sixty male SD rats were randomly divided into five groups: the sham operation group(sham), the cerebral ischemia reperfusion injury group(IR) and low concentration of Cys C group(Cys C1), middle concentration of Cys C group(Cys C2) and high concentration of Cys C group(Cys C3), 12 rats in each group. The model of cerebral ischemia reperfusion injury was established by a suture method. After ischemia for 2 hours and reperfusion 24 hours, the modified neurological severity scores were detected. Using Western blotting method to detect the expression of HSP70, LC3-Ⅱ/LC3-Ⅰprotein. The number of HSP70 and LC3 positive cells were detected by immunohistochemical SP method . The average absorbance of autophagy related protein LC3/neuronal nuclei (NeuN) in corticalneurons was detected by immunofluorescence double labeling assay. The structural changes of the neurons were observed under transmission electron microscope. Results Compared with the IR group, the mNSS score significantly reduced(P<0.05), and upregulated the expression of HSP70 in Cys C1, Cys C2 group(P<0.01). While the mNSS score obviously increased in Cys C3 group, there was no significantly different(P>0.05) between Cys C3 group and IR group; the expression of HSP70 decreased in the Cys C3 group(P<0.01); the expression of LC3-Ⅱ/LC3-Ⅰprotein in each concentration cystatin C group increased gradually(P<0.01). The average absorbance of LC3/NeuN in the each concentration cystatin C group increased gradually (P<0.01). The results of transmission electron microscope showed that the ultrastructure pathological changes of cerebral cortex neurons could alleviated in Cys C1 and Cys C2 group, there were many obvious autophagic lysosomes. While the ultrastructural damage of cerebral cortex neurons in Cys C3 group was very serious. Conclusion Within a certain concentration range, Cyst C has a protective effects on ischemia reperfusion injury neurons. Otherwise, excessive autophagy maybe leads to autophagic death.

Key words: Cerebral ischemia reperfusion, Autophagy, Heat shock protein 70, Microtubule-associated protein light chain-3, Western blotting, Immunofluorescence, Rat