Abstract:

Objective To discuss the protective effect of carnosine（CAR） against oxygen-glucose deprivation/reoxygenation（OGD/R）-induced astrocyte（AS） injury，and to clarify its possible mechanism. Methods The AS were divided into control group， model group （OGD/R group）， OGD/R+CAR group （CAR group）， and OGD/R+CAR+AMP-activated protein kinase （AMPK） activator AICAR group（CAR+AICAR group）. MTT assay and green cyanine staining method were used to detect the survival rates and green cyanine staining positive rates of the AS in various groups； Annexin Ⅴ-FITC/PI method and flow cytometry were used to detect the apoptotic rates of the AS in various groups； Western blotting method was used to detect the expression levels of AMPK， phosphorylated AMPK （p-AMPK）， mammalian target of rapamycin （mTOR）， phosphorylated mTOR （p-mTOR）， microtubule-associated protein light chain 3B（LC3B）， Beclin-1， and P62 proteins in the AS in various groups； immunofluorescence staining was used to observe the LC3B positive fluorescence intensities in the AS in various groups. Results Compared with control group， the survival rate and green cyanine staining positive rate of the AS in OGD/R group were decreased （P<0.01）， the apoptotic rate of the AS was increased （P<0.01）， the ratios of p-AMPK/AMPK and LC3BⅡ/LC3BⅠ and the expression level of Beclin-1 protein were increased （P<0.01）， and the ratio of p-mTOR/mTOR and the expression level of P62 protein were decreased （P<0.01）. Compared with OGD/R group， the survival rate and green cyanine staining positive rate of the AS in CAR group were increased （P<0.01）， the apoptotic rate of the AS was decreased （P<0.01）， the ratios of p-AMPK/AMPK and LC3B Ⅱ/LC3BⅠ and the expression level of Beclin-1 protein were decreased （P<0.01）， and the ratio of p-mTOR/mTOR and the expression level of P62 protein were increased （P<0.01）. Compared with CAR group， the survival rate and green cyanine staining positive rate of the AS in CAR+AICAR group were decreased （P<0.01）， the apoptotic rate of the AS was increased （P<0.01）， the ratios of p-AMPK/AMPK and LC3BⅡ/LC3BⅠ and the expression level of Beclin-1 protein were increased （P<0.01）， and the ratio of p-mTOR/mTOR and the expression level of P62 protein were decreased （P<0.01）. The LC3B immunofluorescence staining results were consistent with the Western blotting results. Conclusion CAR has the protective effect on injury of the AS induced by OGD/R， and its molecular mechanism may be related to the inhibition of the AMPK/mTOR signaling pathway， thereby inhibiting autophagy.

Key words: Carnosine, Autophagy, Astrocyte, Adenosine monophosphate activated protein kinase, Mammalian target of rapamycin