Mild hypothermia alleviates cerebral ischemia reperfusion injury via promoting neurons autophagy in mice / 中华急诊医学杂志

ABSTRACT

Objective To investigate the neurons autophagy and apoptosis after cerebral ischemia reperfusion (I/R) injury in mice,and to explore the effect of mild hypothermia on neurons autophagy and apoptosis.Methods The global cerebral ischemia-reperfusion injury model in C57 mice was established with carotid artery ligation method.Ninety-six C57 mice were randomly (random number) divided into 8 groups (n =12 in each group),namely control group (C0),sham group,normal body temperature group (NT,37 ℃) after I/R 6 h (C6 h),normal body temperature group after I/R 12 h (C12 h),normal body temperature group after I/R 72 h (C72 h),mild hypothermia group (MH,34 ℃) after I/R6 h (C6 h +MH),mild hypothermia group after I/R12 h (C12 h + MH),mild hypothermia group after I/R 72 h (C72 h + MH).The protein expressions of Sirt1,P-FoxO1,Rab7,P53 and autophagy related genes such as Beclin1,LC3 were detected by Western blot at given intervals.The LC3 granules were assayed by immunofluorescence.The neurons apoptosis was detected by TUNEL.The software of SPSS13.0 was used for statistical analysis.Measurement data was expressed with mean ± SD,and comparison between two groups was carried out with Student's t test,One-way ANOVA was used for comparisons among groups,and P ＜ 0.05 was considered statistically significant.Results The global cerebral ischemia-reperfusion injury model in C57 mice was established successfully with bilateral carotid arteries ligation method.Compared with the control group,the protein expressions of Sirt1,P-FoxO1,Rab7,Beclin1 and LC3 Ⅱ / Ⅰ were gradually reduced,especially at 12 h after I/R in NT group (P ＜ 0.05),while the expression of P53 was obviously increased (P ＜0.05).In MH group,the expressions of Sirt1,P-FoxO1,Rab7,Beclin1,LC3 Ⅱ / Ⅰ were higher than those in NT group (P ＜ 0.05).And the expression of P53 was lower than that in NT group (P ＜0.05).Immuno-fluorescence test showed that compared with the control group,the LC3 particles of neurons cells were decreased significantly in group C12 (at 12 h after I/R 6.0 ± 1.5 vs.18.1 ±2.5,P ＜0.05).Nevertheless,LC3 particles were increased in MH group compared with NT group (36.1 ± 4.5 vs.6.0 ± 1.5,P ＜ 0.05).The results of TUNEL test showed that compared with the control group,neurons cells apoptosis were significantly increased in C72 group (at 72 h after I/R,54.8％ ±7.5％ vs.5.5％ ±1.2％,P ＜ 0.05).However,compared with NT group,neurons apoptosis were decreased in MH group (28.8％ ±4.5％ vs.54.8％ ±7.5％,P＜0.05).Conclusions The neuron autophagy was significantly reduced and apoptosis was significantly increased after ischemia reperfusion injury (I/R) in mice.However,mild hypothermia could increase the expression of Sirt1,FoxO1,beclin1 and LC3,so as to promote neurons autophagy and reduce apoptosis,which would provide therapy target for neurons injury after hypoxia and provide soundly theoretical basis for mild hypothermia for clinical application.