Role of autophagy in survival of hypoxia-preconditioned BMSCs in damaged tissues resulting from spinal cord ischemia-reperfusion injury in rats: in vitro and in vivo experiments
10.3760/cma.j.issn.0254-1416.2015.11.023
- VernacularTitle:自噬在低氧预处理BMSC在大鼠脊髓缺血再灌注损伤组织中存活的作用:离体和在体实验
- Author:
Liping WANG
;
Guozhong CHEN
;
Xiaoming DENG
- Publication Type:Journal Article
- Keywords:
Autophagy;
Cell hypoxia;
Ischemic preconditioning;
Mesenchymal stem cell transplantation;
Spinal cord;
Reperfusion injury
- From:
Chinese Journal of Anesthesiology
2015;35(11):1370-1376
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the role of autophagy in survival of hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) in damaged tissues resulting from spinal cord ischemiareperfusion (I/R) injury in rats.Methods In vitro experiment Rat BMSCs were seeded in 12-well plates at a density of 1× 106 cells/ml (1 ml/well) , and randomly divided into 6 groups (n =30 wells each) : control group (group C) , normoxia-incubated group (group N) , hypoxic preconditioning (HP) group (group H), HP + AMP-activated protein kinase (AMPK) inhibitor Compound C group (group HC), HP + autophagy inhibitor 3-methyladenine group (group HM) and HP + mammalian target of rapamycin (mTOR) inhibitor rapamycin group (group HR).In HC, HM and HR groups, 10 mmol/L Compound C, 5 mmol/L 3-methyladenine and 10 nmol/L rapamycin were added to the culture medium, respectively, at 3 h before HP.Twelve wells in each group were selected, and the expression of phosphorylated AMPK (p-AMPK) , phosphorylated mTOR (p-mTOR), microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ), and LC3 Ⅱ in BMSCs was determined.Eighteen wells in each group were selected, and BMSCs were co-cultured with 500 μ mmol/L H2O2 for 24 h, the survival and apoptotic rate of BMSCs were measured, and the activities of caspase-9 and caspase-3 were detected.In vivo experiment Adult male Sprague-Dawley rats, weighing 300-350 g, aged 3 months, underwent spinal cord I/R.A total of 192 rats with spinal cord I/R injury were randomly divided into C, N, H, HC, HM and HR groups (n =32 each) using a random number table.At 30 min of reperfusion, BMSC suspension 5 μ l (1×106 cells/ml) processed in N, H, HC, HM and HR groups of in vitro experiment was implanted into the lumbar segment (L1-5) of the spinal cord in N, H, HC, HM and HR groups, respectively.Neurological function was scored at 4, 12, 24 and 48 h of reperfusion.The lumbar segment of spinal cord was removed for detection of apoptosis in BMSCs.Results In vitro experiment Compared with group N, the p-AMPK expression, LC3 Ⅱ/LC3 Ⅰ and survival rate were significantly increased, and p-mTOR expression, apoptotic rate and activities of caspase-9 and caspase-3 were decreased in group H (P＜0.05).Compared with group H, the p-AMPK expression, LC3 Ⅱ/LC3 Ⅰ and survival rate were significantly decreased, and p-mTOR expression, apoptotic rate and activities of caspase-9 and caspase-3 were increased in group HC, LC3 Ⅱ/LC3 Ⅰ and survival rate were decreased, and apoptotic rate and activities of caspase-9 and caspase-3 were increased in group HM, and p-mTOR expression, apoptotic rate and activities of caspase-9 and caspase-3 were decreased, and LC3 Ⅱ/LC3 Ⅰ and survival rate were increased in group HR (P＜0.05).In vivo experiment Compared with group N, the neurological function scores were significantly increased, and the number of apoptotic BMSCs was decreased in group H (P＜0.05).Compared with group H, neurological function scores were significantly decreased,and the number of apoptotic BMSCs was increased in HC and HM groups (P＜O.05).Conclusion Enhanced autophagy is involved in survival of hypoxia-preconditioned BMSCs in damaged tissues resulting from spinal cord I/R injury, and the mechanism is associated with activated AMPK/mTOR pathway in rats.
