Effect of activating AMPK on sepsis in aged mice and the relationship with autophagy / 中华麻醉学杂志

ABSTRACT

Objective To evaluate the effect of activating adenylate-activated protein kinase (AMPK) on sepsis in aged mice and the relationship with autophagy.Methods Experiment [Twentyeight SPF female C57BL/6 mice,aged 16-19 months,weighing 25-35 g,were divided into sepsis group (group S,n=14) and sepsis plus AMPK agonist AICAR group (S+A group,n=14) by using a random number table method.In group S+A,AICAR 0.5 ml (dissolved in 5％ dimethyl sulfoxide) was administered by intragastric gavage.In group S,5％ dimethyl sulfoxide 0.5 ml was injected by intragastric gavage once a day for 7 consecutive days.The body weight of each mouse before and after administration was recorded.Sepsis was induced by intraperitoneal injection with cecal slurry 200 μl at the end of administration.Nine mice were selected in each group and observed for 7 days after establishing the model,and the survival rates were recorded.At 24 h after establishing the model,5 mice were sacrificed in each group,and spleen tissues were obtained for determination of the expression of AMPK,phosphorylated AMPK (p-AMPK),microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ) and LC3 Ⅱ (by Western blot).The ratios of p-AMPK/AMPK and LC3 Ⅱ/LC3 Ⅰ were calculated.Experiment Ⅱ Ten SPF female C57BL/6 mice,aged 16-19 months,weighing 25-35 g,were studied.The peritoneal macrophages obtained from 5 mice were extracted,cultured primarily and then randomized into control group (group C,n =5) and EG-FP E.Coli group (group E,n=5) using a random number table method.AICAR 0.5 ml was injected by intragastric gavage once a day for 7 consecutive days in the other 5 mice.The peritoneal macrophages were extracted after the end of intragastric administration,cultured primarily and then divided into 2 groups (n=5 each) using a random number table method:AICAR plus EGFP E.Coli group (group A+E) and AICAR plus autophagy inhibitor 3-methyladenine plus EGFP E.Coli group (group A+M+E).In group A+M+E,5 mmol/L 3-methyladenine 100 μl was added,and the cells were incubated for 1 h.EGFP expressingE.Coli was then added,and the cells were incubated for 1 h in E,A+E and A+E+M groups.Flow cytometry was used to detect the phagocytic ability of macrophages.Results Experiment Ⅰ The body weight was significantly lower after the end of administration than before administration in group S+A (P＜0.05).Compared with group S,the body weight was significantly decreased at the end of administration,the survival rate was increased at 7 days after establishing the model,the expression of LC3 Ⅱ in spleen tissues was up-regulated and the ratios of p-AMPK/AMPK and LC3 Ⅱ/LC3 Ⅰ were increased in group S+A (P＜0.05).Experiment Ⅱ Compared with group C,the phagocytic ability of macrophages was significantly enhanced in the other three groups (P＜0.05).Compared with group E,the phagocytic ability of macrophages was significantly enhanced in group A+E (P＜0.05),and no significant change was found in the phagocytic ability of macrophages in group A+M+E (P＞0.05).Compared with group A+E,the phagocytic ability of macrophages was significantly weakened in group A+M +E (P＜0.05).Conclusion Activating AMPK can increase the survival rate of aged mice with sepsis,and the mechanism is associated with enhancing autophagy of macrophages.