| Part1 Effects of adiponectin on the inflammatory response and autophagy in septic miceObjective:To investigate whether adiponectin could regulate the inflammatory response in septic mice by regulating autophagy and its possible mechanism.Methods:32 male, clean C57BL/6 mice, weighing 20-25 g, at the age of 2 months, were randomly divided into 4 groups (8 mice in each group):control group (C group), CLP group, Adiponectin group (APN group) and APN pre-treatment group (CLP/APN group). Animals in CLP group and CLP/APN group underwent CLP; Animals in CLP/APN group received APN 6mg/kg before underwent CLP; And APN group received APN 6mg/kg at the same time with CLP/APN group. The general situation of mice in each groups were observed in 24h, and after anesthesia, the blood was taken from mice's abdominal aorta. Later the mice were sacrificed and the liver and lung tissue samples were collected. HE stained sections were used to observe the liver and lung injury in mice; Elisa method was used to detect the levels of IL-6 and IL-1? in mice serum; The content of LC3B in the liver and lung tissues of mice was observed by immunofluorescence staining.Results:1. Compared with C group, the liver and lung tissue injury was more serious in CLP Group and CLP/APN group, the level of IL-1?and IL-6 in serum were higher (P< 0.05) and immunofluorescence staining showed LC3B particle area increased (P< 0.05).2. Compared with CLP Group, the damage to lung and liver tissue in CLP/APN group was more alleviated, the level of IL-1?and IL-6 in serum were lower (P< 0.05); However, immunofluorescence staining showed a further increase of LC3B particle area (P< 0.05).3. There was no significant difference in liver and lung tissue injury between C group and APN group. And then, levels of IL-6 ?IL-1?and the area of LC3B granules between C group and APN group had no significant difference (P> 0.05). Conclusion:Adiponectin could significantly alleviate the pathological injury of liver and lung tissue, reduce the level of serum IL-1? and IL-6, also improve the level of autophagy in liver and lung tissue of mice with sepsis.Part2 Effects of Adiponectin on inflammatory reaction in septic mice through enhancement of autophagyObjective:To investigate whether adiponectin could regulate the expression of inflammatory cytokines in murine peritoneal macrophages induced by lipopolysaccharide via AMPK/ULK1 pathway.Methods:1. Collecting the peritoneal macrophages of male, clean, C57BL/6 mice.2. To investigate the effects of autophagy inhibitor on the expression of inflammatory cytokines in the peritoneal macrophages induced by LPS in mice. Set the groups:Control group (C group); LPS group (LPS group); Adiponectin Group (APN group); LPS+adiponectin group (LA group); LPS+autophagy inhibitor 3-mA (L3 group); LPS+adiponectin+3-MA group (LA3 group).3. To investigate the role of ULK1 in the regulation of autophagy signaling pathway by adiponectin. Set the groups:Control group (C group); LPS intervention group (LPS group); Adiponectin Group (APN group); LPS+adiponectin group (LA group); LPS+ULK1 inhibitor SBI-0206965 group (LS group); LPS+adiponectin+ SBI-0206965 group (LAS group).4. To investigate whether adiponectin regulates the level of autophagy by AMPK pathway to inhibit the inflammatory response. Set the groups:Control group (C group); LPS group (LPS group); Adiponectin Group (APN group); LPS+ adiponectin group (LA group); LPS+AMPK inhibitor compound C group (LC group); LPS+adiponectin and AMPK inhibitor compound C group (LAC group).Results1. Compared with C group, after LPS induced mouse peritoneal macrophages 16h, the level of IL-1? and IL-6 in mouse peritoneal macrophages was increased, also with the level of ROS?LC3II/LC3I and AMPK (P< 0.05).2. Compared with the LPS group, the levels of IL-1?and IL-6 in mouse peritoneal macrophages in group LA were decreased, the level of ROS was decreased, and the level of LC3II/LC3I was further increased, and the level of AMPK and ULK1 were increased (P< 0.05).3. Compared with the LA group, the IL-1? and IL-6 levels of mouse peritoneal macrophages were increased, and the level of ROS was increased, and the level of LC3II/LC3I was decreased in the LA3 group (P< 0.05).4. Compared with the LA group, the IL-1?and IL-6 levels of mouse peritoneal macrophages were increased and the level of ROS was increased, and the level of LC3II/LC3I was decreased after the ULK1 inhibitor was used in the LAS group (P< 0.05).5. Compared with the LA group, the IL-1? and IL-6 levels of mouse peritoneal macrophages were increased and the level of ROS was increased, and the level of ULK1 and LC3II/LC3I were decreased after the AMPK inhibitor was used in the LAC group (P< 0.05).Conclusion:It might through AMPK/ULK1 pathway that Adiponectin pre-treatment inhibited the expression of oxidative stress, the level of IL-1?, IL-6 and improve autophagy in LPS induced mouse peritoneal macrophage cells. |
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