| Small for gestational age infants growth retardation has been closely watched,and GH/IGF-1 axis is a key in the whole process of growth and development.Insufficient intrauterine energy supply energy supply is the primary factors of small for gestational age infants,in order to ensure energy supply of important organs,fetal programming by changing its own metabolism to adapt to this calorie restriction environment.Studies have found that GH/IGF-1 axis dysfunction exists in Small for gestational age infants,and this state may have been persistent,but it is not clear whether its occurrence is related to calorie restriction.SIRT1 is an energy receptor in the body and plays an important role in adaptive metabolism and endocrine response by changing the acetylation status of various proteins.JAK2/STAT5 is a key pathway for GH to play a biological role,but how it is regulated in the process of GH/ IGF-1 axis disorder has not been fully elucidated.Starting from the clinical findings,this study is divided into three chapters: In the first chapter,in vivo animal experiments were conducted to study the effect of SIRT1 on GH/ IGF-1 axis under calorie restriction.Chapter 2 in vitro cell experiments to investigate the regulatory mechanism of SIRT1 on GH/ igf-1 axis resistance of hepatocytes.In the third chapter,animal in vivo experiments were conducted to verify SIRT1 mediates the resistance of the liver GH/ IGF-1 axis by regulating the activity of STAT5 under calorie restriction.Chapter I The expression of SIRT1 and its regulation of GH/ IGF-1 axis under calorie restriction.Objective To investigate the expression of SIRT1 and its regulation of GH/ IGF-1 axis under calorie restriction.Methods First,a mouse model with calorie restriction was prepared after treatment with 30% limited energy for 48 h.Male C57BL/6J mice at the age of 6 weeks were randomly divided into control group(Control,n=5)and calorie restriction group(CR,n=5).Then,siRNA interfering with SIRT1 was prepared,and the concentration of GH ?IGF-1 in serum samples was detected by ELISA in mice transfected with tail vein.The expression of IGF-1 GHRmRNA in mouse liver tissue was detected by RT-PCR.The expression of SIRT1 protein in liver tissue was detected by Western Blot.Results(1)The serum GH concentration of CR group was significantly higher than that of control group,but the serum IGF-1 concentration was lower than that of control group.The serum GH concentration did not change significantly after SIRT1 interference,while the serum IGF-1 concentration increased.(2)The expression of IGF-1 mRNA in the CR group was significantly lower than that in the control group,but there was no significant difference in the expression of GHRmRNA.After SIRT1 interference,the expression of IGF-1 mRNA in liver tissue was significantly increased,and there was no statistical difference in the expression of GHR mRNA.(3)the expression of SIRT1 protein in CR group was significantly higher than that in control group.Conclusion The expression of SIRT1 in the liver was up-regulated under calorie restriction,and negatively regulated the expression of IGF-1 mRNA in livers.Chapter II Mechanisms by which SIRT1 regulates JAK2/STAT5 signaling pathways affecting GH/ IGF-1 axis resistance in hepatocytesObjective To investigate the mechanism by which SIRT1 regulates JAK2/STAT5 signaling pathway affecting GH/ IGF-1 axis resistance in hepatocytesMethods(1)Hep G2 cells were treated with SIRT1 activator resveratrol(Res)and inhibitor nicotinamide(NAM),respectively,and the expression of GH-dependent IGF-1 mRNA in Hep G2 cells was observed.(2)SIRT1 interfering siRNA and overexpressed plasmid were constructed and Hep G2 cells were transfected to observe the expression of GH-dependent IGF-1 mRNA after SIRT1 interference and overexpression?(3)Hep G2 cells were treated with SIRT1 activator resveratrol(Res)and inhibitor nicotinamide(NAM)respectively,to observe the effect of SIRT1 on key proteins in the JAK2/STAT5 signaling pathway of liver cells.(4)Use the protein interactions to see if there is a direct effect between SIRT1 and STAT5.(5)HepG2 cells were transfected with SIRT1 siRNA and overexpressed plasmid to observe the regulation of SIRT1 on acetylation and phosphorylation of STAT5.Results(1)The expression of GH-dependent IGF-1mRNA in Hep G2 cells decreased after treatment with the SIRT1 activator Res,while the expression of GH-dependent IGF-1mRNA in Hep G2 cells increased after treatment with the inhibitor NAM.(2)The expression of GH-dependent IGF-1 mRNA in Hep G2 cells increased after SIRT1 interference,while the expression of GH-dependent IGF-1mRNA in Hep G2 cells decreased after SIRT1 overexpression.(3)Phosphorylation of GH-dependent STAT5 in Hep G2 cells decreased after treatment with SIRT1 activator Res,while phosphorylation of GH-dependent STAT5 in Hep G2 cells increased after treatment with inhibitor NAM.(4)Interactions between proteins indicate a direct interaction between SIRT1 and STAT5.(5)STAT5 lysine(Lys681)was acetylated with SIRT1 interference,accompanied by increased phosphorylation of STAT5 tyrosine(Tyr699).Acetylation of STAT5 lysine(Lys681)was reduced by overexpression of SIRT1,accompanied by phosphorylation of STAT5 tyrosine(Tyr699)?Conclusion(1)SIRT1 negatively regulates the expression of GH-dependent IGF-1 mRNA in hepatocytes;(2)SIRT1 changes acetylation of Lys681 and phosphorylation of tyrosine Tyr699 by direct interaction with STAT5.Chapter III SIRT1 mediates GH/ IGF-1 axis resistance by regulating the activity of STAT5 under calorie restrictionObjective In vivo validation of calorie restriction SIRT1 mediated GH/ IGF-1 axis resistance of the liver by regulating STAT5 activity.Methods The 6-week-old male C57BL/6J mice were randomly divided into the Control group(Control),calorie restriction group(CR)and the CR+NAM group.The calorie restriction group were treated with 30% restricted energy for 48 h,the CR+NAM group was treated with intraperitoneal injection of nicoamide(150mg/kg).Serum GH and IGF-1 levels were detected by enzyme-linked immunosorbent assay.The expression level of IGF-1 and GHRmRNA in ls was detected by fluorescence real-time quantitative PCR.The acetylation and phosphorylation of STAT5,the key protein of JAK2/STAT5 pathway,were detected by IP Western Blot.Results(1)The serum GH concentration of CR group was higher than that of control group,and the serum IGF-1 concentration was significantly lower than that of control group.The serum GH concentration in CR+NAM group was higher than that in the control group,but lower than that in the CR group,while the serum IGF-1 concentration was significantly higher than that in the CR group.(2)The expression of IGF-1mRNA in the CR group was lower than that in the control group,and the expression of IGF-1mRNA in the CR+NAM group was also lower than that in the control group,but significantly higher than that in the CR group.There was no significant difference in GHR mRNA expression between the three groups.(3)The acetylation and phosphorylation levels of STAT5 Lys681 and tyrosine Tyr699 were significantly lower in the CR group than in the control group,while the acetylation and phosphorylation levels of STAT5 Lys681 and tyrosine Tyr699 were significantly higher in the CR+NAM group than in the CR group.Conclusion(1)The SIRT1 inhibitor NAM restores GH/ IGF-1 resistance due to calorie restriction.(2)The SIRT1 inhibitor NAM can restore the acetylation and phosphorylation activity of STAT5 due to calorie restriction. |
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