Study On Effects Of RNAi-mediated Gene Silencing Of SOCS3 On JAK2-STAT3 Signal Transduction In Hypothalamus Of Obese Rats Fed With High-fat Diet

Background: For the past few years, the case rate of obesity in children has shown an upward trend step by step. Obesity not only influences the health of children, but also becomes a high dangerous factor for diabetes 2, cardiac and cerebrovascular disease of adult. Leptin plays an important role in inhibiting food intake, promoting energy expenditure and decreasing body fat. Leptin acts through its receptor and many signal transduction pathways in which the JAK2–STAT3 pathway is the major pathway of leptin signaling. Many researches have been indicated, however, that the phenomenon of low or no reaction to leptin called leptin resistance has been presented in majority of obese patients and suppressor of cytokine signaling 3(SOCS3) , an endogenous inhibitor, plays inhibitory feedback action during leptin signaling. Over-expression of SOCS3 may be involved in the development of leptin resistance in obesity. RNAi-mediated gene silencing strategy to treat many kinds of disease has been tested in recently years. It is possible to activate the JAK2–STAT3 pathway of leptin signaling and enhance leptin sensitivity and then attenuate leptin resistance in obesity ultimately through down-regulation of SOCS3 expression by RNAi-mediated gene silencing of SOCS3.Objectives: To imitate children, 5-week-old (equal to juvenile stage) SD rats were selected as rodent model in the experiment. Using RNAi-mediated gene silencing strategy of SOCS3, we observed body weight, serum leptin concentration as well as levels of phospho-STAT3 and levels of STAT3 DNA-binding activity in hypothalamus of SD rats fed with standard chow or high-fat diet in order to confirm that the JAK2–STAT3 pathway of leptin signaling can be activated and leptin resistance in obesity can also be alleviated.Methods: Forty eight 5-week-old healthy male Sprague-Dawley (SD) rats were adopted to our research and divided into three groups randomly. In the control lenti + standard chow group(n=15), designated as CS, the control lenti were stereotaxically injected into the hypothalamus of rats which then fed with standard chow; In the control lenti+high-fat diet group(n=15), designated as CHF, the control lenti were stereotaxically injected into the hypothalamus of rats which then fed with high-fat diet containing 20% lard; In the SOCS3 lenti + high-fat diet group(n=18), designated as SHF, the SOCS3 lenti were stereotaxically injected into the hypothalamus of rats which then fed with high-fat diet containing 20% lard. Animal weight was measured weekly. Up to 8 weeks after treatment, serum leptin concentration of rats were determined with radioimmunity assay; Levels of phospho- STAT3 and levels of STAT3 DNA-binding activity in hypothalamus of rats were investigated with western blot and electrophoretic mobility shift assay (EMSA) respectively.Results:1. Comparisons of body weight in rats of the three groups for 8 weeksSHF rats gained smaller weight than CHF rats(P<0.01)and showed larger weight than CS rats (P<0.05).2. Comparisons of serum leptin concentration in rats of the three groupsLevels of serum leptin were lower in SHF rats than CHF rats (P<0.01). There were no significant differences in comparison of levels serum leptin in SHF rats with CS rats (P>0.05).3. Comparisons of levels of phospho-STAT3 in rats of the three groupsLevels of phospho-STAT3 were higher in hypothalamus of SHF rats than CHF rats (P<0.01) and lower than CS rats (P<0.01).4. Comparisons of levels of STAT3 DNA-binding activity in rats of the three groupsSHF rats showed enhanced levels of STAT3 DNA-binding activity in hypothalamus compared with CHF rats (P<0.01). They also showed depressed levels of STAT3 DNA-binding activity in hypothalamus compared with CS rats (P<0.01).5. Correlations among serum leptin concentration, levels of phospho- STAT3 as well as levels of STAT3 DNA-binding activity in hypothalamus of SHF ratsThere were marked negative correlations between serum leptin concentration and levels of phospho-STAT3 as well as levels of STAT3 DNA-binding activity in hypothalamus of SHF rats, R =-0.682, -0.660 respectively (P<0.05). There was a obviously positive correlation between levels of phospho-STAT3 and levels of STAT3 DNA-binding activity in hypothalamus of SHF rats, R=0.785 (P<0.01).Conclusions:1. Levels of phospho-STAT3 and levels of STAT3 DNA-binding activity in hypothalamus were reduced through over-expression of SOCS3 induced by high-fat diet, indicating the impairment of JAK2–STAT3 pathway of leptin signaling and the development of leptin resistance in obesity.2. Levels of phospho-STAT3 and levels of STAT3 DNA-binding activity in hypothalamus were elevated through down-regulation of SOCS3 expression by RNAi-mediated gene silencing of SOCS3, indicating the activation of JAK2–STAT3 pathway of leptin signaling and the improvement of leptin sensitivity.3. Body weight and serum leptin concentration were decreased through down-regulation of SOCS3 expression by RNAi-mediated gene silencing of SOCS3, suggesting that down-regulation of SOCS3 expression is a promising therapeutic approach for leptin resistance in obesity.