| Obesity gradually becomes a medical condition with an increasing prevalence,obesity also is associated with several chronic diseases,including fatty liver,heart disease,type 2 diabetes mellitus,certain types of cancer,and osteoarthritis.The pathogenesises of obesity are involved in inheritance,diet and physical activities,and the balance between energy intake and energy utilization maintains body weight.Uncoupling proteins(UCPs)2 and 3 belong to the family of mitochondrial transporter proteins that may play an important role in energy homeostasis and have become prominent in the fields of thermogenesis,obesity,diabetes and free-radical biology hence they have been considered candidate genes for obesity and insulin resistance.Vitamin D3(VD3)is a fat-soluble vitamin which most active form is 1,25-dihydroxyvitamin D[1,25(OH)2D3]—calcitriol.Recent epidemiological investigation found that low serum 1,25-dihydroxyvitamin D levels were associated with higher Body Mass Index(BMI)and greater body fat,so the influence and mechanism of VD3 for obesity remain to be elucidated.Forming a therapeutic method from kidney is one of the treatments of Traditional Chinese Medicine for obesity,which nourishing and warming kidney-yang.Therefore we speculated the potential relationship with 1,25(OH)2D3 biothysis in kidney.Based on the reports and basic theories of Traditional Chinese Medicine above,we performed this research in order to investigate the effects and mechanism of VD3 and Jinkuishenqiwan for obesity,furthermore,clarified the potential relationship between enhance kidney-yang and VD3 biothysis in kidney.Part 1.The effects of VD3/VDR on genetic transcription of energy metabolism in high fat diet feeding mice.Objective:To investigate the influence of VD3/VDR on body weight energy metabolism in obese mice feeding high fat diet,explored the potential mechanism by uncovered the possible consensus sequence between VDR and UCP2/UCP3 promoter ulteriorly.Method:We constructed the obesity models using high fat diets[45 kcal%fat(lard)],then observed the effects of 50μg/kg body wt/d VD3(cholecalciferol)to the body weight,food consumption and adipose mass,additionally checked the level of mRNA of UCP2 and UCP3 in adipose and muscle tissue.In vitro,the mRNA expressions of genes which involved in energy and lipid metabolism were analyzed by RT-PCR in 3T3-L1,C2C12 and HepG2 cells.Furthermore,the potential promoter of 1,25(OH)2D3/VDR were filtrated by Dual luciferase report assay,and the binding site consensus sequence of 1,25(OH)2D3/VDR on UCP2 and UCP3 promoter also been identified.Results:1.VD3 decreased the body weight of mice which feeding high fat diet(P<0.05),but non-alter the food consumption.VD3 also reduced the fat mass of mesentery adipose(P<0.05)and epididymis adipose tissue(P<0.01),additionally,VD3 increased mRNA expression of UCP2 in mesentery adipose(P<0.01)and UCP3 in muscle tissue(P<0.01),while had no effect on FXR,LXR in liver.2.In vitro,UCP2 and UCP3 mRNA was elevated by 1,25(OH)2D3 in 3T3-L1 and C2C12 cells(P<0.05),the effect were more powerful in the cells which been transfected VDR(P<0.01).1,25(OH)2D3 altered the LXRβ and FXR mRNA in HepG2 cells which been transfected VDR(P<0.05).3.The UCP2 and UCP3 were the promoter of 1,25(OH)2D3/VDR since the relative luciferase activity were dose-dependently elevated by 1,25(OH)2D3 in HEK 293 cells which been transfected VDR by dual luciferase repoter assay(P<0.01).4.The transcriptional activity of UCP3 were dose-dependently increased by 1,25(OH)2D3 in C2C12,L6 and H-EMC-SS cells which been transfected VDR(P<0.05).5.The binding site consensus sequence of 1,25(OH)2D3/VDR on UCP2 and UCP3 promoter VDRE were identified located on-1021～-1001 bp,-941～-892bp region and-1867～-1561bp,-1561～-634bp,+162～+314bp region by promoter deletion model assay.Conclusion:In sumary,we have provided here evidence that the 1,25(OH)2D3/VDR is able to regulate the body weight and white adipose tiuuse weight although non-changed intake food weight.The promotion activity of mitochondria and fatty acid oxidation by increasing expression of UCP2 and UCP3 might be the primary potential mechanism,and we confirmed the presence of activating binding sites consensus sequences on UCP2 promoter within the-1021～-100 1bp and-941～-892bp region while UCP3 promoter might located on-1867～-1561bp,-1561～-634bp,and+162～+314bp.Part 2.The effects and mechanism of Jinkuishenqiwan on mice body weight which feeding high fat diet.Objective:To observe the effect of Jinkuishenqiwan on mice body weight which feeding high fat diet,in addition,explored the potential relationship between decreasing body weight and VD3 biothysis in kidney.Methods:We constructed the obesity models using high fat diets[45 kcal%fat(lard)],then observed the effects of Jinkuishenqiwan 2g/kg body wt/d and 50μg/kg body wt/d VD3(cholecalciferol)to the body weight,food consumption and adipose mass,the protein expression of CYP27B1 and CYP24A1 in kidney were checked by Western blot,the level of serum IL-4,IL-10,IL-15 and PTH were measured by ELISA,the content of serum 25(OH)D3,1,25(OH)2D3,24,25(OH)2D3 were analyzed using HPLC-MS.Results:1.Jinkuishenqiwan 2g/kg body wt/d decreased the mice body weight which feeding high fat diet and had the stronger effect combine with VD3 50 μg/kg body wt/d,but had non-influnence on food consumption;Jinkuishenqiwan reduced the fat mass of mesentery adipose(P<0.01)and epididymis adipose tissue(P<0.01),especially had synergistic effect with VD3 on epididymis adipose tissue.2.From the Western blot,we observed Jinguishenwiwan increased the protein expression of CYP27B1(P<0.01)and down-regulated the protein expression of CYP24A1(P<0.01).3.Compared with high fat diet group,Jinkuishenqiwan reduced the level of serum IL-10(P<0.01)and elevated the level of serum IL-15(P<0.01).4.The level of 25(OH)D3 was decreased in LFD group(P=0.065),while VD3 could increased the serum level of 25(OH)D3(P<0.01);Although the levels of 1,25(OH)2D3,24,25(OH)2D3 were elevated in both VD3 and VD3 plus Jinkuishenqiwan,when compared with VD3 group,VD3 plus Jinkuishenqiwan had the capability to increase the level of 1,25(OH)2D3 and down-regulate the level of 24,25(OH)2D3.Conclusion:Jinkuishenqiwan could alleviate the mice body weight which feeding high fat diet,inaddition,the mechanism involved in enhance the biosynthesis of 1,25(OH)2D3 in kidney,increased the level of serum 1,25(OH)2D3 to play the role in weight loss. |
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