The Effect Of Plant Extract Embelin On Obesity Of Mice And The Mechanisms Involved In The Processes

Backgrounds: Obesity is a metabolic disease caused by a variety of factors. It is characterized by an increase of fat cell size and cell number, leading to an abnormal increase of fat mass and too much fat accumulation in mesentery, epididymis and other organs. Obesity increases the risk of various diseases, such as diabetes, cardiovascular disease and fatty liver, etc. Embelin is a quinone compound extracted from plants. Existing studies show that Embelin can inhibit obesity, hyperlipemia and oxidative stress in obese rats induced by high fat diet. In the present study, we mainly focus on whether Embelin inhibits obesity through the inhibition of adipogenic differentiation from bone marrow mesenchymal stem cells, and explored the mechanism.Methods: 1, Mesenchymal cell line C3H10T1/2 and bone marrow stromal cell line ST2 were treated with dimethyl sulfoxide(DMSO) or different concentrations of Embelin, then treated with adipogenic medium to induce adipocyte differentiation. Oil red O staining was done to test the degree of adipogenesis, and q RT-PCR and Western blotting were performed to detect the m RNA and protein expression of adipogenic transcription factors and maker genes. These experiments help to determine the effect of Embelin on bone marrow stromal cell differentiation. 2, Mesenchymal C3H10T1/2 cells were treated with DMSO or Embelin. Immunofluorescence staining was done to detect the translocation of β-catenin from the cytoplasm into nucleus. This experiment helps to determine the effect of Embelin on canonical Wnt signaling. 3, C3H10T1/2 cells were treated with dimethyl sulfoxide(DMSO) or different concentrations of Embelin. After adipogenic induction, the nuclear protein content was extracted. β-catenin and T cell factor-4(TCF-4) levels within the nuclear part were detected by Western blotting. This further helps to clarify the effect of Embelin on Wnt signaling pathway. 4, C3H10T1/2 cells were treated with either dimethyl sulfoxide(DMSO) or 10 μM Embelin, and adipogenic medium was supplemented to induce adipogenic differentiation. Total RNA was extracted to detect the expression of Wnt10 b, Lrp5, Dkk1 and Sfrp1.Wnt10 b and Lrp5 are the ligand and co-receptor of Wnt pathway, respectively, while Sfrp1 and Dkk1 are inhibitory factors of Wnt signaling. 5, 4-week-old male C57BL/6 mice were fed with high fat diet to build obese mice model. The body weights of mice in each group were recorded and mice were subcutaneously injected different doses of Embelin. After the model was successfully built, body fat content was measured by using a bone densitometer. Moreover, biochemical kits were used to examine glucose, triglyceride, free fatty acid and total cholesterol levels in serum.These experiments help to determine the impact of Embelin on obesity; 6, Total RNA was extracted from adipose tissue in the experimental groups and the control group mice. Expression levels of adipogenic factors were quantified by q PCR. Western blotting was used to detect β-catenin and TCF-4 protein levels; 7, The size of adipocyte was observed in Paraffin sections stained with HE in the adipose tissue in experimental group and control group.Results:1, Oil red O staining showed that Embelin inhibited adipogenic differentiation of C3H10T1/2 and ST2 cell lines. Consistent to this, q RT-PCR and Western blotting showed that Embelin dose dependently suppressed the expression levels of adipogenic transcription factor including peroxisome proliferator activated receptor γ(PPAR) and CCAAT enhancer binding protein α(C/EBPα), and marker gene adipocyte fatty acid-binding protein factor 2(a P2) and adipsin. 2, Cell immunofluorescence showed that Embelin promoted the translocation of β-catenin from the cytoplasm into the nucleus in C3H10T1/2 cells. Western blotting showed that the nuclear protein levels of β-catenin and TCF-4 in was upregulated, which suggests that Embelin activates canonical Wnt signaling. Furthermore, Dickkopf-1(DKK1), a soluble inhibitor of Wnt signaling, was downregulated by Embelin, suggesting that Embelin activates Wnt pathway by repressing the expression of Dkk1. 3, Obesity mouse model was successfully built after 3 months of feeding with high fat diet. By contrast, subcutaneous injection of Embelin dose dependently reduced the weight gain. Furthermore, high fat diet significantly increased the serum level of glucose, triglycerides, free fatty acid and total cholesterol, while Embelin decreased them. 4, HE staining of the Paraffin sections showed that the size of adipocytes in high fat diet group was bigger than those in Embelin treated groups. 5, Expression levels of the adipogenic factors were increased in adipose tissue in high fat diet fed mice, while Embelin blocked them.Conclusion:1, In vitro study showed that Embelin inhibits adipogenic differentiation of adipogenic progenitor cells by activating the Wnt pathway.2, In vivo study showed that Embelin inhibits high fat diet induced obesity in mice through repressing adipogenesis in adipose tissue.