Effects Of (-)-Epigallocatechin-3-gallate On Cell Proliferation, Differentiation, And Angiogenesis In 3T3-L1 Cells

Background:Obesity is a complex multifactorial chronic disease associated with risks of diabetes, hypertension, certain forms of cancer and cardiovascular diseases. Therefore, many nutrition and health researches have focused on prevention and treatment of obesity and related chronic diseases. The development of obesity is described as increased number of fat cells and lipid accumulation. As is known, the growth and expansion of adipose tissues requires the formation of new blood vessels or angiogenesis to enable delivery of oxygen and nutrients. The inhibition of angiogenesis in adipose tissue may be a new pathway for suppression of adipogenesis. The adipose tissue is considered as one of the largest endocrine tissues in the body, which is mediated by adipose tissue secretion of adipokines. Vascular endothelial growth factor(VEGF) is the most important factor of pro-angiogenesis in adipose tissue. The latest evidence shows that modulators of angiogenesis affect the expansion and metabolism of fat mass by regulating the growth and remodelling of the adipose tissue vasculature. Thus, regulation of angiogenesis in the adipose tissue may offer a new therapeutic option for treatment of obesity.(-)-Epigallocatechin-3-gallate(EGCG), which is the most abundant and active component of green tea, has been reported to have chemopreventive and chemotherapeutic properties for obesity. Several studies demonstrated that EGCG inhibited tumor growth by anti-angiogenesis. However, there is no report about the potential inhibiting angiogenesis of EGCG in adipose tissue. We therefore hypothesized that EGCG inhibits the growth and function of adipose tissues by anti-angiogenesis. The hypothesis will be verified at cellular level in this study. Objectives:1. To know the effect of EGCG on the growth of adipose tissue through observing the effect of EGCG on the proliferation and differentiation of 3T3-L1 preadipocytes.2. To know the role of EGCG on angiogenesis by observing the effect of EGCG on VEGF expression during the proliferation and differentiation of 3T3-L1 preadipocytes and the effect of VEGF secreted from adipocytes on the proliferation and tube formation of human umbilical vein endothelial cells(HUVEC).3. To explore the molecular mechanism of EGCG's regulating angiogenesis by observing the role of EGCG on the expression of adipocyte transcription factor PPAR?, C/EBP? and VEGF. Methods:1. The effect of EGCG on the growth and differentiation of 3T3-L1 preadipocytes to adipocytes was observed by using the ACEA x CELLigence Real-Time Cell Analyser dual Plate(RTCA DP) system.2. The anti-adipogenic effect of EGCG on 3T3-L1 cells was analyzed by Oil O red staining.3. The VEGF concentration of the conditioned medium was measured using VEGF enzyme-linked immunosorbent kit. The VEGF expression in 3T3-L1 preadipocytes and adipocytes are measured by immunofluorescence staining. The m RNA level of VEGF in the 3T3-L1 preadipocytes and adipocytes was analyzed by quantitative real-time PCR.4. Western blot analysis was used to detect the protein level of PPAR? and C/EBP?. The m RNA level of PPAR? and C/EBP? in the 3T3-L1 adipocytes was analyzed by quantitative real-time PCR.5. Coculture system of 3T3-L1 cells and human umbilical vein endothelial cells(HUVEC) were performed to observe the effect of 3T3-L1 preadipocytes and adipocytes on the proliferation of HUVEC by using the ACEA x CELLigence Real-Time Cell Analyser dual Plate(RTCA DP) system. HUVECs were cultured by the conditioned medium from 3T3-L1 adipocytes treated with or without EGCG to study the effect on the tube formation. Results:1. Compared with the control group, EGCG at the concentration range of 0.5?g/ml~50?g/ml reduced the proliferative rate of 3T3-L1 preadipocytes and inhibited the differentiation of 3T3-L1 preadipocytes to adipocytes in a dose-dependent manner(P<0.05).2. Although EGCG had no effect on adipose differentiation up to 10 ?g/ml, a significant inhibition of differentiation of preadipocytes to mature adipocytes was observed at 25 ?g/ml(P<0.05) and it was evident from less accumulation of fat in the cells.3. EGCG at the concentration range of 0.5?g/ml~50?g/ml inhibited the expression of VEGF in 3T3-L1 preadipocytes and adipocytes( P<0.05). The fluorescence intensity of VEGF in adipocytes was higher than that in preadipocytes by immunofluorescence staining. EGCG inhibited the m RNA level of VEGF by quantitative real-time PCR.4. EGCG inhibited the protein and m RNA level of PPAR? and C/EBP? by Western blotting and quantitative real-time PCR.5. The HUVEC cocultured with 3T3-L1 cells showed a clear increase compared with HUVEC alone. The conditioned medium from 3T3-L1 adipocytes treated with EGCG inhibited the tube formation of HUVEC. Conclusions:Firstly, EGCG has the ability to inhibit the proliferation of preadipocytes and adipocytes, decrease differentiation of preadipocytes, and reduce accumulation of lipids in adipocytes. Secondly, EGCG has the role of suppressing angiogenesis in adipose tissue at cellular levels.