| Type 2 Diabetes Mellitus (T2DM), as the main characteristics of the clinical syndrome, which strongly harms people’s Health and is caused by the interaction between genetic and environmental factors and a set of sugar metabolic disorder. As the world’s population aging and the diet lifestyle changes, diabetes has become a kind of common disease, frequently-occurring disease, and seriously harms Health of human being. So at present, research and development of new anti-diabetic drugs is valuable and needed desperately. The purpose of this study is to seek and discover new leading compound in the natural products for treatment drug of diabetes and its effect and mechanism in vitro.Background and ObjectiveThe existing literature and our previous experimental Results data showed that B21, from Viburnum odoratissimum, have a good hypogLycemic effect in HepG2 Cells. So, further study of B21 on hypoglycemic activity and mechanism of action was needed to discover a new leading drug for anti-diabetes. In this study, B21 was investigated for its activity of glycoLipid metabolism in models of 3T3-L1 preadipocytes, C2C12 myoblast, HWP-visceral, and mechanism in 3T3-L1 cells.Methods1. Document ResearchIn this part, we try to understand the lasted progresses in DM, throughliterature search and summarizing the reLated studies published in core Chinese and English journals.2. Experimental Studies2.1. Activity study2.1.1. ProLiferation assay:CCK-8 assay was applied to investigate the effects of different amounts of B21 on proLiferation of 3T3-L1 preadipocytes and HWP-visceral.2.1.2. Differentiation assay:3T3-L1 preadipocytes and HWP-visceral were induced for adipocyte differentiation in the presence or absence of various concentrations of B21. OiL red 0 staining method was applied to detect differentiated adiocytes and differentiation rate.2.1.3. Glucose consumption assay:3T3-L1 preadipocytes, HWP-visceral, C2C12 myoblasts were induced to differentiate into adipocytes and myocytes, respectively. Insulin resistant (IR) modeLs were estabolished by treating Cells with 1 μM dexamethasone (DXM). IR Cells were exposed to different concentrations of B21 for 48 h. Glucose consuming ability was evaluated by residual Glucose in culture medium measured by GOD-POD detecting method.2.2. Mechanism research:2.2.1. Detection of Cellar Triglycerides:3T3-L1 preadipocytes were induced for adipocyte differentiation in the presence of different concentrations of B21. Content of Glucose and free fatty acid (FFA) in the culturaL medium on day 6, and Cellar Triglycerides (TG) on day 8 were measured respectively by using the kits.2.2.2. Expression of FAS:3T3-L1 preadipocytes were induced for adipocyte differentiation in the presence of different concentrations of B21. Cellar FAS was measured by ELISA assay at day 8.2.2.3. Gene expression of PPARy and C/EBPα:3T3-L1 preadipocytes were induced for adipocyte differentiation in the presence of different concentrations of B21. Expression leveL of PPARy and C/EBP gene was measured by RT-PCR analysis at day 8.2.2.4. Expression of PTP1B gene:Mature 3T3-L1 adipocytes were exposed to different concentrations of B21 for 48h, in the presence of 1 uM dexamethasone (DXM). Cellular PTP1B mRNA level was measured by RT-PCR analysis.2.2.5. Secretion of cytokines:Mature 3T3-L1 adipocytes were exposed to different concentrations of B21 for 48h, in the presence of 1 u M DXM. concentrations of Leptin, adiponectin, interLeukin 6 and tumor neucrosisα (TNF-α) in culture medium were measured by using the ELISA assay kits.2.2.6. Expression of member proteins in insulin signaling pathway:Mature 3T3-L1 adipocytes exposed to different concentrations of B21 for 48h, in the presence of 1μM DXM. Expression of IRa, IRS1.PI-3K, AKT and GLUT4 were detected by western bLotting.Result2.1. Activity study2.1.1. Proliferation assay:B21 had no effect on proliferation of 3T3-L1 preadipocytes or HWP-visceral at experimental drug concentrations (P>0.05).2.1.2. Differentiation assay:B21 dose-dependentLy inhibited the differentiation of 3T3-L1 preadipocytes and HWP-visceral into adipocytes.2.1.3. Glucose consumption assay:B21 promoted Glucose uptake in DXM-induced IR 3T3-L1 adipocytes, human white adipocytes and C2C12 mouse rayocytes in dose-dependent way.2.2. Mechanism study:2.2.1. Adipogenesis assay:In 3T3-L1 Cells in the process of adipocyte differentiation, B21 dose-dependently reduced the consumption of Glucose and consequently the production of FFA and TG.2.2.2. Expression of Cellar FAS:In 3T3-L1 Cells in the process of adipocyte differentiation, B21 reduced the expression of Cellular FAS, to some extent.2.2.3. Expression of PPARy and C/EBP α gene:In 3T3-L1 Cells in the process of adipocyte differentiation, B21 at 20μM dwon-reglutaed mRNA level of PPARy and C/EBPα (P<0.01 vs Control).2.2.4. Expression PTP1B gene:In DXM-induced IR 3T3-L1 adipocytes, B21 at 20μM dwon-regulated mRNA level of PTP1B (P<0.05 vs Control).2.2.5. Secretion of cytokines:In DXM-induced IR 3T3-L1 adipocytes, B21 redubed the section of TNF-α, IL-6, Leptin but promoted the section of Adippnectin (P<0.05).2.2.6. Expression of member proteins in insulin pathway:In DXM-induced IR 3T3-L1 adipocytes, B21 at 20μM elevated the expression leveL of GLUT4, AKT, IRα,IIRS1.Conclusion and InnovationIn Our study, B21 significantly inhbited adipocyte differentiation of 3T3-L1 preadipocytes and HWP-visceral without affecting Cell profiliferation, in addition, B21 restored Glucose consuming abiLity of DXM-induced IR 3T3-L1 adipocyes, human white adipocytes and C2C12 myocytes, showing activity in insulin sensitizing and lipid reglulation. Mechanistic study reveaLed that B21 achieved inhibitory effect on lipogenesis by dwon-reguLating the transcription of PPARγå'ŒC/EBPα, and restored insulin sensitivity of IR Cells by repairing insulin signaLing pathway. Positive regulation to lipolysis and secretion of cytokines might also contribute to restoration of insulin sensitivity.B21 has the potential to be developed as an insulin sensitizer with lipid regulatory effect. |
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