The Effect Of Bean Tea Cassia And Its Effective Component Luteolin On The Differentiation Of Preadipocytes

Chapter One Inhibitory effect of luteoloside on the differentiation of 3T3-L1 preadipocytesObjective:To observe the effects of luteoloside on the proliferation and the differentiation of 3T3-L1 preadipocytes and investigate the possible mechanism of luteoloside on the differentiation of 3T3-L1 preadipocytes.Methods:1.3T3-L1 preadipocytes were cultured and treated with the absence (0?mol/L) or presence of 5?10?20?40?80?160 and 320?mol/L luteoloside. Methyl thiazolyl tetrazolium method (MTT)assay was used to detect the effect of luteoloside on proliferation of 3T3-L1 preadipocytes at 24?48?72 and 96 hours,respectively.2.Cells were routinely induced to differentiate by the hormonal cocktail , containing O.5mmol/L 3-isobutyl-1-methylxanthine (IBMX), l?mol/L dexamethasone (DEX) and 10?g/mL insulin (Ins). Oil red staining was used to identify the fully differentiated adipocytes.3.3T3-L1 preadipocytes were induced to differentiation in both treated groups containing luteoloside with different concentrations (5 ? 20 ? 80?mol/L) and control groups (without luteoloside). Effects of luteoloside on the lipid synthesis during 3T3-L1 preadipocytes differentiation were detected by Oil red O staining and triglyceride (TG) determination.4.The mRNA expression of CCAAT enhancer binding protein a (C/EBPa) and Peroxisome proliferator-activated receptor ? (PPARy) during 3T3-L1 differentiation was measured by reverse transcription polymerase chain reaction (RT-PCR) when adipocytes were induced to differentiation for 8 days.5.The protein expression of C/EBPa and PPARy during 3T3-L1 differentiation was measured by western blotting when 3T3-L1 preadipocytes were induced to differentiation for 8 days.Results:1. According to the results of the time-effects and dose-effects experiment, compared with the control group (O?mol/L),luteoloside at the concentration of 5?10?20?40?80 ?mol/L had no significant influence on the proliferation of 3T3-L1 preadipocytes (P>0.05), luteoloside at the concentration of 160 and 320?mol/L significantly inhibited the proliferation of 3T3-L1 preadipocytes (P<0.05).2.Compared with the control group (0?mol/L) , 5, 20 and 80?mol/L concentration of luteoloside dose-dependently inhibited differentiation of 3T3-L1 preadipocytes into mature adipocytes (P<0.01). Both Oil red O staining and TG accumulation determination revealed that the formation of lipid droplets especially the size of the lipids drolets was significantly decreased.3.On the 8th day of differentiation, the expression level of mRNA and protein expressions of C/EBPa and PPARy significantly decreased in the group treated with 5, 20 and 80?mol/L concentration of luteoloside compared with the control group (P<0.01).Conclusion:1.Luteoloside significantly inhibited the adipogenic differentiation of 3T3-L1 preadipocytes,reduced lipid accumulation.2.Luteoloside significantly inhibited the adipogenic differentiation of 3T3-L1 preadipocytes,and the process is probably through down-regulation of PPARy and C/EBPa to achieve.Chapter TwoInhibitory effect of Cassia nomame Containing Serums on the differentiation of 3T3-L1 preadipocytesObjective:To study the effects of cassia nomame containing serums on the proliferation and differentiation of 3T3-L1 preadipocytes. Through this research, we clear the intervention of cassia nomame containing serums on 3T3-L1 preadipocytes proliferation and differentiation,which is one of the mechanism of cassia nomame to reduce weight,so as to find a new target for the treatment of obesity.Methods:1.Rat drug-containing serums were prepared for cell culture. 3T3-L1 preadipocytes were cultured in the presence of cassia nomame containing serums (5%?10%?20%). MTT assay was applied to evaluate the cell proliferation.2.By the high pressure liquid chromatography (HPLC), the concentron of luteoloside in the cassia nomame containing serums was determined.3.3T3-L1 preadipocytes were induced to differentiate to adipocytes in the treated groups of cassia nomame containing serums (5%?10%?20%) and the control group. Oil red O staining method was used to assess the lipid accumulation in adipocytes.4.Effects of cassia nomame containing serums (5%?10%?20%) on the lipid synthesis during 3T3-L1 preadipocytes differentiation were detected by triglyceride (TG) content.5.The mRNA expression of CCAAT enhancer binding protein (C/EBP? and C/EBP?) and Peroxisome proliferator-activated receptor y (PPAR?) were detected by reverse transcription polymerase chain reaction (RT-PCR) when adipocytes were induced to differentiation on the sixth days.6.The protein expression of C/EBP?? C/EBP? and PPAR? were detected by western blotting when 3T3-L1 preadipocytes were induced to differentiation on the sixth days.Results:1.The luteoloside content of serum containing cassia nomame is 0.16%.2.Compared with the control group, there was no significant effect of cassia nomame containing serum on the proliferation of 3T3-L1 preadipocytes (P>0.05).3.3T3-L1 preadipocytes were induced into mature adipicytes by hormonal cocktail successfully. Oil red O staining was used to clarified matural adipocytes. The preadipocytes treated with serum containing cassia nomame (5%? 10%? 20%) showed that the lipid droplets in the cytoplasm and TG content were significantly decreased compared with the control group (P<0.05).4.Compared with the control group, the mRNA and protein expressions of C/EBP?.C/EBP?and PPAR? significantly decreased when the preadipocytes was treated with the various concentration of serum containing cassia nomame (5%?10%?20%) (P<0.05).Conclusion:1. Serum containing cassia nomame decreased the accumulation of lipid droplets during the differentiation of adipicytes,and inhibited the differentiation of adipocytes.2.Inhibitory effect of serum containing cassia nomame on the differentiation of 3T3-L1 preadipocytes,which may be related to the decrease of the mRNA and protein expressions of C/EBP?? C/EBP? and PPAR?.3.Serum containing cassia nomame inhibited the differentiation of 3T3-L1 preadipocytes,which might be associated with its main components of luteoloside.