| Obesity is a high degree of overweight caused by excessive accumulation of adipose tissue,a global epidemic that can cause multiple complications,including cardiovascular disease,type 2 diabetes,cancer and neuropsychiatric disorders.FA is a phenolic acid from a variety of plants.It binds to polysaccharides and proteins in the cell wall and becomes the backbone of the cell wall.Its toxicity is very low,and it has the pharmacological function of reducing blood lipids.At present,many studies about the lipid-lowering effect of FA on obesity remain in vivo,and there are few reports on the effect of FA on the differentiation of adipocytes through cell model in vitro,especially lack of the research about mechanism on differentiation of FA.In this study,we established the adipocytes differentiation model with 3T3-L1 preadipocytes and studied the effect and mechanism of FA on the differentiation of 3T3-L1 preadipocytes,providing an experimental basis for FA as a new type of weight-loss drug in vitro.1.WST-1 assay and LDH assay were used to detect the effect of FA on proliferation and cytotoxicity in 3T3-L1 preadipocytes.Compared with the control group(PBS),FA showed no effect on the proliferation of 3T3-L1 preadipocytes(p>0.05)after treated with different concentrations of FA for 24 h and 48 h.50 and 100 μg/mL FA significantly inhibited the release of LDH(p<0.01)after treated for 24 h;but 10 μg/mL FA showed no effect on release of LDH(p>0.05).After treated with FA for 48 h,FA significantly inhibited the release of LDH(p<0.05)at the concentration of 100 μg/mL.2.Effect of FA on differentiation in 3T3-L1 preadipocytes:3T3-L1 preadipocytes were induced to adipocytes with "cocktail" method,the content of triglyceride was detected by Nile Red staining,and the cells were photographed after oil red O staining.After cells treated with 5,10,25,50,100 μg/mL of FA for differentiating 8 days,the results showed that FA could inhibit differentiation in a concentration-dependent manner,and the inhibition rates were 6.43%,31.99%,38.60%,47.61%and 55.70%,respectively.3T3-L1 preadipocytes treated with 10 μg/mL of FA showed the inhibition rates were 0.6%,3.31%,10.37%,31.99%on day 2,4,6,8 of differentiation;and treated with 50,100μg/mL of FA,the inhibition rates were-2.41%,38.02%,29.88%,47.61%and 16.81%,47.52%,50.62%,55.70%on day 2,4,6,8 of differentiation,respectively.and the inhibitory effect of FA on differentiation of adipocytes was in a time-dependent manner.3.Effect of FA on mRNA expression of adipogenic genes in 3T3-L1 preadipocytes:m RNA expression of PPARγ,C/EBP α,C/EBP β,C/EBP 8 and ACC a,FAS,AP2,SCD-1 were detected by quantitative real-time PCR.After differentiation for 2,4,6,8 days,3T3-L1 preadipocytes treated with 10 μg/mL of FA showed no significant effect on mRNA expression of PPAR y;and FA significantly down-regulated the mRNA expression of PPAR y at concentration of 100 μg/mL(p<0.01).The mRNA expression of PPAR y on the day 2,4,6,8 of differentiation were reduced by 40%,30%,53%,33%,respectively. The mRNA expression of C/EBPa showed no significant change(p>0.05)on day 2,4 of differentiation in 3T3-L1 preadipocytes;And compared with control group,the mRNA expression of C/EBPa decreased significantly on day 6 of differentiation(p<0.05).3T3-L1 preadipocytes treated with FA showed no significant change in the mRNA expression of C/EBPβ and C/EBP 8(p>0.05)on the day 2 of differentiation,and decreased significantly with the increasing concentration of FA(p<0.05)on the day 4,6,8 of differentiation. After 8 days of differentiation,compared with the control group,mRNA expression of ACCa,AP2 and FAS were significantly down-regulated by FA at 100 μg/mL(p<0.05),and decreased by 27%,32%,21%,respectively.There was no significant change in the mRNA expression of SCD-1(p>0.05).4.Effect of FA on the key protein expression during 3T3-L1 preadipocytes differentiation:western blot was used to detect protein expression of PPAR γ,C/EBP α,C/EBP β and FAS.After differentiation for 2 days,the protein expression of PPARy and C/EBP α in FA treatment group increased slightly compared with control group(p>0.05);the protein expression of C/EBP β was significantly decreased(p<0.01);and protein expression of FAS decreased with the increasing concentration(p>0.05);protein expression of PPARγ,C/EBP α,C/EBP β and FAS were significantly decreased(p<0.01)on the day 4,6,8 of differentiation,with exception of PPARy on the day 6 of differentiation(p>0.05).5.Effect of FA on MAPK signal pathway:western blot was used to detect the expression of MAPK signal pathway proteins p38,ERK1/2,JNK and its phosphorylation.Compared with the control group,FA significantly increased the phosphorylation level of ERK1/2(p<0.01);the phosphorylation level of JNK increased with the increasing concentration,and the difference was statistically significant at concentration of 100μg/mL(p<0.01);FA had no significant effect on p38 phosphorylation(p<0.05).6.Effect of FA on lipolysis of adipocytes:the concentration of NEFA was detected by colorimetric method;and the mRNA expression of HSL was detected by quantitative real-time PCR.After adipocytes were treated with FA for 24 h,compared with control group,the content of NEFA increased slightly at concentration of 10 and 50 μg/mL(p>0.05),and significantly increased at concentration of 100 μg/mL(p<0.01).Compared with control group,the mRNA expression of HSL had no significant change in the treatment with 10 and 50 μg/mL of FA(p>0.05),and was remarkably up-regulated in the treatment of 100 μg/mL of FA(p<0.01).The results of this study showed that FA had no effect on the proliferation of 3T3-L1 preadipocytes and no cytotoxicity in the concentration rang of 5-100 μg/mL;FA inhibited the differentiation of 3T3-L1 preadipocytes in a concentration-and time-dependent manner.The mechanism that FA suppressed adipogenesis may be to activate MAPK signal pathway,down-regulate the expression of PPAR y.FA could promote the lipolysis in adipocytes and up-regulate the mRNA expression of HSL in the concentration rang of 10-100 μg/mL. |
