| Object: In order to study the molecular mechanisms of chlorogenic acid resisting inflammation and improving obesity in adipose tissue,we investigated the effects of chlorogenic acid on the differentiation and lipid metabolism with mouse 3T3-L1 preadipocytes in vitro,to provide meaningful reference data for the rational use of chlorogenic acid and the improvement of dietary structure。Methods: Mouse 3T3-L1 preadipocytes were cultured,and the effects of chlorogenic acid in different concentrations were detected by CCK-8 method.Induced differentiation of mouse 3T3-L1 preadipocytes by classic cocktail method,divided into 4 groups: control group(CG),the positive control group of rosiglitazone group(RG),the negative control group of GW9662 group(GG)and chlorogenic acid group(CGA).The cell morphological changes and formation of lipids droplet of mouse 3T3-L1 preadipocytes during differentiation were observed by inverted microscope and oil red O staining method.The triacylglyceride(TAG)accumulation of differentiation cells in different groups was determined by tissue cell triglyceride enzyme method.The expressions of the lipid metabolism related genes mRNA and the important factor of PPARγ2 mRNA during the differentiation process of mouse 3T3-L1 preadipocytes was determined qPCR.The expressions and localization of PPARγ2 protein during the differentiation process of mouse 3T3-L1 preadipocytes was determined by western blot and laser confocal.Results: When the concentration of CGA ≤20μM,after the treatment with CGA,during 48 h,the proliferation of mouse 3T3-L1 preadipocytes was normal,the survival rate of the cells was about 100%,and there was no significantly difference compared with CG group(p>0.05).As differentiation progressed,in CGA group,the area of oil red O staining was larger than that of CG group and GG group,but it was significantly less than that of RG group.The results showed that TAG accumulation in preadipocytes in CGA group was lower than that in CG group and RG group,and compared with CG group it was not significantly(P >0.05),but compared with RG group it was significantly(P <0.05).Compared with CG group,after chlorogenic acid intervention,the expressions of key genes of differentiation related PPARγ2 mRNA and protein were significantly up-regulated,SREBP-1 mRNA was up-regulated,Pref-1 mRNA was down regulated during the differentiation process of mouse 3T3-L1 preadipocytes.The expressions of differentiation related genes C/EBPα mRNA was slightly up-regulated during the metaphase differentiation process of mouse 3T3-L1 preadipocytes.The expressions of differentiation related genes C/EBPβ mRNA was up-regulated during the anaphase differentiation process of mouse 3T3-L1 preadipocytes.The expressions of differentiation related genes RXRα mRNA was no significantly change during the differentiation process of mouse 3T3-L1 preadipocytes.The results showed that the lipid metabolism related genes mRNA by qPCR,after chlorogenic acid intervention,the expressions of Fas mRNA and Adrp mRNA were up-regulated during the whole differentiation process of mouse 3T3-L1 preadipocytes.The expressions of HSL mRNA was up-regulated during the prophase differentiation process of mouse 3T3-L1 preadipocytes.The expressions of Plin mRNA was slightly up-regulated during the anaphase differentiation process of mouse 3T3-L1 preadipocytes.The expressions of lipid synthesis related genes DGAT-1 mRNA was no significantly change during the differentiation process of mouse 3T3-L1 preadipocytes.However,the effect of chlorogenic acid on the expression of DGAT-1 mRNA was significantly different from that of rosiglitazone,the expression of mRNA DGAT-1 was significantly upregulated by rosiglitazone.After chlorogenic acid intervention,there was no excessive accumulation of intracellular TAG after the differentiation of mouse 3T3-L1 preadipocytes.Conclusion: Chlorogenic acid can promote the differentiation of mouse 3T3-L1 preadipocytes,the mechanism is that chlorogenic acid can up-regulated the expression of differentiation related genes PPARγ2 mRNA and protein,C/EBPα and C/EBPβ mRNA,and down-regulated the expression of Pref-1 mRNA.Chlorogenic acid and rosiglitazone is different,although both them can improve the expression of PPARγ2,but after chlorogenic acid intervention,the accumulation of intracellular TAG after the differentiation of mouse 3T3-L1 preadipocytes is significantly less than that in RG group.The mechanism is that chlorogenic acid can up-regulated the expression of lipolysis related genes HSL,Adrp and Plin mRNA,and did not increase the expression of lipid synthesis related genes DGAT-1 mRNA.The results implied that the effects of chlorogenic acid on mouse 3T3-L1 preadipocytes is not exactly same as rosiglitazone. |
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