Effect Of Water Extract Of Radix Isatidis On Cell Differentiation And Lipid Metabolism In 3T3-L1 Cells

Objective:To explore the possible mechanisms of water extract of Radix Isatidis(WERI)on proliferation,differentiation and lipid metabolism of murine 3T3-L1 preadipocytes.Methods:(1)Model establishment:To observe the morphological change of 3T3-L1 preadipocytes during the process of induction.(2)Effect of WERI on proliferation of 3T3-L1 preadipocytes:The viability of cell was detected by MTT after treating with different concentration of WERI for 24、48、72h.(3)Effect of WERI on 3T3-L1 preadipocytes differentiation:Different concentration of WERI was added during the process of induction,the level of differentiation and lipid content were detected by Oil Red O Staining method.(4)Effects of WERI on lipid and glucose metabolism:The content of Triacylglyceride and Glucose were detected after 8 days of induction.(5)Effect of WERI on genes:Total RNA was isolated and the mRNA levels were quantitatively analyzed by Real-time fluorescence-based quantitative PCR.(6)Effect of WERI on protein expression:Total protein was extracted after 8 days of induction and was detected by Western-blot.Results:(1)The cell morphology was changed from fibroblast to circular or oval during the induction of 3T3-L1 preadipocytes,as well as the accumulation of lipid droplets.(2)Treated with the concentration range of 0～800mg·L-1 for 24h and 48h,it showed the proliferative effect except the 800mg·L-1 of WERI treated with 48h;treated with 72h,the cell viability was significantly inhibited(P<0.01).(3)WERI significantly inhibited the differentiation of 3T3-L1 preadipocytes and reduced the fat accumulation.(4)WERI remarkably reduced the content of Triacylglyceride and decreased glucose uptake.(5)WERI remarkably down-regulated mRNA levels of PPARγ、C/EBPα、SREBP-1c、FAS、ACC and GPDH.(6)After WERI treatment,the expression of PPARγ、C/EBPα、FAS、FABP4、Adiponectin、AMPK、HMGCR and AceCS1 were significantly decreased and the expression of p-AMPK was increased.Conclusions:(1)WERI effectively inhibited the adipogenic differentiation of 3T3-L1 preadipocytes,reduced lipid synthesis and glucose uptake.(2)WERI effectively inhibited adipocyte differentiation and lipid synthesis by down regulating the expression of transcription factors and downstream target genes.(3)WERI inhibited adipocyte differentiation and lipid synthesis by down-regulating the expression of protein which related to the pathway of differentiation,fatty acid synthesis and cholesterol synthesis and activating the expression of protein related to energy metabolism.