Effect Of Vaccarin On 3T3-L1 Adipocytes And Its Regulation Mechanism

The development of adipose tissue includes the proliferation and differentiation of adipocytes,and its growth is regulated by many internal and external factors,such as endogenous insulin and exogenous environment.Studies have shown that Vaccarin is flavonoids extracted from the seeds of the Vaccaria,and can promote angiogenesis.In addition,Vaccarin are also involved in the regulation of important biological processes such as glucose and lipid metabolism disorders in the liver.A number of studies have found that adipocyte differentiation can accelerate wound healing.Whether Vaccarin could regulate the proliferation and differentiation of fat cells and the healing of wounds are still unclear.In this study,3T3-L1 adipocytes were treated with Vaccarin,and its effect on the proliferation,differentiation and migration of 3T3-L1 cells was explored.The potential regulatory mechanism of Vaccarin in the development of adipocytes was elucidated.The role of Vaccarin in would healing is also investigated and the potential mechanism involves the accumulation of fat cells.We mainly get the following results:1.The effect of Vaccarin on the proliferation of 3T3-L1 adipocytes.3T3-L1 cells were treated with different concentrations of Vaccarin(0,50,100 ?M).The cytotoxicity was studied by CCK-8.The result showed Vaccarin does not have any cytotoxicity on 3T3-L1 cells under the studied concentration(P>0.05).Then,cell proliferation was investigated by EdU staining.There was no significant difference in the rate of EdU positive cells compared with the control after treatment with 100 ?M Vaccarin(P>0.05),indicating that it had no significant effect on the proliferation of 3T3-L1 cells.In the scratch test of 3T3-L1 cells,it was found that 100 ?M of Vaccarin could not accelerate the migration of cells to accelerate the recovery of scratches within 24 h treatment.2.The effect of Vaccarin on the differentiation of 3T3-L1 adipocytes.The 3T3-L1 cells were treated with 100 ?M of Vaccarin and then cells were induced to differentiation for 6 days.The results of oil red O staining showed that Vaccarin significantly promoted the differentiation of adipocytes(P<0.05).The results of RT-qPCR and Western blot showed that Vaccarin significantly promoted the mRNA and protein expression levels of adipocyte differentiation-related genes(P<0.05).Also,we found Vaccarin significantly activated PI3K-Akt signaling pathway(P<0.05).3.The effect of Vaccarin on 3T3-L1 adipocyte differentiation is GPR120-dependent.3T3-L1 cells were treated with 100 ?M of Vaccarin,and the cells were harvested for 0,2,4,and 6 days.The mRNA levels of GPR120 were detected by RT-qPCR.The expression level increased significantly with differentiation(P<0.05).The results of oil red O staining showed that treatment of cells with GPR120 inhibitor(50 nM,AH7614)significantly inhibited the effect of Vaccarin on adipocyte differentiation(P<0.05).Western blot analysis showed that AH7614 significantly inhibited the activation of PI3K-Akt signaling pathway by Vaccarin(P<0.05).4.Vaccarin accelerates wound healing by promoting adipogenesis in mice.Eight-week-old white mice with free access to water and diet were used for wound healing experiment.Skin wounded mice were treated with 50 ?M of Vaccarin mixed with 5000 U of streptomycin to prevent infection.The result showed that Vaccarin significantly promoted wound healing(P<0.05)(showing by reduced wound area)after 4 days of treatment.The skin tissue of the wound area after 12 days of treatment was taken for H&E staining.It was found that Vaccarin accelerated the arrangement of the skin tissue at the wound area,and accumulated a large number of fat cells at the wound healing area.Moreover,RT-qPCR showed that Vaccarin significantly promoted the mRNA expression levels of angiogenesis-related genes including Vegf,Fgf,Pdgfa and Igf(P<0.05).In summary,in this study we showed Vaccarin promotes adipogenesis by activation of the PI3K-Akt-GLUT4 signaling pathway in a GRP120 dependent way.We also showed that Vaccarin can accelerate wound healing by promoting angiogenesis related gene expression in vivo.