Pomegranate flower extract bidirectionally regulates the proliferation, differentiation and apoptosis of 3T3-L1 cells through regulation of PPARγ expression mediated by PI3K-AKT signaling pathway

Abstract

ObjectivesPomegranate flower is a kind of uygur medicine with anti - type 2 diabetes, anti - lipid, anti - inflammation, anti - oxidation. We investigated the effect of pomegranate flower extract (PFE) on the proliferation, differentiation and apoptosis of 3T3-L1 preadipocytes, as well as the effects of five compounds in PF on cell differentiation. Methods3T3-L1 preadipocytes were treated with PFE (0.5, 1, 2, 5, 10, 20, 50, 100 μg/mL), quercetin, luteolin, ursolic acid, apigenin and kaempferol (5, 10, 20, 40, 80 μM), and cell viability was measured at 24, 48 and 72 h by Cell Counting Kit-8. The modified cocktail induction method induced the differentiation of 3T3-L1 preadipocytes, and treated them with PFE and the compounds. The lipid accumulation was determined by oil red O staining, and the intracellular triglyceride content was determined by commercial kit. The expressions of PPARγ, C/EBP, LPL, DGAT and aP2 mRNA in mature adipocyte were determined by q-PCR, and the expressions of PPARγ, Akt, p-akt and PI3K protein were determined by western blot. 3T3-L1 preadipocytes were treated with PFE (5, 10, 20 μg/mL) while induced apoptosis by palmitate (300 μM), Hoechst staining to observe apoptosis morphology, Annexin Ⅴ- FITC/PI staining with flow cytometry instrument to detect the number of early and late apoptosis cells, the q-PCR and western blot for determining the Bcl-2, Bax, caspase 3 mRNA and protein expression. ResultsPFE (5, 10, 20 μg/mL) promoted or did not affect the proliferation and differentiation of 3T3-L1 preadipocytes, and reduced the number of early and late apoptotic cells, increased the expression of Bcl-2 mRNA and protein, and inhibited the expression of Bax and caspase-3 mRNA and protein. Furthermore, PFE (40, 60 μg/mL), quercetin (10, 20, 40 μM), luteolin (5, 10, 20 μM), apigenin(20,40 μM), kaempferol (20, 40 μM) significantly restrain the 3T3-L1 different extent proliferation and differentiation of preadipocyte, reduce the accumulation of lipids in adipocyte, reduce expression of adipogenesis factor, PFE(40, 60 μg/mL) inhibited the activation of the PI3K-Akt pathway by inhibiting the expression of PI3K and p-Akt proteins, and inhibited preadipocyte differentiation by reduce the expression of PPARγ protein. ConclusionPFE has a concentration-related bidirectional effect on the proliferation, differentiation and apoptosis of 3T3-L1 preadipocytes, which depends on the regulation of PI3K-Akt pathway, which is of guiding role for PFE in the treatment of type 2 diabetes, hyperlipidemia, obesity and other diseases.