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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 - 28/10/20

Doi : 10.1016/j.biopha.2020.110769 
Tong Li a, Ling Zhang a, Chen Jin b, , Yao Xiong a, Yu-Yao Cheng a, Kang Chen a
a School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, China 
b Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, China 

Corresponding author.

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Highlights

Pomegranate flower extract regulates the PI3K-Akt signaling pathway bidirectionally.
Different doses of pomegranate flowers have the opposite effect on preadipocyte.
Pomegranate flower extract inhibits adipocyte differentiation.
The components in the pomegranate flower act synergistically in cell differentiation.

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Abstract

Objectives

Pomegranate 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.

Methods

3T3-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.

Results

PFE (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.

Conclusion

PFE 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.

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Keywords : Pomegranate flowers, Akt-PI3K signaling pathway, PPARγ, Lipid accumulation, Apoptosis, Active component


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