Berberine Inhibits Preadipocyte Differentiation,Proliferation And Adiposity Through Down-regulating Galectin-3:an Experimental Study

Berberine(BBR),a natural product,has good pharmacological effects on anti-obesity both in animal experiments and clinical trial.The occurrence of obesity involves the hypertrophy(differentiation)and hyperplasia(proliferation)of preadipocytes in white adipose tissue,and galectin-3(Gal-3)is involved in regulating the differentiation and proliferation of preadipocytes.This study aims to investigate the effects of BBR on inhibiting on preadipocyte differentiation,proliferation and adiposity and the roles of Gal-3 in these effects.Firstly,to explore the effects of BBR on inhibiting the differentiation of preadipocytes and the roles of Gal-3 in these effects,BBR was added when the primary preadipocytes were indued to differentiate at the same time.Oil red O staining,triglyceride(TG)content in cells was measured,and the expression levels of adipogenic genes such as peroxisome proliferators-activated receptors-?(PPAR-?),CCAAT/enhancer binding protein-?(C/EBP-?)and fatty acid-binding protein-4(FABP-4 or aP2)were detected.Quantitative real-time PCR(qPCR)and Western blot were used to detect Gal-3 expression levels.Gal-3 siRNA(Gal-3 siRNA)and Gal-3 lentiviral activation particles(Gal-3 lentiviral)were used to determine the roles of Gal-3 in the effects of BBR above.Secondly,to explore the effects of BBR on preadipocyte proliferation and the roles of Gal-3 in these effects,primary preadipocytes were treated with BBR during proliferation.BrdU incorporation and cell number counts were performed.Gal-3 mRNA and protein expressions were also determined.Gal-3 siRNA and Gal-3 CRISPR activation plasmids(Gal-3 CRISPR)were transfected into primary preadipocytes to explore the roles of Gal-3 in the effects of BBR above.Thirdly,to explore the detailed molecular mechanisms under the regulation of BBR on Gal-3 expression,fluorescent reports plasmids containing Gal-3 gene promoter was constructed to detect the effects of BBR on Gal-3 promoter activities and also fluorescent report plasmids containing wild-type and point mutation of Gal-3 3'UTR region were constructed to detect the effect of BBR on the stability of Gal-3 mRNA.The effects of BBR on some microRNAs(miRNAs)expression which could regulate Gal-3 gene expression were also detected.Finally,to explore the effects of BBR on inhibiting high-fat diet induced obesity(DIO)and the roles of Gal-3 in these effects.Normal mice were infected with recombinant adenovirus encoding the mouse Gal-3 gene(Ad-Gal-3),and given high-fat diet and treated with BBR.To determine the adiposity of mice,body weight and fat tissue weight were record,and magnetic resonance imaging(MRI)was performed.Immunohistochemistry was performed to detect the Gal-3 protein expression,qRT-PCR was used to determine the expression levels of Gal-3 mRNA and related miRNAs in epididymal white adipose tissue(eWAT).Our results showed that BBR greatly suppressed the differentiation and proliferation of mouse primary preadipocytes isolated from eWAT,during which the expression level of Gal-3 was down-regulated significantly.Gal-3 silencing could reproduce the effects of BBR.In vitro overexpression of Gal-3 totally abolished the suppressive activities of BBR on preadipocyte differentiation and proliferation.BBR reduced Gal-3 promoter activity,destabilized its mRNA and inhibited firefly luciferase activity of a recombinant plasmid containing the Gal-3 3'untranslated region(UTR).Furthermore,BBR up-regulated miRNA let-7d expression and the suppressive activity on Gal-3 3'UTR was abolished by point mutation on the let-7d binding site.In mice fed HFD,BBR up-regulated let-7d and downregulated Gal-3 expression in eWAT;it also suppressed adipocyte differentiation and proliferation and reduced adiposity greatly.In vivo overexpression of Gal-3 totally abolished the suppressive activities of BBR on preadipocyte differentiation and proliferation as well as adiposity.In summary,our study proves that BBR inhibits the differentiation and proliferation of preadipocytes through down-regulating Gal-3,which is closely associated with its anti-obesity effect.Our results may support the future clinical application of BBR for the treatment of obesity or related diseases.