An In Vitro Screen Of Traditional Chinese Medicine Attenuating Adipocyte Proliferation Via Regulating The Expression Of Adiponectin Gene

Objectives: To explore the molecular mechanisms underlying traditional Chinesemedicine (TCM) for the treatment of patients with obesity and provide pharmacologi-cal evidence for clinical practice, we identify the TCM with the ability to inhibit theexpression of adiponectin (ADI) gene.Methods: The experimental protocols are described below. Firstly, cell morpho-logy and MTT assay were used to evaluate the changes in the proliferation of mouse3T3-L1adipocyte treated with the extracts of30different TCMs at the concentrationof0.01,0.1,1, or10mg/ml for24hours. Based on the results, we determined theappropriate concentration at which the extracts of the TCM have minimally effects oncell morphology with the greatest effect on cell proliferation. Secondly,quantitativereal-time PCR was used to determine the effects of the extracts of various TCMs onthe expression of adiponectin gene in3T3-L1adipocytes and mature3T3-L1adipocy-tes. Finally, the1.1kb or2.1kb promoter of human adiponectin gene was amplifiedfrom human genomic DNA derived from blood cells by PCR, and then they weresubcloned into pGL3-Basic vector with luciferase reporter gene. The pGL3-Basicvector including ADI promoter1.1-pGL3-Basic vector, ADI promoter2.1-pGL3-Basic vector and pcDNA3.1-EGFP (negative control) were transferred into3T3-L1adipocytes using lipofectamine2000, respectively. We identified the traditionalChinese medicine with the ability to inhibit the expression of ADI gene by determina-tion of the luciferase activity48hours after incubation of the transferred adipocyteswith the extracts of the TCMs.Results: Based on the results of morphology and MTT assay, we found that thebest concentration of the TCMs was1mg/ml. Using quantitative real-time PCR, wealso found that the TCMs including Fanxieye,Shouwu, Huangqin,Daihuang,Heye, Chaihu,Gouqi,Shangzha and Luobuma enhanced the expression of ADI gene in3T3-L1adipocytes. Moreover, our results showed that the TCMs including Fanxieye,Huangqin,Daihuang and Shanzha increased luciferase activity in the3T3-L1adipocy-tes transferred with ADI promote1.1-pGL3-Basic vector.Conclusions:The TCMs including Fanxieye,Daihuang,Huangqin,and Shanzhanot only inhibited adipocyte proliferation, but also regulated the expression of ADIgene and the luciferase activity representing the expression of ADI promoter. Ourresults clarify the mechanisms underlying TCM for the treatment of patients withobesity and would provide theoretical evidence and a novel intervention to improvethe outcomes of patients with obesity.