Rehmannia Glutinosa Inhibits Differentiation Of Preadipocyte And Adipogenesis

Obesity is characterized by an increase in the size of the adipocytes as a result of lipid accumulation and an increase in the number of adipocytes.It is considered to be one of the most important risk factors for noninsulin-dependent diabetes mellitus(NIDDM),diabetes,cardiovascular diseases and cerebrovascular diseases.The research on obesity is very popular in current medical research area because of its high incidence and profound social effects.Rehmannia Glutinosa is a Traditional Chinese Medicine(TCM),which has been used for enhancing physical strength.The chemical compositions of Rehmannia have been studied since 1970's.Tomoda reported the classical method to separate the water-soluble constituents of Rehmannia.Ever since then,it was reported that iridoids,sugars and amino acids have been isolated from Rehmannia.According to its effect of enhancing physical strength,we investigated the effect of Rehmannia extract(RE) on the differentiation of preadipocyte and HFD induced obesity,the effective component and molecular mechanism of RE to inhibit the differentiation of preadipocyte,the effect of betaine,a component of Rehmannia,on the differentiation of preadipocyte and adipogenesis.PartⅠEffect of RE on the differentiation of preadipocyte and HFD induced fat rat modelIn our experiment,different concentration of RE was added to the 3T3-L1 cell culture medium to observe the effect of RE on the differentiation of preadipocyte.Our findings show that RE inhibits the differentiation of preadipocyte in a dose dependent manner.At the molecular level,RE inhibits the expressions of key transcriptional regulator C/EBPβand C/EBPα,and adipocyte phenotype protein 422/aP2.C/EBPβhas two kinds of isoform.The 38kD form of C/EBPβhas DNA binding activity and transcriptional active activity,while the 18kD form only has DNA binding activity.Our results showed that the expression of 18kD was not affected by RE while the 38kD form was nearly inhibited completely by RE.Furthermore,both the expression level of 30kD form and 42kD form of C/EBPαwas inhibited by RE.Serum pharmacology of traditional Chinese medicine(TCM) has been used to study the TCM pharmacology in vitro since 1980's.In our research, rats were orally administrated with RE or water twice a day and lasted for 3 days,one hour after the last administration,blood was separated and serum was got,after inactivated,the serum was added to the cell culture system to test the effect of RE contained serum on the preadipocyte differentiation.The results showed that RE contained serum could inhibit adipocyte differentiation compared with the control serum at a dose dependent manner.Then we established HFD induced fat rats model and tested the effect of RE on the HFD induced obesity.The results showed that rats fed with HFD plus RE had reduced body weight,body weight index,weight of fat tissue,TG level,and LDL level,and increased level of HDL.These results suggest that RE prevented the development of HFD-induced adipogenesis in vivo.PartⅡEffective component of RE to inhibit the differentiation of preadipocyteHaving confirmed that RE could inhibit preadipocyte differentiation,we used LC-MS to investigate the effective component involved in RE to inhibit preadipocyte differentiation.Among 18 fractions,HPLC identified a fraction named Hx that contains 50%of the RE activity.Then Hx was separated by Atlantis HILIC Silica column,which is a kind of hydrophilic column.A combined fraction named Hx-9a which had nearly half activity of Hx was got.KS-802 sugar column was used to separate Hx-9a and mass spectrometry showed that this fraction contains a series of carbohydrate,which including monosaccharide,sugar alcohol,disaccharide and trisaccharide.PartⅢRE inhibited the mitotic clonal expansion during differentiation of preadipocyte and its molecular mechanismIn order to explain why RE inhibited the differentiation,we investigated the molecular mechanism by which the differentiation of preadipocyte was inhibited.Treatment of growth-arrested 3T3-L1 preadipocytes with differentiation inducers initiates mitotic clonal expansion(MCE),which is characterized by synchronous re-entry of the cells into the cell cycle.Our results showed that the time window for RE to inhibit the differentiation is between 0～16 hours after induction.We provided evidence that the inhibition of adipogenesis by RE occurs with the cell number not increasing.The expression level of cdk2 was down regulated by Rehmannia.after induction of differentiation,C/EBPβin RE-treated cells exhibits loss acquisition of DNA-binding activity and centromeric localization as observed with 3T3-L1 adipocytes.Rehmannia inhibited the differentiation of preadipocyte by impaired cell cycle and the expression and localization to the centromers of C/EBPβ.PartⅣBetaine inhibits the differentiation of preadipocyte and adipogenesisIn the process of using Atlantis HILIC silica column to separate Hx,the mass spectrum of one fraction was especially similar with that of betaine.It suggested that betaine or betaine like compound or betaine related compound might be involved in Hx.And we proved that betaine inhibits preadipocyte differentiation in vitro and prevents HFD induced body and fat tissue weight increase as well.At the molecular level,we provide evidence that betaine inhibits expression of the key adipocyte differentiation regulators C/EBPβ, C/EBPαand the terminal marker protein 422/aP2 during the differentiation of preadipocyte to adipocyte.Using HFD induced fat rats model,we confirmed that betaine could prevent the HFD induced obesity. Taken together our results suggest that RE and betaine function to inhibit preadipocyte differentiation in vivo and adipogenesis in vitro.The oligosaccharide involved in Hx could inhibit the differentiation of preadipocyte as well.Therefore,we used modern molecular biology technology and cellular biology to test that the traditional Chinese medicine Rehmannia could inhibit adipocyte differentiation and adipogenesis and its effective component and molecular mechanism to inhibit the differentiation of preadipocyte.