Exploring The Molecular Basis For The Nature Of Cold And Hot Herbs Related With The Pathway Of UCP1/PGC-1a

Objective:Cold and hot herbal theory has been drawn from the experience of clinical medication for thousands of years.It still exists guide meaning in the process of modern clinical medication.But how to explore the molecular mechanism and elucidate the essence of the cold and hot natures of traditional Chinese herbs with application of modern scientific and technological achievements and methods,remains the difficulties and breakthrough points of traditional Chinese herbs researches.Brown adipose tissue(BAT),a special tissue which translates energy to generate heat through a series of decoupling reactions,plays an important part in the regulation of energy balance.BAT is composed of numerous brown adipocytes that contain multilocular lipids and large mitochondrias with uncoupling protein 1(UCP1),a marker uncoupling protein of BAT found in the inner membrane of mitochondria.Meanwhile,the expression UCP1 is modulated by PPARy coactivator-1?(PGC-1?)as a coactivator of PPARy.In our previous integral experiment,we found that cold and hot herb have a certain impact on the gene,protein expression of UCP1 and PGC-1? of BAT with thermocouple technique.Based on this,we try to detect that the typical cold herb and hot herb have some effects on the gene and protein expression of UCP1 and PGC-1 a in vitro study,to reveal the molecular basis and explore evaluation method of the cold herb and hot herb.Methods:Primary brown precursor of adipocytes on the dorsum of the newborn mice were cultivated in vitro for a period of time,and cell proliferation was measured by MTT method.Then the primary brown precursor were induced into mature adipocytes by the classic"hormone cocktail" method with insulin,IBMX and DEX.Subsequently,cell morphologic changes of the brown precursor were observed.After adding ten kinds of drug containing serum in the cold herbs(Radix Scutellariae,Rhizoma coptidis,Cortex phellodendri,Radix sophorae Flavescentis,Herba houttuyniae,Radix gentianae,Radix puerariae,Fructus gardeniae,Herba artemisiae Annuae,Radix et rhizoma rhei)and simplex components(jasminoidin,baicalin,emodin,berberine,sodium houttuyfonate,gentiopicrin,phellodendrine,artemisinin,matrine,puerarin);ten kinds of drug containing serum in the hot herbs(Radix aconitilateralis Preparata,Rhizoma alpiniae Offcina.rum,Radix angelicae,Cortex cinnamomi,Fructu.s evodiae,Radix notoginseng,Herba ephedrae,Rhizoma curcumae Longae,Fructus piperis,Radix angelicae Sinensis),and simplex components(galangin,curcumin,aconitine,evodiamine,ferulic acid,capsaicin,ephedrine,notoginsenoside,isoimperatorin,paeoniflorin)for 24 hours.After 24 hours,fluorescence quantitative PCR was used to detect the gene expression of UCP1mRNA and PGC-1?mRN A of mature adipocytes in different groups.Western blotting assay was used to detect the protein differential expression of UCP1 and PGC-1? of mature adipocytes in different groups respectively.Results:(1)MTT results showed within certain concentrations some simplex components of cold herb had negative effects on cell activity,inducing cell contracting to round.Some simplex components of hot herb had positive effects on cell activity,increasing the cell density.(2)A large amount of oil droplets were contained in the abundant cytoplasm of differentiated mature adipocytes.(3)PCR results showed compared with the control group some simplex components of cold herb lowered the expression of target gene gradually all along.Gentiopicrin,phellodendrine,matrine,puerarin significantly reduced the gene level of UCP1(P<0.05).Jasminoidin,emodin,matrine significantly reduced the gene level of PGC-1?(P<0.05).Moreover,some simplex components of hot herb increased the expression of target gene gradually all along.Aconitine,evodiamine increased the gene level of UCP1(P<0.05),ephedrine increased the gene level of PGC-1? significantly(P<0.05).In addition,compared with the control group some drug containing serum of cold herb lowered the expression of target gene gradually all along.Radix gentianae,Radix et rhizoma rhei significantly reduced the gene level of UCPI(P<0.05).Radix sophorae Flavescentis,Radix gentianae,Fructus gardenia,Herba artemisiae Annuae,Radix et rhizoma rhei reduced the gene level of PGC-1? significantly(P<0.01 or P<0.05).Furthermore,some drug containing serum of hot herb increased the expression of target gene gradually all along.Herba ephedrae,Rhizoma curcumae Longae increased the gene level of UCP1(P<0.05)significantly,Fructus evodiae,Herba ephedrae increased the gene level of PGC-1?(P<005)significantly.(4)Western blot results showed compared with the control group some simplex components of cold herb lowered the expression of target protein gradually all along.Emodin,matrine,gentiopicrin,artemisinin,sodium houttuyfonate significantly reduced the protein level of UCP1(P<0.01).Some simplex components of hot herb increased the expression of target protein gradually all along.Notoginsenoside,ephedrine,curcumin,ferulic acid,aconitine,capsaicin significantly increased the protein level of UCP1(P<0.05 or P<0.01).Compared with the control group some drug containing serum of Radix sophorae Flavescentis significantly reduced the protein level of UCP1(P<0.01).Some drug containing serum of Fructus evodiae significantly increased the protein level of UCP1(P<0.05).Conclusion:(1)Simplex components of cold herb had negative effect on cell activity,inducing cell contracting to round.And drug containing serum and simplex components of cold herb inhibited the gene differential expression of UCP1 mRNA and PGC-la mRNA,protein differential expression of UCP1.(2)Simplex components of hot herb had positive effect on cell activity,increasing cell density.And drug containing serum and simplex components of hot herb promoted the gene differential expression of UCP1 mRNA and PGC-1? mRNA,protein differential expression of UCP1.