| BackgroundThe acetylation of histone plays an important role in epigenetics through regulating the physiological function of cells and rebuilding chromatin structure.The acetylation and deacetylation of histone is charged by a pair of protease with antistatic function on each other,which are histone acetyltransferase(HAT)and histone deacetylase(HDAC).In mammals,18 subtypes of HDAC have already been discovered.They intimately affect the cell life and are closely related to the development and progression of many diseases,such as tumors and AIDS(Acquired Immure Deficiency Syndrome).For AIDS,HAART(Highly Active Anti-Retroviral Therapy)is the major therapeutic method,but unfortunately,HARRT can only kill the virus in blood and is unable to eliminate all the HIV-1 virus due to the existence of HIV-1 reservoir.Recent years,the development of activation agent for latent HIV-1 virus becomes a hot topic in the study of anti-AIDS.Valproic acid(VPA),Suberoylanilide hydroxamic acid(SAHA)and Trichostatin A(TSA)are HDAC inhibitors found to be able to effectively activate latent virus.In China,traditional Chinese medicines and their compounds are widely used for treatment and adjuvanlt therapy of AIDS.Screening of small molecule components from traditional Chinese medicine should be feasible way to discover HDAC inhibitors that promote the elimination of virus reservoir by activation of latent HIV-1.ObjectiveIn our previous in vitro investigation,Aikeqing 2,a Chinese medicine compound developed by Tropical Medincine Institute,Guangzhou University of Chinese Medicine for treatment and adjuvanlt therapy of HIV/AIDS,showed ability to inhibit HDAC activity.This study investigates HDAC inhibitory effect of compounds contained in Aikeqing 2,in order to identify the active components in Aikeqing 2 that inhibit HDAC.Compounds form Other Chinese medicine were also investigated,for the purpose of finding highly effective HDAC inhibitors.Methods1.Totally 154 single compounds,including 59 Aikeqing 2 containing compounds(17 of them are detectable in blood after oral administration of Aikeqing 2),12 flavonoidal glycosides isolated from the ethyl acetate fraction of Epimedium sagittatum,and 83 compounds of other herbal origin,were assessed for their HDAC inhibitory activity by employing HDAC inhibitor screening kits.2.Compounds showing HDAC inhibitory activity were further assessed their ability to inhibit HDAC3/8 by using HDAC3/8 inhibitor screening kits.3.Ten selected compounds were further investigated the effect on expression of HDAC1～11 proteins in HeLa cells by Western Blot analysis.Results1.HDAC inhibitory effect of the 154 compounds had been evaluated using HDAC inhibitor screening kit.It was found that at the concentration of 100μM,(1)Isoliquiritigenin and salvianolic acid C were the most active components in Aikeqing 2 which inhibited 52.7%and 51.0%of HDAC activity respectively;for compounds of other origin,sanguinarine(from Macleaya cordata)and perperine(from Piper nigrum L)which inhibited 53.1%of HDAC activity(both)exhibited to be the most effective;the other compounds were less active,with inhibitory rates to HDAC activity less than 50%.(2)For flavonoidal glycosides from E.sagittatum,three of them showed moderate ability to inhibit HDAC,with inhibitory rate less than 40%.They are kaempferol-3-O-α-L-arabinopyranoside(38.7%),kaempferol-3-O-β-D-xylopyranoside(31.3%),and kaempferol-3-O-β-D-glucopyranoside(26.5%).2.Eleven compounds had been selected for further evaluation using HDAC3/8 inhibitor screening kit.They are isoliquiritigenin,isoliquiritoside,salvianolic acid A and C(from Aikeqing 2),sanguinarine,piperine,curcumine,isoquercitrin(from other origin),kaempferol-3-O-α-L-arabinopyranoside,kaempferol-3-O-β-D-xylopyranoside and kaempferol-3-O-β-D-glucopyranoside(from E.sagittatum).It was found that at the concentration of 100μM,(1)Isoliquiritoside and isoliquiritigenin(Aikeqing 2 containing components that migrate to blood after oral administration)exhibited to be the most active in inhibiting HDAC3 activity,with inhibitory rate of 99.4%and 98.3%respectively.They also showed to be the most active in inhibiting HDAC8 activity,with inhibitory rate of 63.0%and 68.6%respectively.(2)Isoquercitrin and sanguinarine inhibited 76.0%and 56.4%of HDAC3 activity,and 47.3%and 46.2%of HDAC8 activity,respectively.(3)Kaempferol-3-O-α-L-arabinopyranoside,kaempferol-3-O-β-D-xylopyranoside and kaempferol-3-O-P-D-glucopyranoside inhibited 77.8%,60.6%,53.2%of HDAC3 activity respectively;the inhibitory rates to HDAC8 activity of them were less than 50%.(4)Salvianolic acid A inhibited 43.5%of HDAC3 activity and 52.7%of HDAC8 activity respectively.(5)Salvianolic acid C,piperine and curcummine could not inhibit the activity of HDAC3/8.3.Ten compounds were selected for investigation of the effect on expression of HDAC in HeLa by Western-Blotting.They are isoliquiritigenin,isoliquiritoside,salvianolic acid A and C(in Aikeqing 2);sanguinarine,piperine and isoquercitrin(of other origins);kaempferol-3-O-α-L-arabinopyranoside,kaempferol-3-O-β-D-xylopyranoside and kaempferol-3-O-β-D-glucopyranoside(from E.sagittatum).HeLa cells were treated with a test compound at its low or non-toxic concentration for 24h,and the expression of HDACs,including Class Ⅰ(HDAC1～3 and HDAC8),Class Ⅱ(HDAC4～7 and HDAC9,10)and Class Ⅳ(HDAC11),were detected.Results are as follow:(1)Isoliquiritigenin(25,50μM)dose-dependently reduced the expression level of HDAC4,HDAC7 and HDAC10,the level of HDAC3,HDAC8 and HDAC11(Class Ⅳ)was also reduced to some extent;isoliquiritoside(50,100μM)distinctly inhibited the expression of HDAC3,HDAC5,HDAC7,HDAC9,HDAC 10 and HDAC11.(2)Salvianolic acid A(50,100μM)reduced the expression of HDAC3,HDAC8,HDAC4,HDAC6,HDAC7,HDAC9,HDAC 10 and HDAC 11 in dose-dependent manners;salvianolic acid C(50,100μM)reduced the expression of HDAC5,HDAC 10 and HDAC 11 to some extent.(3)Sanguinarine(3,4μM)dose-dependently inhibited the expression of HDAC1,HDAC3,HDAC8(Class Ⅰ)and HDAC4,HDAC5,HDAC7 and HDAC9(Class Ⅱ).(4)Peperine(100μM)reduced the expression of HDAC6 and HDAC 11.(5)Isoquercitrin(100μM)distinctly reduced the expression of HDAC8 and HDAC11.(6)Kaempferol-3-O-α-L-arabinopyranoside,kaempferol-3-O-β-D-xylopyranoside and kaempferol-3-O-β-D-glucopyranoside(50,100μM)distinctly supressed the expression of HDAC6 and HDAC 10;Kaempferol-3-O-p-D-xylopyranoside(100μM)supressed the expression of HDAC4,HDAC5,HDAC7,HDAC9 and HDAC11.Conclusion1.In our study,isoliquiritrigenin and isoliquiritoside not only significantly inhibited the activity of HDAC3/8,but also suppressed the expression of HDAC proteins.As Aikeqing 2 containing components detectable in blood after oral administration,isoliquiritigenin and isoliquiritoside should be bioactive constituents and might play roles of HDAC inhibitors in vivo.Ability of the two compounds to activate latent HIV should be investigated.2.Salvianolic acid A and C in Aikeqing 2(cannot be detected in blood after oral administration),flavonoids from E.Sagittatum,and sanguinarine also exhibited activity to inhibit HDAC both in activity and protein expression,are also worth further investigating. |
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