Study On Anti HIV-1 Activity And Mechanism Of Bruceine E,a Compound Extracted From Brucea Javanica

Background : HIV/AIDS is a worldwide epidemic disease threatens to human health;however,no eradicative drugs and preventive vaccine were developed at present.The emergence of drug-resistance attributed to high replication and mutation of HIV has been a major problem,which will quickly break through genetic barrier to develop drug-resistant quasi-species.One of the main goals of anti-HIV-1 infection is to find more patent drugs or new drug targets before the invention of vaccine.Natural products molecule are the sources of new drugs and the precursor of structure.The Chinese traditional herbals are precious resources for new drug discovery and development.Compounds extracted from Chinese herbal medicine by modern means have achieved fruitful results in treatment diease such as anti-infection and anti-tumor and so on.This study is a further investigates of anti HIV-1 activity and its mechanism of bruceine E,a compound extracted from Brucea javanica on the basis of preliminary screening.Part one: Isolation and purification of Bruceine EObjective:To isolate and purificate of Bruceine E from the dry fruits of Brucea javanica and its cytotoxicity was determined.Methods:Dry fruits of Brucea javanica was first extracted by 95%ethanol and next extracted by petroleum ether,ethyl acetate and n-butanol alcohol respectively again.The resulted components of the ethyl acetate part was isolated by various column chromatographies and identified structurally by NMR and HR-ESI-MS.The cytotoxicity of the resulted compound was measured by morphology,MTT assay and LDH assay respectively.Results:Eleven compounds of quassioids were isolated and structurally identified: brusatol,bruceantinol,bruceine M,bruceine,bruceine J,bruceantin,bruceene,yadanziolide S,bruceine E,yadanzioside A,bruceoside A.MTT assay on MT2 cells and TZM-bl cells shows a lower cytotoxicity with CC50 at concentration of 1312?M(95% CI:1157-1487?M)and 988.1?M(95%CI:886.6-1101?M)respectively.Conclusion: Eleven compounds of quassioids were isolated and structurally identified;Brucine E was demonstrated with a low cytotoxicity.Part2.Inhibitory effects of Bruceine E on HIV-1Objective: To evaluate the anti-HIV-1 effect of bruceine E.Methods: The protection effects of bruceine E on MT2 cells infected with HIV-1 were determined by morphology.Luciferase assay was employed to evaluate the inhibitory effects of HIV-1 infectivity of TZM-bl cells.HIV-1p24 in the culture supernatants of MT2 cells infected with HIV-1were measured by ELISA.TI50 was calculated by furmula CC50/IC50.Results:Syncytia development were inhibited by Bruceine E when MT2 infected HIV-1IIIB were exposed to concentration at higher than 80?M.Luciferase assay of TZM-bl cells infected HIV-1IIIB shows an inhibitory effects of bruceine E with IC50 value of 79.76?M(95% CI:61.37 to 103.7?M)and TIvalue of 13.39(TI=988.1/79.76).P24 production of HIV-1 was inhibited with IC50 value of 34.55?M(95% CI:30.48 to 39.17?M)and TI value of 19.43(TI=671.5/34.55).Conclusions : Brucine E can prevent syncytia development and have inhibitory effects on HIV-1 replication.Part3.Study on the inhibitory steps of HIV-1 replication by bruceine EObjective: To further investigate the effect steps of bruceine E.Methods: Time of addition(TOA)approach was carried out to narrow down the targets of action of bruceine E.Step of entry into cell was evaluated by means of addion drugs before infection.TZM-bl cells were employed to evaluate the inhibitory effects on the early stage of HIV-1 replication.Persistent infection was employed to investigate the inhibitory effects on late phase of HIV-1.Reverse transcriptase and protease inhibitory effects were determined by ELISA in vitro.Results:TOA assay shows inhibitory effects both on early and late phase of HIV-1 replication cycle moderately.Inhibitory effects of bruceine E on early phase of HIV-1 replication cycle was also conformed by luciferase assay of TZM-bl cells infected HIV-1 and late phase of HIV-1 replication cycle was also conformed by persistent infection.Further,entry inhibition assay shows no inhibitory effects.Reverse transcriptase and protease inhibitory effects were also shown an inhibitory effects.Conclusions:Bruceine E inhibits HIV-1 replication at early and lately steps,which may be as results of reverse trancriptase and protease inhibition.Part 4:Impact of bruceine E on cellular genes associated with HIV-1replicationObjective : To evaluate the impact of bruceine E on cellular genes associated with HIV-1 replication.Methods : The PCR array included 84 genes associated with HIV-1replication was performed to evaluate their expression differences.Results:Up-regulation genes in MT2 cells treated with bruceine E include APOBEC3 F,APOBEC3G,CDK7,CXCR4,GADD45 A,HTATSF1,IFNG and NFKBIA,among which GADD45 A and IFNG are shown the highst increase as5 fold as untreated MT2 cells.On the contrary,APEX1,BCL11 B,CASP3,CASP8,CCL2,CCL4,CCL5,CD4,CR2,EP300,FOS,HMGA1,STAT1,STAT3,TNF,TNFSF10 and TRIM5 are down regulated,among which seven gene expressions are reduced by 80%,especially,CCL4,CCL5 and CR2 are reduced by 90%.Transcription factors and regulators genes are the commonest genes down regulated.Conclusions : The compound bruceine E may inhibit HIV-1 replication through inducing APOBEC expression and inhibiting cellular transcriptor expression.