Study On The Effect And Mechanism Of Anti-SIV/HIV Virus Of Icariin And Anhydroicaritin In Vitro

ObjectiveThis study is designed to explore:①The effects of Icariin and its metabolite Anhydroicaritin on the inhibition of Monkey Immunodeficiency Virus and Human immunodeficiency virus in Vitro.②The target of action for Icariin and its metabolite Anhydroicaritin inhibited Human immunodeficiency virus in vitro.③Observe the synergistic effects of between Icariin,Anhydroicaritin and Integrase inhibitors,respectively.Method①Icariin and Anhydroicaritin were tested for their cytotoxicity using CCK-8.②The inhibitory effects of Icariin and Anhydroicaritin on SIV /HIV-1 replication were assessed by Cytopathic effect(CPE)and real-time quantitative PCR(RT-PCR).③The mechanism of Icariin and Anhydroicaritin on anti-HIV-1 was explored by means of Computer Assisted Technology-Molecular Docking Method.④Further Icariin and Anhydroicaritin were detected for their ability to inhibit HIV-1 reverse transcriptase(RT)(HIV-1 RT)and HIV-1 protease(HIV-1 PR)by ELISA method and fluorescence method respectively.⑤Synergistic interaction was evaluated between Icariin,Anhydroicaritin and Integrase inhibitors by observation of cytopathic effect(CPE)and real-time quantitative RT-PCR.Results①The maximum non-toxic concentration of Icariin and and its metabolite Anhydroicaritin is 50μ g//mL,16μ g/mL,respectively.②For SIVmac251,in the cells,the viral load of Icariin(50,25,12.5μ g/mL)and Anhydroicaritin(16,8,4μ g/mL)are significantly lower than that of the virus control group(P<0.01,P <0.05).In the supernatant,the viral load of Icariin(50,25μ g/mL)and Anhydroicaritin(16,8μ g/mL)are also significantly lower than that of the virus control group(P <0.01,P <0.05).③For HIV-1,in the cells,the viral load of Icariin(50,25μ g/mL)and Anhydroicaritin(16,8μ g/mL)are significantly lower than that of the virus control group(P<0.01,P<0.05).In the supernatant,the viral load of Icariin(50,25μ g/mL)and Anhydroicaritin(16μ g/mL)are also significantly lower than that of the virus control group(P<0.01,P<0.05).④The result of molecular docking indicated that the anti-HIV activity is mediated through inhibition of HIV-1 reverse transcriptase and HIV-1 protease activity.⑤The results of ELISA and Fluorescence showed that Icariin(500μ g/mL)and Anhydroicaritin(50μ g/mL)could significantly inhibit the activity of HIV-1 RT,the inhibition rates are 83% and 89 %,respectively.And the positive drug EFV inhibition rate is 90%.Icariin(2mg/mL)and Anhydroicaritin(2mg/mL)significantly inhibited HIV-1 protease activity,the inhibition rates are 83% and 79%,respectively,and the positive inhibition rate is 85%.⑥In the aspect of synergistic interaction,Icariin,Anhydroicaritin and integrase inhibitor Raltegravir on HIV-1 inhibition are higher than the single drug group.Conclusions①Icariin and its metabolite Anhydroicaritin inhibited the replicatio n of SIV/HIV-1 virus,respectively.②The mechanism of Icariin and Anhydro icaritin on anti-HIV-1 are HIV-1 reverse transcriptase(HIV-1 RT)and HIV-1 protease(HIV-1 PR),respectively.③Icariin,Anhydroicaritin and integr ase inhibitor Raltegravir on HIV-1 inhibition are higher than the single drug group.