Effect Of The Compound Folium Isatidis Injection On HIV Entry And Replication

Background and ObjectiveAcquired immunodeficiency syndrome (AIDS), a severe infectious disease, is caused by human immunodeficiency virus (HIV) infection. The virus have the ability to reduce or break the body's immune function and ultimately to result in various opportunistic infections and malignant tumors. In the course of HIV infection, expressing CD4 receptor T lymphocytes are the main attacked target cells, and the effect of co-receptors CXCR4 and CCR5 is also critical for HIV entry. In recent years, designing anti-AIDS drugs targeting co-receptors has become a novel hotspot, expecting to work during the early phase of the infection.The traditional Chinese medicine of Folium isatidis belongs to Cruciferous herba- ceous plant, which has several characteristics-bitter in taste, coldnatured, clearing heat and neutralizing poison to name a lot. The effective components of Folium Isatidis consist of indigotin, indirubin, tryptanthrin,4(3)-quinazoline-dione, organic acids, etc. Nowadays Folium isatidis has been widely used in clinical practice contributed to its function of anti-inflammatory, anti-virus and immunity enhancement. It is reported that there exists evident effect of Folium isatidis on anti various virus, such as influenza virus, coxsackie virus, Japanese encephalitis virus, herpes simplex virus and verruca plana virus. As the effective medicine against various virus, it is required to explore and study its effect on anti-AIDS and its possible mechanisms.Herein we investigate the effect of the compound Folium isatidis injection on the activity of CXCR4/CCR5 promotors in HT-H9 cells cultivated in vitro, and the probable mechanism of the compound Folium isatidis injection on CXCR4/CCR5 promotors, which may downregulate the expression of CXCR4/CCR5 protein and finally result in blocking of HIV entry. Furthermore, pseudotyped virus pNL4-3.luc.R-E- was applied to verify the effect of the compound Folium isatidis injection and to study the activity of inhibiting HIV replication. The two index referring to the relative luciferase activity and p24 protein level was served as the basis for assessing the effect on viral replication in order to investigate if there exists inhibitory effect of the compound Folium isatidis injection on HIV replication.Methods:Using Liposomal Transfection technology to transfect two kinds of luciferase reporter vectors called pGL4.17-CXCR4 and pGL4.17-CCR5 into HT-H9 cells. Then screened by G418 for two weeks to obtain the lasting transfected cell lines and for further expanding culture. The transfected cells were divided into four groups:the compound Folium isatidis injection high dose group, low dose group, physiological saline group and the control group, and each grouped cells were handled with the compound Folium isatidis injection for 24 hours. The effect of the compound Folium isatidis injection on CXCR4/CCR5 promotors was evaluated by measuring the level of the luciferase activity.293T cells were co-transfected by pseudotyped virus pNL4-3.luc.R-E- and plasmid phRL-SV40 and were divided into the following groups after expanding:the compound Folium isatidis injection high dose group, low dose group, physiological saline group, the control group and AZT positive control group. After treated with the compound Folium isatidis injection for 24 hours, the effect on viral replication was evaluated by analyzing the value of relative luciferase unit (RLU), and ELISA assay was used to quantify the level of p24 protein expression for further validating the inhibitory effect of the compound Folium isatidis injection on viral replication. Data obtained was analyzed by statistical software SPSS 13.0.Results:1. The stable transfected cell lines respectively including pGL4.17-CXCR4 and pGL4.17-CCR5 were successfully constructed through G418 screening.2. The luciferase activity of HT-H9 cells showed that for CXCR4 promotor, the level of luciferase activity of the compound Folium isatidis injection groups, both high dose group and low dose group, were remarkably lower than that of control (P<0.01); For CCR5 promotor, there have no change in low dose group compared to the control group (P>0.05), but the fluorescent value in high dose group was decreased when compared with that of the control group (P<0.05).3. The luciferase activity of 293 T cells showed:the value of relative luciferase unit of the compound Folium isatidis injection low dose group showed no difference compared to the control group (P>0.05); the fluorescent value of the compound Folium isatidis injection high dose group were all significantly decreased when compared with that of the control group (P<0.01); and the value in high dose group was higher than that of AZT group (P<0.05).4. Results of ELISA assay were:the level of p24 protein in the compound Folium isatidis injection low dose group showed no difference compared to the control group (P>0.05); and p24 protein in the compound Folium isatidis injection high dose group were significantly decreased when compared with that of the control group (P<0.01); and compared with AZT group, the level was higher (P<0.05).Conclusion1. The compound Folium isatidis injection can suppress the activity of both CXCR4 and CCR5 promotors in HT-H9 cells cultivated in vitro, which were thus presumed to posses the ability to inhibit HIV entry indirectly together with the potential anti-AIDS effect.2. The compound Folium isatidis injection can depress the value of relative luciferase unit in the transient transfected 293 T cells with pNL4-3.luc.R-E-, which may have inhibitory effect on pseudotyped HIV replication consistent with the potential anti-AIDS effect.