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High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing
Published on: January 22, 2019
[Euphorbia fischeriana extract reactivates latent HIV through nuclear factor-κB pathway]
Xiaoyan Pan1, Mingjiao Zhang, Xiaoyun Zeng
1School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.E-mail: xiaoyanpan1201@163.com.
Objective:
To investigate the effect of Euphorbia fischeriana extract on latent HIV reactivation and the pathway involved in this process and discuss the value of Euphorbia fischeriana extract in eliminating HIV.
Methods:
Fresh tissues of Euphorbia fischeriana root were crushed into powder after quick freezing with liquid nitrogen and extracted with acetone followed by a three-day vacuum freeze-drying for dehydration of the extract. The extract (EFE) was separated using RP-C18 column with high-performance liquid chromatography (HPLC) and identified with mass spectrometry (MS). The activity of reactivated latent HIV was analyzed by fluorescence-activated cell sorting in a J-Lat 10.6 cell model treated with EFE (50 µg/mL) for 24 h, using TNF-α (10 ng/mL) as the positive control. The effect of a NF-κB pathway inhibitor (Bay 11-7082) on EFE activity was tested. The changes in P65 expression in the cell nuclei within 2 h and HIV protein p24 expression within 24 h were analyzed by Western blotting in cells treated with EFE.
Results:
EFE was obtained by one-step acetone extraction, and the concentration of prostratin in the extract was around 0.53 mmol/L. About 50% of the cells showed HIV reactivation after treatment with 50 µg/mL EFE for 24 h accompanied by a significantly increased p24 expression. The activity of EFE in reactivating latent HIV was inhibited by Bay 11-7082 in a concentration-dependent manner, and p65 accumulation was detected in the cell nuclei within 2 h.
Conclusion:
EFE we obtained contains the active compounds of prostratin and its analogues and shows a strong capacity to reactivate latent HIV through classical NF-κB pathway.
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