Latent HIV Activator From Wikstroemia Chamaedaphne Meisn. And Its Mechanism

HIV latency is a major obstacle to the cure of AIDS,and the discovery of highly potent HIV latent activators is an important way to solve this problem.Natural products are important sources for the discovery of latent activators of HIV.Wikstroemia chamaedaphne Meisn.is a medicinal herb in Shanxi province.It is traditionally used to treat diseases such as infectious hepatitis.In tour previous work,many tigliane-type and daphnane-type diterpenes natural products were obtained from Wikstroemia chamaedaphne Meisn.Therefore,this study intends to test the latent activation activity of the diterpenes that have been obtained,to screen out high-potential latent activators,and to study their activation mechanism.This thesis mainly includes the following:1.J-Lat A2 cells and He La-NH2 cells were used to evaluate the latent HIV activation activity of 17 diterpenoid compounds in Wikstroemia chamaedaphne Meisn.It was found that the three diterpenoids,pimelea factor P2,pimelea factor P8 and Wikstroelide E exhibited extremely strong activation at a concentration of 10 n M: a)The activation multiples of latent HIV in A2 cells are 5.85,6.32 and 6.96,respectively;b)The activation folds in NH2 cells were 15.91,14.99,and 20.58,respectively.At the same activation multiple,the concentration of Wikstroelide E is only 1/500 of the positive drug prostratin.2.He La-NH1,He La-NH2,J-Lat A2 and J-Lat 2D10 cells were used to test the activation of latent HIV by Wikstroelide E under different concentrations and different treatment time.The results showed that Wikstroelide E activated the latent HIV in a concentration-and-time-dependent manner.The optimal activation concentration was 5-20 n M,and the optimal activation treatment time was 24 h.3.The transcriptomics and proteomics methods were used to evaluate the gene and protein expression differences of Wikstroelide E-treated J-Lat A2 cells at different times,and GO and KEGG analysis of the differential genes and proteins was performed to explore its biological processes.Transcriptomics results showed that Wikstroelide E treated J-Lat A2 cells for 6 hours,significant cellular processes such as cell message transmission,stimulation information regulation,leukocyte activation,regulation of multicellular tissue processes,positive regulation of biological processes,and signal transduction occurred.Changes,especially MAPK signaling pathway,T cell 17 differentiation via PI3K/AKT signaling pathway,NF-?B signaling pathway and other signal transduction pathways are regulated by Wikstroelide E.Proteomics results showed that after Wikstroelide E treated JLat A2 cells for 24 hours,changes in interleukins,regulation of actin cytoskeleton,mineral absorption,p53-mediated transcriptional regulation,PI3K-Akt signaling pathway have taken place.Further comparison and analysis of differentially expressed genes and proteins showed that the differential genes and proteins had better consistency at the transcription and protein levels.According to KEGG enrichment analysis of transcriptomics and proteomics,it was found that the NF-?B signaling pathway is significantly regulated by Wikstroelide E.4.Cell biology methods were used to verify whether Wikstroelide E could activate latent HIV by affecting the NF-?B signaling pathway.First,western blotting was used to detect the degradation of I?B-? and the nucleation of p65 protein in A2 and NH2 cells after Wikstroelide E treatment.The results showed that Wikstroelide E was used to treat A2 cells and NH2 cells with different concentrations and incubation times,and total protein was extracted and detected by WB.Western blotting pictures showed that the level of i?B-? protein was significantly down-regulated with concentration and time;Wikstroelide E was used to treat A2 cells and NH2 cells at different times,and nuclear and cytosolic proteins were extracted and detected by WB.Western blotting pictures showed that p65 nuclear protein levels were significantly up-regulated and intracellular levels were significantly down-regulated within 60 minutes.Further,NF-?B inhibitors: PDTC and BAY 11-7082 were used to pretreat NH2 cells,respectively,and then treated with Wikstroelide E for 30 min,and it was found that it inhibited the activation of Wikstroelide E on latent HIV.5.DARTS method was used to explore the target of Wikstroelide E.The protein bands showed that Wikstroelide E had obtained a great effect by the teratment of pronase E,and then combined with LC/MS/MS to analysis differential proteins.The results showed that the number of target proteins that Wikstroelide E could bind was 45,of which 18 proteins were stable and 27 proteins were unstable.CETSA successfully verified that Wikstroelide E can stabilize the structure of PKC-?.This result indicates that PKC-? may be the target of Wikstroelide E.