Delayed down regulation was the only group of genes with almost no intersection in expression mode between BF S L Ep and cytopiloyne. The genes in this group were again analyzed using the TRANSPATH our site database, in search of upstream effector molecules that were not present in the other two expression modes. This analysis identified one potential pathway pointing to a key regulator, Lck, a member of the Src family of pro tein tyrosine kinases important in T lymphocyte activa tion and differentiation. Delay in inactivation of the ERK pathway during the mid stage of LPS stimulation Since phosphorylation of ERK1 2 plays a pivotal role during the activation of ERK signaling pathway, we used western blot analysis to test our hypothesis that ERK1 2 is a key molecular target for the actions of both BF S L Ep and cytopiloyne.

During LPS sti mulation in THP 1 cells, the phosphorylation level of ERK1 2 molecules was first induced between 0. 5 and 0. 75 h post treatment, it was then suppressed between 1 and 4 h post treatment. When LPS sti mulated THP 1 cells were co treated with test BF S L Ep or cytopiloyne, a similar trend of activation and suppression of phosphorylation of ERK1 2 was observed, but the exact pattern and the level of dephosphorylation of ERK1 2 in phytocom pound treated cells were substantially different, resulting in a delay in the dephosphorylation time course for ERK1 2 in BF S L Ep or cytopiloyne treated cells. Den sitometer analyses showed a 2 3 fold change in phos phorylated ERK1 2 levels for cytopiloyne and between 1 and 4 h post treatment.

Cascade network activities conferred by emodin and BF S L Ep were hypothesized to be mediated by signaling or function via E6 AP, a key member of the E3 ligase enzymes involved in ubiquiti nation pathways. We therefore also tested the ubiquiti nation activity in THP 1 cells treated with or without these two test phytocompounds. By using a ubiquitin enrichment assay to determine polyubiquitin levels of total proteins in test cell samples, we were unable in this case to detect a difference between phytocom pound treated and untreated cells. This result, however, has not ruled out the possibility that emodin or BF S L Ep can preferentially affect ubiquitination AV-951 activity in a specific manner via E6 AP activity. Possible specific subset of protein sub strates in test cells that may be affected in such a man ner by test phytocompounds will need to be evaluated in future systematic studies. Discussion In this study, we characterized the immunomodulatory pharmacogenomics of phytocompounds and herbal extracts on gene expression profiles in LPS induced THP 1 cells, a well established immune cell line, using a tran scriptome approach.