Comparison of the effects of pachymic acid, moronic acid and hydrocortisone on the polysome loading of RNAs in lipopolysaccharide-treated THP-1 macrophages.

We have proposed that analysis of ribosome-loaded mRNAs (i.e., the translatome) is useful for elucidation of pharmacological effects of phytocompounds in immune cells, regarding the involvement of post-transcriptional regulation mechanisms. In the present study, we compared the effects of pachymic acid from Poria cocos fungus and moronic acid from propolis with those of hydrocortisone on the translatomes of THP-1 macrophages exposed to bacterial lipopolysaccharide (LPS) to find clues to their biological effects. Polysome-associated RNAs collected from cells treated for 3 h with LPS plus each of the compounds were analyzed by DNA microarray followed by analyses of pathways/gene ontologies (GO). Upregulated mRNAs in enriched pathways that were found to contain AUUUA (AU)-rich motifs were checked by real-time PCR, and expression of candidate RNA-binding proteins stabilizing/destabilizing such AU-rich mRNAs was checked by Western blotting. The numbers of upregulated and downregulated genes (fold-changes ± 2.0 versus vehicle-control) were, respectively, 209 and 125 for moronic acid, 23 and 2 for pachymic acid, and 214 and 59 for hydrocortisone treatment. Overlapping with hydrocortisone treatment for upregulation were 158 genes in moronic acid and 17 in pachymic acid treatment; of these, 16 overlapped within all treatments (C-X-C motif chemokine ligands, interferon-induced protein with tetratricopeptide repeats, etc.). Pathway analyses showed GO enrichments such as 'immune response', 'receptor binding', 'extracellular space' etc. The pachymic acid-upregulated mRNAs (highly overlapped with the other 2 treatments) showed the presence of signal peptides and AU-rich motifs, suggesting regulation by AU-rich element (ARE)-binding proteins. The expression of ARE-binding protein HuR/ELAV-1 was increased by the 3 compounds, and AUF1/hnRNP D was decreased by pachymic acid. These results suggested that pachymic acid and moronic acid effects may involve as yet unknown post-transcriptional modulation via ARE-binding proteins resembling that of glucocorticoids.

Shikonin changes the lipopolysaccharide-induced expression of inflammation-related genes in macrophages.

We aimed to find candidate molecules possibly involved in the anti-inflammatory activity of shikonin (active compound of "Shikon") by analyzing its effects on gene expression of lipopolysaccharide (LPS)-treated THP-1 macrophages. Polysome-associated mRNAs (those expected to be under translation: translatome) from cells treated with LPS alone (LPS: 5 µg/mL), shikonin alone (S: 100 nM), or LPS plus shikonin (LPS&S) for 3 h were analyzed by DNA microarray followed by detection of enriched pathways/gene ontologies using the tools of the STRING database. Candidate genes in enriched pathways in the comparison of LPS&S cells vs. LPS cells were analyzed by reverse-transcription quantitative real-time PCR (RT-qPCR; 1, 2, and 3 h). DNA microarray showed shikonin significantly influences gene expression. Gene expression changes between LPS&S cells and LPS cells were compared to detect relevant proteins and/or mRNAs underlying its anti-inflammatory effects: shikonin downregulated pathways which were upregulated in LPS cells, for example, 'innate immune response'. Within changed pathways, three genes were selected for RT-qPCR analyses as key candidates influencing inflammatory responses: CYBA (component of the superoxide-generating Nox2 enzyme), GSK3B (controller of cell responses after toll-like receptor stimulation), and EIF4E (a key factor of the eukaryotic translation initiation factor 4F complex that regulates abundance of other proteins involved in immune functions). All three mRNAs were decreased at 2 h, and CYBA continued low at 3 h relative to LPS cells. Given that shikonin decreased the expression of CYBA gene of Nox2, in addition to the direct inhibition of the Nox2 activity that we have previously shown, it is suggested that one of its anti-inflammatory mechanisms could be attenuation of oxidative stress.