Lucia Satiko Yoshida

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 A mu g/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.

Shikonin, an anti-inflammatory compound of "Shikon", inhibits the neutrophil superoxide (O-2(center dot-)) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca2+ concentration ([Ca2+](i)) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca2+](i) (by fluorescence) and O-2 generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca2+](i) via fluxes such as inositol 1,4,5-trisphosphate-induced Ca2+ release (IICR) and store-operated Ca2+ entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca2+](i) and O-2 generation (IC50 values of 1.45 and 1.12 mu M, respectively) in a synchronized manner. Analyses of specific Ca2+ fluxes showed that shikonin inhibits IICR and IICR-linked O-2 generation (IC50 values: 0.28 and 0.31 mu M for [Ca2+](i) and O-2, respectively), as well as SOCE and SOCE-linked O-2(center dot-) generation (IC50 values: 0.39 and 0.25 mu M for [Ca2+](i) and O-2, respectively). These results suggested that shikonin inhibits the O-2. generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca2+ fluxes such as IICR and SOCE.

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4 alpha) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4 alpha in myofibrils, the effects of TPM4 alpha-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, alpha-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4 alpha-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, alpha-actinin and obvious myofibril striations were undetectable. It is possible that TPM4 alpha-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4 alpha-suppression significantly affects actin filament, and that TPM4 alpha plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

With the aim of developing effective anti-inflammatory drugs, we have been investigating the biochemical effects of shikonin of "Shikon" roots, which is a naphthoquinone with anti-inflammatory and antioxidative properties. Shikonin scavenged reactive oxygen species like hydroxyl radical, superoxide anion (O-2(center dot-)) and singlet oxygen in previous studies, but its reactivity with reactive oxygen species is not completely understood, and comparison with standard antioxidants is lacking. This study aimed elucidation of the reactivity of shikonin with nitric oxide radical and reactive oxygen species such as alkyl-oxy radical and O-2(center dot-). By using electron paramagnetic resonance spectrometry, shikonin was found unable of reacting with nitric oxide radical in a competition assay with oxyhemoglobin. However, shikonin scavenged alkyl-oxy radical from 2,2'-azobis(2-aminopropane) dihydrochloride with oxygen radical absorbance capacity, ORAC of 0.25 relative to Trolox, and showed a strong O-2(center dot-)-scavenging ability (42-fold of Trolox; estimated reaction rate constant: 1.7 x 10(5) M(-1)s(-1)) in electron paramagnetic resonance assays with CYPMPO as spin trap. Concerning another source of O-2(center dot-), the phagocyte NADPH oxidase (Nox2), shikonin inhibited the Nox2 activity by impairing catalysis when added before enzyme activation (IC50: 1.1 mu M; NADPH oxidation assay). However, shikonin did not affect the preactivated Nox2 activity, although having potential to scavenge produced O-2(center dot-). In conclusion, shikonin scavenged O-2(center dot-) and alkyl-oxy radical, but not nitric oxide radical.

Objective: A perception survey of healthcare providers and pharmaceutical industries about the current package insert (PI) was conducted to evaluate whether its layout and issues such as the contents concerning drug-drug interactions are found appropriate.<br>Methods: A questionnaire was sent via the Internet to physicians of various subspecialties, or via the postal service to pharmacy-employed pharmacists and pharmaceutical industries.  It consisted of questions regarding the PI layout, the information contents on drug-drug interactions and other matters about PI revision.<br>Results: The survey showed that the PI is a major source of drug information for physicians (82.4%) and pharmacists (98.7%).  The layout (order of appearance of headings and information about drug interactions in a tabular format) of the current PI is widely accepted by physicians, pharmacists, and pharmaceutical industries.  There was, however, some degree of disagreement within these three groups in the perceptions about the presentation/contents of the several drug interactions, as well as about the timing for its updating.  Around 24% of physicians and 35% of pharmacists view that the content of drug interactions is insufficient, and that information about adverse drug reactions and drug interactions is not enough updated in the PIs.  On the other hand, near 86% of pharmaceutical industries were prone to wait for accumulation of enough data until revising the information in PIs.<br>Conclusions: Differences of perception were found between healthcare providers (i.e., PI users) and industries.  Our survey revealed that the basic layout of the current PI should be preserved, but there are issues such as the contents and updating of information regarding drug interactions and adverse drug interactions that may require modifications according to the healthcare providers’ point of view.