6-Shogaol and 10-Shogaol Synergize Curcumin in Ameliorating Proinflammatory Mediators via the Modulation of TLR4/TRAF6/MAPK and NFκB Translocation.

Extensive research supported the therapeutic potential of curcumin, a naturally occurring compound, as a promising cytokinesuppressive anti-inflammatory drug. This study aimed to investigate the synergistic anti-inflammatory and anti-cytokine activities by combining 6-shogaol and 10-shogaol to curcumin, and associated mechanisms in modulating lipopolysaccharides and interferon-É£-induced proinflammatory signaling pathways. Our results showed that the combination of 6-shogaol-10-shogaol-curcumin synergistically reduced the production of nitric oxide, inducible nitric oxide synthase, tumor necrosis factor and interlukin-6 in lipopolysaccharides and interferon-γ-induced RAW 264.7 and THP-1 cells assessed by the combination index model. 6-shogaol-10-shogaol-curcumin also showed greater inhibition of cytokine profiling compared to that of 6-shogaol-10-shogaol or curcumin alone. The synergistic anti-inflammatory activity was associated with supressed NFκB translocation and downregulated TLR4-TRAF6-MAPK signaling pathway. In addition, SC also inhibited microRNA-155 expression which may be relevant to the inhibited NFκB translocation. Although 6-shogaol-10-shogaol-curcumin synergistically increased Nrf2 activity, the anti-inflammatory mechanism appeared to be independent from the induction of Nrf2. 6-shogaol-10-shogaol-curcumin provides a more potent therapeutic agent than curcumin alone in synergistically inhibiting lipopolysaccharides and interferon-γ induced proinflammatory mediators and cytokine array in macrophages. The action was mediated by the downregulation of TLR4/TRAF6/MAPK pathway and NFκB translocation.

Synergistic Inhibition of Pro-Inflammatory Pathways by Ginger and Turmeric Extracts in RAW 264.7 Cells.

Synergy plays a prominent role in herbal medicines to increase potency and widen the therapeutic windows. The mechanism of synergy in herbal medicines is often associated with multi-targeted behavior and complex signaling pathways which are challenging to elucidate. This study aims to investigate the synergistic mechanism of a combination (GT) of ginger (G) and turmeric (T) extracts by exploring the modulatory activity in lipopolysaccharides (LPS)-induced inflammatory pathways and key molecular targets. A Bioplex ProTM mouse cytokine 23-plex assay was utilized to assess the broad anti-cytokine activity of GT in LPS and interferon (IFN)-É£ (both at 50 ng/mL)-activated RAW 264.7 cells. The inhibitory effects of individual and combined G and T on major proinflammatory mediators including nitric oxide (NO), tumor necrosis factor (TNF) and interleukin (IL)-6 were tested using Griess reagents and ELISA assays, respectively. Immunofluorescent staining and Western blot were used to investigate the modulatory effect of GT on key proteins in the LPS/TLR4 signaling transduction. The regulation of murine microRNA miR-155-5p was tested using real-time PCR. The IC50 value and combination index (CI) values were used to demonstrate potency and synergistic interaction, respectively. GT synergistically attenuated a range of pro-inflammatory mediators including inducible NO, major cytokines (TNF and IL-6) and secondary inflammatory cytokines (GM-CSF and MCP-1). GT significantly inhibited LPS-induced NF-kB p65 translocation, the activation of TLR4, TRAF6, and phosphorylation of JNK and c-JUN. Moreover, the suppressive effect of GT on each of the protein targets in this axis was stronger than that of the individual components. Real-time PCR analysis showed that GT suppressed miR-155-5p to a greater extent than G or T alone in LPS-stimulated cells. Our study demonstrates the synergistic mechanism of GT in downregulating LPS-induced proinflammatory pathways at the miRNA and protein levels. Our results establish a scientific basis for the combined application of G and T as an advanced therapeutic candidate in inflammatory diseases with broad and synergistic anti-inflammatory activity and multi-targeted mechanisms.

Sensitivity and specificity of in vitro diagnostic device used for influenza rapid test in Taiwan.

The pandemic influenza A/H1N1 outbreak resulted in 18,449 deaths in over 214 countries. In Taiwan, the influenza rapid test, an in vitro diagnostic device (Flu-IVD), only requires documented reviews for market approval by the Taiwan Food and Drug Administration. The purpose of this study was to investigate the analytical sensitivity and specificity of Flu-IVDs used in Taiwan. Analytical sensitivity and specificity tests were performed for influenza antigens A/California/7/2009 (H1N1) virus, A/Victoria/210/2009 (H3N2) virus, B/ Brisbane/60/08 virus, and human coronavirus OC43. A total of seven domestic and 31 imported Flu-IVD samples were collected, of which, 20 samples had inadequate labeling, including those with removed package inserts or incorrect insert information. The analytical sensitivity of Flu-IVDs for A/H1N1, A/H3N2, and Flu B was 500-1000 ng/mL, 1000 ng/mL, and 1000 ng/mL, respectively. For the 50% cell culture infective dose (CCID50) label, the average A/H1N1 and A/H3N2 sensitivity for Flu-IVDs was log10 5.8 ± 0.5 and log10 6.6 ± 0.5 CCID50/mL, respectively. As to the specificity test, no product cross-reacted with human coronavirus OC43. This study provides important information on the Flu-IVD regulation status and can thus help the government formulate policies for the regulation of in vitro diagnostic devices in Taiwan.

The growth-promoting effect of KGF on limbal epithelial cells is mediated by upregulation of DeltaNp63alpha through the p38 pathway.

Corneal epithelial stem cells are thought to reside in the limbus, the transition zoon between cornea and conjunctiva. Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are two paracrine factors that regulate the proliferation, migration and differentiation of the limbal epithelial cells; however, the underlying mechanisms are still poorly understood. In an ex vivo limbal explant culture, we found that KGF is a more potent growth stimulator for the epithelial outgrowth than HGF. Immunofluorescence studies of the epithelial outgrowth from cells treated with HGF or KGF showed similar expression patterns of keratin-3 and keratin-14. Interestingly, p63 was highly expressed in KGF-treated limbal epithelial sheets but not in those treated with HGF. Kinase inhibitor studies showed that induction of DeltaNp63alpha expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous DeltaNp63alpha was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of DeltaNp63alpha. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with DeltaNp63alpha expression as one of the downstream targets.

Regulation of limbal keratinocyte proliferation and differentiation by TAp63 and DeltaNp63 transcription factors.

PURPOSE: To examine the effects of TAp63 and DeltaNp63 on the proliferation and differentiation of rabbit limbal keratinocytes cultured on human amniotic membrane. METHOD: Real-time Q-RT-PCR was used to quantify the relative abundance of TAp63 and DeltaNp63 transcripts in limbal, peripheral corneal, and central corneal epithelia. Antisense oligonucleotides were designed specifically to suppress the expression of TAp63 or DeltaNp63 in limbal keratinocytes, and their effects on cell proliferation and differentiation were examined. Immunofluorescence was used to examine the expressions of p63 and keratin-3 and -14. RESULTS: The expressions of TAp63 and DeltaNp63 transcripts appeared to be site specific. TAp63 was expressed at the highest level in limbus, decreased by approximately 10-fold in peripheral cornea and was undetectable in the central cornea. DeltaNp63 was also expressed at the highest level in limbus, decreased by approximately 35% in peripheral cornea, and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promoted differentiation. Suppression of DeltaNp63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. CONCLUSIONS: TAp63 and DeltaNp63 affect the proliferation of limbal keratinocytes by a different mechanism. The inhibition by TAp63 antisense oligos appeared to be secondary to the promotion of cell differentiation. In contrast, the inhibition by DeltaNp63 antisense oligos appeared to be independent of cell differentiation.