moracin-p and Inflammation

Accumulating evidence suggests that inflammation is linked to multiple pathological processes and induces cellular and molecular damage through the activation of inflammatory signaling pathways, including the NF‑κB pathway. The aim of the present study was to identify natural anti‑inflammatory products that can target NF‑κB activity, in order to establish a novel therapeutic approach for inflammatory diseases. Using a 4T1 breast cancer cell line that expresses the firefly luciferase gene under the control of an NF‑κB response element, 112 natural products were tested for their anti‑inflammatory properties. Sohakuhi (Morus alba Linn. bark) extract was observed to strongly suppress NF‑κB activity without affecting cell viability. To further examine the anti‑inflammatory effect of Sohakuhi, tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑induced cellular damage of human HaCaT keratinocytes was evaluated. While TRAIL triggered the phosphorylation of the p65 subunit of NF‑κB, leading to cellular damage in HaCaT cells, treatment with Sohakuhi extract protected HaCaT cells against TRAIL‑induced cellular damage. Moreover, Sohakuhi treatment also upregulated the anti‑apoptotic proteins Bcl‑xL and Bcl‑2. Importantly, through chemical fractionation of Sohakuhi extract, moracin O and P were confirmed to mediate its anti‑inflammatory effects. Collectively, the present results indicated that Sohakuhi and moracin may represent potential candidates for the development of novel anti‑inflammatory drugs.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis Regulatory Proteins; Benzofurans; Cell Line, Tumor; Cell Survival; Humans; Inflammation; Keratinocytes; Mice; Morus; NF-kappa B; Phosphorylation; Signal Transduction

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature