No abstract

Mitogen‐activated protein kinase‐activated protein kinase 2 (MAPKAPK2) or MK2 is a downstream effector of the MAPK family member p38. The p38‐MK2 signaling axis is well known to regulate the inflammatory response, thus it is an important target of anti‐inflammatory drugs. For MK2, as with other intracellular targets, delivering the drug across the plasma membrane barrier represents a major challenge. Here we conjugate an anti‐inflammatory peptide (AIP‐1) selective for MK2 to a novel cell‐penetrating peptide (CPP‐AIP‐1) to enhance intracellular delivery. We evaluated the cytotoxicity, efficacy, and intracellular uptake of these therapeutic peptides in an in vitro model of liver inflammation using HepG2 human hepatoma cells challenged with lipopolysaccharides (LPS). HepG2 cells were treated with 0.1–1000 μM AIP‐1 or CPP‐AIP‐1 for 24 hours to assess cytotoxicity. A significant decrease in cell viability was observed in response to 1000 μM CPP‐AIP‐1, whereas 1000 μM AIP‐1 had no significant effect on cell viability. Treatment with either peptide at 300 μM did not alter cell viability relative to untreated control. We selected the next highest noncytotoxic dose tested ‐ 100 μM ‐ as the therapeutic dose. HepG2 cells were treated for 24 hours with 1 ng/mL LPS to induce an inflammatory response and co‐incubated with 100 μM AIP‐1 or 100 μM CPP‐AIP‐1. We observed no changes in mRNA expression of the housekeeping genes ACTB, B2M, and RPLP0 under any of the treatment conditions. We found that LPS significantly upregulated the mRNA expression of the proinflammatory cytokines CXCL8 and TNF, and that CPP‐AIP‐1 (but not AIP‐1) co‐incubation decreased expression to basal levels. Consistent with these mRNA expression data, LPS significantly increased CXCL8 secretion, and this was inhibited by CPP‐AIP‐1 only. Inflammation clearly plays an important role in the development and progression of many diseases. Our results demonstrate the functionality of a cell‐penetrating peptide in inhibiting the MK2‐mediated inflammatory response. Support or Funding Information Research reported in this abstract was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103424‐18, Louisiana Biomedical Research Network grant number LSU Subcontract No. PO‐0000002131 (formerly 99989), and Board of Regent – Research Competitiveness Subprogram grant number LEQSF(2018‐21)‐RD‐A‐13

Oxidative stress, an excess of endogenous or exogenous reactive oxygen species (ROS) in the human body, is closely aligned with inflammatory responses. ROS such as hydrogen peroxide (H2O2), superoxide, and radical hydroxyl ions serve essential functions in fighting infection; however, chronic elevation of these species irreversibly damages cellular components. Given the central role of inflammation in a variety of diseases, including Alzheimer’s disease and rheumatoid arthritis, a low-cost, extracellular, non-invasive assay of H2O2 measurement is needed. This work reports the use of a platinum microelectrode array (Pt MEA)-based ceramic probe to detect time- and concentration-dependent variations in H2O2 production by activated RAW 264.7 macrophages. First, these cells were activated by lipopolysaccharide (LPS) to induce oxidative stress. Chronoamperometry was then employed to detect the quantity of H2O2 released by cells at various time intervals up to 48 h. The most stimulatory concentration of LPS was identified. Further experiments assessed the anti-inflammatory effect of dexamethasone (Dex), a commonly prescribed steroid medication. As expected, the probe detected significantly increased H2O2 production by LPS-doped macrophages, subsequently diminishing the pro-inflammatory effect in LPS-doped cells treated with Dex. These results strongly support the use of this probe as a non-invasive, robust, point-of-care test of inflammation, with a high potential for multiplexing in further studies.