Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages

Abstract

Accumulating evidence indicates that Clara cell protein-16 (CC16) has anti-inflammatory functions, although the involved molecular pathways have not been completely elucidated. Here, we evaluated the effect of recombinant rat CC16 (rCC16) on the expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8 in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells) and explored the underlying molecular mechanisms. It was found that rCC16 inhibited LPS-induced TNF-α, IL-6, and IL-8 expression at both the messenger ribonucleicacid (mRNA) level and protein level in a concentration-dependent manner, as demonstrated by real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Such suppressive effects were accompanied by the inhibition of transcriptional activity and the deoxyribonucleic acid binding activity of nuclear factor (NF)-κB but not activator protein (AP)-1. Western blot analysis further revealed that rCC16 inhibited the increase of nuclear NF-κB and the reduction of cytosolic NF-κB, the phosphorylation and reduction of NF-κB inhibitory protein IκBα, and the p38 mitogen-activated protein kinase (MAPK)-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. Furthermore, rCC16 was found to have no effect on the phosphorylation of c-Jun N-terminal kinase, c-Jun, or the nuclear translocation of c-Jun. In addition, reduction of TNF-α, IL-6, and IL-8 were reversed when the level of endogenous uteroglobin-binding protein was reduced by RNA interference in rCC16- and LPS-treated RAW264.7 cells. Our data suggest that rCC16 suppresses LPS-mediated inflammatory mediator TNF-α, IL-6, and IL-8 production by inactivating NF-κB and p38 MAPK but not AP-1 in RAW264.7 cells.

Curcumin alleviates macrophage activation and lung inflammation induced by influenza virus infection through inhibiting the NF‐κB signaling pathway

“…Previous studies have revealed that NF‐κB signaling pathway is one of the most important pathways that are activated in macrophages under the pathogen challenge . Thus, we aimed to study whether the anti‐inflammatory ability of curcumin is associated with NF‐κB signaling pathway.…”

“…Previous studies have revealed that NF-κB signaling pathway is one of the most important pathways that are activated in macrophages under the pathogen challenge. [34][35][36][37][38] Thus, we aimed to study whether the anti-inflammatory ability of curcumin is associated with NF-κB signaling pathway. By examining the protein expression levels via Western Blotting, we found that IAV infection leads to the decrease in IκBα and cytosolic p65, while enhanced the levels of p-IκBα and nuclear p65.…”

Anti-inflammatory potency of Locusta migratoria manilensis cyclopeptides in mast cells and macrophages

“…The nuclear translocation of NF-kB, which dissociated from its inhibitory protein IkB, is the most crucial step for NF-kB signaling activation. 33 It has been found that LCP-3 suppresses LPS activated NF-kB signaling by inhibiting phosphorylation of IkBa. Moreover, LCP-3 attenuates the phosphorylation of IKKa/b in both types of cells.…”

Renal dysfunction decreases specificity and overturns cutoff value of serum CC16 while prohibiting 7-day mortality prediction in diagnosing acute respiratory distress syndrome in intensive care unit.

“…As an immunoregulatory protein, CC16 executes an anti-inflammatory function by inhibiting phospholipase A2 activation and promoting the expression of inflammatory cascades (IL-1b, IL-8, et al), TH2 cell differentiation, and the migration of neutrophils and monocytes [23,24]. Consistent with these studies, Pang demonstrated that recombinant rat CC16 protein inhibits LPS-induced MMP-9 expression and the production of pro-inflammatory cytokines via NF-κB pathway in model of tracheal epithelial cells and RAW264.7 macrophages [25,26]. She suggested that exogenous supplementation of recombinant CC16 ameliorates cigarette smoke-induced lung inflammation in a murine disease model of chronic obstructive pulmonary disease [27].…”