Anti-inflammatory activities of Waltheria indica extracts by modulating expression of IL-1B, TNF-α, TNFRII and NF-κB in human macrophages.

In Hawaiian traditional medicinal practices, the indigenous 'uhaloa, Waltheria indica var. Americana is one of the most recognized plants. Waltheria is also known in various cultures as a medicinal plant for the treatment of inflammatory conditions. Results in human subjects and cell and animal models supported anti-inflammatory activity for the Waltheria flavonoid quercetin, and for crude plant extracts, limited animal studies also confirmed anti-inflammatory effects. Yet no systematic studies have examined immune or inflammatory responses affected by these extracts. In order to gain insight into inflammatory cascades modulated by Waltheria extracts, and to uncover the mechanistic bases for the effective use of this medicinal plant as a natural anti-inflammatory agent, we have undertaken analyses of LPS and TNF-α/IF-γ-stimulated human macrophages treated with Waltheria extracts using targeted qRT-PCR and Inflammation Panels to test differential mRNA expression of two hundred immune-related genes, furthermore, ELISA assays and Inflammatory Protein arrays to determine extracts-modulated intracellular and secreted levels of prominent cytokines. Results demonstrate that Waltheria extracts inhibit key inflammatory cytokines and cytokine receptors including protein levels of IL-1B, IL-1ra, IL-8 and IL-6, reduce both mRNA and protein levels of TNF-α and protein levels of its receptor, TNF RII, predicting diminished TNF-α-associated inflammatory signaling that, together with significant reduction of NF-κB mRNA and protein, can effectively diminish activities of multiple pro-inflammatory signaling pathways and mitigate key processes in diseases with inflammatory components.

Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation.

Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (K(d)) of 2.5 nm. This was comparable with our measured K(d) of LOX binding to tropoelastin (1.9 nm) and type I collagen (5.2 nm), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity.