Low-level Cu-fortification of bovine lactoferrin: Focus on its effect on in vitro anti-inflammatory activity in LPS-stimulated macrophages

Bovine lactoferrin (LF) per 1 g was reacted with 0.16, 0.32, and 0.64 mg CuCl(2) to reach 10%, 20%, and 40% copper-saturation, respectively, aiming to assess their anti-inflammatory activities to lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The macrophages treated with CuCl(2) at 0.051 μg/mL dose did not have obvious change in cell viability, lactate dehydrogenase (LDH) release, and intracellular reactive oxygen species (ROS) production. However, LF and Cu-fortified LF products (10−80 μg/mL doses) mostly showed inhibitory effects on the stimulated macrophages dose-dependently. Moreover, Cu-fortified LF products of lower Cu-fortifying levels at lower doses exerted weaker inhibition on the stimulated macrophages than LF, leading to higher cell viability but decreased LDH release. Meanwhile, LF and Cu-fortified LF products at 10 and 20 μg/mL doses showed different activities to the stimulated cells, via partly decreasing or increasing the production of inflammatory mediators namely prostaglandin E(2) (PGE(2)), nitric oxide, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and ROS production, depending on the used Cu-fortifying and dose levels. Compared with LF, Cu-fortified LF product (Cu-fortifying level of 0.16 mg/g LF) at 10 μg/mL dose showed enhanced inhibition on the production of PGE(2), ROS, IL-1β, and TNF-α, evidencing increased anti-inflammatory activity. However, the inhibition of Cu-fortified LF product (Cu-fortifying level of 0.32 mg/g LF) at 20 μg/mL dose on the production of these inflammatory mediators was mostly reduced. It is thus proposed that both Cu-fortifying and dose levels could affect LF's anti-inflammatory activity in LPS-stimulated macrophages, while the Cu-fortifying level of LF could govern activity change.