Effects Of The Supplemented Copper/Zinc Ions On Immuno-Modulatory And Anti-Inflammatory Activities Of Bovine Lactoferrin

Lactoferrin(LF)is a native iron-binding glycoprotein and has anti-microbial,anti-carcinogenic,anti-inflammatory,and immuno-modulatory activities.With increased attention paid on the physiological properties of lactoferrin,the bovine lactoferrin,as one of the protein components of food source,now is commercially produced and used as a food supplement to benefit human health.Many trace elements also have multiple physiological properties and play critical roles in the immune system for optimal immune function.However,whether the trace elements-supplemented lactoferrin has changed bioactivities including immuno-modulatory and anti-inflammatory activity is less studied so far.In this study,a commercial bovine lactoferrin was supplemented with Cu2+/Zn2+ at three contents(0.16,0.32,and 0.64 mg/g LF),and their in vitro immuno-modulatory effects in two immune cells murine splenocytes and RAW264.7 macrophages and anti-inflammatory effect in lipopolysaccharide(LPS)-induced RAW264.7 macrophage cells were assayed.CCK-8 method was used to evaluate the inhibitory effect of bovine lactoferrin and Cu/Zn-supplemented lactoferrin at three chosen dose levels(10,20 and 40 μg/m L)on splenocytes as well as the concanavalin A(Con A)-or lipopolysaccharide(LPS)-stimulated splenocytes.Neutral red uptake was used to evaluate macrophage phagocytosis.Flow cytometry assay was used to evaluate T lymphocyte subpopulations(CD4+ and CD8+),while ELISA method was used to evaluate cytokine secretion,including interleukin-2(IL-2),IL-4,interferon-γ(in splenocytes),IL-1β,and tumor necrosis factor-α(in macrophages).The effect of the supplemented copper ion on immuno-modulatory activities of lactoferrin was thus assayed using these four evaluation reflectors.The results indicated that bovine lactoferrin and Cu/Zn-supplemented lactoferrin products had suppression on splenocytes as well as Con A-or LPS-stimulated splenocytes;however,using lower Cu/Zn supplementation level(i.e.,0.16 mg/g LF)and lower dose(10 μg/m L)alleviated this suppression significantly(P < 0.05).Compared to bovine lactoferrin,Cu/Zn-supplemented lactoferrin product of lower Cu/Zn-supplementation content at lower dose level yielded slightly enhanced macrophage stimulation,increased CD4+/CD8+ ratio of T lymphocyte subpopulations in the Con A-stimulated splenocytes,and significant secretion enhancement for IL-2,IL-4,IFN-γ(in splenocytes),IL-1β,and TNF-α(in macrophages)(P < 0.05).Furthermore,Cu/Zn-supplemented lactoferrin product of higher Cu/Zn-supplementation level(i.e.,0.64 mg/g LF)at higher dose mostly showed opposite effects in the cells,in comparison with its counterpart at lower dose.It is thus concluded that Cu/Zn-supplementation of bovine lactoferrin was able to alleviate or increase bovine lactoferrin’s effects on the two immune cells.CCK-8 method was used to evaluate the effect of bovine lactoferrin and Cu-supplemented lactoferrin at four chosen dose levels(10,20,40 and 80 μg/m L)on cell viability of LPS-stimulated RAW264.7 macrophages.Lactate dehydrogenase(LDH)assay was used to evaluate cytotoxicity of chosen samples to macrophage.The Griess reaction method was used to evaluate nitric oxide(NO)production.DCFH/DA method was used to assay intracellular reactive oxygen species(ROS)production;ELISA method was used to assay the secretion of inflammatory mediators including PGE2,IL-1β,TNF-α,and IL-6.The effect of the supplemented copper ion on anti-inflammatory activities of bovine lactoferrin was assayed using these five evaluation reflectors.The results indicated that bovine lactoferrin and Cu-supplemented lactoferrin products did significantly have inhibitory effect on LPSinduced production of NO,PGE2,TNF-α,IL-6,IL-1β,and ROS,and thus exerted an anti-inflammatory effect.Compared with bovine lactoferrin,Cu-supplemented lactoferrin product of lower Cu-supplementation level at lower dose most led to enhanced inhibition on these inflammatory mediators.In conclusion,Cu/Zn supplementation of bovine lactoferrin can suppress or increase its immuno-modulatory and anti-inflammatory activities.Cu/Zn-supplemented lactoferrin of lower Cu/Zn supplementation level at lower dose can enhance immuno-modulatory effects and anti-inflammatory activity and moreover,Cu2+/Zn2+ content of the supplemented lactoferrin is a key factor that modulates these effects.