Mechanisms Of Osteogenesis Activity Of Lactoferrin And The Effects Of Thermal-treatment On The Activity

Lactoferrin, a kind of iron-binding glycosidoprotein, is widely consist in themilk of mammals. It has many kinds of functions such as antibacterium, antivirusand immunoregulation. There are little lactoferrin consist in the eccrineincluding tears, synovia and saliva. The effect of Lactoferrin on bone growth andmetabolize gradually attracts more and more attentions. In the present study, theisolation and purification procedures of lactoferrin from fresh bovine milk hasbeen optimized, the effects of lactoferrin on the proliferation of osteoblast andthe effects on mitogen activated protein kinase(MAPK) signaling transduction ofosteoblast induced by lactoferrin have been investigated. Moreover, the effects ofheat-treatment on the osteogenesis activity of lactoferrin and the MAPKsignaling transduction have been determined. The results will establish sometheoretical basement of the research and development of lactoferrin in foodindustry, especially for the determination of suitable processing parameters oflactoferrin added in different food products as a functional components oradditives.The lactoferrin from fresh bovine milk was purified by some basicpre-treatment such as sterilizing by microfiltration membrane, defatting andcasein-removing. The crude lactoferrin was purified by two sequentchromatography, SP Sepharose Big Bead ion exchange chromatography (16×50cm) and Superdex200gel chromatography (1.6×60cm), the ultrafiltration andvacuum freeze drying. During the refine process, the elution parameters ofSuperdex200chromatography has been optimized. The molecular weight ofpurified Lactoferrin was76kD estimated by SDS-PAGE, the purity was93.82%,the yield was68.19%. The denaturation temperature of lactoferrin was70.4℃and88.2℃determined by Differential Scanning Calorimetry. Theheat-processed parameter has been set:62-65℃/30min,72℃/10s,85℃/10minand95℃/10min according to the the denaturation temperature companied withthe parameter of heat-processed of milk in production.Osteoblast cells were extracted from newborn SD rats. The methods toidentify osteoblasts were morphological observation, HE staining, alkalinephosphatase staining and mineralized nodules staining. To compare differentheating processing of lactoferrin on the proliferation of osteoblasts, cellproliferation assay was done by using MTT assay. Experimental results wereanalysed by software SPSS17.0. Results showed that unheated lactoferrin cansignificantly promote the proliferation of osteoblasts in a dose-dependent manner at physiological concentration range from1μg/mL to100μg/mL. There were nosignificant changes of osteoblasts proliferation in the group of lactoferrin treatedby62-65℃/30min. However, the relative high concentration of lactoferrin (1000,1250,1500μg/mL) significantly reduced the proliferative of osteoblasts.72℃heat treatment lactoferrin on osteoblasts, for24h, the low concentration (1,10μg/mL), the proliferation rate of osteoblasts was significantly improved, but therewas no significant changes by treating for24h,48h.72℃heat treatment on thehigh concentration of lactoferrin (1000,1250,1500μg/mL) can significantlyreduce the proliferative of osteoblasts in24h,48h. But72h do not.85℃and95℃heat treatment onlactoferrin (≥100μg/mL) will significantly reduce theproliferative effect on osteoblasts.Researched of the effects of lactoferrin on MAPK signaling pathwayshowed that un-heated treatment of lactoferrin can mediated by ERK1/2、p38MAPK and JNK signaling pathway to promote the proliferation of osteoblasts,and can mediated by ERK1/2to improve the activity of ALP. But different heattreatments of lactoferrin can be mediated by ERK1/2、p38MAPK and JNKsignaling pathway to inhibit the proliferation of osteoblasts and the activity ofALP.