Lactoferrin (LF) is an iron-binding glycoprotein that plays an important role in promoting bone formation and inhibiting bone resorption; however, its effects on senile osteoporosis remain unknown. This study aimed to investigate the effects and mechanism of LF intervention using a senile osteoporosis model (SAMP6 mice) and senescent osteoblasts. Micro-CT and hematoxylin and eosin staining demonstrated that the intragastric administration (2 g/kg/day) of LF could improve the bone mass and microstructure of SAMP6 mice. Furthermore, LF treatment improved bone metabolism and increased insulin-like growth factor 1 (Igf1) mRNA expression and activated phosphorylation status of AKT. Using osteoblasts passaged for ten generations as an in vitro senescence model, various markers associated with osteoblast formation and differentiation, as well as related indices of oxidative stress were analyzed. Our results revealed that after multiple generations, osteoblasts entered senescence, in conjunction with increased oxidative stress damage, reduced bone metabolism and enhanced expression of aging-related markers. While inhibiting oxidative stress, LF improved osteoblast proliferation by promoting the expression of osteogenesis markers, including alkaline phosphatase (ALP) activity, Igf1, bone gla protein (Bglap) and osteoprotegerin/receptor activator of nuclear factor-kB ligand (Opg/Rankl) mRNA and delayed senescence by decreasing the level of p16 and p21 expression. RNAI-mediated downregulation of IGF1 attenuated the effect of LF on osteogenesis. Therefore, the findings of the present study indicate that LF may promote osteogenesis via IGF1 signaling, thereby preventing senile osteoporosis.
Supplementary Figures 1. Identification of passage 3 and passage 10 rat osteoblasts. (A)Alkaline phosphatase staining of the cells at passage 3 and passage 10, magnification ×100. (B)Type-I collagen immunohistochemical staining of the cells at passage 3 and passage 10, magnification ×400.
Supplementary Figures 2. Effects of 2 g•kg−1•d−1 lactoferrin treatment on biochemical analysis in SAMP6 mice.(A-C) Analysis of serum Ca, P, Fe concentration in mice in SAMR1, SAMP6 and SAMP6 + LF group. *P < 0.05, **P<0.01 vs the SAMR1 group; #P < 0.05, ##P < 0.01 vs the SAMP6 group.