Lactoferrin Promotes Proliferation And Differentiation Of Senescent Osteoblasts Via IGF-1 Signaling

Objective:We aim to investigate the improvement effects and mechanism of lactoferrin on senescent osteoblasts.Method:(1)Primary osteoblasts were isolated from the calvaria of newborn Sprague-Dawley male rats and cultured up to ten passages.The cell proliferation was determined by MTT method.The ALP activity was determined by pNPP method.Quantitative RT-PCR was used to detect the related mRNA expression of bone metabolism genes,such as Igf-1,Opg,Rankl,Bglap and cell aging genes,like p16 and p21.And we also detect oxidative stress injury.(2)With the treatment of lactoferrin,we observe the effects of lactoferrin on senescent osteoblasts by detecting the changes of the above indicators.And the cell morphology was observed by transmission electron microscope.(3)We used RNAi method to decrease the expression of IGF-1.After LF treatment,the IGF-1 shRNA transfected senescent osteoblasts were detected the above indicators.We also used western blot to detect the IGF-1/PI3K/Akt signaling.Results:(1)There was a negative correlation between the number of passages and the cell proliferative ability or ALP activity.The mRNA expression levels of the bone metabolism genes Igf-1,Opg,Rankl,Bglap were significantly decreased with increasing passages of subcultures.In contrast,expression of the aging related genes p16,p21 and oxidative stress injury were increased with passages.(2)Transmission electron microscopy exhibited more elongated morphology with fewer cell organelles and more secondary lysosomes in the cytoplasm with the passage number increased.The cells treated with LF had a plump cytoplasm with abundant organelles,expanded rough endoplasmic reticulum,and fewer secondary lysosomes.While inhibiting oxidative stress,LF improved osteoblasts proliferation by promoting the expression of osteogenesis markers,including ALP activity,the ressions of Igf1,Bglap and Opg/Rankl mRNA,and delayed senescence by decreasing the level of p16 and p21 expression..(3)RNAI-mediated downregulation of IGF-1 attenuated the effects of LF on osteogenesis.The western blot analysis indicated that the aging group exhibited low levels of IGF-1/PI3K/Akt activation.LF intervention at a concentration of 100 ?g/mL significantly induced the expression of both p-PI3 K and p-Akt.Moreover,LF-treated shIGF1 cells exhibited significantly decreased levels of p-PI3 K and p-Akt.Conclusion:Therefore,the findings of the present study indicate that lactoferrin may promote proliferation and differentiation ofsenescent osteoblasts via IGF-1 signaling.