[Impact of hypoxia-reoxygenation environment on autophagy level of osteoblasts]

Objective: To investigate the impact of hypoxia-reoxygenation environment on the level of autophagy in osteoblasts. Methods: Osteoblasts were purified from the skulls of newborn SD rats within 24-48 hours by tissue block adherence culture and differential centrifugation. The osteoblasts were identified by alizarin red staining and alkaline phosphatase staining. The third generation osteoblasts were cultured in normal state and randomly divided into four groups: group A was cultured under normal condition for 36 hours; group B was cultured under normal condition for 18 hours, then under hypoxia for 18 hours; group C was cultured under hypoxia for 36 hours; group D was cultured under hypoxia for 18 hours, and then under normal condition for 18 hours. The ability to form calcium nodules of osteoblasts in the four groups was observed after culture. The proliferation activity of osteoblasts was detected by CCK-8 assay. The expressions of autophagy specified gene Beclin 1, microtubule-associated protein light chain 3(LC3) and collagen Ⅰ(COL-Ⅰ), bone morphogenetic protein 2 (BMP-2) genes were detected by real time polymerase chain reaction (RT-PCR), and the protein expressions of Beclin 1, LC3-Ⅰ,LC3-Ⅱ and P62 were detected by immunoblotting. Results: Alizarin red staining showed that osteoblasts in group A had the strongest calcification ability, and calcification ability of osteoblasts in group B,C and D lowered gradually, and it was lowest in group D. The proliferative activity under the CCK-8 detection in group A, B, C and D was 98%±8%, 90%±8%,82%±9%,76%±8%, respectively (F=35.764, P=0.000). The mRNA expression of Beclin 1, LC3-Ⅱthe 4 groups increased gradurally (group D> group C> group B> group A)(F=38.327, 16.583, both P<0.05); and the mRNA expression of COL-Ⅰ, BMP-2 decreased gradually in the 4 groups (group A> group B> group C> group D) (F=20.387, 12.426, both P<0.05). The protein expression of Beclin 1,LC3-Ⅱ/LC3-Ⅰ increased gradually in the groups (group D>group C>group B>group A) (F=26.843, 28.576, bothP<0.05), and the expression of P62 protein decreased gradually (F=18.946, P=0.011). Conclusions: Hypoxia-reoxygenation environment can reduce the proliferation activity of osteoblasts and up-regulate the expression of autophagy-related genes in osteoblasts. Anoxic reoxygenation environment promotes the increasing of autophagy levels in osteoblasts.