Abstract: BACKGROUND: Because of its anti-inflammatory and antioxidant activities, Apelin-13 plays an effective role in the treatment of common clinical diseases such as neuroinflammation, cardiovascular injury and pneumonia. However, there is no relevant basic research on whether Apelin-13 also has a good effect in the treatment of inflammatory bone loss.
OBJECTIVE: To explore the therapeutic effect and mechanism of Apelin-13 on inflammatory bone loss, in order to find potential drugs for the treatment of inflammatory bone loss.
METHODS: (1) In vitro experiment: RAW264.7 cells were divided into three groups: control group, lipopolysaccharide group and treatment group. The control group was only added with DMEM complete medium; lipopolysaccharide group was added with lipopolysaccharide (100 ng/mL) induced inflammation DMEM medium; and the treatment group was added with 10 nmol/L Apelin-13+lipopolysaccharide induced inflammation DMEM medium. Then, 24 hours after lipopolysaccharide induced inflammation, western blot was used to detect the marker proteins inducible nitric oxide synthase and CD86 of M1 macrophages, and cell immunofluorescence was extracted to detect the expression of inducible nitric oxide synthase. Finally, the same amount of receptor activator of nuclear factor-κB ligand (RANKL; 50 ng/ml) was added to the control group, lipopolysaccharide group and treatment group to induce osteoclasts. The results of osteoclast induction were evaluated by tartrate-resistant acid phosphatase staining and F-actin staining after 6 days of induction. (2) In vivo experiment: Eighteen male C57bl/6 mice were randomly divided into three groups: sham group, lipopolysaccharide group and treatment group. The sham group received intraperitoneal injection of 0.1 mL of PBS; the lipopolysaccharide group was injected with 0.1 mL of PBS diluent containing lipopolysaccharide (5 mg/kg); and the treatment group was injected with 0.1 mL of PBS diluent containing lipopolysaccharide (5 mg/kg)+Apelin-13 (100 µg/kg). After 7 days of continuous intraperitoneal injection, the mice in each group were killed on the 8th day, and two femurs of each mouse were collected. Half of them were scanned by micro-CT and analyzed by bone mineral density, and the other half were stained by hematoxylin-eosin staining
RESULTS AND CONCLUSION: (1) In vitro experiment: Western blot results showed that the expressions of inducible nitric oxide synthase and CD86 in the lipopolysaccharide group were significantly higher than those in the control group, and Apelin-13 could significantly inhibit the M1 polarization of macrophages induced by lipopolysaccharide. Cell immunofluorescence results also showed that the expression of inducible nitric oxide synthase in the treatment group was lower than that in the lipopolysaccharide group. Besides, tartrate-resistant acid phosphatase staining and F-actin staining results showed that Apelin-13 inhibited the abnormal activation and bone resorption of lipopolysaccharide induced osteoclasts. (2) In vivo experiment: The results of micro-CT showed that systemic inflammation led to significant bone loss in the distal femur, while Apelin-13 could significantly inhibit bone loss in vivo. Hematoxylin-eosin staining results also showed that Apelin-13 could effectively alleviate inflammation induced bone loss in the distal femur of mice. To conclude, Apelin-13 can alleviate bone loss induced by systemic inflammation by inhibiting M1 polarization of macrophages, inhibiting abnormal activation of osteoclasts and bone resorption.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Apelin-13, macrophage polarization, osteoclast activation, systemic inflammatory bone loss