Dcfh da

Intracellular ROS was detected using a ROS assay kit (Genmed Scientifics Inc.) containing an oxidation-sensitive fluorescent probe (DCFH-DA) in a spectrofluorometer (excitation 490 nm, emission 520 nm). Assessment of relative ATP contents was performed using the ATP bioluminescence assay kit (Beyotime Institute of Biotechnology) following the manufacturer’s instructions.

Rats (n = 6 for each group) were sacrificed immediately postanesthesia (at PND 7), and the hippocampi were removed quickly. Intracellular ROS were detected using a ROS assay kit (Genmed Scientifics Inc., Shanghai, China) containing an oxidation-sensitive fluorescent probe (DCFH-DA) with a spectrofluorometer (excitation 490 nm, emission 520 nm). Malondialdehyde (MDA) is an end-product of ROS-induced peroxidation. Superoxide Dismutase (SOD) is an important enzyme that participates in the removal of ROS from the cellular environment. The extent of lipid peroxidation was estimated by MDA levels, which were measured by using the spectrophotometric diagnostic kits (Jiancheng Biological Technology Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. The SOD activity was determined using a SOD assay kit (Jiangsu KeyGEN BioTECH Co., Ltd., Nanjing, China) according to manufacturer’s instructions. Enzyme activity was converted to units per milligram of protein. One unit of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%.

Bone marrow monocytes (BMMs) isolated from rat femurs were induced to differentiate towards osteoclasts by incubation in α‐MEM media supplemented with 50 ng/mL receptor activator of nuclear factor‐kappa B ligand (RANKL) (PeproTech, London, UK) and 50 ng/mL macrophage colony‐stimulating factor（M‐CSF）in the presence or absence of isoproterenol (1 μM). Osteoclast differentiation was confirmed using the Tartrate‐Resistant Acid Phosphatase Kit (Sigma‐Aldrich) as described in section 2.9. Formation of F‐actin rings, a marker of osteoclasts, was detected using rhodamine‐phalloidin fluorescent staining (Cytoskeleton, Denver, CO, USA); nuclei were counterstained with DAPI（Sigma‐Aldrich Co., Taufkirchen, Germany. To detect intracellular reactive oxygen species (ROS), BMMs cultured in conditioned or control media with isoprenaline were measured with the fluorescent oxidation‐sensitive probe 2’,7’‐dichlorodihydrofluorescein diacetate (DCFH‐DA; Genmed, Shanghai, China). Expressions of osteoclast‐related factors tartrate‐resistant acid phosphatase (Trap) and cathepsin k (Ctsk) were also determined by Western blotting using standard techniques and incubated anti‐Trap (ab96372, Abcam), anti‐Ctsk (ab19027, Abcam) and anti‐β‐tublin (ab6046, Abcam).