Bone homeostasis is a dynamic balance maintained by bone formation and resorption. An increase in the number and activity of osteoclasts leads to excessive bone resorption, which in turn results in bone disease, including osteoporosis. Therefore, inhibiting the differentiation and activity of osteoclasts is important for maintaining bone mass. Several studies have revealed that the use of a low-frequency pulsed electromagnetic field (PEMF) is an effective method to treat osteoporosis. However, its exact mechanism remains to be fully clarified. Therefore, the present study was designed to examine the effects that PEMF exerts on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and intracellular reactive oxygen species (ROS) production in RAW264.7 cells. The viability of cells was determined using a Cell Counting Kit-8 assay, and gene and protein expression were investigated via reverse transcription-quantitative polymerase chain reaction and western blot analyses. Furthermore, microscopy was performed to detect the levels of intracellular ROS and tartrate-resistant acid phosphatase (TRAP). Following the culture of RAW264.7 cells with RANKL (50 ng/ml) for 4 days (3 h/day) under PEMF (75 Hz, 1 mt) exposure, it was observed that PEMF had an inhibitory effect on RANKL-induced osteoclastic differentiation. Multinucleated osteoclast formation, the activity of TRAP and the expression of osteoclastogenesis-associated genes, including cathepsin K, nuclear factor of activated T cells cytoplasmic 1 and TRAP, were significantly reduced by PEMF. Furthermore, PEMF effectively decreased the generation of intracellular ROS during osteoclastic differentiation. In addition, the results demonstrated that ROS are the key factor in osteoclast differentiation and formation. Reducing intracellular ROS with diphenylene-iodonium chloride significantly inhibited RANKL-induced osteoclast differentiation. Taken together, the results of the present study demonstrated that PEMF may inhibit RANKL-induced osteoclastogenesis by scavenging intracellular ROS. These results may provide the groundwork for future PEMF clinical applications in osteoclast-associated bone disease.
Plants have developed complex mechanisms to adapt to the changing nitrate (NO3-) levels and can recruit microbes to boost nitrogen absorption. However, little is known about the relationship between functional genes and the rhizosphere microbiome in NO3- uptake of apple rootstocks. Here, we found that variation in MdNRT2.4 expression contributes to nitrate-uptake divergence between two apple rootstocks. Overexpression of MdNRT2.4 in apple seedlings significantly improved tolerance to low nitrogen via increasing net NO3- influx at root surface. But when inhibited the root plasma membrane H +-ATPase activity, which abolished NO3- uptake and led to NO3- release, suggesting that MdNRT2.4 encodes an H +-coupled nitrate transporter. Surprisingly, the nitrogen concentration of MdNRT2.4-overexpressing apple seedlings in unsterilized nitrogen-poor soil was higher than that in sterilized nitrogen-poor soil. Using 16S ribosomal RNA gene profiling to characterize rhizosphere microbiota, we found that MdNRT2.4-overexpressing apple seedlings recruited more bacterial taxa with nitrogen metabolic functions, especially Rhizobiaceae. We isolated a bacterial isolate AR11 from apple rhizosphere soil and identified it as Rhizobium. Inoculation with ARR11 improved apple seedling growth in nitrogen-poor soil compared with uninoculated seedlings. Together, our results highlight the interaction of host plant genes with the rhizosphere microbiota for host plant nutrient uptake.
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