Hesperetin affects osteoclast differentiation via MAPK signaling pathway.

Abstract

The number and activity of osteoblasts and osteoclasts play an important role in skeletal biology, especially in bone reconstruction. Scientific and rational regulation of osteoclast formation and activity has become a critical strategy aimed at inhibiting the loss of bone mass in the body and alleviating the occurrence of bone diseases. Currently, there are only a few reports related to hesperetin-regulated osteoclast differentiation. To investigate the influence of hesperetin on osteoclast-like cell differentiation and formation, and determine whether the MAPK signaling pathway is involved in the differentiation process. The RAW264.7 cells were induced and cultured in vitro to promote their differentiation into osteoclast-like cells. Tetrazolium bromide was utilized to determine the effects of different concentrations (100, 200, 400, and 600 μM) of hesperetin on the proliferation of osteoclast-like cell precursors. Osteoclast-like cell differentiation was conducted using tartrate-resistant acid phosphatase (TRAP) staining assay. The status of nuclei and actin filaments of differentiated osteoclast-like cells was observed with the use of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and actin-tracker green staining experiments. Changes in key proteins of the MAPK signaling pathway were detected using western blot. The results of TRAP staining experiments showed that the number of osteoclast-like cells decreased with the increase in hesperetin concentration. The DAPI and actin-tracker green staining demonstrated that the nuclei of differentiated osteoclast-like cells reduced in size with the increase in hesperetin concentration, and the osteoclast-like cells became smaller. Western blot for key MAPK signaling pathway proteins revealed that phospho-ERK and phospho-p38 protein levels were not significantly inhibited, but phospho-JNK protein levels were reduced. Hesperetin inhibits the differentiation of osteoclast-like cells. Further studies revealed that hesperetin also affects the activation level of phospho-JNK, a key signaling protein of the MAPK signaling pathway, in the induced differentiation of osteoclast-like cells.