The antiresoptive effects of recombinant Lingzhi-8 protein against retinoic acid-induced osteopenia.

The antiresorptive agents still are the mainstay of osteoporosis treatment. This study aimed to investigate the efficacy of recombinant Lingzhi-8 (rLZ-8) on osteoclast in vitro and bone resorption in vivo. The rLZ-8 protein was derived from Ganoderma lucidum transformation and produced by a genetic system. Receptor activator of nuclear factor kappa-Β ligand induced RAW 264.7 cells to differentiate into osteoclastic cells in vitro. Cells were exposed to different doses of rLZ-8 for 7 days to measure differences of osteoclastic differentiation, apoptosis rate and gene expression. rLZ-8 was labeled with Alexa Fluor 568 to observe its intracellular distribution under super-resolution light microscopy. In addition, retinoic acid was administered to female rats for 14 days to develop osteopenia changes. Different doses of rLZ-8 were simultaneously administered to rats treated with retinoic acid to observe changes of bone mineral density, biochemical parameters and organ weight ratio. Results indicated that rLZ-8 regulated receptor activator of nuclear factor kappa-Β (RANK) - tumor necrosis factor receptor-associated factor 6 (TRAF6) - c-Jun N-terminal kinase (JNK) signaling pathway, by which rLZ-8 inhibited osteoclastic differentiation and promoted osteoclastic apoptosis. Through 3D-structured illumination microscopy, it was observed that rLZ-8 entered RAW264.7 cells and accumulated gradually into the cytoplasm but little into nucleus. Administration with rLZ-8 reversed loss of bone mass and improved ALP activity in osteoporotic rats. Low-to high-dose rLZ-8 treatments displayed little toxic effects on rat organs and did not seem to impact their overall health. All data suggested that rLZ-8 has possible action against osteoporosis.