N16 suppresses RANKL-mediated osteoclatogenesis by down-regulating RANK expression.

N16 is an active protein existing in Pinctada martensi. Our previous studies have demonstrated that N16 inhibited osteoclast differentiation in vitro. To better understand how N16 regulates osteoclast differentiation, RAW264.7 cells, a murine monocytic cell line and murine bone marrow-derived macrophages (BMMs) were adopted. Treatment of RAW264.7 cells with RANKL activated osteoclastogenesis and N16 inhibited the formation of multinucleated osteoclasts and TRAP activity. The suppression occurred at the early stage of osteoclastogenesis. Moreover, we found that N16 inhibited PU.1 and MITF expressions, mirroring the inhibition of RANK expressions, indicating that N16 inhibited RANK expression by down-regulating the expressions of MITF and PU.1, thus preventing osteoclastogenesis.

Transcriptome analysis reveals functional roles of nacreous protein N16 in prednisolone-induced osteoporotic zebrafish.

N16 is an active protein isolated and screened from nacre proteins. Our previous studies have shown that N16 exerted a good effect on osteoporosis caused by dexamethasone on female rats. In order to further confirm the action of N16 against glucocorticoid-induced osteoporosis (GIOP) and clarify its possible mechanisms, prednisolone-treated larval zebrafish (Danio rerio) model was adopted. Administration of N16 resulted in a significant increase of vertebral bone density of osteoporotic zebrafish, indicating a good effect of N16 against GIOP. Transcriptomic sequencing analysis was then performed to investigate the mechanisms of the action of N16 against GIOP. It was observed that N16 modulated the osteoporotic phenotype by up-regulating the osteoblastic characteristic genes and down-regulating the osteoclastic characteristic genes in zebrafish.

Naringin and Naringenin Relax Rat Tracheal Smooth by Regulating BKCa Activation.

Naringin and its aglycone, naringenin, occur naturally in our regular diet and traditional Chinese medicines. This study aimed to detect an effective therapeutic approach for cough variant asthma (CVA) through evaluating the relaxant effect of these two bioactive herbal monomers as antitussive and antiasthmatic on rat tracheal smooth muscle. The relaxant effect was determined by measuring muscular tension with a mechanical recording system in rat tracheal rings. Cytosolic Ca2+ concentration was measured using a confocal imaging system in primary cultured tracheal smooth muscle cells. In rat tracheal rings, addition of both naringin and naringenin could concentration dependently relax carbachol (CCh)-evoked tonic contraction. This epithelium-independent relaxation could be suppressed by BaCl2, tetraethylammonium, and iberiotoxin (IbTX), but not by glibenclamide. After stimulating primary cultured tracheal smooth muscle cells by CCh or high KCl, the intracellular Ca2+ increase could be inhibited by both naringin and naringenin, respectively. This reaction was also suppressed by IbTX. These results demonstrate that both naringin and naringenin can relax tracheal smooth muscle through opening big conductance Ca2+-activated K+ channel, which mediates plasma membrane hyperpolarization and reduces Ca2+ influx. Our data indicate a potentially effective therapeutic approach of naringin and naringenin for CVA.

Preparative expression and purification of a nacreous protein N16 and testing its effect on osteoporosis rat model.

N16, a nacreous protein isolated from Pinctada martensii, is related to nacreous layer formation. Our previous study indicated that N16 showed dual regulatory effects by inducing osteoblast biomineralization as well as inhibiting osteoclast formation. In order to obtain large quantity of N16 for animal experiment and clinical trial, a fermentation and preparative purification method was established. The N16 cDNA was cloned to a BL21(DE3)plysE-pET32a vector and grown in a 20 L fermenter. The medium, temperature, pH and dissolved oxygen (DO) were optimized. N16 was expressed in inclusion bodies. It was denatured and refolded in 8 M urea buffer and purified to 97% purity by passing through a gel filtration column. The glucocorticoid induced osteoporosis (GIO) rat model was used to investigate the anti-osteoporosis activity of N16 in vivo. Results showed that the decrease of the bone mineral density (BMD) and the ultimate load was significantly relieved after N16 treatment. N16 displayed dual regulatory effects by promoting osteogenesis as well as inhibiting bone resorption in vivo. Our work will contribute to further clinical studies on N16 for osteoporosis treatment.