Silencing Proteasome 26S Subunit ATPase 2 (PSMC2) Protects the Osteogenic Differentiation In Vitro and Osteogenesis In Vivo.

Osteoporosis is a commonly seen degenerative bone disorder in the elderly and postmenopausal women, with a low bone mineral density as a major risk factor. The osteogenic potential of bone marrow stromal cells (BMSCs) showed to be impaired during osteoporosis. We established a postmenopausal osteoporosis model in ovariectomized (OVX) mice and found the upregulation of proteasome 26S subunit ATPase 2 (PSMC2) in OVX mice. PSMC2 silencing improved OVX-impaired biomechanical properties of mice femur, OVX-decreased BMD, and OVX-destroyed bone structure. Histopathological analysis indicated that PSMC2 silencing improved bone trabecular structure and increased the contents of collagen fibers and newly formed bone or cartilage in OVX mice. In the meantime, PSMC2 silencing increased Runx2, PI3K, Wnt3a, and ß-catenin protein contents while reduced CTSK protein. Within BMSCs isolated from OVX mice, PSMC2 silencing promoted BMSC osteogenic differentiation and elevated osteogenic markers' protein contents, including HOXA10, Runx2, OCN, OPN, and COL1A2. In conclusion, PSMC2 expression is upregulated in the postmenopausal osteoporosis model in OVX mice. PSMC2 silencing promotes the osteogenic differentiation of BMSCs in vitro, promotes bone formation, and inhibits bone resorption in vivo.

Apigenin, a Single Active Component of Herbal Extract, Alleviates Xerostomia via ERα-Mediated Upregulation of AQP5 Activation.

Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.

Anti-Osteoporotic Effects of the Herbal Mixture of Cornus officinalis and Achyranthes japonica In Vitro and In Vivo.

Osteoporosis is a porous bone disease caused by bone density loss, which increases the risk of fractures. Cornus officinalis (CO) and Achyranthes japonica (AJ) have been used as traditional herbal medicine for various disorders in East Asia. Although the anti-osteoporotic effects of single extract of CO and AJ have already been reported, the synergistic effect of a combined mixture has not been studied. In this study, we investigated the effects of a CO and AJ herbal mixture on osteoporosis in in vitro and in vivo models. The results demonstrate that treatment with the CO and AJ mixture significantly promoted osteoblast differentiation of MC3T3-E1 mouse preosteoblasts through the upregulation of osteoblastic differentiation-associated genes such as alkaline phosphatase (Alpl), runt-related transcription factor 2 (Runx2), and bone gamma-carboxyglutamic acid-containing protein (Bglap), while the mixture significantly inhibited differentiation of osteoclasts isolated from primary-cultured mouse monocytes. In addition, oral administration of CO and AJ mixture significantly prevented bone mineral density loss and trabecular bone structures in an ovariectomy-induced osteoporotic mouse model. These results suggest that the combination treatment of CO and AJ mixture might be a beneficial therapy for osteoporosis.

Kefir Peptides Prevent Estrogen Deficiency-Induced Bone Loss and Modulate the Structure of the Gut Microbiota in Ovariectomized Mice.

Osteoporosis is a major skeletal disease associated with estrogen deficiency in postmenopausal women. Kefir-fermented peptides (KPs) are bioactive peptides with health-promoting benefits that are produced from the degradation of dairy milk proteins by the probiotic microflora in kefir grains. This study aimed to evaluate the effects of KPs on osteoporosis prevention and the modulation of the composition of the gut microbiota in ovariectomized (OVX) mice. OVX mice receiving an 8-week oral gavage of 100 mg of KPs and 100 mg of KPs + 10 mg Ca exhibited lower trabecular separation (Tb. Sp), and higher bone mineral density (BMD), trabecular number (Tb. N) and bone volume (BV/TV), than OVX groups receiving Ca alone and untreated mice, and these effects were also reflected in bones with better mechanical properties of strength and fracture toughness. The gut microbiota of the cecal contents was examined by 16S rDNA amplicon sequencing. α-Diversity analysis indicated that the gut microbiota of OVX mice was enriched more than that of sham mice, but the diversity was not changed significantly. Treatment with KPs caused increased microbiota richness and diversity in OVX mice compared with those in sham mice. The microbiota composition changed markedly in OVX mice compared with that in sham mice. Following the oral administration of KPs for 8 weeks, the abundances of Alloprevotella, Anaerostipes, Parasutterella, Romboutsia, Ruminococcus_1 and Streptococcus genera were restored to levels close to those in the sham group. However, the correlation of these bacterial populations with bone metabolism needs further investigation. Taken together, KPs prevent menopausal osteoporosis and mildly modulate the structure of the gut microbiota in OVX mice.

Nobiletin-loaded micelles reduce ovariectomy-induced bone loss by suppressing osteoclastogenesis.

BACKGROUND: Nobiletin (NOB), a polymethoxy flavonoid, possesses anti-cancer and anti-inflammatory activities, has been reported that it played role in anti-osteoporosis treatment. However, previous research did not focus on practical use due to lack of hydrophilicity and cytotoxicity at high concentrations. The aim of this study was to develop a therapeutic formulation for osteoporosis based on the utilization of NOB. METHODS: In this study, NOB-loaded poly(ethylene glycol)-block-poly(e-caprolactone) (NOB-PEG-PCL) was prepared by dialysis method. The effects on osteoclasts and anti-osteoporosis functions were investigated in a RANKL-induced cell model and ovariectomized (OVX) mice. RESULTS: Dynamic light scattering and transmission electron microscopy examination results revealed that the NOB-PEG-PCL had a round shape, with a mean diameter around 124 nm. The encapsulation efficiency and drug loading were 76.34±3.25% and 7.60±0.48%, respectively. The in vitro release of NOB from NOB-PEG-PCL showed a remarkably sustained releasing characteristic and could be retained at least 48 hrs in pH 7.4 PBS. Anti-osteoclasts effects demonstrated that the NOB-PEG-PCL significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells stimulated by RANKL. Furthermore, the NOB-PEG-PCL did not produce cytotoxicity on bone marrow-derived macrophages (BMMs). The mRNA expressions of genetic markers of osteoclasts including TRAP and cathepsin K were significantly decreased in the presence of NOB-PEG-PCL. In addition, the NOB-PEG-PCL inhibited OC differentiation of BMMs through RANKL-induced MAPK signal pathway. After administration of the NOB-PEG-PCL, NOB-PEG-PCL prevented bone loss and improved bone density in OVX mice. These findings suggest that NOB-PEG-PCL might have great potential in the treatment of osteoporosis. CONCLUSION: The results suggested that NOB-PEG-PCL micelles could effectively prevent NOB fast release from micelles and extend circulation time. The NOB-PEG-PCL delivery system may be a promising way to prevent and treat osteoporosis.

Effect of shaking and vibration stimulation on lumbar vertebrae in ovariectomized mice.

OBJECTIVES: Bone fractures affect the activities of daily living and lower quality of life, so investigating preventative measures is important. We developed novel stimulation equipment that combined a vibration stimulus with a shaking stimulus for preventing osteoporosis (one of the causes of bone fractures). We aimed to investigate the effect of this equipment on ovariectomized mice. METHODS: Oophorectomy of 8-week-old female mice was done. The stimulation group was stimulated for 10 consecutive weeks. RESULTS: The stimulation group showed significantly higher values (p<0.05) for osteoid thickness, osteoid volume-to-bone volume ratio and mineral apposition rate than those in the non-stimulation control group. The stimulation group showed significantly higher values (p<0.05) compared with the non-stimulation for expression of bone morphogenetic protein-2, interleukin-1ß, interleukin-6 and myogenic determination gene in quadriceps femoris muscles (QFMs). CONCLUSIONS: These data suggest that cytokine secretion by QFMs carried a humoral factor throughout the body via the blood and blood vessels and acted on bone and various organs. Development of this stimulation method and its clinical application, new methods for preventing and treating osteoporosis could ensue.