Different methionine to cysteine supplementation ratios altered bone quality of broilers with or without Eimeria challenge assessed by dual energy X-ray absorptiometry and microtomography.

Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

Icariin attenuates thioacetamide­induced bone loss via the RANKL­p38/ERK­NFAT signaling pathway.

There is an increasing incidence of destructive bone disease caused by osteoclast proliferation. This is characterized by reduced bone mass and imbalance of bone homeostasis. Icariin (ICA), a flavonoid compound isolated from Epimedium, has anti­osteoporosis activity and inhibits the formation of osteoclasts and bone resorption. The purpose of the present study was to investigate the protective effect of ICA on osteoclastic differentiation induced by thioacetamide (TAA) and its possible mechanism in Sprague Dawley (SD) rats. In the present study, SD rats were intraperitoneally injected with TAA (300 mg/kg) for the bone loss model, treated with ICA (600 mg/kg, intragastric gavage) in the ICA group and TAA+ICA group for treatment of bone loss for 6 weeks. Indexes associated with bone metabolism, such as alkaline phosphatase, N­terminal telopeptide of type­I collagen (NTX­I), calcium (Ca), phosphorus (P) and magnesium (Mg) in the serum, were detected. Osteoclast differentiation of femoral tissues was detected by hematoxylin and eosin and tartrate­resistant acid phosphatase staining. The femoral bone mass was evaluated using a three­point bending test and micro computed tomography. Western blotting was used to detect the expression levels of osteoclast­related proteins in each group. In the rats treated with TAA, the serum concentrations of Ca, P and Mg were decreased, the serum concentration of NTX­I was increased, osteoclast differentiation of the femur was increased, femur bone stress and bone mass were decreased and the bone loss and osteoclast formation were reduced after ICA treatment. In addition, ICA inhibited the protein expression of receptor activator of nuclear factor κ­Β ligand (RANKL), receptor activator of nuclear factor κ­B (RANK), p38, ERK, c­Fos and nuclear factor of activated T cells 1 (NFATc1) in the femur of rats treated with TAA. The results suggested that ICA may inhibit osteoclast differentiation by downregulating the RANKL­p38/ERK­NFAT signaling pathway and prevent TAA­induced bone loss. The results are helpful to understand the mechanism of osteoclast differentiation induced by TAA, as well as the antiresorptive activity and molecular mechanism of ICA, and to provide new ideas for the treatment of osteolytic diseases.

Peiminine Suppresses RANKL-Induced Osteoclastogenesis by Inhibiting the NFATc1, ERK, and NF-κB Signaling Pathways.

Osteoclasts (OCs) play an important role in osteoporosis, a disease that is mainly characterized by bone loss. In our research, we aimed to identify novel approach for regulating osteoclastogenesis and thereby treating osteoporosis. Previous studies have set a precedent for screening traditional Chinese herbal extracts for effective inhibitors. Peiminine is an alkaloid extracted from the bulb of Fritillaria thunbergii Miq that reportedly has anticancer and anti-inflammatory effects. Thus, the potential inhibitory effect of peiminine on OC differentiation was investigated via a series of experiments. According to the results, peiminine downregulated the levels of specific genes and proteins in vitro and consequently suppressed OC differentiation and function. Based on these findings, we further investigated the underlying molecular mechanisms and identified the NF-κB and ERK1/2 signaling pathways as potential targets of peiminine. In vivo, peiminine alleviated bone loss in an ovariectomized mouse model.

Supplementation of Morin Restores the Altered Bone Histomorphometry in Hyperglycemic Rodents via Regulation of Insulin/IGF-1 Signaling.

Pathological mechanisms underlining diabetic bone defects include oxidative damage and insulin/IGF-1 imbalance. Morin is a bioflavonoid with antioxidant and anti-diabetic effects. This study evaluates morin's protective effects against altered bone histomorphometry in diabetic rats through assessing insulin/IGF-1 pathway as a potential mechanism. Diabetic animals were administered two morin doses (15 and 30 mg/kg) for 5 weeks. Different serum hepatic and renal functions tests were assessed. Bone density and histomorphometry in cortical and trabecular tissues were evaluated histologically. The expressions of insulin, c-peptide and IGF-1 were estimated. In addition, the enzymatic activities of the major antioxidant enzymes were determined. Diabetic-associated alterations in serum glucose, aminotransferases, urea and creatinine were attenuated by morin. Diabetic bone cortical and trabecular histomorphometry were impaired with increased fibrosis, osteoclastic functions, osteoid formation and reduced mineralization, which was reversed by morin; particularly the 30 mg/kg dose. Insulin/IGF-1 levels were diminished in diabetic animals, while morin treatment enhanced their levels significantly. Diabetes also triggered systemic oxidative stress noticeably. The higher dose (30 mg/kg) of morin corrected the endogenous antioxidant enzymatic activities in diabetic rats. Findings indicate the potential value of morin supplementation against hyperglycemia-induced skeletal impairments. Activation of insulin/IGF-1 signaling could be the underlining mechanism behind these effects.

Co-Treatment of Purified Annatto Oil (Bixa orellana L.) and Its Granules (Chronic®) Improves the Blood Lipid Profile and Bone Protective Effects of Testosterone in the Orchiectomy-Induced Osteoporosis in Wistar Rats.

This study aimed to evaluate and compare the effects of co-treatment with purified annatto oil (PAO) or its granules (GRA, Chronic®) with that of testosterone on the orchiectomy-induced osteoporosis in Wistar rats. After surgery, rats were treated from day 7 until day 45 with testosterone only (TES, 7 mg/kg, IM) or TES + PAO or GRA (200 mg/kg, p.o.). The following parameters were evaluated: food/water intake, weight, HDL, LDL, glucose, triglycerides (TG), total cholesterol (TC), alkaline phosphatase levels, blood phosphorus and calcium contents, femur weight, structure (through scanning electron microscopy), and calcium content (through atomic absorption spectrophotometry). Our results show that orchiectomy could significantly change the blood lipid profile and decrease bone integrity parameters. Testosterone reposition alone could improve some endpoints, including LDL, TC, bone weight, and bone calcium concentration. However, other parameters were not significantly improved. Co-treatment with PAO or GRA improved the blood lipid profile and bone integrity more significantly and improved some endpoints not affected by testosterone reposition alone (such as TG levels and trabeculae sizes). The results suggest that co-treatment with annatto products improved the blood lipid profile and the anti-osteoporosis effects of testosterone. Overall, GRA had better results than PAO.

Inhibitory effect of Astragalus Membranaceus on osteoporosis in SAMP6 mice by regulating vitaminD/FGF23/Klotho signaling pathway.

Spontaneous senile osteoporosis severely threatens the health of the senior population which has emerged as a severe issue for society. A SAMP6 mouse model was utilized to estimate the impact of intragastrically administered Astragalus Membranaceus (AR) on spontaneous senile osteoporosis. Bone mineral density (BMD) and bone microstructure were measured using Micro-CT; contents of calcium and phosphorus were determined with the colorimetric method; and gene and protein expressions of fibroblast growth factor 23 (FGF23), Klotho, Vitamin D receptor (VDR), CYP27B1 and CYP24A1 were detected using qPCR, Western blot and ELISA assays, respectively. The findings indicated that AR could improve the femoral BMD and bone microstructure, elevate the contents of calcium and phosphorus, and increase the expression of Klotho, VDR, and CYP27B1 whereas decreasing the expression of FGF23 and CYP24A1 in SAMP6 mice in a dose independent manner. The present study has demonstrated that AR can promote osteogenesis and alleviate osteoporosis. It is also expected to provide a new insight for the treatment of spontaneous senile osteoporosis and to serve as a research basis for AR application.

Zhuanggu Zhitong Capsule alleviates osteosarcopenia in rats by up-regulating PI3K/Akt/Bcl2 signaling pathway.

BACKGROUND AND AIMS: Osteosarcopenia (OS), characterized by the coexistence of osteoporosis (OP) and sarcopenia (SP), is associated with high morbidity and mortality in the elderly. Nevertheless, its pathogenesis and treatment remain unclear. The aim of this study was to investigate the effect and mechanism of Zhuanggu Zhitong Capsule (ZGZT) in OS rats. METHODS: All the related targets of OS, corresponding targets for bioactive ingredients of ZGZT, intersection targets of ZGZT against OS, and signaling pathways were predicted and analyzed by network pharmacology. Next, a rat OS model was established by ovariectomy (OVX) and injection of dexamethasone (DXM). Rats were then randomly divided into a Control group, a Sham operation group, an OS model group, an OS+ZGZT group, and an OS+E2 group. The drug was given for 12 weeks. During treatment, body weight, forelimb grip and body composition were measured. In addition, bone mineral density (BMD) and micro CT were used to assess the left femur. After treatment, the left femur, left gastrocnemius, and left soleus, as well as uterus, liver, and kidney, were separated and analyzed using Histomorphology, Western blot, and quantitative real-time PCR (qRT-PCR). RESULTS: ZGZT could effectively improve the phenotypes of OS by increasing forelimb grip strength, percentage lean mass of the whole body (SMI) or appendicular lean (RSMI), BMD, levels of bone formation markers, improving the microstructure of femur, and decreasing apoptotic rate in femur and gastrocnemius in OS rats by up-regulating PI3K/Akt/Bcl2 signal pathway. CONCLUSIONS: ZGZT may be a new treatment option for the prevention and treatment of OS.

Development of selective bispecific Wnt mimetics for bone loss and repair.

The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.

FK506 ameliorates osteoporosis caused by osteoblast apoptosis via suppressing the activated CaN/NFAT pathway during oxidative stress.

OBJECTIVE: Osteoporosis is affecting the health of postmenopausal women in the world. In case of that, we explored whether FK-506 could ameliorate osteoporosis by inhibiting the activated CaN/NFAT pathway during oxidative stress. METHODS: First, the castrated rat model is constructed through the bilateral ovariectomy. Hologic Discovery (S/N 80347) dual-energy X-ray absorptiometry assessed bone mineral density (BMD) implemented at left femur of rats. Next, hematoxylin-eosin (H&E) staining observed and calculated the changes of bone trabecular, mean trabecular plate separation (Tb.Sp), mean trabecular plate thickness (Tb.Th), and bone volume fraction (BV/TV). Then, CCK-8 assay, TUNEL assay, ALP kit and alizarin red staining detected the viability, apoptosis, alkaline phosphatase (ALP) activity, and capacity of mineralization respectively. At last, commercially available kits detected the levels of ROS and SOD in transfected MC3T3-E1 cells and bone tissues, and Western blot analysis detected proteins related to apoptosis and CaN/NFAT pathway. RESULTS: FK-506 increased the BMD and changes of bone trabecular in female castrated rats. FK-506 inhibited the oxidative stress and apoptosis by suppressing the activated CaN/NFAT pathway. Low dose of FK-506 improved the viability, ALP activity, and mineralization capacity. What's more, it suppressed the apoptosis of H2O2-induced MC3T3-E1 cells, which was deteriorated by the high dose of FK-506. Briefly, low dose of FK-506 inhibited the oxidative stress by suppressing the activated CaN/NFAT pathway, while high dose of that further inhibited the oxidative stress by suppressing the CaN/NFAT pathway. CONCLUSION: FK-506 ameliorates osteoporosis resulted from osteoblastic apoptosis which caused by suppressing the activated CaN/NFAT pathway during oxidative stress.

Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice.

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

Modulation of gut microbiota by chondroitin sulfate calcium complex during alleviation of osteoporosis in ovariectomized rats.

Although chondroitin sulfate calcium complex (CSCa) was claimed to have the bioactivity for bone care in vitro, its anti-osteoporosis bioactivity was little reported in vivo. Here, the effects of CSCa on osteoporosis rats were investigated. Results showed that, compared with the osteoporosis rats, CSCa could improve the bone mineral density and microstructure of femur, and change the bone turnover markers level in serum. 16S rRNA sequencing and metabolomics analysis indicated CSCa intervention altered the composition of gut microbiota along with metabolite profiles in ovariectomized rat faeces. The correlation analysis showed some gut microbiota taxa were significantly correlated with osteoporosis phenotypes and the enriched metabolites. Taken together, dietary CSCa intervention has the potential to alleviate the osteoporosis and related symptoms probably involving gut microbiota or the metabolite profiles as demonstrated in rats. This study provides some scientific evidence for the potential effects of CSCa as the food supplement on the osteoporosis.

Aloperine improves osteoporosis in ovariectomized mice by inhibiting RANKL-induced NF-κB, ERK and JNK approaches.

Presently, postmenopausal osteoporosis mainly caused by excessive activation of in vivo osteoclasts has become a global public health burden. Natural compounds have gradually become the potential drugs for the treatment of postmenopausal osteoporosis. Aloperine is a new alkaloid extracted from the leaves and seeds of sophora bean. The current studies have proved that aloperine has many biological activities, including anti-inflammatory, antiviral and anticancer activities. This study shows that aloperine can inhibit activity and formation of osteoclast mediated by RANKL in a dose-dependent manner without affecting the activity of bone marrow macrophages (BMM). In addition, it is found that aloperine can inhibit the expression of osteoclast specific marker genes, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP), matrix metallopeptidase 9 (MMP9), cathepsin K (Ctsk), V-ATPase d2 and calcitonin receptor. The in vitro experiment of aloperine proved that aloperine can inhibit the degradation of IκBα and the phosphorylation of P65, ERK and JNK. Additionally, aloperine improves bone loss in ovariectomized (OVX) mice by inhibiting osteoclast activity. This project proved that aloperine can affect the formation of osteoclasts by inhibiting RANKL signaling channel, and it is indicated that aloperine has the potential to be developed as a new drug for the prevention and treatment of postmenopausal osteoporosis.

ß-Hydroxy-ß-Methylbutyrate (HMB) Supplementation Prevents Bone Loss during Pregnancy-Novel Evidence from a Spiny Mouse (Acomys cahirinus) Model.

The aim of this study was to determine the effects of ß-hydroxy-ß-methylbutyrate (HMB) supplementation during pregnancy on postpartum bone tissue quality by assessing changes in trabecular and compact bone as well as in hyaline and epiphyseal cartilage. The experiment was carried out on adult 6-month-old female spiny mice (Acomys cahirinus) divided into three groups: pregnant control (PregCont), pregnant HMB-treated (supplemented with 0.02 g/kg b.w of HMB during the second trimester of pregnancy, PregHMB), and non-pregnant females (NonPreg). Cross-sectional area and cortical index of the femoral mid-shaft, stiffness, and Young modulus were significantly greater in the PregHMB group. Whole-bone mineral density was similar in all groups, and HMB supplementation increased trabecular number. Growth plate cartilage was the thinnest, while the articular cartilage was the thickest in the PregHMB group. HMB supplementation increased the content of proteoglycans in the articular cartilage and the percentage of immature collagen content in metaphyseal trabeculae and compact bone. In summary, dietary HMB supplementation during the second trimester of pregnancy intensifies bone metabolic processes and prevents bone loss during pregnancy.

Bionic Tiger-Bone Powder Improves Bone Microstructure and Bone Biomechanical Strength of Ovariectomized Rats.

OBJECTIVE: To study the curative effect of bionic tiger-bone powder on osteoporosis in ovariectomized rats and investigate its mechanism. METHODS: Overall, a 120 female Wistar rats were randomly divided into Sham (sham-operated group), ovariectomy (OVX, ovariectomized group), TB (bionic tiger-bone powder treatment group after ovariectomy) and TB + VD groups (bionic tiger-bone powder + vitamin D treatment group after ovariectomy). The osteoporotic rat model was established 3 months after ovariectomy, and rats were intragastrically administrated with the corresponding drugs. Serum and bone tissue samples were collected from 10 rats in each group at weeks 4, 12 and 24 after intragastric administration. The bone microstructure of L6 vertebrae was analyzed by MicroCT, the biomechanical strength of left femurs was measured by the three-point bending test, and serum bone metabolism markers (P1NP and CTX) were detected by ELISA. Changes in bone collagen were analyzed by Masson's trichrome staining and hydroxyproline detection, and members of the BMP2/SMAD/RUNX2 and OPG/RANKL/RANK signal pathways were detected by immunoblotting. RESULTS: Compared with the OVX group, the serum level of P1NP in the TB and TB + VD groups was higher (P < 0.05), while the CTX level was lower (P < 0.05). Bone collagen fiber structures in the TB and TB + VD groups were repaired, and the collagen content was significantly higher than that in the OVX group (P < 0.05). In the TB group, BMP-2, P-SMAD1/5, RUNX2 and OPG levels were increased in bone tissue (P < 0.01), RANKL levels were decreased (P < 0.01), and the bone microstructure and biomechanical strength were improved. CONCLUSION: Bionic tiger-bone powder promotes osteogenesis by activating the BMP2/SMAD/RUNX2 signaling pathway, suppresses osteoclasts by downregulating the OPG/RANK/RANKL signaling pathway, increases bone collagen content, and improves bone microstructure and bone biomechanical strength.

In vitro and in vivo study of naturally derived alginate/hydroxyapatite bio composite scaffolds.

Biogenic bioceramics scaffolds are receiving considerable attention for bone restoration applications. Compared with scaffolds of chemical origin, biogenic scaffolds exhibit greater biocompatibility and enhanced bioactive features. In the present study, porous biogenic hydroxyapatite (bHA) was prepared via a polymeric infiltration route and was subsequently coated with alginate to produce alginate/biogenic hydroxyapatite (Alg/bHA) composites. Alginate was used to enhance the mechanical properties as well as the bioactivity and biodegradability of the HA scaffolds. A coating of 3%w/v alginate applied for 10 min was found to result in the best coating for the HA porous scaffolds. The in vitro study demonstrated that the prepared composites had acceptable bioactivity and biodegradability characteristics. The histological study in femur bone of rats indicated that the 3Alg/HA scaffolds capable of supporting both endochondral and intramembranous bone formation. The defect was fully regenerated and mostly filled with the mature lamellar bone after 6 months, with Ca/P atomic ratio similar to the rat's normal bone. The studied scaffolds provide a promising therapeutic option to enhance local bone healing because they do not damage liver or kidney functions and do not induce carcinogenic or inflammatory effects. Accordingly, 3Alg/HA scaffolds are recommended for the tissue engineering applications.

Ginsenoside Rg3 attenuates ovariectomy-induced osteoporosis via AMPK/mTOR signaling pathway.

Ginsenoside Rg3, a ginsenoside isolated from Panax ginseng, can regulate autophagy via AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. AMPK/mTOR signaling and autophagy have been reported to be involved in osteogenesis. Here, the effect of Rg3 on ovariectomy (OVX)-induced osteoporosis is explored. In vivo, rats were treated with 20 mg/kg Rg3 after OVX and the body weight (BW) was monitored. Bone mineral density (BMD), hematoxylin-eosin staining of femur tissues, osteogenesis, autophagy, and AMPK/mTOR signaling were analyzed. In vitro, MC3T3-E1 cells were treated with 0, 1, 5, 10, 20, and 100 µmol/L Rg3. 10 and 20 µmol/L Rg3, which had no significant effect on cell viability and significantly affected AMPK/mTOR signaling, were chosen for further analysis. Then osteogenic differentiation was induced with Rg3 or/and AMPK inhibitor (Compound C). AMPK/mTOR signaling, autophagy, osteogenic differentiation, and mineralization by Alizarin Red staining were analyzed. The expression or activity of AMPK/mTOR signaling-related proteins, autophagy markers, and osteogenesis markers was measured by western blotting or commercial kits, and cell viability by cell counting kit-8 assay kits. Rg3 significantly alleviated OVX-induced BW increases, BMD declines and histological changes of femur tissues, promoted osteogenesis, autophagy, and AMPK signaling, but inhibited mTOR signaling in vivo. Moreover, Rg3 significantly enhanced AMPK signaling, autophagy, osteogenic differentiation, and mineralization, but suppressed mTOR signaling in vitro. However, Compound C significantly reversed Rg3-induced alterations in vitro, indicating that Rg3 regulated autophagy, osteogenic differentiation, and mineralization via AMPK/mTOR signaling. Hence, it was speculated that Rg3 might attenuate OVX-induced osteoporosis via AMPK/mTOR signaling pathway.

Therapeutic potential of Liuwei Dihuang pill against KDM7A and Wnt/ß-catenin signaling pathway in diabetic nephropathy-related osteoporosis.

The effects of Liuwei Dihuang pill (LWDH) on diabetic nephropathy-related osteoporosis (DNOP) are unclear. The present study aimed to evaluate the effects of LWDH on KDM7A and Wnt/ß-catenin signaling pathway in DNOP rats and the high glucose-induced MC3T3-E1 cells. A DNOP model was prepared by streptozotocin in 9-week-old male Sprague-Dawley (SD) rats to evaluate the effects of LWDH. The cell viability and differentiation capacity of high glucose-induced MC3T3-E1 cells were determined by CCK-8 assay, Alizarin Red staining, and alkaline phosphatase (ALP) staining, respectively. Furthermore, the expressions of KDM7A and Wnt1/ß-catenin pathway-related proteins were determined by Western blot analysis. Treatment of DNOP rats with LWDH could significantly ameliorate the general state, degradation of renal function, and renal pathological changes. LWDH decreased the levels of TNF-α, IL-6, IL-8, IL-1ß, ALP, and TRAP, and increased the calcium, phosphorus in serum, as well as decreased the level of the calcium and phosphorus in the urine. Besides, LWDH significantly improved bone mineral density (BMD), bone volume (BV), and the bone microstructure of DNOP rats. Moreover, LWDH increased the levels of the elastic modulus, ultimate load, and bending strength in the femurs. In MC3T3-E1 cells, serum-containing LWDH significantly increases in cell viability and osteoblastic differentiation capability. The expression of α-SMA, vimentin, KDM7A, Wnt1 and ß-catenin were significantly down-regulated, and the E-cadherin, H3K9-Me2, H3K27-Me2, BMP-4, BMP-7, Runx2, osteocalcin, and Col1a1 were significantly up-regulated with LWDH treatment. The present study shows that LWDH has a therapeutic effect on DNOP, in part, through down-regulation of KDM7A and Wnt/ß-catenin pathway.

Bioassay-guided isolation and evaluation of anti-osteoporotic polysaccharides from Morinda officinalis.

ETHNOPHARMACOLOGICAL RELEVANCE: Morinda officinalis is a well-known Chinese tonic herb that has shown clinical efficacy in the treatment of bone disease. However, its anti-osteoporotic potential and the M. officinalis polysaccharides (MOPs) responsible for activity require further investigation. AIM OF THE STUDY: This study aimed to investigate the anti-osteoporotic effects of different MOP fractions in ovariectomized (OVX) rats, and to identify the osteoprotective components by bioassay-guided isolation. MATERIALS AND METHODS: MOPs were prepared by hot water and alkali extraction, separated into three fractions (MO50, MO70, and MOB) and evaluated in the classic OVX rat model and in MC3T3-E1 cells for anti-osteoporotic activity. RESULTS: Administration of MOPs (400 mg/kg/day) provided significant protection against ovariectomy-induced bone loss and biomechanical dysfunction in rats. Treated animals exhibited reduced deterioration of trabecular microarchitecture and lower levels of bone turnover markers. Bioactivity-guided fractionation led to the isolation of two inulin-type fructans from MO50, MOW50-1 and MOP50-2, with potential anti-osteoporotic activities. These consisted of (2 → 1)-linked ß-D-fructosyl residues with degrees of polymerization (DP) of 7 and 13, respectively. Furthermore, MOW50-1 promoted osteogenic differentiation of MC3T3-E1 cells by increasing alkaline phosphatase activity. CONCLUSIONS: These data suggest very strongly that MOPs, especially MO50 and MOW50-1, may play important roles in the prevention and treatment of osteoporosis.

Lactobacillus plantarum GKM3 and Lactobacillus paracasei GKS6 Supplementation Ameliorates Bone Loss in Ovariectomized Mice by Promoting Osteoblast Differentiation and Inhibiting Osteoclast Formation.

Osteoporosis, an imbalance in the bone-forming process mediated by osteoblasts and the bone-resorbing function mediated by osteoclasts, is a bone degenerative disease prevalent among the aged population. Due to deleterious side effects of currently available medications, probiotics as a potential treatment of osteoporosis is an appealing approach. Hence, this study aims to evaluate the beneficial effects of two novel Lactobacilli strain probiotics on bone health in ovariectomized (OVX) induced osteoporotic mice model and its underlying mechanisms. Forty-five 9-week-old Institute of Cancer Research (ICR) mice underwent either a sham-operation (n = 9) or OVX (n = 36). Four days after the operation, OVX mice were further divided into four groups and received either saline alone, Lactobacillus plantarum GKM3, Lactobacillus paracasei GKS6 or alendronate per day for 28 days. After sacrifice by decapitation, right distal femur diaphysis was imaged via micro-computed tomography (MCT) and parameters including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and bone mineral density (BMD) were measured. Moreover, GKM3 and GKS6 on RANKL-induced osteoclast formation and osteoblast differentiation using in vitro cultures were also investigated. The results showed that both probiotics strains inhibited osteoporosis in the OVX mice model, with L. paracasei GKS6 outperforming L. plantarum GKM3. Besides this, both GKS6 and GKM3 promoted osteoblast differentiation and inhibited RANKL-induced osteoclast differentiation via the Bone Morphogenetic Proteins (BMP) and RANKL pathways, respectively. These findings suggested that both strains of Lactobacilli may be pursued as potential candidates for the treatment and management of osteoporosis, particularly in postmenopausal osteoporosis.

The effect of fructan-enriched diet on bone turnover parameters in ovariectomized rats under calcium restriction.

INTRODUCTION: Osteoporosis, the "quiet epidemic", is one of the most serious threats to public health. It is known that estrogen plays a significant role in the regulation of bone turnover, and its loss at menopause causes osteoporosis. Added to this, insufficient calcium intake accelerates bone mass loss, increasing the risk of fractures. OBJECTIVE: The study aimed to answer the question whether a fructan-enriched diet could be helpful in preventing from disturbances in bone turnover caused by calcium restriction combined with ovariectomy-induced estrogen deficiency. The differences related to the kind of fructan and 'matrix effect' of fructan action (form of addition) were also evaluated. MATERIAL AND METHODS: The study was conducted using sham-operated (control groups) or ovariectomized (OVX) rats fed a calcium restricted diet. The treatment diets contained one of three fructan sources - Jerusalem artichoke, yacon and Beneo Orafti Synergy1 - added alone or as an ingredient of strawberry sorbet, all in the amount providing 8% fructans. Analyses of biological material included: serum Ca, Mg and P concentrations, alkaline phosphatase activity (ALP), osteocalcin (OC) and C-telopeptide degradation products from type I collagen (CTX). Densitometric parameters of femora were also assayed. RESULTS: Among markers of bone turnover, the ALP activity depended both on the kind of fructan and the form of addition. The highest value was shown in the OVX group fed a low-calcium diet, whereas administration of diet enriched with Jerusalem artichoke led to an almost 50% decrease in the value of this parameter. Dietary fructans also lowered the OC level. Feeding rats with diet containing sorbet enriched in yacon or Jerusalem artichoke resulted in a decrease of CTX, compared to the diet containing yacon alone or fructan formulation in both forms No significant differences were observed in densitometric parameters between treatment groups. CONCLUSIONS: The obtained findings suggest that fructan administration with a calcium-restricted diet might exert a positive effect on bone turnover parameters. Regarding the form of their addition, it is possible that other constituents of sorbets contributed to the fructan action. It remains open whether this impact would be significant over a longer period of time.