Formula Milk Supplementation and Bone Acquisition in 4-6 Years Chinese Children: A 12-Month Cluster-Randomized Controlled Trial.

Dairy foods are crucial for adequate calcium intake in young children, but scarce data are available on the effects of formula milk on bone acquisition. This cluster-randomized controlled trial investigated the effects of the supplementation of formula milk on bone health in rural children accustomed to a low-calcium diet between September 2021 and September 2022. We recruited 196 healthy children aged 4-6 years from two kindergartens in Huining County, Northwest China. A class-based randomization was used to assign them to receive 60 g of formula milk powder containing 720 mg calcium and 4.5 µg vitamin D or 20-30 g of bread per day for 12 months, respectively. Bone mineral density (BMD) and bone mineral content (BMC) at the left forearm and calcaneus, bone biomarkers, bone-related hormones/growth factors, and body measures were determined at baseline, 6, and 12 months. A total of 174 children completed the trial and were included in the analysis. Compared with the control group, formula milk intervention showed significant extra increments in BMD (3.77% and 6.66%) and BMC (4.55% and 5.76%) at the left forearm at 6th and 12th months post-intervention (all p < 0.001), respectively. Similar trends were observed in BMD (2.83%) and BMC (2.38%) in the left calcaneus at 6 months (p < 0.05). The milk intervention (vs. control) also showed significant changes in the serum concentrations of osteocalcin level (-7.59%, p = 0.012), 25-hydroxy-vitamin-D (+5.54%, p = 0.001), parathyroid hormone concentration (-15.22%, p = 0.003), and insulin-like growth factor 1 (+8.36%, p = 0.014). The percentage increases in height were 0.34%, 0.45%, and 0.42% higher in the milk group than in the control group after 3-, 6-, and 9-month intervention, respectively (p < 0.05). In summary, formula milk supplementation enhances bone acquisition at the left forearm in young Chinese children.

Slowly Digestible Carbohydrate Diet Ameliorates Hyperglycemia and Hyperlipidemia in High-Fat Diet/Streptozocin-Induced Diabetic Mice.

Objective: Given that the prevalence rate of type 2 diabetes mellitus (T2DM) continues to increase, it is important to find an effective method to prevent or treat this disease. Previous studies have shown that dietary intervention with a slowly digestible carbohydrate (SDC) diet can improve T2DM with almost no side effects. However, the underlying mechanisms of SDC protect against T2DM remains to be elucidated. Methods: The T2DM mice model was established with a high-fat diet and streptozocin injection. Then, SDC was administered for 6 weeks. Bodyweight, food intake, organ indices, fasting blood glucose (FBG), oral glucose tolerance test (OGTT), homeostasis model assessment for insulin resistance (HOMA-IR), and other biochemical parameters were measured. Histopathological and lipid accumulation analyses were performed, and the glucose metabolism-related gene expressions in the liver and skeletal muscle were determined. Lastly, colonic microbiota was also analyzed. Results: SDC intervention alleviated the weight loss in the pancreas, lowered blood glucose and glycosylated hemoglobin levels, and improved glucose tolerance and HOMA-IR. SDC intervention improved serum lipid profile, adipocytokines levels, and lowered the lipid accumulation in the liver, subcutaneous adipose tissue, and epididymal visceral adipose tissue. In addition, SDC intervention increased the expression levels of IRS-2 and GLUT-2 in liver tissues and elevated GLUT-4 expression levels in skeletal muscle tissues. Notably, SDC intervention decreased the Bacteroidetes/Firmicutes ratio, increased Desulfovibrio and Lachnospiraceae genus levels, and inhibited the relative abundance of potentially pathogenic bacteria. Conclusions: SDC intervention can improve hyperglycemia and hyperlipidemia status in diabetic mice, suggesting that this intervention might be beneficial for T2DM.

[Status analysis and thinking on experimental study on efficacy of Mori Fructus in treatment of cardiovascular diseases].

Cardiovascular diseases are the most important diseases that endanger national health, and its development process is complex and diverse. Various cardiovascular diseases caused by obesity, such as hyperlipidemia, hyperglycemia and atherosclerosis, are interrelated and interacted each other. Diet, as the main means of prevention and treatment, plays an important role in the occurrence and development of cardiovascular disease. Mori Fructus is one of the first ingredients that are listed in medicinal and edible food. With a wide range of applications in daily life, it contains polysaccharides(polysaccharide, APS), anthocyanins(anthocyanin, LCRA), flavonoids and other bioactive ingredients. With a wide range of antioxidant, anti-aging, hypoglycemic and hypolipidemic activities, these materials exert effects in alleviating diabetes, hyperglycemia, hyperlipidemia and other cardiovascular diseases. In this paper, we retrieved such databases as PubMed, Web of science, CNKI, VTTMS, Wan Fang, and collected literatures about the effect of single administration of mulberry on cardiovascular diseases in the past 15 years, with "mulberry and cardiovascular disease" as the key word, and summarized the latest progress. The results of many experimental studies have showed that different forms of mulberry can significantly alleviate obesity, diabetes, atherosclerosis, hyperlipidemia and hypertension, suggesting that the scope of action of Mori Fructus covers different pathological stages of cardiovascular diseases. This paper systematically analyzes and summarizes the application forms, efficacy and the existing problems of these experiments, and provides study thinking and development direction for the utilization and new product design of Mori Fructus-related products in the treatment of cardiovascular diseases.

Therapeutic Potential and Outlook of Alternative Medicine for Osteoporosis.

Osteoporosis, a bone disease resulting in the loss of bone density and microstructure quality, is often associated with fragility fractures, and the latter imposes a great burden on the patient and society. Although there are several different treatments available for osteoporosis such as hormone replacement therapy, bisphosphonates, Denosumab, and parathyroid hormone, some concerns have been raised regarding the inherent side effects of their long term use. It would be of great relevance to search for alternative natural compounds, which could complementarily overcome the limitations of the currently available therapy. Herein, we review current literature on natural compounds that might have therapeutic values for osteoporosis. Search terms included bone resorption, bone density, osteoporosis, postmenopausal, osteoporosis or bone density conservation agents, and any of the terms related to traditional, herbal, natural therapy, natural health, diet, or phytoestrogens. All the compounds and herbs included in the review are naturally bioactive or are used in folk herbal medicine and have been reported to be capable of attenuating osteopenia or osteoporosis in vivo or in vitro, through various mechanisms - estrogen-like activity, antioxidant and anti-inflammatory properties, or by modulating the key signaling pathways in the pathogenesis of osteoporosis. Through our assessment of the therapeutic potential and outlook of alternative medicine, we aim to provide an appealing perspective for the consideration of the application of a complementary anti-osteoporotic treatment option and prevention strategy for osteoporosis or osteolytic bone disorders.

Nitidine chloride prevents OVX-induced bone loss via suppressing NFATc1-mediated osteoclast differentiation.

Nitidine chloride (NC), a bioactive alkaloid isolated from Zanthoxylum nitidum, has been used as a herbal ingredient in toothpaste that prevents cavities for decades. It also displays potential antitumor and anti-inflammation properties. However, its anticatabolic effect on bone is not known. We investigated the effect of NC on osteoclastogenesis, bone resorption and RANKL-induced NF-κB and NFATc1 signalling. In mouse-derived bone marrow monocytes (BMMs), NC suppressed RANKL-induced multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation and bone resorption in a dose dependent manner. NC attenuated the expression of osteoclast marker genes including cathepsin K, D2, calcitonin receptor, NFATc1, and TRAP. Further, NC inhibited RANKL-activated NF-κB and NFATc1 signalling pathways. In vivo study revealed that NC abrogated oestrogen deficiency-induced bone loss in ovariectomized mice. Histological analysis showed that the number of osteoclasts was significantly lower in NC-treated groups. Collectively, our data demonstrate that NC suppressed osteoclastogenesis and prevented OVX-induced bone loss by inhibiting RANKL-induced NF-κB and NFATc1 signalling pathways. NC may be a natural and novel treatment for osteoclast-related bone lytic diseases.

Ethanol Extract of Fructus ligustri lucidi Increased Circulating 1,25(OH)2D3 Levels, but Did Not Improve Calcium Balance in Mature Ovariectomized Rats.

Our previous studies found that different extracts or fractions of Fructus ligustri lucidi (FLL) played different roles in altering the regulation of bone and mineral metabolism in different animal models. The present study was designed to compare the actions of FLL ethanol (EE) and water extracts (WE) on bone and mineral metabolism in a 6-month-old mature ovariectomized (OVX) rat model. Our results showed that FLL extracts did not significantly improve systematic Ca balance in mature OVX rats. However, EE, but not WE treatment, significantly increased serum 1,25(OH)2D3 levels in mature OVX rats. An in vitro study using human proximal tubule (HKC-8) cells showed that EE, but not WE, significantly enhanced renal 25-dihydroxyvitamin D3-1[Formula: see text]-hydroxylase (1-OHase) mRNA expressions and simultaneously repressed renal 25-dihydroxyvitamin D3-24-hydroxylase (24-OHase) mRNA expressions. Further investigation indicated that EE could significantly induce the protein expression of 1-OHase, but did not alter 24-OHase expression in HKC-8 cells. Our results demonstrated that EE increased circulating 1,25(OH)2D3 levels in OVX rats, possibly via upregulation of renal 1-OHase expressions in renal proximal tubule cells. Our study indicates that FLL is a natural oral agent that could directly regulate renal vitamin D metabolism in vivo and in vitro.

Bafilomycin A1 Attenuates Osteoclast Acidification and Formation, Accompanied by Increased Levels of SQSTM1/p62 Protein.

Vacuolar proton pump H(+)-adenosine triphosphatases (V-ATPases) play an important role in osteoclast function. Further understanding of the cellular and molecular mechanisms of V-ATPase inhibition is vital for the development of anti-resorptive drugs specifically targeting osteoclast V-ATPases. In this study, we observed that bafilomycin A1, a naturally-occurring inhibitor of V-ATPases, increased the protein level of SQSTM1/p62, a known negative regulator of osteoclast formation. Consistently, we found that bafilomycin A1 diminishes the intracellular accumulation of the acidotropic probe lysotracker in osteoclast-like cells; indicative of reduced acidification. Further, bafilomycin A1 inhibits osteoclast formation with attenuation of cell fusion and multi-nucleation of osteoclast-like cells during osteoclast differentiation. Taken together, these data indicate that bafilomycin A1 attenuates osteoclast differentiation in part via increased levels of SQSTM1/p62 protein, providing further mechanistic insight into the effect of V-ATPase inhibition in osteoclasts.

Deficiency of sorting nexin 10 prevents bone erosion in collagen-induced mouse arthritis through promoting NFATc1 degradation.

OBJECTIVE: Periarticular and subchondral bone erosion in rheumatoid arthritis caused by osteoclast differentiation and activation is a critical index for diagnosis, therapy and monitoring of the disease. Sorting nexin (SNX) 10, a member of the SNX family which functions in regulation of endosomal sorting, has been implicated to play an important clinical role in malignant osteopetrosis. Here we studied the roles and precise mechanisms of SNX10 in the bone destruction of collagen-induced arthritis (CIA) mice. METHODS: The role of SNX10 in bone destruction was evaluated by a CIA mice model which was induced in male SNX10(-/-) mice and wild type littermates. The mechanism was explored in osteoclasts induced by receptor activator of nuclear factor κB ligand from bone marrow mononuclear cells of wild type and SNX10(-/-) mice. RESULTS: SNX10 knockout prevented bone loss and joint destruction in CIA mice with reduced serum levels of TNF-α, interleukin 1ß and anticollagen IgG 2α antibody. SNX10 deficiency did not block osteoclastogenesis, but significantly impaired osteoclast maturation and bone-resorption function by disturbing the formation of actin belt. The production of TRAP, CtsK and MMP9 in SNX10(-/-) osteoclasts was significantly inhibited, and partially restored by SNX10 overexpression. We further demonstrated that the degradation of NFATc1 was accelerated in SNX10(-/-) osteoclasts causing an inhibition of integrin ß3-Src-PYK2 signalling. CONCLUSIONS: Our study discloses a crucial role and novel mechanism for SNX10 in osteoclast function, and provides evidence for SNX10 as a promising novel therapeutic target for suppression of immune inflammation and bone erosion in rheumatoid arthritis.

Combination treatment with Fructus Ligustri Lucidi and Puerariae radix offsets their independent actions on bone and mineral metabolism in ovariectomized rats.

OBJECTIVE: The aim of this study was to evaluate the efficacy of Fructus Ligustri Lucidi (FLL) and Puerariae radix (PR) combination treatment in bone and mineral metabolism in ovariectomized (OVX) rats in our search for an alternative regimen for the management of postmenopausal osteoporosis. METHODS: Six-month-old OVX rats were used as postmenopausal osteoporotic models, and PR water extract (PR) and FLL water extract (WE) were added to commercial diets individually or in combination and administered to OVX rats for 12 weeks. Bone properties, calcium and phosphorus absorption, and bone biochemical markers were measured to investigate the potential interactions between the actions of PR and the actions of WE on bone and mineral metabolism in OVX rats. RESULTS: Long-term treatment with PR did not significantly improve bone properties but greatly ameliorated the secondary hyperparathyroidism induced by ovariectomy in the animals. WE significantly enhanced the intestinal calcium absorption rate and decreased the enlarged trabecular bone surface at the site of metaphysic tibia in OVX rats. However, the positive effects of WE or PR alone on bone and mineral metabolism were diminished when OVX rats were cotreated with WE and PR. CONCLUSIONS: The combination of these two herbs offsets their independent actions on bone and mineral metabolism in vivo. The results of the present study could provide insights to medical professionals to further their understanding of the potential negative impact of herb-herb interactions when a combination of herbal mixtures is used for the management of osteoporosis.

Disruption of the dynein-dynactin complex unveils motor-specific functions in osteoclast formation and bone resorption.

Osteoclastic bone resorption requires strict interplay between acidified carrier vesicles, motor proteins, and the underlying cytoskeleton in order to sustain the specialized structural and functional polarization of the ruffled border. Cytoplasmic dynein, a large processive mechanochemical motor comprising heavy, intermediate, and light chains coupled to the dynactin cofactor complex, powers unilateral motility of diverse cargos to microtubule minus-ends. We have recently shown that regulators of the dynein motor complex constitute critical components of the osteoclastic bone resorptive machinery. Here, by selectively modulating endogenous dynein activity, we show that the integrity of the dynein-dynactin motor complex is an essential requirement for both osteoclast formation and function. Systematic dissection of the osteoclast dynein-dynactin complex revealed that it is differentially localized throughout RANKL-induced osteoclast formation and activation, undergoing microtubule-coupled reorganization upon the establishment of cellular polarization. In osteoclasts actively resorbing bone, dynein-dynactin intimately co-localizes with the CAP-Gly domain-containing microtubule plus-end protein CLIP-170 at the resorptive front, thus orientating the ruffled border as a microtubule plus-end domain. Unexpectedly, disruption of the dynein-dynactin complex by exogenous p50/dynamitin expression retards osteoclast formation in vitro, owing largely to prolonged mitotic stasis of osteoclast progenitor cells. More importantly, loss of osteoclastic dynein activity results in a drastic redistribution of key intracellular organelles, including the Golgi and lysosomes, an effect that coincides with impaired cathepsin K secretion and diminished bone resorptive function. Collectively, these data unveil a previously unrecognized role for the dynein-dynactin motor complex in osteoclast formation and function, serving not only to regulate their timely maturation but also the delivery of osteolytic cargo that is essential to the bone resorptive process.

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL-induced activation of NF-κB and ERK.

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.

Tctex-1, a novel interaction partner of Rab3D, is required for osteoclastic bone resorption.

Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function.

Sesquiterpene lactone parthenolide blocks lipopolysaccharide-induced osteolysis through the suppression of NF-kappaB activity.

UNLABELLED: Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity. INTRODUCTION: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity. MATERIALS AND METHODS: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6. RESULTS: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6. CONCLUSION: The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.