Effects of undercarboxylated osteocalcin and voluntary running on bone tissue in aged mice

Type 1 diabetes (T1D) represents a significant health burden worldwide, with associated complications including bone fragility. Current clinical methods and biomarkers for assessing bone health and predicting fracture risk in T1D are limited and lack accuracy. MicroRNAs (miRNAs) have emerged as potential biomarkers for predicting T1D-induced bone loss, although comprehensive profiling studies are lacking. Previous investigations have indicated a link between dysregulated miRNA expression levels and impaired bone health in T1D. Therefore, in this study, we explored differential miRNA expression levels in serum and bone tissue of mice with T1D-induced bone loss using Next Generation Sequencing (NGS). T1D was induced using streptozotocin in male wild-type mice. Serum and bone tissues were analyzed at 14 weeks of age, following the prior characterization of bone loss in this mouse model. MiRNA profiling was conducted using two-independent NGS analyses and validated through quantitative RT-PCR. NGS profiling identified differential expression of miRNAs in serum and bone tissue of T1D mice compared to controls. The first NGS analysis revealed 24 differentially expressed miRNAs in serum and 13 in bone tissue. Especially, miR-136-3p was consistently downregulated in both serum and bone tissue. However, the second NGS analysis presented a distinct set of dysregulated miRNAs, with miR-206-3p overlapping in both tissues but exhibiting differential expression patterns. Surprisingly, miR-144-5p, miR-19a-3p, and miR-21a-5p displayed contrasting regulatory patterns between NGS and qPCR analyses. Finally, gene network analysis identified associations between dysregulated miRNAs and pathways involved in bone physiology, including TGF-beta, PI3-Akt signaling, and osteoclast differentiation in humans. In conclusion, our study offers initial insights into dysregulated miRNAs associated with T1D-induced bone loss, but also highlights the lack of consistency in the results obtained from miRNA sequencing in different cohorts. Thus, further investigation is needed to better understand the complexities of miRNA analyses before they can be established as reproducible biomarkers for predicting bone health in T1D.

Age-related osteoporosis is characterized by reduced bone mineralization and reduced bone strength, which increases the risk of fractures. We examined metabolic changes associated with age-related bone loss by profiling lipids and polar metabolites in tibia and femur bone tissues from young (5 months old) and old (28 months old) male C57BL/6J mice using ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry. Partial least-squares discriminant analysis showed clear differences in metabolite levels in bone tissues of young and old mice. We identified 93 lipid species, including free fatty acids, sphingolipids, phospholipids, and glycerolipids, that were significantly altered in bone tissues of old mice. In addition, the expression of 26 polar metabolites differed significantly in bone tissues of old mice and young mice. Specifically, uremic toxin metabolite levels (p-cresyl sulfate, hippuric acid, and indoxylsulfate) were higher in bone tissues of old mice than in young mice. The increase in p-cresyl sulfate, hippuric acid, and indoxylsulfate levels were determined using targeted analysis of plasma polar extracts to determine whether these metabolites could serve as potential osteoporosis biomarkers. This study demonstrates that LC-MS-based global profiling of lipid and polar metabolites can elucidate metabolic changes that occur during age-related bone loss and identify potential biomarkers of osteoporosis.

Objective To observe the effect of fluoride on fibroblast growth factor 23 (FGF23) in bone tissue of mice, and to explore the role of FGF23 in fluoride-induced bone injury. Methods Sixty-four Balb/c mice, half male and female, were divided into 4 groups based on body weight via the random number table method and 16 mice were in each group. The mice in control group, low fluoride group, middle fluoride group and high fluoride group were treated with 0, 25, 50, and 100 mg/L F- distilled water, respectively. After three months, the mice were put to death and the prevalence of dental fluorosis was calculated. The fluoride contents in spine were detected via the fluoride-ion selective electrode method, serum content of calcium and phosphorus were detected by micro enzyme labeled method. The levels of FGF23, parathyroid hormone (PTH) and 1,25 dihydroxy vitamin D3 [1,25(OH)2D3] in serum were measured by enzyme-linked immunosorbent assay. The FGF23 protein expression levels in bone tissue were determined by immunohistochemistry and Western blotting. Results The rates of dental fluorosis in low, medium and high fluoride groups were 75% (4/16), 100% (16/16) and 100% (16/16), respectively. Compared with control group [0(0/16)] the differences were statistically significant (P 0.05). The contents of phosphorus in the serum of the medium and the high fluoride groups [(2.46 ± 0.32), (2.48 ± 0.73) mmol/L] were lower than those in the control and the low fluoride groups [(2.89 ± 0.45), (3.25 ± 0.69) mmol/L, P < 0.05]. The serum PTH and 1,25 (OH)2D3 content increased first and then decreased. The expression of FGF23 in middle and high fluoride groups [(660.84 ± 64.18), (638.74 ± 121.23) ng/L] was up-regulated compared with that of control group [(613.53 ± 98.18) ng/L]. The expression of FGF23 protein in cortical bone increased gradually with the dose of fluoride. Western blotting results showed that the content of FGF23 protein in the bone tissue of mice was significantly increased in the low fluoride group (1.58 ± 0.46) and the middle fluoride group (1.40± 0.41) compared with that of control group (1.00 ± 0.41), the differences were statistically significant (P < 0.05). Conclusions The phosphorus, FGF23, PTH, and 1,25 (OH)2D3 levels in the serum and FGF23 protein levels in the bone tissue of fluorosis mice have changed. It may be suggested that FGF23 interacts with PTH and 1,25 (OH)2D3 to influence the level of calcium and phosphorus metabolism in the body and participate in the formation of skeletal fluorosis. Key words: Fluorosis; Fluoride on fibroblast growth factor 23; Parathyroid hormone; 1,25 dihydroxy vitamin D3; Phosphorus; Calcium

BackgroundIt is well known that exercise has positive effects on bone. It has been reported that voluntary exercise increase bone formation and bone density during growing period. However, effects of more long‐term exercise performed until adult on bone tissue are little known.PurposeTo investigate the effects of long‐term voluntary wheel running on histological and mechanical properties of bone tissue in mice.MethodsFour‐week‐old male C57BL/6 mice (n=31) were assigned to 1) a sedentary control (CON) and 2) a voluntary running (VE) groups. Mice in VE group were allowed free access a wheel. Femurs were removed at 4, 28, or 40 weeks of age. Bone volume was measured histologically as the bone volume per tissue volume［BV/TV (%)］, and bone strength was measured using three‐point bending test. Bone strength was normalized to body weight［N/g; maximum breaking force (N) per body weight (g)］.ResultsThe body weight in the CON at 4, 28, 40 weeks of age was 18.4±0.5, 30.1±1.3, 29.8±1.4 g, respectively, while it in VE at 28 and 40 weeks of age was 26.7±0.8, and 29.2±2.4 g. BV/TV (%) significantly decreased with aging from 4 to 28 weeks of age in both groups. There were no significant differences in BV/TV (%) between CON and VE. Also, bone strength significantly increase with aging from 4 to 28 weeks of age in both groups. There were no significant differences between CON and VE.ConclusionOur data suggests that long‐term voluntary running do not effect on histological and mechanical properties of bone tissue in mice.This work was supported by grants from the MEXT‐Supported Program for the Strategic Research Foundation at Private Universities (S1101008).

Objective To observe the expression of interleukin-6 (IL-6), glycoprotein 130 (gp130), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in bone tissue of fluorosis mice and explore the mechanisms of IL-6/gp130 signaling pathway in the pathogenesis of bone injury caused by fluoride. Methods Sixty-four male Balb/c mice were divided into 4 groups based on body weight by random number table method and 16 mice were in each group. The mice in control group were fed with distilled water, experimental animals in low, middle and high dose groups were fed with distilled water containing NaF 25, 50 and 100 mg/L, respectively. The mice were fed for three months to establish a drinking water fluorosis model. Mice dental fluorosis was observed and the fluoride contents in spine were detected by fluorine-ion selective electrode method. The changes of morphology and cell ultra structure in fluorosis bone tissue were observed by optical and electronic microscope. The activities of serum alkaline phosphatase (ALP) and acid phosphatase (ACP) were detected by micro enzyme labeled method. The levels of serum IL-6 were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of IL-6, gp130, PI3K and Akt in long bone tissue were detected by real-time fluorescence quantitative PCR (real-time PCR). The protein expression levels and activity of gp130, PI3K and Akt were detected by Western blotting. Results The rates of dental fluorosis in low fluoride group, middle fluoride group and high fluoride group were 62.5% (10/16), 100.0% (15/15) and 100.0% (16/16), respectively, and all the fluoride groups were higher than control group (0, 0/15, all P < 0.05). Fluoride contents of bone were significantly increased with increase of fluoride doses (F= 309.716, P < 0.05). The bone trabecular thickness and density in fluorosis mice were remarkably increased and disorder, and bone sclerosis was observed under optical microscope. The percentage of trabecular bone area was significantly increased with increase of fluoride doses(F= 25.161, P < 0.05). Ultra structurally, swelling of cells, dilation of endoplasmic reticulum, chromatin margination in osteoblasts and osteoclasts of high fluoride group were observed which indicated that cells ultra structure was apparently damaged. The activities of serum ALP and ACP were significantly increased with increase of fluoride doses (F= 19.586, 13.329, all P < 0.05), and both high fluoride group [(8.05 ± 1.10), (10.95 ± 1.85) King unit] and middle fluoride group [(6.48 ± 0.92), (9.18 ± 0.76) King unit] were significantly higher than control group [(5.15 ± 0.73), (7.49 ± 0.66) King unit, all P < 0.05]. The levels of serum IL-6 in control group, low fluoride group, middle fluoride group and high fluoride group were (5.98 ± 1.43), (7.54 ± 2.16), (5.25 ± 1.97), (6.31 ± 1.36) ng/L, respectively, the differences were statistically significant (F= 3.840, P < 0.05), and low fluoride group was significantly higher than control group (P < 0.05), and middle fluoride group was significantly lower than low fluoride group (P < 0.05). The differences of mRNA expression levels of IL-6, gp130, PI3K and Akt in long bone tissue between groups were statistically significant (F= 9.952, 8.954, 6.997, 21.504, all P < 0.05), and high fluoride group (0.59 ± 0.42, 0.46 ± 0.22, 0.55 ± 0.30, 0.61 ± 0.10) was lower than control group (1.10 ± 0.54, 1.16 ± 0.67, 1.20 ± 0.84, 1.06 ± 0.37) and low fluoride group (1.86 ± 0.63, 1.61 ± 0.64, 1.74 ± 0.65, 1.70 ± 0.58, all P < 0.05), and all mRNA expression except gp130 of low fluoride group was higher than that of control group (all P < 0.05). The differences of protein expression levels and activities of gp130, PI3K and Akt in long bone tissue between groups were statistically significant (F= 4.777, 7.479, 3.535, all P < 0.05), and low fluoride group (0.66 ± 0.10, 0.69 ± 0.12, 0.92 ± 0.09) was significantly higher than control group (0.51 ± 0.11, 0.47 ± 0.06, 0.66 ± 0.14, all P < 0.05), and high fluoride group (0.42 ± 0.11, 0.38 ± 0.06, 0.67 ± 0.14) was lower than low fluoride group (all P < 0.05). Conclusion The mRNA and protein expression levels of IL-6, gp130, PI3K and Akt in bone tissue of fluorosis mice have changed, which indicates that IL-6/gp130 signaling pathway may be involved in the pathogenesis of bone injury caused by fluoride. Key words: Fluorine; Mice; Interleukin-6; Glycoproteins; Bone and Bones

Increasing age is the strongest predictor of risk of COVID-19 severity and mortality. Immunometabolic switch from glycolysis to ketolysis protects against inflammatory damage and influenza infection in adults. To investigate how age compromises defense against coronavirus infection, and whether a pro-longevity ketogenic diet (KD) impacts immune surveillance, we developed an aging model of natural murine beta coronavirus (mCoV) infection with mouse hepatitis virus strain-A59 (MHV-A59). When inoculated intranasally, mCoV is pneumotropic and recapitulates several clinical hallmarks of COVID-19 infection. Aged mCoV-A59-infected mice have increased mortality and higher systemic inflammation in the heart, adipose tissue, and hypothalamus, including neutrophilia and loss of γδ T cells in lungs. Activation of ketogenesis in aged mice expands tissue protective γδ T cells, deactivates the NLRP3 inflammasome, and decreases pathogenic monocytes in lungs of infected aged mice. These data establish harnessing of the ketogenic immunometabolic checkpoint as a potential treatment against coronavirus infection in the aged.

Ketogenic diet protects against coronavirus-infected aged mice, which, like humans, are significantly more susceptible to infections, by deactivating NLRP3 inflammasome, reducing pathogenic monotypes, and expanding γδ T cells in the lung.

Wnt signaling is vital for osteoblast differentiation and recently has been associated with aging. Because impaired osteoblastogenesis is a cellular characteristic of age-induced bone loss, we investigated whether this process is associated with an altered expression of Wnt signaling-related proteins in bone and osteoblasts. Bone marrow cells were isolated from male C57BL/6 mice, aged 6 weeks, 6 months, and 18 months, respectively. Osteogenic differentiation was induced for 3 weeks and assessed using alizarin red staining. Gene expression of Wnt1, 3a, 4, 5a, 5b, 7b, 9b, 10b, lipoprotein receptor-related protein (LRP)-5/6, as well as dickkopf-1 (Dkk-1), sclerostin, and secreted frizzled related protein-1 (sFRP-1) was determined in bone tissue and osteoblasts on days 7, 14, and 21 by real-time RT-PCR. Osteoblast differentiation was significantly reduced in aged mice compared with young and adult mice. In bone tissue, expression levels of all genes assessed were decreased in adult and old mice, respectively, compared with young mice. Mature osteoblasts of aged compared with those of young mice showed enhanced expression of Wnt9b, LRP-6, and Dkk-1, and decreased expression of Wnt5a and 7b. In early osteoblasts, mRNA levels of Wnt1, 5a, 5b, and 7b were increased significantly in aged mice. The expression of Wnt3a, 4, LRP-5, and sclerostin was not altered in aged osteoblasts. In conclusion, osteoblastic expression of each Wnt-related protein is regulated individually by aging. The overall decreased expression of Wnt-related proteins in bone tissue of aged mice underlines the newly discovered association of Wnt signaling with aging.

Critical biological processes are under control of the circadian clock. Disruption of this clock, e.g. during aging, results in increased risk for development of chronic disease. Exercise is a protective intervention that elicits changes in both age and circadian pathologies, yet its role in regulating circadian gene expression in peripheral tissues is unknown. We hypothesized that voluntary wheel running would restore disrupted circadian rhythm in aged mice. We analyzed wheel running patterns and expression of circadian regulators in male and female C57Bl/6J mice in adult (~4 months) and old (~18 months) ages. As expected, young female mice ran further than male mice, and old mice ran significantly less than young mice. Older mice of both sexes had a delayed start time in activity which likely points to a disrupted diurnal running pattern and circadian disruption. Voluntary wheel running rescued some circadian dysfunction in older females. This effect was not present in older males, and whether this was due to low wheel running distance or circadian output is not clear and warrants a future study. Overall, we show that voluntary wheel running can rescue some circadian dysfunction in older female but not male mice; and these changes are tissue dependent. While voluntary running was not sufficient to fully rescue age-related changes in circadian rhythm, ongoing studies will determine if forced exercise (e.g. treadmill) and/or chrono-timed exercise can improve age-related cardiovascular, skeletal muscle, and circadian dysfunction.

Aging Aggravates the Progression of Muscle Degeneration After Rotator Cuff Tears in Mice

Age-induced chronic inflammation is prevented by aerobic and resistance exercise training. However, the effects of the mechanism of exercise on chronic inflammation in each tissue remains unclear. The aim of this study was to investigate the effects of resistance and aerobic training on gene expression profiles for macrophage infiltration and polarization (M1/M2 ratio) with chronic inflammation in various tissues of aged model mice. Male 38-week-old SAMP1 (senescence-accelerated prone mouse 1) mice were randomly divided into three groups—sedentary (Aged-Sed-SAMP1), aerobic training (Aged-AT-SAMP1; voluntary running), and resistance training—for 12 weeks (Aged-RT-SAMP1; climbing ladder). Resistance and aerobic exercise training prevented an increase in circulating TNF-α levels (a marker of systemic inflammation) in aged SAMP1 mice, along with decreases in tissue inflammatory cytokine (TNF-α and IL-1β) mRNA expression in the heart, liver, small intestine, brain, aorta, adipose, and skeletal muscle, but it did not change the levels in the lung, spleen, and large intestine. Moreover, resistance and aerobic exercise training attenuated increases in F4/80 mRNA expression (macrophage infiltration), the ratio of CD11c/CD163 mRNA expression (M1/M2 macrophage polarization), and MCP-1 mRNA expression (chemokine: a regulator of chronic inflammation) in the chronic inflamed tissues of aged SAMP1 mice. These results suggested that resistance and aerobic exercise training-induced changes in gene expression for macrophage infiltration and polarization in various tissues might be involved in the prevention of age-related tissue chronic inflammation, and lead to a reduction of the increase in circulating TNF-α levels, as a marker of systemic inflammation, in aged SAMP1 mice.

As bones age in most mammals, they typically become more fragile. This state of bone fragility is often associated with more homogenous collagen fiber orientations (CFO). Unlike most mammals, bats maintain mechanically competent bone throughout their lifespans, but little is known of positional and age-related changes in CFO within wing bones. This study tests the hypothesis that age-related changes in CFO in big brown bats (Eptesicus fuscus) differ from those of the standard mammalian model for skeletal aging, the C57BL/6 laboratory mouse. We used data from quantitative polarized light microscopy (qPLM) to compare CFO across the lifespan of long-lived big brown bats and age matched C57BL/6 mice. Eptesicus and C57BL/6 mice displayed idiosyncratic patterns of CFO. Consistent age-related changes were only apparent in the outer cortical bone of Eptesicus, where bone tissue is more longitudinally arranged and more anisotropic in older individuals. Both taxa displayed a ring of more transversely oriented bone tissue surrounding the medullary cavity. In Eptesicus, this tissue represents a greater proportion of the overall cross-section, and is more clearly helically aligned (arranged at 45° to the bone long axis) than similar bone tissue in mice. Bat wing bones displayed a proximodistal gradient in CFO anisotropy and longitudinal orientation in both outer and inner cortical bone compartments. This study lays a methodological foundation for the quantitative evaluation of bone tissue architecture in volant and non-volant mammals that may be expanded in the future.

Expression of the prostaglandin E 2 (PGE 2) receptor subtype EP 4 and its regulation by PGE 2 in osteoblastic cell lines and adult rat bone tissue

Introduction: The aim was to evaluate the structural and functional changes of bone tissue in mice with null expression of 11β-HSD2 or both 11β-HSD2 and Apolipoprotein E. Materials and methods: The experimental study was performed in 60 male mice, weighting 24–30 g. The animals were kept in accordance with the rules of laboratory practice for preclinical studies on the territory of the Russian Federation. Mice lacking 11β-HSD2 (Hsd2-/-) and male mice lacking 11β-HSD2 and Apolipoprotein E (Hsd2-/-/Apoe-/-) were used in the study. We studied and characterized the state of bone tissue, indicators of bone density, microcircu­lation in bone tissue, endothelial dysfunction coefficient, width of bone trabeculae, as well as serum concentrations of bone alkaline phosphatase, hydroxyproline, deoxyprinoline and expression levels of p53, Bcl2, Bax, eNOS genes. Results and discussion: We showed that mice with the Hsd2-/- genotype with no expression of 11ß-HSD2 by the 6th month of life showed a statistically significant decrease in bone density, which progresses to the 7th and 8th months of life. At the 8th month of animal life, a decrease in bone density is accompanied by a statistically significant decrease in the level of microcirculation in the bone and an increase in the coefficient of endothelial dysfunction. Taking into account the relationship of endothelial dysfunction, atherogenesis and disorders in the processes of bone remodeling, in the framework of this study, we also assessed the state of bone tissue in double transgenes with the genotype Hsd2-/-/Apoe-/-, which lack the expression of both 11ß-HSD2 and Apolipoprotein E. In this study, we also saw increased acti­vation of processes leading to disruption of bone remodeling processes. In the group of the animals with the genotype Hsd2-/-/Apoe-/-, we found statistically significant differences from the mice with no expression of 11ß-HSD2 in bone density and microcirculation, and the width of bone trabeculae. Also, a statistically significant increase in hydroxypro­line and deoxyprinoline was found in the group of double transgenes, in the absence of significant changes in the con­centration of bone alkaline phosphatase. This fact indicates a pronounced activation of bone resorption processes in the absence of activation of osteosynthesis processes, which leads to the detected violation of bone remodeling processes. Conclusion: Thus, we have shown that a violation of the metabolic regulation of steroid hormone metabolism in ani­mals with null expression of the 11ß-HSD2 (Hsd2-/- genotype) leads to the development of signs of osteoporosis – bone density decreases, which is accompanied by a decrease in the width of bone trabeculae, the level of microcirculation in bone tissue decreases simultaneously with an increase in the coefficient of endothelial dysfunction. The additional null expression of ApoE gene in double transgenes with the genotype Hsd2-/-/Apoe-/- leads to an increase in the severity of changes associated with a violation of bone remodeling processes and, in addition to a more pronounced change in bone tissue density, bone trabecular width, microcirculation and the coefficient of endothelial dysfunction leads to an increase in the concentration of biochemical markers of bone resorption. These changes indicate the important role of the enzyme 11ß-hydroxysteroid dehydrogenase type 2 in the processes of bone remodeling disorders. Graphical abstract:

Acute toxic effects of Bothrops atrox venom on calcium homeostasis and bone tissue in mice