ZmSMR10 Increases the Level of Endoreplication of Plants through Its Interactions with ZmPCNA2 and ZmCSN5B.

As a plant-specific endoreplication regulator, the SIAMESE-RELATED (SMR) family (a cyclin-dependent kinase inhibitor) plays an important role in plant growth and development and resistance to stress. Although the genes of the maize (Zea mays) SMR family have been studied extensively, the ZmSMR10 (Zm00001eb231280) gene has not been reported. In this study, the function of this gene was characterized by overexpression and silencing. Compared with the control, the transgenic plants exhibited the phenotypes of early maturation, dwarfing, and drought resistance. Expression of the protein in prokaryotes demonstrates that ZmSMR10 is a small protein, and the results of subcellular localization suggest that it travels functionally in the nucleus. Unlike ZmSMR4, yeast two-hybrid experiments demonstrated that ZmSMR10 does not interact strongly with with some cell cycle protein-dependent protein kinase (CDK) family members ZmCDKA;1/ZmCDKA;3/ZmCDKB1;1. Instead, it interacts strongly with ZmPCNA2 and ZmCSN5B. Based on these results, we concluded that ZmSMR10 is involved in the regulation of endoreplication through the interaction of ZmPCNA2 and ZmCSN5B. These findings provide a theoretical basis to understand the mechanism of the regulation of endoreplication and improve the yield of maize through the use of molecular techniques.

Electromagnetic fields ameliorate hepatic lipid accumulation and oxidative stress: potential role of CaMKKß/AMPK/SREBP-1c and Nrf2 pathways.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, and is related to disturbed lipid metabolism and redox homeostasis. However, a definitive drug treatment has not been approved for this disease. Studies have found that electromagnetic fields (EMF) can ameliorate hepatic steatosis and oxidative stress. Nevertheless, the mechanism remains unclear. METHODS: NAFLD models were established by feeding mice a high-fat diet. Simultaneously, EMF exposure is performed. The effects of the EMF on hepatic lipid deposition and oxidative stress were investigated. Additionally, the AMPK and Nrf2 pathways were analysed to confirm whether they were activated by the EMF. RESULTS: Exposure to EMF decreased the body weight, liver weight and serum triglyceride (TG) levels and restrained the excessive hepatic lipid accumulation caused by feeding the HFD. The EMF boosted CaMKKß protein expression, activated AMPK phosphorylation and suppressed mature SREBP-1c protein expression. Meanwhile, the activity of GSH-Px was enhanced following an increase in nuclear Nrf2 protein expression by PEMF. However, no change was observed in the activities of SOD and CAT. Consequently, EMF reduced hepatic reactive oxygen species (ROS) and MDA levels, which means that EMF relieved liver damage caused by oxidative stress in HFD-fed mice. CONCLUSIONS: EMF may activate the CaMKKß/AMPK/SREBP-1c and Nrf2 pathways to control hepatic lipid deposition and oxidative stress. This investigation indicates that EMF may be a novel therapeutic method for NAFLD.

First-principles investigation of band offset and charge transfer characteristics at the PE/fluorinated layer interface.

Polymers, especially polyethylene (PE), are widely employed as insulating materials in electrical power transmission systems. However, the insulation still faces the problem of space charge, which distorts the electric field distribution and accelerates electrical aging. Experimental results show that after the fluorination process, less charge injection occurs compared with pure PE. To clarify the mechanism, classical molecular dynamics was employed to build a PE/fluorinated layer interfacial model and first principles calculation was utilized to get the band offset at the interface. The results calculated by both the bulk plus band lineup method and the layer-decomposed density of states method show that the energy band of the fluorinated PE layer is overall lower than that of the PE side, and the band offsets are around 2 eV. Charge transport results based on Marcus theory and kinetic Monte Carlo simulations also show that charge can easily accumulate at the interfacial area under an electric field and the band offset can suppress charge injection. The conduction band offset acts as an energy barrier for the excess electrons at the fluorinated layer side to cross the interface, while the valence band offset has the same effect on hole transport because of the energy barrier caused by the inverted region. Our findings provide a fundamental and theoretical basis for material modification and space charge inhibition.

Pulsed electromagnetic fields regulate osteocyte apoptosis, RANKL/OPG expression, and its control of osteoclastogenesis depending on the presence of primary cilia.

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).

Fluid shear stress improves morphology, cytoskeleton architecture, viability, and regulates cytokine expression in a time-dependent manner in MLO-Y4 cells.

The effects of load-induced interstitial fluid shear stress (FSS) on instantaneous signaling response of osteocytes (e.g., calcium signaling) have been well documented. FSS can also initiate the release of many important messenger molecules of osteocytes (e.g., ATP and PGE2 ). However, the effects of FSS on cellular function and bone metabolism-modulating cytokine expression of osteocytes have not been fully identified (some inconsistent/conflicting results have been documented). Herein, osteocyte-like MLO-Y4 cells were stimulated with 1 Pa, 2-h FSS, and the effects of FSS on cellular morphology, cytoskeletal microstructure, biological activity, and gene and protein expression of important cytokines were investigated. SEM and cytoskeleton staining revealed that FSS induced well-organized cytoskeleton and increased filopodia processes. The osteocytic viability was sustained and apoptosis was inhibited via flow cytometry. FSS promoted Wnt3a and ß-catenin gene and protein expression in 0-, 3-, and 6-h (sample collection time post FSS) groups. The FSS-stimulated cells in the 3-h group exhibited more significant effects on the promotion of OCN and Cx43 and inhibition of DKK1 and SOST expression than the 0- and 6-h groups. The 3-h group with FSS stimulation also showed the most prominent effects on suppressing RANKL and RANKL/OPG gene and protein expression. This study revealed a direct regulatory effect of FSS on osteocytic morphology and apoptotic characteristics, and showed that osteocyte-secreted bone metabolism-modulating molecule expression was regulated by FSS in a time-dependent manner. This study not only enriches our basic knowledge for understanding osteocytic mechanotransduction, but also provides important evidence for more scientific experimental design.

Differential intensity-dependent effects of pulsed electromagnetic fields on RANKL-induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells.

Pulsed electromagnetic fields (PEMF) have been proven to be effective for promoting bone mass and regulating bone turnover both experimentally and clinically. However, the exact mechanisms for the regulation of PEMF on osteoclastogenesis as well as optical exposure parameters of PEMF on inhibiting osteoclastic activities and functions remain unclear, representing significant limitations for extensive scientific application of PEMF in clinics. In this study, RAW264.7 cells incubated with RANKL were exposed to 15 Hz PEMF (2 h/day) at various intensities (0.5, 1, 2, and 3 mT) for 7 days. We demonstrate that bone resorbing capacity was significantly decreased by 0.5 mT PEMF mainly by inhibiting osteoclast formation and maturation, but enhanced at 3 mT by promoting osteoclast apoptosis. Moreover, gene expression of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 was significantly decreased by 0.5 mT PEMF, but increased by 3 mT. Our findings reveal a significant intensity window for low-intensity PEMF in regulating bone resorption with diverse nature for modulating osteoclastogenesis and apoptosis. This study not only enriches our basic knowledge for the regulation of PEMF in osteoclastogenesis, but also may lead to more efficient and scientific clinical application of PEMF in regulating bone turnover and inhibiting osteopenia/osteoporosis. Bioelectromagnetics. 38:602-612, 2017. © 2017 Wiley Periodicals, Inc.

Pulsed electromagnetic fields promote in vitro osteoblastogenesis through a Wnt/ß-catenin signaling-associated mechanism.

Substantial evidence indicates that pulsed electromagnetic fields (PEMF) could accelerate fracture healing and enhance bone mass, whereas the unclear mechanism by which PEMF stimulation promotes osteogenesis limits its extensive clinical application. In the present study, effects and potential molecular signaling mechanisms of PEMF on in vitro osteoblasts were systematically investigated. Osteoblast-like MC3T3-E1 cells were exposed to PEMF burst (0.5, 1, 2, or 6 h/day) with 15.38 Hz at various intensities (5 Gs (0.5 mT), 10 Gs (1 mT), or 20 Gs (2 mT)) for 3 consecutive days. PEMF stimulation at 20 Gs (2 mT) for 2 h/day exhibited most prominent promotive effects on osteoblastic proliferation via Cell Counting kit-8 analyses. PEMF exposure induced well-organized cytoskeleton, and promoted formation of extracellular matrix mineralization nodules. Significantly increased proliferation-related gene expressions at the proliferation phase were observed after PEMF stimulation, including Ccnd 1 and Ccne 1. PEMF resulted in significantly increased gene and protein expressions of alkaline phosphatase and osteocalcin at the differentiation phase of osteoblasts rather than the proliferation phase via quantitative reverse transcription polymerase chain reaction and Western blotting analyses. Moreover, PEMF upregulated gene and protein expressions of collagen type 1, Runt-related transcription factor 2 and Wnt/ß-catenin signaling (Wnt1, Lrp6, and ß-catenin) at proliferation and differentiation phases. Together, our present findings highlight that PEMF stimulated osteoblastic functions through a Wnt/ß-catenin signaling-associated mechanism and, hence, regulates downstream osteogenesis-associated gene/protein expressions. Bioelectromagnetics. 37:152-162, 2016. © 2016 Wiley Periodicals, Inc.

Overexpression of the potato VQ31 enhances salt tolerance in Arabidopsis.

Plant-specific VQ proteins have crucial functions in the regulation of plant growth and development, as well as in plant abiotic stress responses. Their roles have been well established in the model plant Arabidopsis thaliana; however, the functions of the potato VQ proteins have not been adequately investigated. The VQ protein core region contains a short FxxhVQxhTG amino acid motif sequence. In this study, the VQ31 protein from potato was cloned and functionally characterized. The complete open reading frame (ORF) size of StVQ31 is 672 bp, encoding 223 amino acids. Subcellular localization analysis revealed that StVQ31 is located in the nucleus. Transgenic Arabidopsis plants overexpressing StVQ31 exhibited enhanced salt tolerance compared to wild-type (WT) plants, as evidenced by increased root length, germination rate, and chlorophyll content under salinity stress. The increased tolerance of transgenic plants was associated with increased osmotic potential (proline and soluble sugars), decreased MDA accumulation, decreased total protein content, and improved membrane integrity. These results implied that StVQ31 overexpression enhanced the osmotic potential of the plants to maintain normal cell growth. Compared to the WT, the transgenic plants exhibited a notable increase in antioxidant enzyme activities, reducing cell membrane damage. Furthermore, the real-time fluorescence quantitative PCR analysis demonstrated that StVQ31 regulated the expression of genes associated with the response to salt stress, including ERD, LEA4-5, At2g38905, and AtNCED3. These findings suggest that StVQ31 significantly impacts osmotic and antioxidant cellular homeostasis, thereby enhancing salt tolerance.

A comparison of the physicochemical properties, digestibility, and expression patterns of starch-related genes of two supersweet corn hybrids (F1) and their parents.

The quality difference of corn largely depends on parental selection. Herein, the structure, digestive characteristics, and expression patterns of starch-related genes of two supersweet maize hybrids and their parents were studied. The structural analysis revealed that the starch of supersweet corn is round or oval, and the particles are smaller compared to those of normal corn. Hybridization changed the grain morphology, crystal, and helical structure of starch. Parents had a significantly different influence on supersweet corn. Notably, hybridization improved the setback value and digestibility of Shantian1500F1 and Shantian2000F1 compared to that of the parents. ZmBEI, ZmPHOH, and ZmAGPL2 genes had a consistent high expression throughout the whole grain formation phase. The results of this study expand our understanding of the breeding of supersweet corn hybrids and the effect of parents on the new strand. These results provide a useful reference for further breeding and studies of supersweet corn for starch production in corn.

Kalman filtering to reduce measurement noise of sample entropy: An electroencephalographic study.

In the analysis of electroencephalography (EEG), entropy can be used to quantify the rate of generation of new information. Entropy has long been known to suffer from variance that arises from its calculation. From a sensor's perspective, calculation of entropy from a period of EEG recording can be treated as physical measurement, which suffers from measurement noise. We showed the feasibility of using Kalman filtering to reduce the variance of entropy for simulated signals as well as real-world EEG recordings. In addition, we also manifested that Kalman filtering was less time-consuming than moving average, and had better performance than moving average and exponentially weighted moving average. In conclusion, we have treated entropy as a physical measure and successfully applied the conventional Kalman filtering with fixed hyperparameters. Kalman filtering is expected to be used to reduce measurement noise when continuous entropy estimation (for example anaesthesia monitoring) is essential with high accuracy and low time-consumption.

Optimizing planting density enhances the multi-scale structural characteristics and in vitro digestibility of maize starch via modulating the size distribution of granules.

The modification of starch through agricultural practices is becoming increasingly significant for producing healthy foodstuffs and raw materials for industrial applications, consequently gaining momentum in academic research. This study examined how three different planting densities influenced the distribution of granule sizes, multi-scale structural characteristics, and in vitro digestibility of maize starch. The results showed that planting density significantly enhanced grain yield and relative crystallinity, and significant increases were also observed in the contents of both rapidly and slowly digestible starch. The surface- and volume-weighted mean diameter of granules significantly increased under the medium level (6.75 × 104 plants ha-1), and then decreased under high planting density level. As planting density level increased, the amylose content, peak viscosity, and hardness varied from 23.3 to 26.4 %, from 1962 to 2659 mPa·s, and from 129.3 to 307.6 g, respectively. However, no change was found in crystalline structure of maize starch. These results indicated that optimizing planting density could effectively improve grain yield and starch characteristics of maize, with the best effect under the level of 6.75 × 104 plants ha-1.

Starch granular size and multi-scale structure determine population patterns in bivariate flow cytometry sorting.

Bivariate flow cytometry (FC) sorting with forward scatter (FSC) and side scatter (SSC) is a recently established novel technique to separate starch granules. However, the forming mechanism of starch FC-dependent population patterns (i.e. the number of subgroups (NS) and FSC/SSC-dependent distribution patterns) remain partly elusive. For this, the correlation of granular size and multi-scale structure of native starches and FC-dependent population patterns was investigated through employing a wide range of native starches originating from different species involving cereal-, pulse-, and tuber crops. Results showed NS was pertinent with particle size, amylose content (AC), amylopectin chains length distribution, lamellar structure, short-range ordered structure. The distinct NS was determined by impacts of native starch FSC / SSC-dependent distribution patterns. Specifically, starch granular size significantly correlated with both FSC and SSC-dependent distribution patterns. The proportion of chains with DP 6-12 was the intra-molecular decisive factor to influence short-range ordered structure, finally leading to FSC-dependent distribution patterns. By contrast, AC was another intra-molecular index to determine SSC-dependent distribution patterns through affecting lamellar structure and short-range ordered structure.

Keratin 18 Depletion as a Possible Mechanism for the Induction of Apoptosis and Ferroptosis in the Rat Hippocampus After Hypobaric Hypoxia.

Memory impairment is one of the neuropsychological effects of hypobaric hypoxia (HH), which can be associated with programmed cell death, such as apoptosis and ferroptosis. Emerging evidence indicates crosstalk between apoptosis and ferroptosis, while the crosstalk between HH-induced apoptosis and ferroptosis in the hippocampus has not been clarified. Here, microarray profiles were extracted to analyze the differentially expressed genes with and without HH exposure, and keratin 18 (Krt18) was found to be a potential gene related to both apoptosis and ferroptosis. Then, we conducted morphological observations that showed that apoptosis and ferroptosis coexisted in the rat hippocampus after HH exposure. Combined with the real-time q-PCR analysis, the mRNA expression of Krt18 decreased significantly after HH exposure for 1 day and 3 days, and Mapk10 (JNK3) was upregulated at the corresponding time points. After exposure for 7 days, Krt18 and JNK3 showed no significant change. In conclusion, Krt18 may regulate apoptosis and ferroptosis simultaneously, possibly via the JNK signaling pathway, which might provide a potential central target for apoptosis and ferroptosis in hippocampal injury after HH exposure.

Oxygen enrichment protects against intestinal damage and gut microbiota disturbance in rats exposed to acute high-altitude hypoxia.

Acute high-altitude hypoxia can lead to intestinal damage and changes in gut microbiota. Sustained and reliable oxygen enrichment can resist hypoxic damage at high altitude to a certain extent. However, it remains unclear whether oxygen enrichment can protect against gut damage and changes in intestinal flora caused by acute altitude hypoxia. For this study, eighteen male Sprague-Dawley rats were divided into three groups, control (NN), hypobaric hypoxic (HH), and oxygen-enriched (HO). The NN group was raised under normobaric normoxia, whereas the HH group was placed in a hypobaric hypoxic chamber simulating 7,000 m for 3 days. The HO group was exposed to oxygen-enriched air in the same hypobaric hypoxic chamber as the HH group for 12 h daily. Our findings indicate that an acute HH environment caused a fracture of the crypt structure, loss of epithelial cells, and reduction in goblet cells. Additionally, the structure and diversity of bacteria decreased in richness and evenness. The species composition at Phylum and Genus level was characterized by a higher ratio of Firmicutes and Bacteroides and an increased abundance of Lactobacillus with the abundance of Prevotellaceae_NK3B31_group decreased in the HH group. Interestingly, after oxygen enrichment intervention, the intestinal injury was significantly restrained. This was confirmed by an increase in the crypt depth, intact epithelial cell morphology, increased relative density of goblet cells, and higher evenness and richness of the gut microbiota, Bacteroidetes and Prevotellaceae as the main microbiota in the HO group. Finally, functional analysis showed significant differences between the different groups with respect to different metabolic pathways, including Amino acid metabolism, energy metabolism, and metabolism. In conclusion, this study verifies, for the first time, the positive effects of oxygen enrichment on gut structure and microbiota in animals experiencing acute hypobaric hypoxia.

Electromagnetic Fields Ameliorate Insulin Resistance and Hepatic Steatosis by Modulating Redox Homeostasis and SREBP-1c Expression in db/db Mice.

PURPOSE: The prevalence of nonalcoholic fatty liver disease (NAFLD), which has recently become known as metabolic-associated fatty liver disease (MAFLD), has risen. However, pharmacotherapies for this disease have not been approved. Electromagnetic fields (EMFs) have excellent bioeffects on multiple diseases. However, the effects of EMFs on NAFLD are unknown. This study investigated the bioeffects of EMF exposure on insulin resistance, liver redox homeostasis and hepatic steatosis in db/db mice. METHODS: Animals were sacrificed after EMF exposure for 8 weeks. The fasting blood glucose and insulin levels in the serum were tested. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated by a formula. The levels of MDA, GSSG and GSH, biomarkers of redox, were assessed. The activities of CAT, SOD and GSH-Px were assessed. The body and liver weights were measured. Hepatic lipid accumulation was observed by Oil Red O staining. Hepatic CAT, GR, GSH-Px, SOD1, SOD2 and SREBP-1 expression was determined by Western blotting. RESULTS: EMF exposure ameliorated insulin resistance and oxidative stress in the liver by downregulating the MDA and GSSG levels, increasing the reduced GSH levels, and promoting the GSH-Px levels in db/db mice. In addition, liver weight and triglyceride (TG) levels were reduced by EMF exposure. Simultaneously, EMF exposure improved hepatic steatosis by downregulating the protein expression of SREBP-1c. CONCLUSION: The present findings suggest that EMF exposure has positive effects in the treatment of NAFLD.

Melatonin suppresses ER stress-dependent proapoptotic effects via AMPK in bone mesenchymal stem cells during mitochondrial oxidative damage.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have been used as important cell-based tools for clinical applications. Oxidative stress-induced apoptosis causes a low survival rate after transplantation, and the underlying mechanisms remain unknown. The endoplasmic reticulum (ER) and mitochondria are vital organelles regulated by adenosine monophosphate (AMP)-activated protein kinase (AMPK), especially during oxidative stress injury. Melatonin exerts an antioxidant effect by scavenging free radicals. Here, we aimed to explore whether cytoprotective melatonin relieves ER stress-mediated mitochondrial dysfunction through AMPK in BMSCs after oxidative stress injury. METHODS: Mouse BMSCs were isolated and exposed to H2O2 in the absence or presence of melatonin. Thereafter, cell damage, oxidative stress levels, mitochondrial function, AMPK activity, ER stress-related proteins, and apoptotic markers were measured. Additionally, the involvement of AMPK and ER stress in the melatonin-mediated protection of BMSCs against H2O2-induced injury was investigated using pharmacologic agonists and inhibitors. RESULTS: Melatonin improved cell survival and restored mitochondrial function. Moreover, melatonin intimately regulated the phosphorylation of AMPK and molecules associated with ER stress pathways. AMPK activation and ER stress inhibition following melatonin administration improved the mitochondrial membrane potential (MMP), reduced mitochondria-initiated oxidative damage, and ultimately suppressed apoptotic signaling pathways in BMSCs. Cotreatment with N-acetyl-L-cysteine (NAC) significantly enhanced the antioxidant effect of melatonin. Importantly, pharmacological AMPK activation/ER stress inhibition promoted melatonin-induced cytoprotection, while pharmacological AMPK inactivation/ER stress induction conferred resistance to the effect of melatonin against H2O2 insult. CONCLUSIONS: Our data also reveal a new, potentially therapeutic mechanism by which melatonin protects BMSCs from oxidative stress-mediated mitochondrial apoptosis, possibly by regulating the AMPK-ER stress pathway.

Spatiotemporal characterization of microdamage accumulation in rat ulnae in response to uniaxial compressive fatigue loading.

Repetitive fatigue loading can induce microdamage accumulation in bone matrix, which results in impaired mechanical properties and increased fracture susceptibility. However, the spatial distribution and time-variant process of microdamage accumulation in fatigue-loaded skeleton, especially for linear microcracks which are known to initiate bone remodeling, remain not fully understood. In this study, the time-varying process of the morphology and distribution of microcracks in rat ulnae subjected to uniaxial compressive fatigue loading was investigated. Right forelimbs of thirty four-month-old male Sprague-Dawley rats were subjected to one bout of cyclic ramp loading with 0.67 Hz at a normalized peak force of 0.055 N/g body weight for 6000 cycles, and the contralateral left ulnae were not loaded as the control samples. Ten rats were randomly euthanized on Days 3, 5, and 7 post fatigue loading. Our findings via two-dimensional histomorphometric measurements based on basic fuchsin staining and three-dimensional quantifications using contrast-enhanced micro-computed tomography (MicroCT) with precipitated BaSO4 staining demonstrated that the accumulation of linear microcracks (increase in the amount of linear microcracks) on Day 5 was significantly higher than that on Day 3 and Day 7 post fatigue loading. Our histological and histomorphometric results revealed that linear microcrack density (Cr.Dn) in the tensile cortex at Days 3, 5 and 7 post fatigue loading was significantly higher than that in the compressive side, whereas linear microcrack length (Cr.Le) in the tensile cortex at Day 3 was significantly lower than that in the compressive cortex. Our findings revealed that microcrack accumulation exhibited a non-linear time-varying process at 3, 5 and 7 days post axial compressive fatigue loading (with observable peak Cr.Dn at Day 5). Our findings also revealed distinct distribution of microcrack density and morphology in rat ulnae with tensile and compressive strains, as characterized by more microcracks accumulated in tensile cortices, and longer cracks shown in compressive cortices.

Low­level mechanical vibration enhances osteoblastogenesis via a canonical Wnt signaling­associated mechanism.

Osteoporosis is a skeletal metabolic disease characterized by reduced bone mass and a high susceptibility to fractures, in which osteoblasts and osteoclasts are highly involved in the abnormal bone remodeling processes. Recently, low­magnitude, high­frequency whole­body vibration has been demonstrated to significantly reduce osteopenia experimentally and clinically. However, the underlying mechanism regarding how osteoblastic activity is altered when bone tissues adapt to mechanical vibration remains elusive. The current study systematically investigated the effect and potential molecular signaling mechanisms in mediating the effects of mechanical vibration (0.5 gn, 45 Hz) on primary osteoblasts in vitro. The results of the present study demonstrated that low­level mechanical stimulation promoted osteoblastic proliferation and extracellular matrix mineralization. In addition, it was also revealed that mechanical vibration induced improved cytoskeleton arrangement in primary osteoblasts. Furthermore, mechanical vibration resulted in significantly increased gene expression of alkaline phosphatase, bone morphogenetic protein 2 and osteoprotegerin, and suppressed sclerostin gene expression, as determined by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analyses. Mechanical vibration was observed to upregulate gene and protein expression levels of osteogenesis­associated biomarkers, including osteocalcin and Runt­related transcription factor 2. In addition, RT­qPCR and western blotting analysis demonstrated that mechanical vibration promoted gene and protein expression of canonical Wnt signaling genes, including Wnt3a, low­density lipoprotein receptor­related protein 6 and ß­catenin. In conclusion, the present study demonstrated that mechanical vibration stimulates osteoblastic activities and may function through a potential canonical Wnt signaling­associated mechanism. These findings provided novel information that improves the understanding of the molecular mechanisms involved in osteoblastic activities in response to mechanical vibration, which may facilitate the scientific application of mechanical vibration for the treatment of osteoporosis in the clinic.

Mechanical Vibration Mitigates the Decrease of Bone Quantity and Bone Quality of Leptin Receptor-Deficient Db/Db Mice by Promoting Bone Formation and Inhibiting Bone Resorption.

Leptin, a major hormonal product of adipocytes, is involved in regulating appetite and energy metabolism. Substantial studies have revealed the anabolic actions of leptin on skeletons and bone cells both in vivo and in vitro. Growing evidence has substantiated that leptin receptor-deficient db/db mice exhibit decreased bone mass and impaired bone microstructure despite several conflicting results previously reported. We herein systematically investigated bone microarchitecture, mechanical strength, bone turnover and its potential molecular mechanisms in db/db mice. More importantly, we also explored an effective approach for increasing bone mass in leptin receptor-deficient animals in an easy and noninvasive manner. Our results show that deterioration of trabecular and cortical bone microarchitecture and decreases of skeletal mechanical strength-including maximum load, yield load, stiffness, energy, tissue-level modulus and hardness-in db/db mice were significantly ameliorated by 12-week, whole-body vibration (WBV) with 0.5 g, 45 Hz via micro-computed tomography (µCT), three-point bending, and nanoindentation examinations. Serum biochemical analysis shows that WBV significantly decreased serum tartrate-resistant acid phosphatase 5b (TRACP5b) and CTx-1 levels and also mitigated the reduction of serum osteocalcin (OCN) in db/db mice. Bone histomorphometric analysis confirmed that decreased bone formation-lower mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone-in db/db mice were suppressed by WBV. Real-time PCR assays show that WBV mitigated the reductions of tibial alkaline phosphatase (ALP), OCN, Runt-related transcription factor 2 (RUNX2), type I collagen (COL1), BMP2, Wnt3a, Lrp6, and ß-catenin mRNA expression, and prevented the increases of tibial sclerostin (SOST), RANK, RANKL, RANL/osteoprotegerin (OPG) gene levels in db/db mice. Our results show that WBV promoted bone quantity and quality in db/db mice with obvious anabolic and anticatabolic effects. This study not only enriches our basic knowledge about bone quality and bone turnover mechanisms in leptin receptor-deficient animals, but also advances our understanding of the skeletal sensitivity of leptin-resistant db/db mice in response to external mechanical stimulation. © 2016 American Society for Bone and Mineral Research.

Whole Body Vibration Improves Insulin Resistance in db/db Mice: Amelioration of Lipid Accumulation and Oxidative Stress.

Insulin resistance (IR) is the hallmark of type 2 diabetes mellitus (T2DM), which is one of the most important chronic noncommunicable diseases. Effective and feasible strategies to treat IR are still urgently needed. Previous research studies reported that whole body vibration (WBV) was beneficial for IR in clinical; however, its underlying mechanisms remains unknown. In the present study, db/db mice were treated with WBV administration 60 min/day for 12 weeks and the impaired insulin sensitivity was improved. Besides, liver steatosis was also ameliorated. Further explorations revealed that WBV could reduce the expression of SREBP1c and increase the expression of GSH-Px and consequently suppress oxidative stress. In conclusion, WBV attenuates oxidative stress to ameliorate liver steatosis and thus improves insulin resistance in db/db mice. Therefore, WBV administration is a promising treatment for individuals who suffered from central obesity and IR.