Identification of an MGST2 gene and analysis of its function in antioxidant processes in Apis cerana cerana.

MGST2 is a member of the MAPEG superfamily, which participates in LTC4 synthesis and plays important roles in the regulation of the oxidative stress pathway and some diseases. Here, we isolated a previously uncharacterized gene in Apis cerana cerana named AccMGST2 by reverse transcription-polymerase chain reaction. The biological characteristics of AccMGST2 were analyzed by bioinformatics. The amino acid sequence similarity between AccMGST2 and AmMGST2 of Apis mellifera reached 96.08%. The expression characteristics of AccMGST2 were explored in several tissues. The quantitative real-time polymerase chain reaction results showed that the AccMGST2 gene was highly expressed in the head and muscle and that AccMGST2 expression responded to oxidative stress caused by different abiotic stresses. AccMGST2 was silenced using RNA interference, which decreased the expression levels of some MAPK and antioxidant genes. Therefore, we conclude that AccMGST2 is involved in the regulation of oxidative stress in A. cerana cerana.

Role of AccMGST1 in oxidative stress resistance in Apis cerana cerana.

As detoxification enzymes, proteins in the glutathione S-transferase (GST) superfamily are reported to participate in oxidative stress resistance. Nevertheless, microsomal GSTs (MGSTs), a unique subclass of the GST superfamily associated with membranes, are rarely studied in insects. Here, we isolated an MGST gene in Apis cerana cerana (AccMGST1) and verified its role in oxidative stress response. We found higher expression of AccMGST1 in protective or defensive tissue, that is, the epidermis, which indicated its role in stress resistance. Real-time quantitative PCR (qRT-PCR) analysis indicated that AccMGST1 was upregulated by oxidative stresses at the transcriptional level. In contrast, AccMGST1 expression was inhibited when the antioxidant vitamin C (VC) was fed to experimental bees. Through western blotting, we found that the protein level of AccMGST1 under oxidative stress corresponded to the transcript level. Disc diffusion and mixed-function oxidation (MFO) assays suggested that AccMGST1 can protect not only cells but also DNA against oxidative damage. Furthermore, we discovered that the expression patterns of known antioxidant genes were changed in A. cerana cerana after AccMGST1 was silenced by RNA interference (RNAi). Thus, we concluded that the gene AccMGST1 exerts a significant role in the antioxidant mechanism.

Effects of electromagnetic fields on bone loss in hyperthyroidism rat model.

Optimal therapeutics for hyperthyroidism-induced osteoporosis are still lacking. As a noninvasive treatment, electromagnetic fields (EMF) have been proven to be effective for treating osteoporosis in non-hyperthyroidism conditions. We herein systematically evaluated the reduced effects of EMF on osteoporosis in a hyperthyroidism rat model. With the use of Helmholtz coils and an EMF stimulator, 15 Hz/1 mT EMF was generated. Forty-eight 5-month-old male Sprague-Dawley rats were randomly divided into four different groups: control, levothyroxine treated (L-T4), EMF exposure + levothyroxine (EMF + L-T4), and EMF exposure without levothyroxine administration (EMF). All rats were treated with L-T4 (100 mg/day) except those in control and EMF groups. After 12 weeks, the results obtained from bone mineral density analyses and bone mechanical measurements showed significant differences between L-T4 and EMF + L-T4 groups. Micro CT and bone histomorphometric analyses indicated that trabecular bone mass and architecture in distal femur and proximal tibia were augmented and restored partially in EMF + L-T4 group. In addition, bone thyroid hormone receptors (THR) expression of hyperthyroidism rats was attenuated in EMF + L-T4 group, compared to control group, which was not observed in L-T4 group. According to these results, we concluded that 15 Hz/1 mT EMF significantly inhibited bone loss and micro architecture deterioration in hyperthyroidism rats, which might occur due to reduced THR expression caused by EMF exposure. Bioelectromagnetics. 38:137-150, 2017. © 2016 Wiley Periodicals, Inc.

The effect of electromagnetic fields on the proliferation and the osteogenic or adipogenic differentiation of mesenchymal stem cells modulated by dexamethasone.

Although glucocorticoids provide benefits for inflammation or autoimmune disorders, high-dose and long-term use could cause osteonecrosis or osteoporosis as adverse effect for patients. Electromagnetic field (EMF) treatments have been clinically used for many years to promote fracture healing, but whether EMF can attenuate the deleterious effects of glucocorticoids is not clear. In this study, the effects of different concentrations of dexamethasone (DEX) on proliferation and adipogenic or osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were detected and compared, and the effects of EMF treatment (15 Hz, 1 mT, 4 h/day) on 0.1 µM DEX-modulated BMSCs' proliferation and adipogenic or osteogenic differentiation were investigated. Higher concentrations of DEX (0.1 and 1 µM) inhibited proliferation of BMSCs but promoted expression of adipogenic-related genes, increasing the number of lipid droplets. In the early stage of differentiation, DEX restrained expression of RUNX2 and alkaline phosphatase (ALP), but amplified expression of ALP and osteopontin (OPN) in the late stage. EMF treatment of BMSCs influenced by 0.1 µM DEX inhibited the high expression of adipogenic-related genes, stimulated the expression of RUNX2, ALP, OPN, and osteocalcin, and increased the activity of ALP. EMF exposure augmented the expression of p-ERK, which DEX reduced. After using mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway inhibitor, U0126, the effect of EMF was reduced. In conclusion, EMF exposure accelerates BMSCs proliferation, inhibits adipogenic differentiation, and promotes osteogenic differentiation of BMSCs modulated by DEX, and these effects are mediated at least in part by MEK/ERK signaling pathway.

The time-dependent manner of sinusoidal electromagnetic fields on rat bone marrow mesenchymal stem cells proliferation, differentiation, and mineralization.

Electromagnetic fields (EMFs) are used clinically to promote fracture healing and slow down osteoporosis without knowledge of optimal parameters and underlying principles. In the present study, we investigate the effects of irritation for different durations with 15 Hz 1 mT sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs) proliferation, differentiation, and mineralization potentials. Our results show that SEMFs irritation promote rat BMSCs proliferation in a time-dependent manner, and the expression of osteogenic gen [Cbfa 1/RUNX2, bone sialoprotein (BSP), osteopontin (OPN)], alkaline phosphatase activity, and calcium deposition were enhanced after SEMFs treatment depending on the time duration of treatment. To determine the role of MEK/ERK signaling pathway, U0126, a MEK/ERK inhibitor was used. It can suppress rat BMSCs' proliferation with or without SEMF exposure, and partly attenuate the expression of osteogenesis related proteins (RUNX2, BSP, OPN) which were improved by SEMF. This finding suggests that the effects of SEMF on rat BMSCs' proliferation differentiation and mineralization are time duration dependent and MEK/ERK signaling pathway plays important role.

Effect of 1 mT sinusoidal electromagnetic fields on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stromal cells.

Electromagnetic field (EMF) stimulation is clinically beneficial for fracture nonunion and a wide range of bone disorders. However, no consensus has been reached on the optimal parameters of the EMF. The exact mechanism by which EMFs enhance osteogenesis has also not been defined. In the present study, a sinusoidal 1 mT EMF at frequencies of 10, 30, 50, and 70 Hz were administered to rat bone marrow mesenchymal stromal cells (rBMSCs) in the cyclic mode of 2 h exposures followed by 4 h of culture without exposure. The cell viability, proliferation, expression of some osteogenic genes, and mineralization of the extracellular matrix were investigated. It was found that the cell viability was decreased by EMF exposures of 50 and 70 Hz. The proliferation of rBMSCs was elevated significantly in the 10 Hz EMF-treated group during the culture periods. The expression of alkaline phosphatase (ALP) and osteocalcin (OC), two early-phase osteogenic differentiation markers, was up-regulated by the 1 mT, 10 Hz EMF after 1 week. However, the expression of genes that marked the later-phase osteogenic differentiation and maturation of osteoblasts was elevated by the stimulation of 50 Hz EMFs after 2 weeks. In addition, it was observed that the mineralization of the extracellular matrix was enhanced by 50 Hz EMF exposure. These results indicated that the 1 mT EMF at different frequencies had disparate effects on the viability, proliferation and osteogenic differentiation of rBMSCs, and may be beneficial for developing novel therapeutic approaches in bone regenerative medicine.

EMF acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes.

The use of electromagnetic fields (EMFs) to treat nonunion fractures developed from observations in the mid-1900s. Whether EMF directly regulates the bone marrow mesenchymal stem cells (MSCs), differentiating into osteoblasts or adipocytes, remains unknown. In the present study, we investigated the roles of sinusoidal EMF of 15 Hz, 1 mT in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that EMF promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. EMF increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of RUNX2, ALP, BMP2, DLX5, and BSP. In contrast, EMF decreased adipogenesis and inhibited adipocyte-specific mRNA expression of adipsin, AP-2, and PPARgamma2, and also inhibited protein expression of PPARgamma2. These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is influenced by EMF.

Effect of electromagnetic fields on proliferation and differentiation of cultured mouse bone marrow mesenchymal stem cells.

In order to study the effects of electromagnetic fields (EMFs) on proliferation, differentiation and intercellular cyclic AMP (cAMP) in mouse bone marrow mesenchymal stem cells (MSCs) in vitro, the mouse bone MSCs were isolated and cultured in vitro. The third passage MSCs were divided into 4 groups and stimulated with EMFs. The cellular proliferation (MTT), the cellular differentiation (alkaline phosphatase activity, ALP), and the intercellular cAMP level were investigated at different time points. The results showed that EMF (50Hz pulse burst 2 mT peak) inhibited the cellular proliferation (P < 0.05), enhanced the cellular differentiation (P < 0. 05), and increased the intercellular cAMP level (P < 0.01) in the early time of the stimulation (1-3 days), but the intercellular cAMP level did not increased further in the later days. We are led to conclude that the cAMP may be involved in the mediation of the growth inhibitory and differentiation-inducing signals of specific EMFs in vitro.

[Preliminary research on the proliferation and differentiation of rat bone marrow mesenchymal stem cells with exposure to 50 Hz magnetic fields].

A preliminary experimental research on the proliferation and differentiation of rat bone marrow mesenchymal stem cells with exposure to 50 Hz magnetic fields is done. The experimental results can be well repeated, showing that 50 Hz magnetic fields can evidently affect the proliferation and differentiation of rat bone marrow mesenchymal stem cells (MSCs), besides, the results are not linear with the amplitude of magnetic fields, but existing therein a 'window' phenomenon. At the end of this paper, the domestic and overseas development of the research is discussed on with the emphasis how to cure the bone fracture with the help of magnetic fields, and our investigative thoughts are also presented.