Gengyang Shen

Variance of spinal osteoporosis induced by dexamethasone and methylprednisolone and its associated mechanism

BACKGROUND Glucocorticoid (GC) administration is the most common cause of secondary osteoporosis. Previous studies investigated GCs dose and frequency correlated positively with the side effects of glucocorticoid on bone health, however the impaired effect of various types of GCs on bone has not yet been reported. PURPOSE The aim is to compare the effect of long-acting (dexamethasone) and relatively short-acting glucocorticoid (methylprednisolone) on rat lumbar spine and try to explore the associated mechanism. METHOD Sprague Dawley rats (N=48) were randomly divided into four groups: baseline group (BL), control group (CON), methylprednisolone group (MP) and dexamethasone group (DEXA). BL rats were euthanized to remain as baseline (M0) at the beginning of experiment. CON group were injected daily with vehicle, while the other groups were given a daily subcutaneous injection of 1mg/kg methylprednisolone and were given a subcutaneous injection of 0.6mg/kg dexamethasone per 3days, respectively. CON, MP and DEXA groups were monitored at 4th week (M1), 8th week (M2) and 12th week (M3) after intervention. Dual-energy X-ray, micro-computed tomography, compressive test, enzyme-linked immunosorbent assay have been used for bone mineral density, microarchitecture, biomechanical property of vertebrae and levels of estrogen, PINP and β-CTX, respectively. mRNA expression analysis of Biglycan, Col1a1, MMP9, Cathepsin K, Runx2, OPG, LRP5, Sclerostin were performed. RESULT We found that the bone mineral density (BMD) was significantly lower in DEXA rats at M3 compared with MP rats. The relative surface and trabecular number were significantly lower in DEXA group than that in MP group at M2, while trabecular separation was significantly higher in DEXA group than that in MP group at the same point. The compressive strength was significantly lower in L4 of DEXA than that in MP rats at M2 and M3. The levels of both PINP and estradiol in DEXA group were lower than MP group at M3, even though without statistical significance. The expression of bone formation marker Runx2 was significantly down-regulated at M3 in DEXA group compared with MP, CON and BL groups, while the expression of Col1a1 was significantly up-regulated and biglycan, LRP-5, OPG were significantly down-regulated in GCs intervention groups compared with CON and BL groups. There were no statistical differences in MMP9, Cathepsin K, Sclerostin among CON, MP and DEXA groups. CONCLUSION These results indicate that dexamethasone, the long-acting glucocorticoid, generates more serious osteoporosis of rat lumbar spine than methylprednisolone, which is relatively short-acting glucocorticoid. The discrepancy between the two GCs inducing osteoporosis may be mainly caused by a decrease in bone formation. RUNX2 and Col1a1 may be the two of critical genes inducing the discrepant impairment.

Effects of combined ovariectomy with dexamethasone on rat lumbar vertebrae.

Objective:This study investigated the effects of combined ovariectomy with dexamethasone treatment on rat lumbar vertebrae in comparison with osteoporosis induced via ovariectomy or dexamethasone alone, and analysis of the associated molecular mechanism. Methods:Sixty-two female Sprague–Dawley rats (3 months’ old) were randomly divided into five treatment groups: an untreated baseline (BL) group; those receiving a sham operation (SHAM); those receiving a dexamethasone injection alone (DEXA); those undergoing bilateral ovariectomy (OVX); and those subjected to both ovariectomy and dexamethasone injection (OVX-DEXA). Animals in the BL group were euthanized at the beginning of the experiment, whereas animals in the remaining groups were euthanized at the end of the first month (M1), second month (M2), or third month (M3). Bone mineral density, bone microarchitecture, biomechanical properties of vertebrae, and serum levels of estrogen, amino-terminal propeptide of type I collagen (PINP), and &bgr;-C-telopeptide of type I collagen (&bgr;-CTX) were measured. In addition, we examined biglycan, runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), lipoprotein receptor-related protein-5 (LRP-5), cathepsin K (CTSK), and sclerostin mRNA expression. Results:Bone mineral content and bone mineral density were markedly lower in the OVX-DEXA group compared with the OVX group at all time points examined. The relative bone surface (BS/TV, mm–1), relative bone volume (BV/TV,%), and trabecular number (Tb.N, 1/mm) were markedly lower in the OVX-DEXA group compared with the remaining groups, whereas trabecular separation (Tb.Sp, mm) was markedly higher in the OVX-DEXA group compared with the remaining groups at M2 or M3. The OVX-DEXA group showed lower compressive strength and lower stiffness compared with the other groups at M2 and M3. Compressive displacement and energy absorption capacity were also markedly lower in the OVX-DEXA group compared with the OVX group at M3. Estradiol levels were markedly lower in the OVX-DEXA group compared with the other groups. Biglycan, runt-related transcription factor 2, osteoprotegerin, and lipoprotein receptor-related protein-5 were down-regulated in the DEXA, OVX, and OVX-DEXA groups compared with the BL and SHAM groups, whereas cathepsin K and sclerostin were up-regulated in the OVX-DEXA group compared with the DEXA and OVX groups. Conclusions:Ovariectomy combined with dexamethasone induced more serious osteoporosis in the rat lumbar spine than either ovariectomy or dexamethasone alone. The combined effect may be due to a combination of suppressed bone formation and increased bone resorption related to an estradiol deficit.

Animal models for glucocorticoid-induced postmenopausal osteoporosis: An updated review

Glucocorticoid-induced postmenopausal osteoporosis is a severe osteoporosis, with high risk of major osteoporotic fractures. This severe osteoporosis urges more extensive and deeper basic study, in which suitable animal models are indispensable. However, no relevant review is available introducing this model systematically. Based on the recent studies on GI-PMOP, this brief review introduces the GI-PMOP animal model in terms of its establishment, evaluation of bone mass and discuss its molecular mechanism. Rat, rabbit and sheep with their respective merits were chosen. Both direct and indirect evaluation of bone mass help to understand the bone metabolism under different intervention. The crucial signaling pathways, miRNAs, osteogenic- or adipogenic- related factors and estrogen level may be the predominant contributors to the development of glucocorticoid-induced postmenopausal osteoporosis.

Implications of the Interaction Between miRNAs and Autophagy in Osteoporosis

Abstract Imbalances between bone formation and resorption are the primary cause of osteoporosis. However, currently, a detailed molecular mechanism of osteoporosis is not available. Autophagy is the conserved process characterized by degrading and recycling aggregated proteins, intracellular pathogens, and damaged organelles. MicroRNAs (miRNAs) are novel regulatory factors that play important roles in numerous cellular processes, including autophagy, through the posttranscriptional regulation of gene expression. Conversely, autophagy plays a role in the regulation of miRNA homeostasis. Recent advances have revealed that both autophagy and miRNAs are involved in the maintenance of bone homoeostasis, whereas the role of the interaction of miRNAs with autophagy in osteoporosis remains unclear. In this paper, we review previous reports on autophagy, miRNAs, and their interaction in osteoporosis.

Mammalian Target of Rapamycin As a Therapeutic Target in Osteoporosis.

The mechanistic target of rapamycin (mTOR) plays a key role in sensing and integrating large amounts of environmental cues to regulate organismal growth, homeostasis, and many major cellular processes. Recently, mounting evidences highlight its roles in regulating bone homeostasis, which sheds light on the pathogenesis of osteoporosis. The activation/inhibition of mTOR signaling is reported to positively/negatively regulate bone marrow mesenchymal stem cells (BMSCs)/osteoblasts‐mediated bone formation, adipogenic differentiation, osteocytes homeostasis, and osteoclasts‐mediated bone resorption, which result in the changes of bone homeostasis, thereby resulting in or protect against osteoporosis. Given the likely importance of mTOR signaling in the pathogenesis of osteoporosis, here we discuss the detailed mechanisms in mTOR machinery and its association with osteoporosis therapy.

Promotion effect of extracts from plastrum testudinis on alendronate against glucocorticoid-induced osteoporosis in rat spine

Alendronate (ALN) is a key therapeutic used to treat glucocorticoid-induced osteoporosis (GIOP), but may induce severe side effects. We showed earlier that plastrum testudinis extracts (PTE) prevented and treated GIOP in vivo. However, clinically, PTE is seldom used alone. Herein, we reveal the synergistic effect of ALN and PTE can treat GIOP of the rat spine and define the mechanism. Sprague-Dawley rats were randomly assigned to four groups: a vehicle group, a GIOP group, an ALN group, and an ALN+PTE group. Each group was further divided into two experimental phases, including dexamethasone (DXM) intervention and withdrawal. Bone mass, microarchitecture, biomechanics, bone-turnover markers, and histomorphology were evaluated. The mRNA and protein expression levels of CTSK and Runx2 were detemined. We found that ALN+PTE improved bone quantity and quality, bone strength, bone turnover; and mitigated histological damage during glucocorticoid intervention and withdrawal. The therapeutic effect was better than that afforded by ALN alone. ALN+PTE reduced CTSK protein expression, promoted Runx2 mRNA and protein expression to varying extents, and more strongly inhibited bone resorption than did ALN alone. Overall, the synergistic effect mediated by ALN+PTE reversed GIOP during DXM intervention and withdrawal via affecting CTSK and Runx2 expression at mRNA and protein levels.

Extracts from plastrum testudinis reverse glucocorticoid-induced spinal osteoporosis of rats via targeting osteoblastic and osteoclastic markers

Extracts from plastrum testudinis (PTE), an important traditional Chinese medicine, have been demonstrated promotion of osteoblastic function in vitro. This study aims to investigate the protective effect of PTE on glucocorticoid-induced osteoporosis(GIOP) in vivo and analyze therapeutic targets of PTE on GIOP. SD rats were randomly assigned to two experiments: preventive and therapeutic experiments, in which rats respectively received oral PTE at the same time of glucocorticoid injection or after glucocorticoid injection inducing osteoporosis. BMD, microarchitecture, biomechanics, bone metabolism markers and histomorphology were evaluated. mRNA and protein expression of OPG, Runx2, CTSK and MMP9 were examined.Results showed bone quality and bone quantity were significantly elevated by PTE. Histomorphometry showed thicker and denser bone trabecularsand more osteoblasts and less osteoclasts in group of PTE intervention. The mRNA expression of OPG was significantly upregulated whereas expression of CTSK was significantly downregulatedin different groups of PTE intervention. Stronger immunostaining for Runx2 and weaker immunostaining for CTSK were observed in groups of PTE intervention. This demonstrated that PTE may reverse GIOP in prevention and management via targeting OPG, Runx2 and CTSK in mRNA and protein levels.

Therapeutic potential of microRNAs in osteoporosis function by regulating the biology of cells related to bone homeostasis: ZHAO et al.

MicroRNAs (miRNAs) are novel regulatory factors that play important roles in numerous cellular processes through the posttranscriptional regulation of gene expression. Recently, deregulation of the miRNA‐mediated mechanism has emerged as an important pathological factor in osteoporosis. However, a detailed molecular mechanism between miRNAs and osteoporosis is still not available. In this review, the roles of miRNAs in the regulation of cells related to bone homeostasis as well as miRNAs that deregulate in human or animal are discussed. Moreover, the miRNAs that act as clusters in the biology of cells in the bone microenvironment and the difference of some important miRNAs for bone homeostasis between bone and other organs are mentioned. Overall, miRNAs that contribute to the pathogenesis of osteoporosis and their therapeutic potential are considered.

Autophagy as a target for glucocorticoid-induced osteoporosis therapy

Autophagy takes part in regulating the eukaryotic cells function and the progression of numerous diseases, but its clinical utility has not been fully developed yet. Recently, mounting evidences highlight an important correlation between autophagy and bone homeostasis, mediated by osteoclasts, osteocytes, bone marrow mesenchymal stem cells, and osteoblasts, and autophagy plays a vital role in the pathogenesis of glucocorticoid-induced osteoporosis (GIOP). The combinations of autophagy activators/inhibitors with anti-GIOP first-line drugs or some new autophagy-based manipulators, such as regulation of B cell lymphoma 2 family proteins and caspase-dependent clearance of autophagy-related gene proteins, are likely to be the promising approaches for GIOP clinical treatments. In view of the important role of autophagy in the pathogenesis of GIOP, here we review the potential mechanisms about the impacts of autophagy in GIOP and its association with GIOP therapy.

Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, β-catenin, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, β-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.