Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis.

BACKGROUND: Recently, type 2 diabetic osteoporosis (T2DOP) has become a research hotspot for the complications of diabetes, but the specific mechanism of its occurrence and development remains unknown. Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP. Polycytosine RNA-binding protein 1 (PCBP1), an iron ion chaperone, is considered a protector of ferroptosis. AIM: To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes. METHODS: A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose (HG) and/or ferroptosis inhibitors at different concentrations and times. Transmission electron microscopy was used to examine the morphological changes in the mitochondria of osteoblasts under HG, and western blotting was used to detect the expression levels of PCBP1, ferritin, and the ferroptosis-related protein glutathione peroxidase 4 (GPX4). A lentivirus silenced and overexpressed PCBP1. Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin (OPG) and osteocalcin (OCN), whereas flow cytometry was used to detect changes in reactive oxygen species (ROS) levels in each group. RESULTS: Under HG, the viability of osteoblasts was considerably decreased, the number of mitochondria undergoing atrophy was considerably increased, PCBP1 and ferritin expression levels were increased, and GPX4 expression was decreased. Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1, increased the expression levels of ferritin, GPX4, OPG, and OCN, compared with the HG group. Flow cytometry results showed a reduction in ROS, and an opposite result was obtained after silencing PCBP1. CONCLUSION: PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment. Moreover, PCBP1 may be a potential therapeutic target for T2DOP.

NIPA2 regulates osteoblast function via its effect on apoptosis pathways in type 2 diabetes osteoporosis.

Type 2 diabetes osteoporosis has recently become a hot topic in the study of diabetic complications, but the specific mechanism of its development remains unclear. Non-imprinted in Prader-Willi/Angelman syndrome region protein 2 (NIPA2), a highly-selective magnesium ion transporter, has been found to be associated with type 2 diabetes. In this study we aimed to investigate the specific role and mechanism of NIPA2 in the pathogenesis of type 2 diabetes osteoporosis. We first used western blotting, PCR, immunofluorescence, and magnesium ion probes to detect changes of NIPA2 and intracellular magnesium levels in osteoblasts at different concentrations of advanced glycation end products (AGEs). We then up- or down-regulated NIPA2 using a lentivirus and analyzed apoptotic biomarkers as well as the osteogenic ability of osteoblasts. We found that AGEs dose-dependently down-regulated the expression of NIPA2 in osteoblasts. NIPA2 also regulated osteoblast apoptosis by affecting the intracellular magnesium level and further affecting the osteogenic capacity of osteoblasts. Our study revealed the changes of NIPA2 in response to AGEs in the environment, as well as its function and mechanism in osteoblasts, demonstrating its important role in the pathogenesis of type 2 diabetes osteoporosis. The study suggests that NIPA2 is a potential target for the treatment of type 2 diabetes osteoporosis.

High glucose downregulates the effects of autophagy on osteoclastogenesis via the AMPK/mTOR/ULK1 pathway.

Diabetes is a chronic disease that disrupts the balance between bone formation and bone desorption, which can lead to osteoporosis, increasing the risk of fracture. However, compared with osteoblasts, the biological effects of hyperglycemia on osteoclastogenesis remain to be elucidated. Therefore, we investigated the impact of glucose at different concentrations (5.5, 10.5, 15.5, 20.5, 25.5, and 30.5 mM) on osteoclastogenesis using RAW264.7 cells. Cell proliferation was measured with the cell counting kit-8 assay, and osteoclastogenesis was detected with tartrate-resistant acid phosphatase staining and bone resorption assays, as well as protein cathepsin K expression. Compound C, the AMP-activated protein kinase (AMPK) pathway inhibitor, was used to examine the relationship between the AMPK/mTOR/ULK1 signaling pathway and autophagy in osteoclasts. Autophagy was evaluated with transmission electron microscopy and immunofluorescence microscopy and associated proteins were detected with western blotting. The pharmacological autophagic reagents bafilomycin A1, 3-methyladenine, and rapamycin were used to determine the effect of autophagy on osteoclastogenesis. Our results showed that glucose negatively affected osteoclast formation and function but did not affect the proliferation of RAW264.7 cells. Suppression of the AMPK/mTOR/ULK1 signaling axis decreased autophagy in glucose-mediated osteoclast. Furthermore, High levels of glucose decreased autophagy level in osteoclasts. Additionally, interfering with autophagy affected osteoclast formation and function. These findings clarify the mechanisms underlying the effects of glucose-mediated osteoclastogenesis and will help identify novel therapeutic strategies for the protection of skeletal health in diabetic osteoporosis.

Regulation of DMT1 on autophagy and apoptosis in osteoblast.

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 µmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Role of miR-155 in the regulation of MMP-16 expression in intervertebral disc degeneration.

The molecular mechanisms of intervertebral disc degeneration (IDD) remain elusive. We found that miR-155 is down-regulated in degenerative nucleus pulposus (NP), and more severe degeneration is correlated with higher matrix metallopeptidase 16 (MMP-16) expression. MMP-16 also degraded matrix aggrecan. Here, we addressed the in vivo miR-155-mediated pathological impact on IDD using a classic puncture mouse model. Lentiviral upregulated-miR-155 or downregulated-miR-155 was transduced into the discs of C57 mice, which was validated by real-time polymerase chain reaction (real-time PCR) and in situ hybridization. Immunohistochemistry and western blotting revealed that up-regulation of miR-155 resulted in down-regulation of MMP-16 and an increase in aggrecan and collagen type II in mouse NP; whereas, down-regulation of miR-155 resulted in up-regulation of MMP-16 and a decrease in aggrecan in mouse NP. Radiographic and histological analysis showed that the up-regulation of miR-155 attenuated IDD, while down-regulation of miR-155 resulted in the deterioration of IDD. These findings indicate that decreased miR-155 contributed to the up-regulation of MMP-16 in vivo, and MMP-16 further degraded aggrecan and collagen type II, leading to the dehydration and degeneration of discs. Our findings revealed a therapeutic role for miR-155 in IDD. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1323-1334, 2017.

Melatonin suppresses autophagy in type 2 diabetic osteoporosis.

Type 2 diabetes mellitus is often complicated by osteoporosis, a process which may involve osteoblast autophagy. As melatonin suppresses autophagy under certain conditions, we its investigated the effects on bone autophagy during diabetes. We first assessed different body parameters in a diabetic rat model treated with various concentrations of melatonin. Dynamic biomechanicalmeasurements, bone organization hard slice dyeing and micro-CT were used to observe the rat bone microstructure, and immunohistochemistry was used to determine levels of autophagy biomarkers. We also performed in vitro experiments on human fetal osteoblastic (hFOB1.19) cells cultured with high glucose, different concentrations of melatonin, and ERK pathway inhibitors. And we used Western blotting and immunofluorescence to measure the extent of osteogenesis and autophagy. We found that melatonin improved the bone microstructure in our rat diabetes model and reduced the level of autophagy(50 mg/kg was better than 100 mg/kg). Melatonin also enhanced osteogenesis and suppressed autophagy in osteoblasts cultured at high glucose levels (10 µM was better than 1 mM). This suggests melatonin may reduce the level of autophagy in osteoblasts and delay diabetes-induced osteoporosis by inhibiting the ERK signaling pathway.

Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

Regulation of DMT1 on Bone Microstructure in Type 2 Diabetes.

Diabetic osteoporosis is gradually attracted people's attention. However, the process of bone microstructure changes in diabetic patients, and the exact mechanism of osteoblast iron overload are unclear. Therefore, the present study aimed to explore the function of DMT1 in the pathological process of diabetic osteoporosis. We build the type two diabetes osteoporosis models with SD rats and Belgrade rats, respectively. Difference expression of DMT1 was detected by using the method of immunohistochemistry and western blotting. Detection of bone microstructure and biomechanics and iron content for each group of samples. We found that DMT1 expression in type 2 diabetic rats was higher than that in normal rats. The bone biomechanical indices and bone microstructure in the rat model deficient in DMT1 was significantly better than that in the normal diabetic model. The loss of DMT1 can reduce the content of iron in bone. These findings indicate that DMT1 expression was enhanced in the bone tissue of type 2 diabetic rats, and plays an important role in the pathological process of diabetic osteoporosis. Moreover, DMT1 may be a potential therapeutic target for diabetic osteoporosis.

Radiographic angles in hallux valgus: Comparison between protractor and iPhone measurements.

Radiographic angles are used to assess the severity of hallux valgus deformity, make preoperative plans, evaluate outcomes after surgery, and compare results between different methods. Traditionally, hallux valgus angle (HVA) has been measured by using a protractor and a marker pen with hardcopy radiographs. The main objective of this study is to compare HVA measurements performed using a smartphone and a traditional protractor. The secondary objective was to compare the time taken between those two methods. Six observers measured major HVA on 20 radiographs of hallux valgus deformity with both a standard protractor and an Apple iPhone. Four of the observers repeated the measurements at least a week after the original measurements. The mean absolute difference between pairs of protractor and smartphone measurements was 3.2°. The 95% confidence intervals for intra-observer variability were ±3.1° for the smartphone measurement and ±3.2° for the protractor method. The 95% confidence intervals for inter-observer variability were ±9.1° for the smartphone measurement and ±9.6° for the protractor measurement. We conclude that the smartphone is equivalent to the protractor for the accuracy of HVA measurement. But, the time taken in smartphone measurement was also reduced.

[Simple osteotomy for correct hallux valgus with increased I, II intermetatarsal angle].

OBJECTIVE: To investigate efficacy of simple osteotomy for correct hallux valgus,and explore its scope and condition. METHODS: From December 2009 to April 2011, 20 patients (32 feet) with hallux valgus were treated by simple osteotomy. There were 1 male (1 foot), 19 females (31 feet) with an average age of 40 years (ranged, 22 to 64 years). The course of disease ranged from 2 to 31 years(mean 12 years). Among them, 6 feet were mild, 20 feet were moderate, 6 feet were serious. Patients had symptoms of metatarsophalangeal joint pain, but tensity of lateral soft tissue were normal. Hallux Valgus Angle (HVA) and Intermetatarsal Angle (IMA) were examined before and after treatment. The criteria of hallux valgus was used to evaluate the effects from valgus deformity, activity of metatarsophalangeal joint, satisfaction of patients. RESULTS: Twenty patients were followed up from 6 to 18 months with an average of 8.5 months. The wounds were healed well, no infection and metatarsal head necrosis occurred, 95.1% of patients were satisfied with the efficacy. Average AOFAS score increased from preoperative (53.1 +/- 7.5) points to the final follow-up (93.1 +/- l.9) points (P<0.05), the average correct HVA increased from preoperative (33.4 +/- 7.8) degrees to postoperative (11.9 +/- 3.6) degrees (P<0.05), the average IMA were decreased from preoperative (12.3 +/- 3.0) degrees to postoperative (6.3 +/- 1.9) degrees (P<0.05), tibial sesamoid position improved from 1.9 to 0.9 (P<0.05). CONCLUSION: the main pathological changes of hallux valgus patients with normal tension of lateral soft tissue is metatarsal adduction, simple osteotomy can get satisfactory results, no need to cut adductor muscle.

Curcumin improves bone microarchitecture and enhances mineral density in APP/PS1 transgenic mice.

Alzheimer's disease and osteoporosis are often observed to co-occur in clinical practice. The present study aimed to evaluate the bone microarchitecture and bone mineral density (BMD) of the proximal tibia in APP/PS1 transgenic mice by micro-computed tomography (micro-CT), and to search for evidence that curcumin can be used to reduce bone mineral losses and treat osteoporosis after senile dementia in these transgenic mice. Three-month-old female mice were divided into the following groups (n=9 per group): wild-type mice (WT group); APP/PS1 transgenic mice (APP group); and APP/PS1 transgenic mice with curcumin treatment (APP+Cur group). Between 9 and 12 months of age, the APP+Cur group were administered curcumin orally (600ppm). CT scans of the proximal tibia were taken at 6, 9 and 12 months. At 6 months, there were little differences in the structural parameters. At 9 months, the APP groups displayed loss of bone volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and connectivity density (Conn.D) and increases in trabecular separation (Tb.Sp) and geometric degree of anisotropy (DA) (P<0.05 or P<0.01), with significant changes in the BMD parameters. At 12 months, curcumin treatment led to constant increases in the trabecular bone mass of the metaphysis and clearly improved the BMD. By the same time, we measured the TNF-α and IL-6 in the serum among the different groups at 6, 9 and 12 months by enzyme-linked immunoassay(ELISA). These results suggest that APP/PS1 transgenic mice are susceptible to osteoporosis, and that curcumin can prevent further deterioration of the bone structure and produce beneficial changes in bone turnover. The change of inflammation cytokine, including TNF-α and IL-6, may play an important role in the mechanisms of action of curcumin, but the detail mechanism remains unknown.

Self-excision of bilateral patellae treated by replantation: a report of one case.

Absence of patella may be caused by congenital factors, trauma and surgical operations (patellectomy, etc). Complete absence of bilateral patellae is rare in clinical case. We report a case of posttraumatic bilateral patella excision. To the best of our knowledge, absence of bilateral patellae caused by self-mutilation has never been reported. Our patient and his family members were informed that the data concerning this case would be submitted for publication.

Effect of fluvastatin on vascular endothelial growth factor in rats with osteoporosis in process of fracture healing / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To explore the effect of fluvastatin on vascular endothelial growth factor (VEGF) in rats with osteoporosis in the process of fracture healing.</p><p><b>METHODS</b>Fractures at the intermediate piece of the femur were made on 72 Sprague Dawley (SD) rats (weighing initially 290-340 g and aged 6 months) with osteoporosis after ovariectomy for three months, then these rats were divided randomly into the medication administration group (the experimental group) and the control group, 36 rats each. In the experimental group, the rats received fluvastatin lavage (10 mg/kg per day) since the next day of operation lasting for 6 weeks, and the rats in the control group received placebo. Then the expression of VEGF and VEGF mRNA in bony callus of the two groups was measured respectively with immunohistochemistry and in situ hybridization on days of 3rd, 7th, 14th, 21st, 28th, and 42nd, and image analysis was made with real-color image analysis machine.</p><p><b>RESULTS</b>No difference was found in the cellular localization of VEGF and VEGF mRNA gene expression between the experimental group and the control group in process of fracture healing and their expression modes were almost similar. On the 14th day postoperatively, the positive extent of positive cells in the experimental group was higher than that of the control group (P < 0.05).</p><p><b>CONCLUSION</b>Fluvastatin can promote the VEGF level in rats with osteoporosis in process of fracture healing.</p>