Restrained Mitf-associated autophagy by Mulberroside A ameliorates osteoclastogenesis and counteracts OVX-Induced osteoporosis in mice.

Bone and mineral metabolism homeostasis accounts for the maintenance of normal skeletal remodeling. However, with aging and changes in hormone levels, over-activated osteoclasts disrupt homeostasis, induce osteoporosis, and even cause osteoporotic fractures, leading to an enormous economic burden. Despite the rapid development of pharmacological therapy for osteoporosis, safer and more effective treatments remain to be explored. Here, we demonstrate that Mulberroside A (Mul-A), a natural component extracted from mulberry bark and branches, effectively suppresses osteoclastogenesis in vitro and counteracts bone loss caused by ovariectomy (OVX). The mechanism underlying this effect involves the repression of autophagic flux during osteoclastogenesis by Mul-A, which can be attributed to the restrained expression of microphthalmia-related transcription factor (Mitf) and its nuclear translocation. Importantly, Mitf overexpression partially reverses the inhibitory effects of Mul-A on autophagy and osteoclastogenesis. Moreover, applying two autophagy agonizts, rapamycin and Torin 1, attenuates the osteoclastogenic regulatory role of Mul-A. Collectively, our study demonstrates that Mul-A damages osteoclast differentiation and ameliorates osteoporosis caused by estrogen deficiency by modulation of Mitf-associated autophagy, indicating its therapeutic potential against osteoporosis.

Metabolite asymmetric dimethylarginine (ADMA) functions as a destabilization enhancer of SOX9 mediated by DDAH1 in osteoarthritis.

Osteoarthritis (OA) is a degenerative disease with a series of metabolic changes accompanied by many altered enzymes. Here, we report that the down-regulated dimethylarginine dimethylaminohydrolase-1 (DDAH1) is accompanied by increased asymmetric dimethylarginine (ADMA) in degenerated chondrocytes and in OA samples. Global or chondrocyte-conditional knockout of ADMA hydrolase DDAH1 accelerated OA development in mice. ADMA induces the degeneration and senescence of chondrocytes and reduces the extracellular matrix deposition, thereby accelerating OA progression. ADMA simultaneously binds to SOX9 and its deubiquitinating enzyme USP7, blocking the deubiquitination effects of USP7 on SOX9 and therefore leads to SOX9 degradation. The ADMA level in synovial fluids of patients with OA is increased and has predictive value for OA diagnosis with good sensitivity and specificity. Therefore, activating DDAH1 to reduce ADMA level might be a potential therapeutic strategy for OA treatment.

Correlation Analysis Between Basic Diseases and Subsequent Vertebral Fractures After Percutaneous Kyphoplasty (PKP) for Osteoporotic Vertebral Compression Fractures.

BACKGROUND: Percutaneous kyphoplasty (PKP) is a widely accepted surgical treatment modality for painful osteoporotic vertebral compression fractures. The risk factors cause of subsequent vertebral fractures after PKP are debated. OBJECTIVES: To evaluate risk factors for the occurrence of new vertebral compression fractures after PKP. STUDY DESIGN: A retrospective study. SETTING: A single-center inpatient population. METHODS: A total of 921 patients (1,152 vertebrae) with PKP were investigated. Among those patients, 111 patients (155 levels) incurred refractures after PKP. RESULTS: The average bone mineral density was -3.27 in the "refracture"group and -3.00 in the "no fracture" group (P = 0.031). Morbidities of women were significantly higher in the "refracture" group (90.99%) compared with the "no fracture" group (81.73%) (P = 0.015). Among the basic diseases, several diseases (history of previously fracture, previously osteoporosis, gallstone disease, stomach disease, and ovariectomy) are associated with refractures after PKP (P < 0.05). And antiosteoporotic treatment (calcium + vitamin D or zoledronate) after PKP can also significantly reduce the occurrence of refracture (P < 0.000). In addition, logistic regression analysis also showed that most of the above contents had significant correlation with the refracture after PKP (P < 0.05), except for gallstone disease (P = 0.362). LIMITATIONS: Retrospective study, single center. CONCLUSION: Osteoporosis is the main cause of refracture after PKP. Elderly women were found to be more susceptible than elderly men to refracture. Patients with a history of previously fracture, previously osteoporosis, stomach ulcer, and ovariectomy are more likely to be refracture. Antiosteoporosis treatment (calcium + vitamin D or zoledronate) after PKP can reduce the risk of refracture.

Lycorine Induces Apoptosis and G1 Phase Arrest Through ROS/p38 MAPK Signaling Pathway in Human Osteosarcoma Cells In Vitro and In Vivo.

STUDY DESIGN: Xenograft osteosarcoma mouse model. OBJECTIVE: We determined the effect of lycorine on osteosarcoma. SUMMARY OF BACKGROUND DATA: Osteosarcoma is an aggressive malignant neoplasm, is most prevalent in teenagers and adults and current treatment approaches have reached a survival plateau and attempts to improve osteosarcoma prognosis have proven unsuccessful. Thus there is clear evidence that development of new agents with high efficacy and fewer side effects to provide better prognostic outcome is urgently needed. METHODS: The toxicity, function and mechanism of lycorine (LY) on osteosarcoma were accessed in vitro by CCK-8 assay, flow cytometry, and western blotting and in vivo by the xenograft osteosarcoma mouse model. RESULTS: In this study, we found that LY exhibited dose-dependent and time-dependent cytotoxic effects on human osteosarcoma cell-lines SJSA-1 and U2OS, inducing G1 phase cell cycle arrest and cellular death via apoptosis. Mechanistically, LY treatment elevated ROS generation that activates the p38 mitogen-activated protein kinases (MAPKs) and p53-dependent apoptotic program. Inhibition of ROS generation by NAC or p38 MAPK signaling by SB203580 attenuated the p53-mediated cell cycle arrest and apoptosis induced by LY. In vivo administration of LY markedly reduced tumor growth with little organ-related toxicity in a mouse xenograft model of osteosarcoma. CONCLUSION: Collectively, our data suggests that LY exhibit therapeutic potential for the treatment of osteosarcoma. LEVEL OF EVIDENCE: N/A.

The PPAR-γ antagonist T007 inhibits RANKL-induced osteoclastogenesis and counteracts OVX-induced bone loss in mice.

BACKGROUND: Osteoclasts are key determinant cellular components implicated in the development and progression of disorders driven by bone damage. Herein, we studied the upshot of T007, an antagonist of peroxisome proliferator-activated receptor-gamma (PPARγ), on osteoclastogenesis using cell and animal models. RESULTS: The in vitro assays revealed that T007 hindered the osteoclastogenesis caused by the treatment with the receptor activator of nuclear factor-κB ligand (RANKL) through inhibiting the levels of PPARγ in cells. The PPARγ siRNA partially reproduced the inhibitory action of T007. The opposite findings were produced after PPARγ overexpression. Furthermore, T007 prevented from bone loss in a mouse model of osteoporosis induced by ovariectomy (OVX). These findings implied that T007 is a potential efficient drug for the prophylaxis and cure of osteoclast-related disorders. CONCLUSIONS: Taken together, our findings demonstrated that T007 impedes osteoclastogenesis and will be useful for the therapy of bone related diseases, essentially osteoporosis.

The GLP-1 agonist, liraglutide, ameliorates inflammation through the activation of the PKA/CREB pathway in a rat model of knee osteoarthritis.

BACKGROUND: Inflammation is a common pathological phenomenon of osteoarthritis (OA). Accumulated evidence indicates that ameliorating or suppressing inflammation might be a promising and effective therapeutic strategy for the treatment of OA. Notably, glucagon-like peptide-1 (GLP-1)-based drugs are being successfully used to control glucose levels in patients with diabetes mellitus. In addition, recent findings have indicated that GLP-1 agonists, such as liraglutide have therapeutic potential in preventing inflammation-related disorders through the regulation of protein kinase A (PKA)/ cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signals. Intra-articular injection of monoiodoacetate (MIA) has been widely used to induce OA. Thus, the present study aimed to investigate whether liraglutide has anti-inflammatory effects on MIA-induced OA rats and uncover its underlying molecular mechanisms. METHODS: Intra-articular injection of MIA was used to induce knee OA in a rat model. Subcutaneous injection of liraglutide was used to upregulate the expression of GLP-1 receptor (GLP-1R). Western blot analysis was utilized to measure the expression of GLP-1R, PKA/CREB pathway components and inflammation-related proteins, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6. Immunoprecipitation techniques were used to detect the interactions between GLP-1R and the PKA/CREB pathway. RESULTS: The levels of GLP-1R decreased significantly in the knees of OA rats, accompanied by the downregulation of PKA /CREB signals and upregulation of inflammation-related proteins. We also found that GLP-1R interacted with the PKA/CREB pathway and that liraglutide could activate PKA/CREB signals, thereby inhibiting the expression of inflammation-related proteins. CONCLUSIONS: Together our results suggest that liraglutide exhibits anti-inflammatory activity through the activation of the PKA/CREB pathway in an OA rat model.

The Novel p38 Inhibitor, Pamapimod, Inhibits Osteoclastogenesis and Counteracts Estrogen-Dependent Bone Loss in Mice.

Pamapimod (PAM) is a novel selective p38 mitogen-activated protein (MAP) kinase inhibitor proved to be effective in rheumatoid arthritis in phase 2 clinical trial. However, its effect on osteoclast-associated osteoporosis and the underlying mechanisms remain unclear. In this study, we showed that PAM suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation via inhibition of p38 phosphorylation and subsequent c-Fos and nuclear factor of activated T cells c1 (NFATc1) expression. In addition, the downregulated NFATc1 leads to reduced expression of its targeting gene disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), which was further proven to be critical for osteoclastic bone resorption. Therefore, we treated ovariectomized (OVX) mice with PAM and revealed a protective effect of PAM on osteoporosis in vivo. In conclusion, our results demonstrated PAM can prevent OVX-induced bone loss through suppression of p38/NFATc1-induced osteoclast formation and NFATc1/ADAM12-associated bone resorption. © 2018 American Society for Bone and Mineral Research.

MicroRNA-543 promotes ovariectomy-induced osteoporosis through inhibition of AKT/p38 MAPK signaling pathway by targeting YAF2.

The present study aimed to determine the roles of miRNA-543 in osteoporosis in rats induced by ovariectomy. The osteoporosis rat model was established by ovariectomy induction. MiRNA-543 expression in osteoblasts was measured by quantitative real-time polymerase chain reaction. The cell proliferation and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays, respectively. Western blot analysis was conducted to examine the expression of YAF-2 and AKT signaling. TargetScan analysis and dual-luciferase reporter assay were performed to determine the target gene of miRNA-543. MiRNA-543 was significantly upregulated in osteoporosis rat model. Overexpression of miRNA-543 significantly suppressed cell growth and promoted apoptosis in osteoblasts, whereas downregulation of miRNA-543 significantly enhanced cell growth and inhibited apoptosis. MiRNA-543 upregulation significantly inhibited YAF-2 expression and suppressed the phosphorylation and expression of AKT and p38 mitogen-activated protein kinases (MAPK) in osteoblasts. Furthermore, YAF-2 knockdown enhanced the effects of miRNA-543 on apoptosis in osteoblasts. AKT inhibitor MK2206 and p38 MAPK inhibitor SB203580 also enhanced the effects of miRNA-543 on apoptosis in osteoblasts. Our findings revealed that inhibition of miRNA-543 could protect osteoblasts against ovariectomy-induced osteoporosis through AKT/p38 MAPK signaling pathway by targeting YAF2.

New Levels of Vertebral Compression Fractures after Percutaneous Kyphoplasty: Retrospective Analysis of Styles and Risk Factors.

BACKGROUND: The causes of subsequent vertebral fractures after kyphoplasty are debated. It is reported that most new vertebral fractures after kyphoplasty develop in adjacent vertebrae. OBJECTIVES: We explored whether kyphoplasty increases the incidence of adjacent vertebral fractures and identified risk factors for new vertebral compression fractures (VCFs) after kyphoplasty. STUDY DESIGN: Retrospective study. SETTING: Inpatient population of a single center. METHODS: We studied 356 patients treated with kyphoplasty from January 2008 to March 2012. Among those patients, there were 35 new VCFs after kyphoplasty. Subsequently, these patients were divided into 2 groups: an "adjacent fracture" group and a "nonadjacent fracture" group. In addition, all patients treated with kyphoplasty were further assigned to either a "new fracture" group or a "no fracture" group. RESULTS: The occurrence of new VCFs in the "nonadjacent fracture" group was significantly higher than that in the "adjacent fracture" group. The average bone mineral density (BMD) of the spine was -3.95 in the "new fracture" group and -2.86 in the "no fracture" group. The risk of new vertebral fracture increased as the bone mineral density decreased (P < 0.05). The morbidity of women was significantly higher in the "new fracture" group (94.29%) than in the "no fracture" group (77.88%) (P = 0.025). LIMITATIONS: Retrospective study at a single center. CONCLUSION: New VCFs after kyphoplasty occurred most often in nonadjacent vertebrae. VCFs after kyphoplasty were common in patients with low bone mineral density and in women, suggesting that osteoporosis is an underlying mechanism. INSTITUTIONAL REVIEW: This study was approved by the institutional review board.

Re-fractures of non fracture vertebral body after percutaneous kyphoplasty / 中华骨科杂志

Objective To investigate the causes of re-fractures of non fracture vertebral body after percutaneous kyphoplasty (PKP).Methods 512 patients (618 vertebral bodies) treated with PKP because of osteoporosis VCFs were recruited from June 2010 to June 2014.There were 107 males (121 vertebral bodies) and 405 females (497 vertebral bodies) with the mean age of 70.38±7.59 years old (51 to 91 years).There were 406 single segment fracture and 106 double segment fractures cases,and the fracture segments were T4 to L5.The T value of the patients' bone mineral density (BMD) was from-1.0 to-5.2 SD.The clinic characteristics of all the patients including age,sex,body weight,body height,body mass index (BMI),BMD score of the spine,volume of bone cement,restoration rate of anterior/middle vertebral height,postoperative complications (pulmonary embolism,bone cement leakage,nerve injury),and treated vertebral level were analyzed.Results 52 patients (10.16％,52/512) experienced refractures of non fracture vertebral body after kyphoplasty,and 4 experienced re-fracture of the fracture vertebral body after kyphoplasty.The average age of the 52 patients was 71.88±7.74 years old,meanwhile,the ratio of female was 94.23％ (49/52),the mean T value of BMD-4.03±0.60 SD,the ratio of initial double segment fractures 51.92％ (27/52).In addition,among the 456 cases with no fracture,the average age was 70.21±7.56 years,the ratio of female was 77.19％ (352/456);the mean T value of BMD was-2.89±0.55 SD;the ratio of initial double segment fractures was 17.32％(79/456).The data above (age,T value of BMD and initial double segment fractures) were all with statistical significant differences.Whereas the BMI,volume of bone cement,intervertebral disc leakage and restoration rate of anterior/middle vertebral height had no significant difference between the two groups.Furthermore,in the re-fracture of non fracture vertebral body group,32 cases (61.54％,32/52) were nonadjacent fractures,and 20 (38.46％,20/52) were adjacent fractures.Conclusion Osteoporosis degree,female and initial double segment fractures were major risk factors in the development of re-fracture of non fracture vertebral body after PKP.

Is unilateral kyphoplasty as effective and safe as bilateral kyphoplasties for osteoporotic vertebral compression fractures? A meta-analysis.

BACKGROUND: An osteoporotic vertebral compression fracture is a common condition in elderly people, especially women. The percutaneous kyphoplasty is an effective treatment for osteoporotic vertebral compression fractures. Controversy remains regarding whether a unilateral or a bilateral approach is superior, and to our knowledge, there have been no large studies comparing these two approaches, therefore a meta-analysis synthesizing the data on this question is warranted. QUESTIONS/PURPOSES: We asked the following questions: (1) Is there evidence to suggest a benefit in clinical outcome as assessed by the VAS and Oswestry Disability Index of a unilateral kyphoplasty or bilateral kyphoplasties? (2) Are the complications associated with the two approaches different? (3) Do the procedures result in different kyphosis angle reduction or anterior vertebral height restoration? (4) Is the surgical time for the procedures different? METHODS: We searched the Cochrane Library, PubMed MEDLINE, EMBASE, Web of Knowledge MEDLINE (January 1980 to June 2013), and reference lists of eligible prospective studies. The levels of the evidence and recommendations were assessed using the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) system. Five studies encompassing 253 patients met the inclusion criteria. RESULTS: The short- and long-term clinical outcomes as assessed by the VAS and Oswestry Disability Index showed no differences between unilateral and bilateral kyphoplasties (p = 0.41, p = 0.60 for VAS; p = 0.10, p = 0.36 for Oswestry Disability Index). There were no differences in complications such as cement leakage and adjacent vertebral fractures associated with the two approaches (p = 0.43 and p = 0.95). The kyphosis angle reduction and anterior vertebral height restoration showed no difference between the two approaches (p = 0.34 and p = 0.46). The unilateral approach was shorter in terms of surgical time (mean difference, -24.98; p < 0.0001). The overall GRADE system evidence quality was very low, with only high evidence for operation time, which lessens our confidence in recommendations. CONCLUSIONS: Unilateral and bilateral percutaneous kyphoplasties appear to be safe and effective for treating osteoporotic vertebral compression fractures. No clinically important differences were found between them. Considering less operation time and less cost, we suggest that a unilateral percutaneous kyphoplasty is advantageous, but because of the poor quality of the evidence, high-quality randomized controlled trials are required to resolve this issue.

Spontaneous epidural hematoma of thoracic spine presenting as Brown-Séquard syndrome: report of a case with review of the literature.

BACKGROUND: Spontaneous spinal epidural hematoma (SSEH) is an uncommon clinical entity. It produces a severe neurological deficit and prompt decompression is usually the first choice of treatment. Brown-Séquard syndrome is commonly seen in the setting of spinal trauma or an extramedullary spinal neoplasm, but rarely caused by SSEH. METHODS: Case report and literature review. FINDINGS: A previously healthy man presented with Brown-Séquard syndrome below T5-T6 cord segment secondary to spontaneous epidural hematoma. He opted for conservative treatment, which was followed by rapid resolution. CONCLUSIONS: Although Brown-Séquard syndrome as a presenting feature of SSEH is rare, it does exist in exceptional case, which should be taken into consideration for differential diagnosis. Prompt surgical decompression is an absolute surgical indication widely accepted for patient with progressive neurological deficit. However, SSEH presenting with incomplete neurological insult such as Brown-Séquard syndrome might have a benign course. Successful non-operative management of this problem does not make it a standard of care, and surgical decompression remains the standard treatment for SSEH.

Percutaneous vertebroplasty versus balloon kyphoplasty for treatment of osteoporotic vertebral compression fracture: a meta-analysis of randomised and non-randomised controlled trials.

PURPOSE: There is still debate over whether vertebroplasty (VP) or kyphoplasty (KP) is superior for the treatment of osteoporosis vertebral compression fractures (VCFs). We performed a systematic review and meta-analysis of randomised and non-randomised controlled trials comparing VP with KP to reach a relatively conclusive answer. METHODS: We searched computerised databases comparing efficacy and safety of VP and KP in osteoporotic fractures. These trials reported pain relief (Visual Analogue Scale), disability (Oswestry disability score) and complications (i.e., cement leakage, incident fractures) as the primary outcome. RESULTS: Eight studies involving 848 patients were identified. The outcome showed that VP is more effective in the short-term (no more than seven days) pain relief. Kyphoplasty had a superior capability for intermediate-term (around three months) functional improvement. As for long-term pain relief and functional improvement, there is no significant difference between these two interventions. Consistently, both interventions have similar risk for subsequent fracture and cement leakage. CONCLUSION: Thus considering the higher cost of the KP procedure, we recommend VP over KP for the treatment of osteoporotic VCFs.

Stronger expression of CHOP and caspase 12 in diabetic spinal cord injury rats.

OBJECTIVES: It is suggested that the injury-induced cell death of neurons within the spinal cord is related with endoplasmic reticulum (ER) stress. In this work, we explored that diabetes induce more severe spinal cord injury (SCI) and stronger ER stress in rats. METHODS: Forty-five Sprague-Dawley rats were divided into three groups at random (the sham operation control group, SCI group and diabetic SCI group). Streptozotocin was injected into the caudal vein (30 mg/kg) to induce diabetes; 4 weeks after diabetes model induction, using a weight-drop contusion model of SCI in SCI group and diabetic SCI group; then, rats were killed at 24 hours and 7 days, and the level of C/EBP homologous transcription factor protein (CHOP), a proapoptotic protein, and caspase 12 were determined by immunohistochemistry staining and real-time reverse transcription quantitative polymerase chain reaction analysis. RESULTS: We observe that both CHOP and caspase 12 were higher in diabetic SCI group than in the SCI group. Diabetes also caused severe locomotor dysfunction and slowly recovered as their Basso, Beattie and Bresnaha scores lowered. Pathological hematoxylin-eosin staining observation also showed progressive disruption of the dorsal white and few neurons regeneration in diabetic SCI rats. DISCUSSION: These results suggest that stronger ER stress in diabetic rats may be the reason for severe SCI observed.

[Enhancement effect of caffeine on chemotherapy of osteosarcoma in Fischer 344/N rats].

OBJECTIVE: To determine the enhancement effects of caffeine on chemotherapy of transplanted osteosarcoma in Fischer 344/N rats. METHODS: Osteosarcoma-bearing Fischer 344/N rats were treated with cisplatin 2.5 mg/kg (Group DDP), caffeine 90 mg/kg x 2 d (Group caffeine), and cisplatin 2.5 mg/kg plus caffeine 90 mg/kg x 2 d (Group DDP+caffeine), and the control group was treated with normal saline in the same volume. All drugs were given by intra-peritoneum injection with micro-pump, in the rate of 0.5 ml/h. The tumor volume was measured and evaluated. The tumors were stained in TUNEL, and PCNA was detected with immunohistochemistry. The tumor growth inhibition rate, PCNA index and apoptosis index were calculated, and the survival time were recorded. RESULTS: The tumor inhibition rate was -0.5219 +/-0.1429 in control group, 0.0362 +/-0.0957 in Group DDP, -0.4193 +/-0.1345 in Group caffeine, and 0.3646 +/-0.1313 in Group DDP+caffeine (P <0.01). PCNA index was 0.4587 +/-0.1312 in control group, 0.1847+/-0.0535 in Group DDP, 0.4381 +/-0.0706 in Group caffeine, and 0.0314 +/-0.0231 in Group DDP+caffeine (P <0.01). Apoptosis index was 0.0008 +/-0.0005 in control group, 0.0077 +/-0.0060 in Group DDP, 0.0011 +/-0.0003 in Group caffeine, and 0.0295 +/-0.0069 in Group DDP+caffeine (P <0.01). And the survival time was (33.63 +/-4.63)d in control group, (52.13 +/-11.74)d in Group DDP, (35.63 +/-5.15)d in Group caffeine, and (55.13 +/-16.23)d in Group DDP+caffeine (P <0.01). CONCLUSION: Caffeine could enhance the anti-tumor effect of cisplatin in rat osteosarcoma.

[Cytotoxic effect of thermo-chemotherapy with cisplatin on osteosarcoma OS-732 cell line].

OBJECTIVE: To observe the cytotoxic effect of thermo-chemotherapy with cisplatin on osteosarcoma OS-732 cell line and to explore its mechanism. METHODS: The osteosarcoma OS-732 cell line was treated with different temperature, cisplatin alone and 43 degrees C +cisplatin for 1 h, respectively and the in vitro cytotoxic effect was observed with MTT assay. The cell cycle and the apoptotic rate were analyzed with flow cytometry (FCM); the cellular apoptosis was observed also with electron microscope. RESULT: The cytotoxicity index increase markedly as the temperature elevated or the concentration of cisplatin increased. While treated with 43 degrees C hyperthermia and cisplatin simultaneously, the cytotoxicity index increased to 72.37%;the cell cycle of the treated OS-732 cells line was changed with marked increase in S phase and decreasing in G(2)/M phase. The apoptotic rate increased markedly with the highest of 56.47%. Electron microscope showed the characteristic apoptotic alterations. CONCLUSION: Hyperthermia with cisplatin enhance cytotoxicity on osteosarcoma OS-732 cell line and the induced cell apoptosis may be one of the mechanisms of enhanced cytotoxicity by thermo-chemotherapy.