Wenbin Hua

Aberrant CpG Islands' Hypermethylation of ABCB1 in Mesenchymal Stem Cells of Patients with Steroid-associated Osteonecrosis

Objective. Patients carrying an ABCB1 polymorphism have a higher risk of developing osteonecrosis of the femoral head (ONFH). We investigated whether aberrant dinucleotide CpG islands’ hypermethylation of ABCB1 gene existed in mesenchymal stem cells (MSC) of patients with ONFH, which results in cell dysfunction. Methods. Bone marrow was collected from the proximal femur of patients with glucocorticoid (GC)-associated ONFH (n = 22) and patients with new femoral neck fractures (n = 25). MSC were isolated by density gradient centrifugation. We investigated cell viability, intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), the amount of P-glycoprotein (P-gp) and ABCB1 transcripts, and methylation at CpG islands of ABCB1 promoter from both the femoral neck fractures group and the GC-associated ONFH group treated with or without the DNA methyltransferase inhibitor, 5′-Aza-2-deoxycytidine (5′-Aza-dC). Results. We observed that MSC from GC-associated ONFH groups showed reduced proliferation ability, elevated ROS levels, and depressed MMP when compared with the other 2 groups. Low levels of P-gp and ABCB1 transcript, as well as ABCB1 gene hypermethylation, in patients with GC-associated ONFH were also noted. Treatment with 5′-Aza-dC rapidly restored ABCB1 expression. Analysis of general expression revealed that aberrant CpG islands’ hypermethylation of ABCB1 caused sensitivity to GC and induced changes in the proliferation and oxidative stress of MSC under GC administration. Conclusion. These data suggest that aberrant CpG islands’ hypermethylation of ABCB1 gene may be responsible for individual differences in the development of GC-associated ONFH.

MicroRNA-494 promotes apoptosis and extracellular matrix degradation in degenerative human nucleus pulposus cells

Purpose This study investigated the expression and function of the microRNA-494 in intervertebral disc degeneration (IDD). Results MicroRNA-494 expression was upregulated during IDD progression; its overexpression increased the expression of ECM catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in NP cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it also induced the apoptosis of NP cells, as determined by flow cytometry. These effects were reversed by microRNA-494 inhibitor treatment. SOX9 was identified as a target of negative regulation by microRNA-494. Promoter hypomethylation and NF-κB activation were associated with microRNA-494 upregulation in IDD. Materials and Methods MicroRNA-494 expression in degenerative nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The effect of microRNA-494 on extracellular matrix (ECM) metabolism and NP cell apoptosis was evaluated by transfection of microRNA-494 mimic or inhibitor. The regulation of SRY-related high mobility group box (SOX)9 expression by microRNA-494 was assessed with the luciferase reporter assay, and the methylation status of the microRNA-494 promoter was evaluated by methylation-specific PCR and bisulfite sequencing PCR. The role of activated nuclear factor (NF)-κB in the regulation of microRNA-494 expression was evaluated using specific inhibitors. Conclusions MicroRNA-494 promotes ECM degradation and apoptosis of degenerative human NP cells by directly targeting SOX9.

Simvastatin Inhibits IL-1β-Induced Apoptosis and Extracellular Matrix Degradation by Suppressing the NF-kB and MAPK Pathways in Nucleus Pulposus Cells

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway. Some studies have shown that statins may have the potential to inhibit intervertebral disk (IVD) degeneration (IDD). Interleukin (IL)-1β, a catabolic cytokine, is a key regulator of IDD. This study aimed to investigate the mechanism underlying the effect of simvastatin on IDD. The viability of nucleus pulposus (NP) cells was determined by the methyl-thiazolyl-tetrazolium (MTT) assay. The apoptosis of NP cells was measured by flow cytometric analysis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blotting of relevant apoptotic proteins. The protein levels of catabolic factors and anabolic factors were determined by western blotting. The cells were stimulated with IL-1β in the absence or presence of simvastatin to investigate the effects on matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, type II collagen, and aggrecan expression. Our findings indicate that simvastatin considerably inhibited IL-1β-induced apoptosis in NP cells. We also found that simvastatin attenuated IL-1β-induced expression and MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 activities and also reduced the decrease in type II collagen and aggrecan expression. In addition, simvastatin considerably suppressed the nuclear translocation and activation of nuclear factor-kappa B (NF-KB) by inhibiting p65 phosphorylation and translocation and blocking inhibitor kB-α degradation. It also inhibited MAPK pathway activation by blocking c-Jun N-terminal kinase (JNK), p38, and ERK phosphorylation. The results of our study revealed that simvastatin is a potential agent for IDD prevention and treatment.

Analysis of Sagittal Parameters in Patients Undergoing One- or Two-Level Closing Wedge Osteotomy for Correcting Thoracolumbar Kyphosis Secondary to Ankylosing Spondylitis

Study Design. Retrospective analysis of clinical records. Objective. To assess and compare the improvement in sagittal balance after one- or two-level closing wedge osteotomy for correcting thoracolumbar kyphosis secondary to ankylosing spondylitis (AS). Summary of Background Data. Closing wedge osteotomy represents a common approach to correct kyphosis in AS. Although several reports have described the outcomes of one- or two-level closing wedge osteotomy in terms of sagittal parameters, data comparing the outcomes of these procedures are scarce. Methods. Between January 2010 and December 2014, 22 patients with AS underwent closing wedge osteotomy (one-level, 12 patients; two-level, 10 patients) for correcting thoracolumbar kyphosis (mean follow-up, 24.8 months; range, 12–60 months). Preoperative and postoperative chin-brow vertical angle, and the sagittal parameters of the vertebral osteotomy segment were documented and compared. Perioperative and postoperative complications were also recorded. Results. The chin-brow vertical angle improved significantly, from 55.0° ± 27.3° to 4.7° ± 4.9° and from 38.2° ± 14.9° to 3.2° ± 5.4° in the one-level and two-level groups, respectively. The total correction (thoracic kyphosis and lumbar lordosis) was 32.8° ± 18.2° and 53.7° ± 9.4° in the one-level and two-level groups, respectively. No death, complete paralysis, or vascular complications occurred during the procedure, but cerebrospinal fluid leak was noted in one and two patients from the one-level and two-level groups, respectively. A distal pedicle screw adjacent to the osteotomy segment became loose during surgery in one patient (one-level group). Postoperatively, no transient neurological deficit, infection, delay union, or loosening or breaking of the internal fixation devices was observed. Osteotomy site fusion was achieved in all patients, and the Oswestry Disability Index scores improved significantly. Conclusion. Closing wedge osteotomy is effective and safe for correcting thoracolumbar kyphosis in patients with AS. Significant correction and improvement in all sagittal parameters were noted in both groups, but two-level closing wedge osteotomy provided better correction. Level of Evidence: 3

Methylation of microRNA-129-5P modulates nucleus pulposus cell autophagy by targeting Beclin-1 in intervertebral disc degeneration

MicroRNAs play an important role in the etiology and progression of many diseases, including intervertebral disc degeneration (IVDD). The miRNA miR-129-5P regulates autophagy in various cancers, but its role in human nucleus pulposus (NP) cells is unclear. The present study investigated whether miR-129-5p regulates the expression of Beclin-1 which is known to induce autophagy in NP cells by evaluating their levels in normal and degenerative disc tissues and human NP cells transfected with miR-129-5P mimic or inhibitor by quantitative real-time (qRT-)PCR, western blotting, flow cytometry, and immunofluorescence analysis. A bioinformatics analysis was used to predict the relationship between miR-129-5P and Beclin-1, which was confirmed by the dual luciferase assay. DNA methylation status was assessed by methylation-specific PCR, and the effect of demethylation on miR-129-5P level and autophagy was examined by qRT-PCR, western blotting, and flow cytometry. We found that miR-129-5P expression was downregulated while that of Beclin-1 and LC3-II was upregulated in degenerative disc NP cells. Meanwhile, autophagy was reduced in human NP cells transfected with miR-129-5P mimic, whereas the opposite result was observed upon treatment with miR-129-5P inhibitor. Bioinformatics analysis and the luciferase reporter assay revealed that Beclin-1 is a target of and is inhibited by miR-129-5P. We also found that CpG islands in the miR-129-5P promoter region were hypermethylated in degenerative as compared to normal disc tissue. Thus, miR-129-5P blocks NP cell autophagy by directly inhibiting Beclin-1, a process that is dependent on miR-129-5P promoter methylation.

Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration

Accumulating evidence indicates that microRNAs can regulate the apoptosis of various cells. Apoptosis of nucleus pulposus cells plays an important role in the progression of intervertebral disc degeneration. The aim of this study is to investigate whether microRNA-143 (miRNA-143) is involved in the progression of intervertebral disc degeneration. In this study, the expression of miRNA-143 and its biological modulatory effects were examined. Messenger RNA and protein expression of miRNA-143 and B-cell lymphoma-2 (BCL2) in both normal and degenerative disc tissues was determined by using RT-PCR and western-blot assays. After miRNA-143 transfection, BCL2 expression and NP cell apoptosis were assessed by using RT-PCR, western-blot, and flow cytometry. The relationship between miRNA-143 and BCL2 was assessed by bioinformatics and dual luciferase assays. Epigenetic regulation of miRNA-143 was determined by methylation-specific PCR and the effect of hypomethylation using 5-AZA. In this study, miRNA-143 expression significantly increased, while that of BCL2 decreased in degenerative disc specimens. In addition, CpG islands in the promoter region of miRNA-143 were hypomethylated in degenerative disc tissues. Furthermore, bioinformatics analysis and luciferase reporter assay indicated that BCL2 was a target gene of miRNA-143, and miRNA-143 suppressed BCL2 messenger RNA (mRNA) and protein expression. MiRNAmiRNA-143 overexpression enhances apoptosis of nucleus pulposus cells, while miRNA-143 inhibitor had the opposite effect. BCL2 knockdown reversed the effects of the miRNA-143 inhibitor on nucleus pulposus apoptosis. Our results suggest that miRNA-143 promotes the progression of nucleus pulposus apoptosis by directly targeting BCL2, providing a potential therapeutic target for the treatment of intervertebral disc degeneration disease.

Icariin Attenuates Interleukin-1β-Induced Inflammatory Response in Human Nucleus Pulposus Cells

BACKGROUND Low back pain is a common problem, mainly caused by intervertebral disc degeneration (IDD). An important pathophysiological characteristic of IDD is the loss of homeostatic balance of the extracellular matrix metabolism. Interleukin-1β (IL-1β) is one of the inflammatory mediators stimulating the degradation of extracellular matrix in the nucleus pulposus (NP) and contributing to IDD pathogenesis. Icariin, which is isolated from Epimedium brevicornum, acts as an anti-inflammatory drug. OBJECTIVE This study aimed to explore the pharmacological effects of icariin in IDD by simulating NP inflammation in vitro. METHOD Human NP cells were isolated and cultured in vitro. NP cells were pretreated with icariin (0.1, 1 and 10 µM) and stimulated by IL-1β (10 ng/ml). The concentration of Prostaglandin E2 was determined by enzymelinked immunosorbent assay. Nitric oxide was determined by Griess reagent assay. The expression of cyclooxygenase- 2 (COX-2), inducible nitric oxide synthase (iNOS), degrading enzymes, collagen II, aggrecan, mitogenactivated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB)-related signaling molecules was assessed via western blotting. RESULTS IL-1β induced pronounced expression of COX-2 and iNOS, and stimulated production of prostaglandin E2 and nitric oxide. Icariin exhibited significant anti-inflammatory effect, inhibiting IL-1β-induced production of degrading enzymes, as well as extracellular matrix reduction. Finally, icariin suppressed IL-1β-induced activation of MAPK- and NF-κB-related signaling pathways. CONCLUSION The present findings suggest that icariin may have a protective effect on NP cells. The antiinflammatory effect may contribute to the therapeutic action of icariin in IDD.

Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

BACKGROUND Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. OBJECTIVE We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. METHODS miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. RESULTS We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. CONCLUSION Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD.

Total hip arthroplasty with subtrochanteric femoral shortening osteotomy for high hip dislocation.

To evaluate the outcomes of total hip arthroplasty (THA) with subtrochanteric femoral shortening osteotomy for high hip dislocation.

Initial CT-guided percutaneous biopsy of vertebral lesions: Evaluation of its diagnostic accuracy and clinical value

This study aimed to examine the diagnostic accuracy and clinical efficacy of initial CT-guided percutaneous biopsy of the vertebral lesions. A total of 305 percutaneous biopsies of the vertebral lesions were performed under either CT guidance (n=127) or C-arm guidance (n=178). The diagnostic accuracy rate was evaluated by comparing the histopathological diagnosis with the ultimate diagnosis. The histopathological diagnosis was consistent with the ultimate diagnosis in 108 (85.0%, 108/127) cases of CT-guided biopsy and in 135 (75.8%, 135/178) cases of C-arm guided biopsy and there was a significant difference. The accuracy of diagnosis based on biopsies varied with different diseases, including primary benign or malignant tumors, metastatic tumors, inflammatory lesions and fractures. A second biopsy or further examinations were required for patients with negative result obtained in the initial biopsy. The complication rate was 3.1% (4/127) in CT-guided biopsy and 7.3% (13/178) in C-arm guided biopsy. In conclusion, CT-guided percutaneous biopsy is an accurate and safe technique for biopsy of the vertebral lesions.SummaryThis study aimed to examine the diagnostic accuracy and clinical efficacy of initial CT-guided percutaneous biopsy of the vertebral lesions. A total of 305 percutaneous biopsies of the vertebral lesions were performed under either CT guidance (n=127) or C-arm guidance (n=178). The diagnostic accuracy rate was evaluated by comparing the histopathological diagnosis with the ultimate diagnosis. The histopathological diagnosis was consistent with the ultimate diagnosis in 108 (85.0%, 108/127) cases of CT-guided biopsy and in 135 (75.8%, 135/178) cases of C-arm guided biopsy and there was a significant difference. The accuracy of diagnosis based on biopsies varied with different diseases, including primary benign or malignant tumors, metastatic tumors, inflammatory lesions and fractures. A second biopsy or further examinations were required for patients with negative result obtained in the initial biopsy. The complication rate was 3.1% (4/127) in CT-guided biopsy and 7.3% (13/178) in C-arm guided biopsy. In conclusion, CT-guided percutaneous biopsy is an accurate and safe technique for biopsy of the vertebral lesions.