Zhenming Hu

SIRT1 alleviates senescence of degenerative human intervertebral disc cartilage endo-plate cells via the p53/p21 pathway

Cartilage end plates (CEP) degeneration plays an integral role in intervertebral disc (IVD) degeneration resulting from nutrient diffusion disorders. Although cell senescence resulting from oxidative stress is known to contribute to degeneration, no studies concerning the role of senescence in CEP degeneration have been conducted. SIRT1 is a longevity gene that plays a pivotal role in many cellular functions, including cell senescence. Therefore, the aim of this study was to investigate whether senescence is more prominent in human degenerative CEP and whether SIRT1-regulated CEP cells senescence in degenerative IVD as well as identify the signaling pathways that control that cell fate decision. In this study, the cell senescence phenotype was found to be more prominent in the CEP cells obtained from disc degenerative disease (DDD) patients than in the CEP cells obtained from age-matched lumbar vertebral fractures (LVF) patients. In addition, the results indicated that p53/p21 pathway plays an important role in the senescence of CEP cells in vivo and vitro. Furthermore, SIRT1 was found to be capable of alleviating the oxidative stress-induced senescence of CEP cells in humans via p53/p21 pathway. Thus, the information presented in this study could be used to further investigate the underlying mechanisms of CEP.

IL-1β induces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells

IL-1β has been reported highly expressed in degenerative intervertebral disc, and our previous study indicated IL-1β facilitates apoptosis of human degenerative nucleus pulposus (NP) cell. However, the underlying molecular mechanism remains unclear. We here demonstrate that IL-1β played a significantly pro-apoptotic effect under serum deprivation. IL-1β decreased Bcl-2/Bax ratio and enhanced cytochrome C released from mitochondria to cytosol, which proved mitochondria-meidated apoptosis was induced. Subsequently, mitochondria damage was detected under IL-1β stimualtion. In addition, IL-1β-mediated injuried mitochondria contributes to activate autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine showed the potential in further elevating the apoptosis rate induced by IL-1β in NP cells. Our results indicated that the mitochondrial pathway was involved in IL-1β-induced apoptosis of NP cells. Meanwhile, the damaged mitochondria-induced autophagy played a protective role against apoptosis, suggesting a postive feedback mechanism under inflammatory stress.

Low Intensity Pulsed Ultrasound Promotes the Extracellular Matrix Synthesis of Degenerative Human Nucleus Pulposus Cells Through FAK/PI3K/Akt Pathway.

Study Design. In vitro experimental study Objective. To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on the extracellular matrix (ECM) synthesis of degenerative human nucleus pulposus cells and explore the molecular mechanism. Summary of Background Data. LIPUS has been used successfully for bone fracture healing and been proved to be effective in stimulating ECM metabolism in animal intervertebral disc cells. However, whether LIPUS also exerts an anabolic effect on degenerative human nucleus pulposus cells and the possible molecular mechanism is still unclear. Methods. The degenerative human nucleus pulposus cells were cultured in calcium alginate beads. In the LIPUS group, cells were exposed to an average temporal intensity of 30 mW/cm2 and a frequency of 1.5 MHz of LIPUS 20 minutes daily for 1 week. The control group was cultured in the same way but without LIPUS stimulation. The LY294002 group was stimulated by LIPUS and treated with LY294002 simultaneously. The expression of aggrecan, collagen-II, Sox9, tissue inhibitor of metalloproteinase-,1and matrix metalloproteinase-3 were evaluated by Enzyme-Linked Immunosorbent Assay, Western blot or RT-PCR. Expression of signaling proteins involved in FAK/PI3K/Akt pathway was studied by Western blot analysis. Results. LIPUS significantly upregulated expression of aggrecan, collagen-II, Sox9, and tissue inhibitor of metalloproteinase-1 compared with control group, but inhibited secretion of matrix metalloproteinase-3. The study further demonstrated that the upregulation of aggrecan, collagen-II, and Sox9 was related to the activation of focal adhesion kinase (FAK)//PI3K/Akt pathway caused by LIPUS. Moreover, inhibition of PI3K/Akt significantly suppressed the special biological effect activated by LIPUS. Conclusion. LIPUS promotes the ECM synthesis of degenerative human nucleus pulposus cells through activation of FAK/PI3K/Akt pathway. Level of Evidence: N/A

SDF‑1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF‑κB pathway.

Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF-1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF-1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor-κB (NF-κB) signaling pathway was investigated using CXCR4-siRNA and NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF-1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF-1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post-transfection with CXCR4 siRNA compared with SDF-1 stimulation alone. Furthermore, SDF-1 treatment increased the level of phosphorylated NF-κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF-1. Taken together, SDF-1-mediated apoptosis was suppressed by NF-κB inhibition using PDTC. In conclusion, the SDF-1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF-κB pathway, thus suggesting that SDF-1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases.

Percutaneous Fibrin Gel Injection under C-Arm Fluoroscopy Guidance: A New Minimally Invasive Choice for Symptomatic Sacral Perineural Cysts

BACKGROUND Symptomatic sacral perineural cysts are a common cause of chronic pain. Surgery is one choice for symptom relief but has a high risk of cyst recurrence and complications. As a simple and safe method to manage symptomatic sacral perineural cysts, C-arm fluoroscopy-guided fibrin gel injection may represent a new minimally invasive alternative. To evaluate the efficacy of this new method, we conducted a retrospective study of 42 patients. METHODS AND FINDINGS From June 2009 to August 2012, a total of 42 patients with symptomatic sacral perineural cysts underwent C-arm fluoroscopy-guided percutaneous fibrin gel injection therapy. Patient outcomes in terms of improvements in pain and neurologic function were evaluated during a follow-up period of 13-39 months. The preoperative and postoperative pain severity were assessed according to a 10-cm visual analog pain scale, and imaging changes were evaluated by magnetic resonance imaging. We also assessed postoperative complications. Most patients experienced benefit from the procedure: twenty-five patients (59.5%) reported excellent recovery, eleven (26.2%) reported good recovery, three (7.1%) reported fair recovery, and three (7.1%) reported poor recovery. The overall effectiveness rate (excellent and good recoveries) was 85.7%. No serious postoperative complications were observed. CONCLUSION Percutaneous fibrin gel injection under C-arm fluoroscopy guidance could be a simple, safe and effective treatment option for symptomatic sacral perineural cysts.

Significant Associations Between the A163G and G1181C Polymorphisms of the Osteoprotegerin Gene and Risk of Osteoporosis, Especially in Postmenopausal Women: A Meta-Analysis

OBJECTIVE Whereas some studies have reported that the osteoprotegerin (OPG) gene is associated with osteoporosis risk in some studies, their results have proved inconclusive. We performed a meta-analysis of studies on the associations between OPG A163G and G1181C polymorphisms and the risk of osteoporosis. METHODS A literature search in PubMed, Embase, Web of Science, Cochrane Library, and China Biological Medicine (CBM) databases was conducted to identify all eligible case-control studies published before August 15th, 2013. Pooled odds ratios with their corresponding 95% confidence intervals were used to evaluate the strength of the association under either a fixed- or random-effect model according to the heterogeneity test. RESULTS Ten case-control studies were included with a total of 1673 osteoporosis cases and 1554 healthy controls in this meta-analysis. For the OPG A163G polymorphism, the combined results showed that the G allele of the A163G polymorphism may be associated with an increased risk of osteoporosis. Stratified analyses showed that the magnitude of the effect was similar among the Caucasian and postmenopausal women subgroups. Unlike the A163G polymorphism, the meta-analysis results revealed that the C allele of the G1181C polymorphism may be associated with a decreased risk of osteoporosis, especially in the Asian and postmenopausal women subgroups. No publication bias was detected for either polymorphism. CONCLUSION Our findings showed that the G allele of the OPG A163G polymorphism may increase osteoporosis risk, whereas the C allele of the G1181C polymorphism may protect individuals from osteoporosis. Both of these effects were observed in postmenopausal women.

Nerve growth factor delivery by ultrasound-mediated nanobubble destruction as a treatment for acute spinal cord injury in rats

Background Spinal cord injuries (SCIs) can cause severe disability or death. Treatment options include surgical intervention, drug therapy, and stem cell transplantation. However, the efficacy of these methods for functional recovery remains unsatisfactory. Purpose This study was conducted to explore the effect of ultrasound (US)-mediated destruction of poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs) expressing nerve growth factor (NGF) (NGF/PLGA NBs) on nerve regeneration in rats following SCI. Materials and methods Adult male Sprague Dawley rats were randomly divided into four treatment groups after Allen hit models of SCI were established. The groups were normal saline (NS) group, NGF and NBs group, NGF and US group, and NGF/PLGA NBs and US group. Histological changes after SCI were observed by hematoxylin and eosin staining. Neuron viability was determined by Nissl staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining was used to examine cell apoptosis. NGF gene and protein expressions were detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Green fluorescent protein expression in the spinal cord was examined using an inverted fluorescence microscope. The recovery of neural function was determined using the Basso, Beattie, and Bresnahan test. Results NGF therapy using US-mediated NGF/PLGA NBs destruction significantly increased NGF expression, attenuated histological injury, decreased neuron loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in rats with SCI. Conclusion US-mediated NGF/PLGA NBs destruction effectively transfects the NGF gene into target tissues and has a significant effect on the injured spinal cord. The combination of US irradiation and gene therapy through NGF/PLGA NBs holds great promise for the future of nanomedicine and the development of noninvasive treatment options for SCI and other diseases.

Cytotoxicity of polymethyl methacrylate cement on primary cultured metastatic spinal cells

Polymethyl methacrylate (PMMA) bone cement has been commonly used for percutaneous injection into collapsed vertebral bodies due to malignant tumor. The purpose of this study was to investigate the possible mechanisms of PMMA’s cytotoxcity on primary cultured spinal metastastic cells (SMCs) in vitro. PMMA specimens were prepared in standard discs made of dough and polymerization stages, and the eluates were prepared following the ISO standard. Primary SMCs were obtained and isolated from 7 patients with spinal metastatic tumors undergoing vertebroplasty. Primary cultured SMCs were treated with PMMA specimens of different stages for 24 h, or co-cultured with extracted medium for successive 3 days. The temperatures in two locations from cement discs were recorded by K-type thermocouples. Furthermore, cell proliferation, apoptosis and cycles were determined by MTT and flow cytometry, respectively. The modulation of apoptotic proteins (bcl-2, bax, caspase-3, caspase-8, Fas and PARP) and cell cycle proteins (cyclin D1, P21 and P27) were analyzed by western blot and real time PCR. The results of this study demonstrated that PMMA treatment was able to suppress SMCs proliferation, induce cell apoptosis and inhibit cell cycle arrest when compared to the control group in vitro (P<0.05). Simultaneously, the temperature recorded at the periphery of the PMMA specimen was not high enough to casue thermal injury. Furthermore, molecular markers of apoptosis including Fas, caspase-3 and caspase-9 activated, and Bcl-2/Bax dysregulated in the SMCs with PMMA stimulation. In addition, PMMA treatment showed decreased expression of cyclin D1 that induces cell cycle and increased epxression of inhibitory protein P21, with no significant difference of P27 expression. In summary, the present study confirms that PMMA cement can compromise the vitality and apoptosis of SMCs. This cytotoxic effect may be regulated by the biomarkers Fas, Bcl-2, Bax, caspase-3, caspase-9, cyclin D1 and P21, but not on account of thermal damage.

Dickkopf-1 is involved in BMP9-induced osteoblast differentiation of C3H10T1/2 mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering. [BMB Reports 2016; 49(3): 179-184]

Resveratrol delivery by ultrasound-mediated nanobubbles targeting nucleus pulposus cells

AIM To improve nucleus pulposus cell-targeted therapy for intervertebral disc degeneration (IDD) by fabricating a novel kind of ultrasound (US)-mediated poly(lactic-co-glycolic acid) nanobubbles (NBs) as a means of targeted drug delivery. MATERIALS & METHODS The resveratrol (RES)-embedded NBs were synthesized using a double-emulsion method. The active NP cell-targeting biomarker CDH2 antibody (AbCDH2) was further conjugated to the NBs using a carbodiimide method. Then, this RES/AbCDH2 NBs were examined by physical properties, specifc cell-targeting ability, anticatabolism effect in vitro and in vivo. RESULTS RES/AbCDH2 NBs exhibited high RES-loading efficiency, and US triggered accelerated RES release. Furthermore, RES/AbCDH2 NB treatment exhibited excellent anticatabolic ability in vitro; and in an IDD rabbit model, US-mediated RES/AbCDH2 NB injection effectively retarded the degenerative process of the intervertebral disc in vivo. CONCLUSION The combination of US irradiation and drug delivery through RES/AbCDH2 NBs can be considered as a novel treatment option for IDD.