Resveratrol inhibits inflammation after spinal cord injury via SIRT-1/NF-κB signaling pathway.

Spinal cord injury (SCI) can cause severe trauma to the central nervous system. Resveratrol has been widely studied for several of its medicinal properties, including anti-inflammatory, anti-aging and anti-oxidative effects. The regulation of SIRT-1 is thought to be related to the effects of resveratrol. As a downstream component of SIRT-1, NF-κB is one of the important signaling pathways that regulate the inflammatory response. Herein, we explored how treatment with resveratrol promoted recovery of motor function after SCI by activating the SIRT-1/NF-κB signaling pathway and inhibiting inflammation in rat models. Recovery of hind limb function was observed using the Basso, Beattie, and Bresnahan locomotor rating scale at different time points after SCI. Western blot analysis, immunofluorescence, Nissl staining and HE staining were utilized to observe the morphological characteristics of spinal cord tissue, as well as the expression of SIRT-1, NF-κB, TNF-α, IL-1ß, IL-6 and brain-derived neurotrophic factor. Resveratrol treatment promoted motor function recovery, increased the expression of brain-derived neurotrophic factor, and reduced loss of motor neurons and lesion size among rats after SCI. Meanwhile, inflammatory response was inhibited as the SIRT-1/NF-κB signaling pathway was modulated. These results suggest that resveratrol can help achieve neuroprotective effect by inhibiting inflammation, regulated by the SIRT-1/NF-κB signaling pathway.

Accurate simulation of the herniated cervical intervertebral disc using controllable expansion: a finite element study.

Expansions were carried out in finite element (FE) models of disc hernia including symmetric (median, lateral, paramedian) and asymmetric types. In all models, lubricous disk bulging that applied a linear compression to the anterior part of the cord was observed at the posterior surfaces of the expansion zone, respectively. The shape and position of protrusions varyed with the temperature, magnitude, and location of expanding elements. The geometric deformation and stress distribution of the spinal cord increased as the extent of compression grew. This method is in possession of enormous potential in promoting further individualized research of cervical spondylotic myelopathy.

Role of Smad3 inhibitor and the pyroptosis pathway in spinal cord injury.

The aim of the present study was to investigate the role of Smad3 inhibitors and the pyroptosis pathway in spinal cord injury, and to determine the underlying mechanism. The pyroptosis signaling pathway may be involved in spinal cord injury during the recovery period. Smad3 inhibitor may serve a role in alleviating spinal cord injury by reducing the pyroptosis of neurons, which is induced by caspase-1, absent in melanoma-2 or NOD-like receptors protein-1 during the recovery period of spinal cord injury. In the present study, spinal cord injury was alleviated by caspase-1 and Smad3 inhibitors. Therefore, a Smad3 inhibitor could relieve spinal cord injury in mice by directly downregulating caspase-1 and reducing neuron pyroptosis following spinal cord injury during the recovery period.

Fingolimod inhibits proliferation and epithelial-mesenchymal transition in sacral chordoma by inactivating IL-6/STAT3 signalling.

PURPOSE: To explore the sensitivity of the immunosuppressive agent fingolimod (FTY720) in chordoma and determine whether it can serve as an appropriate alternate treatment for unresectable tumours in patients after incomplete surgery. METHODS: Cell viability assays, colony formation assays and EdU assays were performed to evaluate the sensitivity of chordoma cell lines to FTY720. Transwell invasion assays, wound healing assays, flow cytometry, cell cycle analysis, immunofluorescence analysis, Western blotting analysis and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate cell invasion, epithelial-mesenchymal transition (EMT) and activation of related pathways after treatment with FTY720. The effect of FTY720 was also evaluated in vivo in a xenograft model. RESULTS: We found that FTY720 inhibited the proliferation, invasion and metastasis of sacral chordoma cells (P < 0.01). FTY720 also inhibited the proliferation of tumour cells in a xenograft model using sacral chordoma cell lines (P < 0.01). The mechanism was related to the EMT and apoptosis of chordoma cells and inactivation of IL-6/STAT3 signalling in vitro and in vivo. CONCLUSIONS: Our findings indicate that FTY720 may be an effective therapeutic agent against chordoma. These findings suggest that FTY720 is a novel agent that can treat locally advanced and metastatic chordoma.

Knockdown of macrophage migration inhibitory factor (MIF), a novel target to protect neurons from parthanatos induced by simulated post-spinal cord injury oxidative stress.

Parthanatos is a form of regulated cell death (RCD) that is closely linked to DNA damage, which is a common consequence of oxidative stress due to central nervous trauma, such as spinal cord injury (SCI). The mechanism by which apoptosis-inducing factor (AIF) mediates DNA strand breaks in parthanatos was not clear until the discovery of the nuclease function of MIF. A previous study suggested that observed results may not be reliable if the oxidative stress induced in cells observed under experimental pathological conditions does not accurately replicate the specific pathologies being studied. According to an earlier direct measurement of extracellular oxidative stress in a rat SCI model, post-SCI oxidative stress was approximately the same as exposure to 150 µM H2O2. However, this concentration has been reported as sublethal oxidative stress in other cell types related to senescence, apoptosis, and parthanatos. Using sublethal H2O2 concentrations to induce oxidative stress is equivocal. Also, different cell types have diverse tolerances and responses to oxidative stress, and, therefore, exposure to H2O2. To avoid these limitations, the present study explored the mechanism of neuronal death under this simulated post-SCI oxidative stress and determined the effects of MIF knockdown in parthanatos associated with SCI. Immunofluorescence and flow cytometry were used to reveal typical characteristics of parthanatos that were blocked by PARP-1 inhibitors but not caspase inhibitors. In addition to classic features like PARP-1 and caspase-3 cleavage that were absent, we determined that parthanatos instead of apoptosis played a major role in the cell death caused by oxidative stress following SCI. Flow cytometry analysis of cells transfected by adenovirus with MIF-shRNA then exposed to H2O2 showed a significant decrease in cell death for MIF knockdown cells, even after AIF nuclear translocation. The comet assay also displayed significantly fewer DNA strand breaks after MIF knockdown. This is the first study has verified that MIF knockdown enables to protect neurons from parthanatos under a simulated in vivo oxidative stress following SCI. It suggests that MIF knockdown is a promising therapy to rescue neurons suffering from oxidative stress-induced SCI pathology.

Neuregulin­1 impacting bone marrow mesenchymal stem cell migration is conducive to functional recovery following spinal cord injury.

The present study was designed to investigate the effect of neuregulin­1 (NRG1) on the migration of rat bone marrow mesenchymal stem cells (BMSCs) and evaluate the role of NRG1 in the functional recovery following spinal cord injury (SCI). Firstly, the effect of NRG1 on the mRNA expression of Snail in the BMSCs was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis; secondly, the BMSCs were transfected with a Snail­overexpression plasmid (pBabe­puro­Snail) and the expression levels of Snail and matrix metalloptoreinase­2 (MMP­2) were detected by RT­qPCR and western blot analyses; thirdly, the cell proliferation and migration of BMSCs modified with pBabe­puro­Snail were detected by methyl thiazolyl tetrazolium and migration assays, respectively; finally, functional recovery of SCI was assessed using the Basso, Beattie, and Bresnahan rating scales. The results showed that NRG1 concentration­dependently promoted the expression of Snail with a peak at 40 ng/ml and 48 h; NRG1 enhanced the promoting effect of Snail on the expression of MMP­2; the overexpression of Snail did not enhance the cell growth of the BMSCs. The NRG1­modified BMSCs promoted the functional recovery of SCI. These results suggested that NRG1 significantly promoted the expression of MMP­2 by upregulating the expression of Snail, and enhanced cell migration of the BMSCs conducive to the functional recovery of SCI.

miR­329­3p regulates neural stem cell proliferation by targeting E2F1.

Neural stem cells (NSCs) are a class of self­renewing and undifferentiated progenitor cells that retain the ability to differentiate to neurons, astrocytes and oligodendrocytes. MicroRNAs (miRNAs) are small noncoding RNAs that serve crucial roles in regulating a number of cellular processes, including cell proliferation, differentiation and apoptosis. Our previous GeneChip data indicated that the expression of miR­329­3p was increased in neurons compared with NSCs. However, whether miRNA­329­3p participates in regulating NSC function remains to be elucidated. In the present study, it was identified that the expression of miR­329­3p was upregulated in NSCs during neuronal differentiation, whereas expression of transcription factor E2F1 (E2F1), a putative target gene of miR­329­3p, was downregulated. Using luciferase reporter assays, it was confirmed that miR­329­3p regulated E2F1 expression. As differentiation has been demonstrated to limit the proliferative capacity of NSCs, the effects of miR­329­3p and E2F1 modulation on NSC proliferation were examined. Forced overexpression of miR­329­3p or RNA­mediated silencing of E2F1 inhibited NSC proliferation, and overexpression of miR­329­3p also inhibited E2F1 expression. Notably, ectopic expression of E2F1 reversed the inhibition of NSC proliferation induced by miR­329­3p overexpression. These results indicated that miR­329­3p may serve crucial roles in regulating the proliferation of NSCs, at least in part via inhibition of E2F1 expression. These data improve the understanding of the microRNA­mRNA regulatory network that controls NSC proliferation.

Overexpression of HIPK2 attenuates spinal cord injury in rats by modulating apoptosis, oxidative stress, and inflammation.

HIPK2 is considered to be a tumor suppressor. It also has been implicated in several functions such as apoptosis and inflammation that are linked to spinal cord injury (SCI). However, whether HIPK2 ameliorates the neurological pain of SCI remains unclear. Here, we investigated the effects of HIPK2 on neurological function, oxidative stress, levels of inflammatory cytokines and expression of Bcl-2/Bax in an SCI model. Firstly, we evaluated the therapeutic effects of HIPK2 on neurological pain in the SCI rat using the Basso, Beattie and Bresnahan scores and H & E staining. Overexpression of HIPK2 significantly elevated the levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), and reduced the mRNA expression of Nogo-A and RhoA in SCI rats. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays showed that overexpression of HIPK2 significantly reduced the number of apoptotic cells. Overexpression of HIPK2 also decreased expression of Bax and Caspase-3 and elevated expression of Bcl-2 in the SCI model, indicating that HIPK2 exhibited its protective activity by inhibiting SCI-induced apoptosis. Then, we measured the serum concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). We also determined the mRNA and protein levels of nuclear factor-κB p65 unit, tumor necrosis factor-α (TNF-α), and interleukin (IL)-1ß. HIPK2 overexpression reduced oxidative stress and the levels of inflammatory cytokines compared with SCI control animals. Additionally, acetylation of HIPK2 was reduced in SCI rats. Overexpression of HIPK2 could enhance autophagy by elevating the expression of Beclin-1 and LC3-II while autophagy is regarded as a beneficial regulator to improve spinal cord injury. Together, overexpression of HIPK2 improved contusive SCI induced pain by modulating oxidative stress, Bcl­2 and Bax signaling, and inflammation, and also regulating autophagy.

MEG3 inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells.

Osteosarcoma is known as a malignant tumour with a high mortality rate in orthopaedic settings; however, the factors associated with its degree of malignancy and the biological response remains to be elucidated. Although the essential role of the long noncoding RNA (lncRNA) maternally expressed 3 (MEG3) has been recently reported, its biological functions and regulatory mechanism in osteosarcoma cells have not yet been reported. In the present study, reverse transcription-quantitative polymerase chain reaction analysis revealed that the expression of MEG3 in MG63 cells was lower compared with in hFOB1.19 cells. Furthermore, it was observed that overexpressing MEG3 in MG63 cells resulted in a decline in the proliferation and invasion, and a marked increase in apoptosis. Additionally, western blotting was used to detect the changes in expression of p53 and MDM2 proto-oncogene, which may be regulated by MEG3, and proteins that associated with cell proliferation, invasion and apoptosis. It was demonstrated that the upregulation of MEG3 significantly increased the transactivation of p53 and induced downstream changes in protein expression. In conclusion, these experiments have demonstrated that MEG3 serves an essential regulatory role in the biological processes of human osteosarcoma cells, and imply that MEG3 may be a marker for predicting the occurrence and development of osteosarcoma.

MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway.

MicroRNA-21 (miR-21) contributes to anti-apoptosis in bone marrow mesenchymal stem cells (BMSC), but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible effect of miR-21 on regulating BMSCs directional migration and the expression of MMP-2/MMP-9 in BMSCs in vitro. BMSCs were successfully infected with miR-21-up lentivirus. Cell migration using Transwell assay indicated that upregulated expression of miR-21 could significantly promote BMSCs migration. Western blot analysis indicated that miR-21 significantly upregulated the expression of MMP-2 and MMP-9, which were related to metastasis-associated genes. GM6001, the specific MMPs inhibitor, abrogated the upregulated expression of MMP-2/MMP-9 and abolished the positive effect of miR-21 on promoting BMSCs migration. Meanwhile, miR-21 significantly enhanced Akt phosphorylation, as measured by Western blot analysis. LY294002, an inhibitor of Akt activation, abrogated the phosphorylation of Akt and abolished the positive effect of miR-21 on promoting BMSCs migration and upregulating MMP-2/MMP-9 expression. These results suggest that miR-21 contributes to BMSCs migration by upregulating MMP-2/MMP-9, potentially via the PI3K/Akt pathway.

A Meta-Analysis on the Clinical Significance of Redundant Nerve Roots in Symptomatic Lumbar Spinal Stenosis.

OBJECTIVE: Symptomatic lumbar spinal stenosis (LSS), which leads to severe socioeconomic consequences and places a heavy burden on global healthcare system, is a relatively frequent spine disorder. Redundant nerve roots (RNRs) are a relatively common finding in which slender, serpiginous, or tortuous nerve roots are found in the subarachnoid space of the lumbar spine. Previous studies that evaluated the prognostic assessment of RNRs in patients with symptomatic LSS are composed of doubtful results. Therefore, the clinical significance of RNRs in symptomatic LSS is still uncertain. The aim of this meta-analysis is a systematic assessment of the clinical significance of RNR syndrome in symptomatic LSS. METHODS: This study used a highly sensitive search strategy to identify all published studies in multiple databases up to January 1, 2017. All identified trials were systematically evaluated using specific inclusion and exclusion criteria. Cochrane methodology was also applied to the results of this study. RESULTS: This study identified 4 relevant studies involving 297 patients. Compared with a non-RNR group, the RNR group results included worse clinical outcomes that were assessed using the Japanese Orthopedic Association scores after surgery (weighted mean difference [WMD], -0.78; 95% confidence interval [CI], -1.26 to -0.29; P = 0.002; I2 = 0%), for recovery rate (WMD, -9.87; 95% CI, -15.07 to -4.67; P = 0.0002; I2 = 0%), and for older age (WMD, 2.51; 95% CI, 0.45-4.57; P = 0.02; I2 = 43%). CONCLUSIONS: RNR is an entity in association with symptomatic LSS, which may be viewed as a potentially powerful prognostic indicator of worse postoperative functional recovery for symptomatic LSS.

Effect of problem and scripting-based learning on spine surgical trainees' learning outcomes.

PURPOSE: To assess the impact of problem and scripting-based learning (PSBL) on spine surgical trainees' learning outcomes. METHODS: 30 spine surgery postgraduate-year-1 residents (PGY-1s) from the First Hospital of China Medical University were randomly divided into two groups. The first group studied spine surgical skills and developed individual judgment under a conventional didactic model, whereas the PSBL group used PBL and Scripted model. A feedback questionnaire and the satisfaction of residents were evaluated by the first assistant surgeon immediately following each procedure. At the end of the study, residents filled out questionnaires focused on identifying the strengths of each teaching method and took a multiple-choice theoretical examination. The results were analyzed by t tests. RESULTS: Significant difference was found between the two groups in total mean score of preparedness and performance feedback statement (P = 0.01) and the questionnaire by PGY-1's opinion on the effectiveness of the two teaching methods (P = 0.004). Compared with the non-PSBL group, the PSBL group had significantly higher mean score of pre-operative preparedness (P = 0.01), but there was no significant difference between the two groups in theoretical examination, intra-operative performance, and overall satisfaction with the PGY-1s. The residents found that PSBL could develop their judgment (P = 0.03) and provide greater satisfaction (P = 0.02), and would like to repeat the experience (P = 0.03). CONCLUSIONS: The PSBL method can activate spine residents' prior knowledge and building on existing cognitive frameworks, which is an important tool for improving pre-operative preparedness. We believe that PSBL is an important first step in training spine residents to become confident and safe spine surgeons.

Effects of Reactive Oxygen Species on Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND The low differentiation rates for transplanted stem cells are challenging problems in spinal cord injury (SCI) treatment. Studies have demonstrated that the inhibition of the Notch1 pathway in bone marrow mesenchymal stem cells (BMSCs) contributed to the differentiation of these cells. Research findings that certain antioxidants induce BMSCs to differentiate into neuronal cells suggest that BMSC differentiation is related to the level of reactive oxygen species (ROS) in cells. This study aimed to define the effect of ROS on the differentiation of BMSCs. MATERIAL AND METHODS In this study, after BMSCs were induced with the antioxidant ß-mercaptoethanol (ß-ME), related proteins were analyzed by Western blotting and immunofluorescence. In order to find the role of ROS in the differentiation, DCFH-DA was used to detect ROS levels in antioxidant-treated BMSCs, H2O2-treated BMSCs, and normal BMSCs, and the expression levels of Notch1 and its downstream transcriptional suppressor Hes1 were analyzed. RESULTS Induced with ß-ME, Western blotting and immunofluorescence revealed gradual increases in the expression of Nestin (a neural stem cell-specific protein) and neuron-specific enolase (NSE) but decreases in Notch1 expression. The expression levels of Notch1 and Hes1 were positively correlated with changes in ROS level. CONCLUSIONS These data suggest that the antioxidant-induced differentiation of BMSCs into neurons may be related to ROS-based regulation of the Notch1 signalling pathway.

MicroRNA-210 promotes sensory axon regeneration of adult mice in vivo and in vitro.

Axon regeneration as a critical step in nerve repairing and remodeling after peripheral nerve injury relies on regulation of gene expression. MicroRNAs are emerging to be important epigenetic regulators of gene expression to control axon regeneration. Here we used a novel in vivo electroporation approach to transfect microRNA-210 (miR-210) or siRNAs to adult mice dorsal root ganglion (DRG) neurons, measured the axon length 3days after sciatic nerve crush or dissociated DRG cultures in vitro to detect the effect of miR-210 in sensory axon regeneration. Importantly, we found that miR-210 overexpression could promote sensory axon regeneration and inhibit apoptsosis by ephrin-A3 (EFNA3). In addition, inhibition of endogenous miR-210 in DRG neurons impaired axon regeneration in vitro and in vivo, the regulatory effect of miR-210 was mediated by increased expression of EFNA3 because downregulation of EFNA3 fully rescued axon regeneration. We thus demonstrate that miR-210 is a new physiological regulator of sensory axon regeneration, and EFNA3 may be the functional target of miR-210. We conclude that miR-210 may play an important role in sensory axon regeneration.

Role and mechanism of microRNA-21 in H2O2-induced apoptosis in bone marrow mesenchymal stem cells.

microRNA-21 (miR-21) contributes to anti-apoptosis, proliferation and migration in many cells, but its role in inhibiting apoptosis in bone marrow mesenchymal stem cells (BMSC) remains unclear. The aim of this study was to determine the role of miR-21 in H2O2-induced BMSC apoptosis. We used quantitative real time-polymerase chain reaction (RT-PCR) to demonstrate the level of miR-21 after treatment of BMSC with H2O2. BMSC apoptosis was induced by different concentrations of H2O2 and was decreased in miR-21-upregulated cells. The expression of PTEN, a functional target gene of miR-21 in BMSC, was regulated by miR-21. The RT-PCR results indicated that miR-21 was significantly up-regulated, and western blot analysis indicated that Bcl-2 was up-regulated, whereas the apoptosis-related genes caspase 3/9 and Bax were down-regulated in miR-21-up-regulated cells. The miR-21-up-regulated cells had significantly enhanced Akt phosphorylation, as measured by western blot analysis. LY294002, an inhibitor of Akt activation, abolished the protective effects of miR-21-up-regulated cells. These results suggest that miR-21 contributes to inhibition of apoptosis in BMSC by down-regulating PTEN, potentially via the PI3K/Akt pathway.

MicroRNA-21 promotes proliferation, invasion and suppresses apoptosis in human osteosarcoma line MG63 through PTEN/Akt pathway.

Osteosarcoma, which accounts for 5 % of pediatric tumor, remains the major cause of death among orthopedic malignancies. However, the factors associated with its malignant biological behavior are still poorly understood. MicroRNAs are a class of small noncoding RNAs, which have been considered to associate with malignant progression including cell differentiation, proliferation, apoptosis, invasion, and distant metastasis. In our research, we found that microRNA-21 (miR-21) was significantly overexpressed in human osteosarcoma cell line MG63 compared to human fetal osteoblastic cell line hFOB1.19 by using quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, miR-21 overexpression in MG63 caused a significant raise in cell proliferation and invasion and a significant reduction in cell apoptosis. However, miR-21 underexpression in MG63 caused an opposite result. Western blotting displayed that proteins related with proliferation, apoptosis, and invasion were significantly changed in different groups, respectively. Furthermore, we demonstrated that PTEN may be a potential target of miR-21 in MG63 cells and miR-21 could activate PI3K/Akt pathway by suppressing PTEN expression. In summary, our findings suggested that miR-21 played an active role in osteosarcoma and it could predict the occurrence and development of osteosarcoma.

A meta-analysis of endoscopic discectomy versus open discectomy for symptomatic lumbar disk herniation.

PURPOSE: The purpose of this study was to systematically compare the effectiveness and safety of endoscopic discectomy (ED) with open discectomy (OD) for the treatment of symptomatic lumbar disc herniation (LDH). METHODS: A highly sensitive search strategy was used to identify all published randomized controlled trials up to August 2014. A criteria list taken from Koes et al. was used to evaluate the risk of bias of the included studies. The five questions that were recommended by the Cochrane Back Review Group were used to evaluate the clinical relevance. Cochrane methodology was used for the results of this meta-analysis. RESULTS: Nine relevant RCTs involving 1,092 patients were identified. Compared with OD, ED results in slightly better clinical outcomes which were evaluated by the Macnab criteria without clinical significance (ED group: 95.76 %; OD group: 80 %; OR: 3.72, 95 % CI: [0.76, 18.14], P = 0.10), a significantly greater patient satisfaction rate (ED group: 93.21 %; OD group: 86.57 %; OR: 2.19; 95 % CI: [1.09, 4.40]; P = 0.03), lower intraoperative blood loss volume (WMD: -123.71, 95 % CI: [-173.47, -73.95], P < 0.00001), and shorter length of hospital stay (WMD: -Table 2144.45, 95 % CI: [-239.54, -49.37], P = 0.003). CONCLUSIONS: From the existing outcomes, ED surgery could be viewed as a sufficient and safe supplementation and alternative to standard open discectomy. The cost-effectiveness analyses still remain unproved from the existing data. More independent high-quality RCTs using sufficiently large sample sizes with cost-effectiveness analyses are needed.

Osteoblastic NF-κB pathway is involved in 1α, 25(OH)2D3-induced osteoclast-like cells formation in vitro.

1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3) acts on the osteoblasts to enhance the expressions of receptor activator of nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and induce the formation of osteoclasts. However, the mechanism in osteoblasts by which 1α, 25(OH)2D3 promotes osteoclastogenesis has not yet been completely understood. This study aimed to select the first generation of murine osteoblasts to explore the underlying mechanism of 1α, 25(OH)2D3-induced osteoclastic formation from bone marrow mononuclear cells (BMMNCs). We discovered the activation of osteoblastic NF-κB pathway under 10(-8) mol/L 1α, 25(OH)2D3 treatment, as evidenced by the transfer of NF-κB p65 from cytoplasm to nuclei. Then, the NF-κB p65-siRNA was designed, constructed, and transfected into osteoblastic cells. Immunofluorescence assay confirmed the successfully silenced NF-κB p65 gene in osteoblasts. In the co-culture system of osteoblasts and BMMNCs with 1α, 25(OH)2D3 added, the multinucleated osteoclast-like cells containing 2-3 nuclei were observed in BMMNCs co-cultured with non-transfection osteoblasts, conversely, silencing osteoblastic NF-κB p65 resulted in failed differentiation of BMMNCs along with substantial vacuolar degeneration in cytoplasm. In addition, the expressions of RANKL and M-CSF were notably decreased in NF-κB p65-silenced osteoblasts. Taken together, our data indicated that osteoblastic NF-κB pathway was involved in 1α, 25(OH)2D3-induced osteoclast-like cells formation from BMMNCs through regulating the expression of RANKL and M-CSF. Therefore, our findings further identified the mechanism of 1α, 25(OH)2D3-induced osteoclastogenesis on the basis of prior studies.

Lithium enhanced cell proliferation and differentiation of mesenchymal stem cells to neural cells in rat spinal cord.

Lithium has been shown to inhibit apoptosis of neural progenitor cells (NPCs) and promote differentiation of NPCs. However, there was rare data to discuss the effects of lithium on neural differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential promotion of lithium to MSC proliferation and neural differentiation in vitro and after transplanted into the ventral horn of rat spinal cord in vivo. We found that lithium possesses the ability to promote proliferation of GFP-MSCs in a dose dependent manner as verified by growth curve and bromodeoxyuridine (BrdU) incorporation assays; While in neural induction medium, lithium (0.1 mM) promotes neural differentiation of GFP-MSCs as verified by immunostaining and quantitative analysis. After transplantation of GFP-MSCs into the rat spinal cord, lithium treatment enhanced cell survival and neural differentiation after transplantation as verified by immunohistochemistry. These data suggested that lithium could be a potential drug to augment the therapeutic efficiency of MSCs transplantation therapy in central nervous system (CNS) disorders.

[Association between the expression of Aggrecan and the distribution of Aggrecan gene variable number of tandem repeats with symptomatic lumbar disc herniation].

OBJECTIVE: To study the expression of Aggrecan and the relationship between the variable number of tandem repeats (VNTR) of Aggrecan and lumbar disc herniation (LDH). METHODS: The disease group comprised of 74 patients already diagnosed with symptomatic LDH. The control group consisted of 15 patients restricted to spinal trauma and 113 healthy blood donors without symptoms of LDH who were not diagnosed with LDH. Disc tissue samples were obtained from surgical operations and blood samples were donated from all participants. The Aggrecan expression in isolated tissues was assessed by western blot using specific antibodies. The Aggrecan gene VNTR region was analyzed by PCR. RESULTS: The Aggrecan expression positive rate of control group was statistically and significantly higher (control group:86.67%, disease group:13.51%;χ(2) = 34.83, P < 0.05) than that of the disease group. Moreover, there was a statistically significant higher frequency of Allele 25 or Allele 21 in disease group compared to controls (A25disease group = 22.97%, A25control group = 12.11%, χ(2)A25 = 8.20, PA25 = 0.004; A21disease group = 6.76%, A21control group = 0.39%, χ(2)A21 = 14.35, PA21 = 0.000). Compared to the participants with 2 Alleles>25 repeats, subjects with 1 or 2 Alleles ≤ 25 repeats statistically and significantly over represented the disease group without the expression of Aggrecan (χ(2) = 5.69, P = 0.017). CONCLUSIONS: The findings suggest a relationship between Aggrecan and symptomatic LDH, where symptomatic LDH has a tendency of allele 21 and allele 25 repeats.In addition, an association between the distribution of Aggrecan gene VNTR polymorphism and the expression of Aggrecan is observed in symptomatic LDH.