Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor regulate the interaction between astrocytes and Schwann cells at the trigeminal root entry zone.

The trigeminal root entry zone is the zone at which the myelination switches from peripheral Schwann cells to central oligodendrocytes. Its special anatomical and physiological structure renders it susceptible to nerve injury. The etiology of most primary trigeminal neuralgia is closely related to microvascular compression of the trigeminal root entry zone. This study aimed to develop an efficient in vitro model mimicking the glial environment of trigeminal root entry zone as a tool to investigate the effects of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor on the structural and functional integrity of trigeminal root entry zone and modulation of cellular interactions. Primary astrocytes and Schwann cells isolated from trigeminal root entry zone of postnatal rats were inoculated into a two-well silicon culture insert to mimic the trigeminal root entry zone microenvironment and treated with glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor. In monoculture, glial cell line-derived neurotrophic factor promoted the migration of Schwann cells, but it did not have effects on the migration of astrocytes. In the co-culture system, glial cell line-derived neurotrophic factor promoted the bidirectional migration of astrocytes and Schwann cells. Brain-derived neurotrophic factor markedly promoted the activation and migration of astrocytes. However, in the co-culture system, brain-derived neurotrophic factor inhibited the migration of astrocytes and Schwann cells to a certain degree. These findings suggest that glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor are involved in the regulation of the astrocyte-Schwann cell interaction in the co-culture system derived from the trigeminal root entry zone. This system can be used as a cell model to study the mechanism of glial dysregulation associated with trigeminal nerve injury and possible therapeutic interventions.

Blood-nerve barrier disruption and coagulation system activation induced by mechanical compression injury participate in the peripheral sensitization of trigeminal neuralgia.

Introduction: The aim of this study was to investigate the effect and possible mechanisms of the blood-nerve barrier (BNB) and the coagulation-anticoagulation system in modulating the mechanical allodynia in a trigeminal neuralgia (TN) rat model induced by chronic compression of the trigeminal root entry zone (TREZ). Methods: Von Frey filaments were applied to determine the orofacial mechanical allodynia threshold. The BNB permeability was evaluated by Evans blue extravasation test. Immunohistochemical staining and laser confocal microscopy were used to measure the length of the depletion zones of the nodes of Ranvier in the TREZ, the diameter of nerve fibers and the length of the nodal gap. The transcriptional levels of prothrombin and endogenous thrombin inhibitor protease nexin-1 (PN-1) in the TREZ of TN rats were assessed by RT-qPCR. A Western blotting assay was performed to detect the expression of paranodal proteins neurofascin-155 (NF155) and neurofascin-125 (NF125) in the TREZ. The spatiotemporal expression pattern of thrombin activated receptor (i.e. protease activated receptor 1, PAR1) in TREZ were defined by immunostaining and immunoblotting assays. PAR1 receptor inhibitors SCH79797 were administrated to TN rats to analyze the effect of thrombin-PAR1 on orofacial hyperalgesia. Results: A compression injury of a rat's TREZ successfully induced TN-like behavior and was accompanied by the destruction of the permeability of the BNB and the promotion of prothrombin and thrombin inhibitor protease nexin-1 (PN-1) expression. The expression of the paranodal proteins neurofascin-155 (NF155) and neurofascin-125 (NF125) was increased, while the nodal gap length of the nodes of Ranvier was widened and the length of node-depleted zones was shortened. Moreover, the expression of PAR1 within the TREZ was upregulated at an early stage of TN, and administration of the PAR1 antagonist SCH79797 effectively ameliorated orofacial mechanical allodynia. Conclusion: A compression injury of the TREZ increased the permeability of the BNB and induced disturbances in the local coagulation-anticoagulation system, concomitant with the structural changes in the nodes of Ranvier, thrombin-PAR1 may play a critical role in modulating orofacial mechanical hyperalgesia in a TN rat model.

Extracellular ATP and cAMP signaling promote Piezo2-dependent mechanical allodynia after trigeminal nerve compression injury.

Trigeminal neuralgia (TN) is a type of severe paroxysmal neuropathic pain commonly triggered by mild mechanical stimulation in the orofacial area. Piezo2, a mechanically gated ion channel that mediates tactile allodynia in neuropathic pain, can be potentiated by a cyclic adenosine monophosphate (cAMP)-dependent signaling pathway that involves the exchange protein directly activated by cAMP 1 (Epac1). To study whether Piezo2-mediated mechanotransduction contributes to peripheral sensitization in a rat model of TN after trigeminal nerve compression injury, the expression of Piezo2 and activation of cAMP signal-related molecules in the trigeminal ganglion (TG) were detected. Changes in purinergic P2 receptors in the TG were also studied by RNA-seq. The expression of Piezo2, cAMP, and Epac1 in the TG of the TN animals increased after chronic compression of the trigeminal nerve root (CCT) for 21 days, but Piezo2 knockdown by shRNA in the TG attenuated orofacial mechanical allodynia. Purinergic P2 receptors P2X4, P2X7, P2Y1, and P2Y2 were significantly up-regulated after CCT injury. In vitro, Piezo2 expression in TG neurons was significantly increased by exogenous adenosine 5'-triphosphate (ATP) and Ca2+ ionophore ionomycin. ATP pre-treated TG neurons displayed elevated [Ca2+ ]i and faster increase in responding to blockage of Na+ /Ca2+ exchanger by KB-R7943. Furthermore, mechanical stimulation of cultured TG neurons led to sustained elevation in [Ca2+ ]i in ATP pre-treated TG neurons, which is much less in naïve TG neurons, or is significantly reduced by Piezo2 inhibitor GsMTx4. These results indicated a pivotal role of Piezo2 in peripheral mechanical allodynia in the rat CCT model. Extracellular ATP, Ca2+ influx, and the cAMP-to-Epac1 signaling pathway synergistically contribute to the pathogenesis and the persistence of mechanical allodynia.

Flow cytometry analysis of immune and glial cells in a trigeminal neuralgia rat model.

Microvascular compression of the trigeminal root entry zone (TREZ) is the main cause of most primary trigeminal neuralgia (TN), change of glial plasticity was previously studied in the TREZ of TN rat model induced by chronic compression. To better understand the role of astrocytes and immune cells in the TREZ, different cell markers including glial fibrillary acidic protein (GFAP), complement C3, S100A10, CD45, CD11b, glutamate-aspartate transporter (GLAST), Iba-1 and TMEM119 were used in the TN rat model by immunohistochemistry and flow cytometry. On the post operation day 28, GFAP/C3-positive A1 astrocytes and GFAP/S100A10-positive A2 astrocytes were activated in the TREZ after compression injury, there were no statistical differences in the ratios of A1/A2 astrocytes between the sham and TN groups. There was no significant difference in Iba-1-positive cells between the two groups. The ratios of infiltrating lymphocytes (CD45+CD11b-) (p = 0.0075) and infiltrating macrophages (CD45highCD11b+) (p = 0.0388) were significantly higher than those of the sham group. In conclusion, different subtypes A1/A2 astrocytes in the TREZ were activated after compression injury, infiltrating macrophages and lymphocytes increased, these neuroimmune cells in the TREZ may participate in the pathogenesis of TN rat model.

Using RNA-Seq to Explore the Hub Genes in the Trigeminal Root Entry Zone of Rats by Compression Injury.

BACKGROUND: Mechanical compression on the trigeminal root entry zone (TREZ) by microvascular is the main etiology of primary trigeminal neuralgia (TN). OBJECTIVES: To study the pathogenesis of TN, hub genes screening in the TREZ of TN in an animal model was performed. STUDY DESIGN: A double blind, randomized study was designed in a controlled animal trial. SETTING: The research took place in the Laboratory of Clinical Applied Anatomy at the School of Basic Medical Science of Fujian Medical University. METHODS: Twelve male rats were randomly divided into a sham operation group and a TN animal model group. TN animal model was induced by chronic compression of trigeminal nerve root (CCT) operation. Gene expression in the TREZ were analyzed by RNA sequencing (RNA-Seq) technique. KEGG analysis, GO analysis, and PPI analysis were performed in the DEGs. Key signaling pathways analyzing by GSEA and the hub genes in the DEGs were also studied. Reverse transcription real-time polymerase chain reaction (RT-qPCR) was used to verify the RNA-Seq results. RESULTS: Transcriptome data showed that 352 genes up-regulated and 59 genes down-regulated in DEGs on post-operation day 21, after CCT operation in the TN group. KEGG analysis revealed that, "neuroactive ligand receptor interaction" and "cytokine cytokine receptor interaction" may be related to the pathogenesis of TN. GO analysis showed "regulation of signing receptor activity", "chemokine activity", and "carbohydrate binging" may be related to TN. The RT-qPCR results were consistent with the test results, indicating that the transcriptome sequencing results were reliable. LIMITATIONS: Although the incidence of TN in female rats was higher than in male rats, we only used male SD rats to establish the TN animal model, to avoid the effect of estrogen on experimental results. This study only presents some respects of RNA-Seq technique and, therefore, did not identify the DEGs at the protein level. The relationship between the DEGs at different levels shoud be done in the future. CONCLUSIONS: Based on the results of RNA-seq, this study discovered 6 hub genes in the TREZ that are closely related to the TN animal model, which provide a potential breakthrough point to explore the pathogenesis of TN.

Differential lncRNA/mRNA Expression Profiling and Functional Network Analyses in Bmp2 Deletion of Mouse Dental Papilla Cells.

Bmp2 is essential for dentin development and formation. Bmp2 conditional knock-out (KO) mice display a similar tooth phenotype of dentinogenesis imperfecta (DGI). To elucidate a foundation for subsequent functional studies of cross talk between mRNAs and lncRNAs in Bmp2-mediated dentinogenesis, we investigated the profiling of lncRNAs and mRNAs using immortalized mouse dental Bmp2 flox/flox (iBmp2fx/fx) and Bmp2 knock-out (iBmp2ko/ko) papilla cells. RNA sequencing was implemented to study the expression of the lncRNAs and mRNAs. Quantitative real-time PCR (RT-qPCR) was used to validate expressions of lncRNAs and mRNAs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict functions of differentially expressed genes (DEGs). Protein-protein interaction (PPI) and lncRNA-mRNA co-expression network were analyzed by using bioinformatics methods. As a result, a total of 22 differentially expressed lncRNAs (16 downregulated vs 6 upregulated) and 227 differentially expressed mRNAs (133 downregulated vs. 94 upregulated) were identified in the iBmp2ko/ko cells compared with those of the iBmp2fx/fx cells. RT-qPCR results showed significantly differential expressions of several lncRNAs and mRNAs which were consistent with the RNA-seq data. GO and KEGG analyses showed differentially expressed genes were closely related to cell differentiation, transcriptional regulation, and developmentally relevant signaling pathways. Moreover, network-based bioinformatics analysis depicted the co-expression network between lncRNAs and mRNAs regulated by Bmp2 in mouse dental papilla cells and symmetrically analyzed the effect of Bmp2 during dentinogenesis via coding and non-coding RNA signaling.

Involvement of the Ventrolateral Periaqueductal Gray Matter-Central Medial Thalamic Nucleus-Basolateral Amygdala Pathway in Neuropathic Pain Regulation of Rats.

The central medial nucleus (CM), a prominent cell group of the intralaminar nuclei (ILN) of the thalamus, and the ventrolateral periaqueductal gray matter (vlPAG) are two major components of the medial pain system. Whether vlPAG and CM are input sources of nociceptive information to the basolateral amygdala (BLA) and whether they are involved in neuropathic pain regulation remain unclear. Clarifying the hierarchical organization of these subcortical nuclei (vlPAG, CM, and BLA) can enhance our understanding on the neural circuits for pain regulation. Behavioral test results showed that a CM lesion made by kainic acid (KA) injection could effectively alleviate mechanical hyperalgesia 4, 6, and 8 days after spared nerve injury (SNI) surgery, with the symptoms returning after 10 days. Morphological studies revealed that: (1) the CM received afferents from vlPAG and sent efferents to BLA, indicating that an indirect vlPAG-CM-BLA pathway exists; (2) such CM-BLA projections were primarily excitatory glutamatergic neurons as revealed by fluorescence in situ hybridization; (3) the fibers originated from the CM-formed close contacts with both excitatory and inhibitory neurons in the BLA; and (4) BLA-projecting CM neurons expressed Fos induced by SNI and formed close contacts with fibers from vlPAG, suggesting that the vlPAG-CM-BLA indirect pathway was activated in neuropathic pain conditions. Finally, the vlPAG-CM-BLA indirect pathway was further confirmed using anterograde and monosynaptic virus tracing investigation. In summary, our present results provide behavioral and morphological evidence that the indirect vlPAG-CM-BLA pathway might be a novel pain pathway involved in neuropathic pain regulation.

Brain-derived neurotrophic factor and Glial cell line-derived neurotrophic factor expressions in the trigeminal root entry zone and trigeminal ganglion neurons of a trigeminal neuralgia rat model.

Microvascular compression on the trigeminal root entry zone (TREZ) is the main etiology of trigeminal neuralgia (TN) patients. To investigate brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in the trigeminal ganglion (TG) and TREZ, immunofluorescence staining and Western blot were used in a rat TN model. Both BDNF and GDNF were observed in the TG neurons and TREZ. The expression of the BDNF dimer in the TG was increased in the TN group, while GDNF expression was decreased after compression injury. The BDNF dimer/pro-BDNF ratio in the TREZ of the TN group was higher than that in the sham group, but the GDNF expression in the TREZ was significantly lower than that in the sham group. These results suggested that compression injury in the TREZ of rats induced dynamic changes in BDNF and GDNF in both the TG and TREZ, and these changes are involved in the nociceptive transmission of the TN animal model.

Glial Plasticity in the Trigeminal Root Entry Zone of a Rat Trigeminal Neuralgia Animal Model.

The trigeminal root entry zone (TREZ) is the transitional zone of central and peripheral tissue compartments in the trigeminal root. Microvascular compression on the TREZ is the main etiology of most idiopathic trigeminal neuralgia (TN) patients. However, the pathogenesis of TN is still uncertain. To investigate the glial plasticity changes in oligodendrocytes, Schwann cells, astrocytes and microglia/macrophages in the TREZ in TN, immunohistochemical staining and Western blot methods were performed in rats with TN induced by compression injury. The results showed that mechanical compression injury in the trigeminal nerve of the TN rats induced glial plasticity in the TREZ, which dynamically changed the glial interface of the CNS-PNS transitional zone. Additionally, glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes significantly proliferated and extended distally from the central region to the peripheral side of the TREZ after nerve compression injury in the TN group. Moreover, the expression of p75 in Schwann cells was upregulated on the peripheral side of the TREZ, and activated Iba-1-immunoreactive microglia/macrophages were observed on both sides of the TREZ. A significantly higher number of Schwann cells, astrocytes and microglia/macrophages were found in the TN group than in the sham operation group (p < 0.05). In conclusion, mechanical compression injury in the TN rats activated various glial cells, including oligodendrocytes, astrocytes, Schwann cells and microglia/macrophages, in the CNS-PNS transitional zone of TREZ. Changes in glial cell plasticity in the TREZ after compression injury might be involved in TN pathogenesis.

Immunohistochemical analysis of histone H3 acetylation in the trigeminal root entry zone in an animal model of trigeminal neuralgia.

OBJECTIVE: The trigeminal root entry zone (TREZ) is a transitional zone between the central nervous system (CNS) and peripheral nervous system (PNS), adjacent to the brainstem. Microvascular compression of the TREZ has been considered to be the primary etiology in most cases of trigeminal neuralgia (TN), but whether epigenetic regulation is involved in the pathogenesis of TN is still unclear. Therefore, this study was designed to investigate the epigenetic regulation of histone H3 acetylation in the TREZ in an animal model of TN. METHODS: An animal model of TN was established, and adult male Sprague-Dawley rats were randomly assigned to a TN group with trigeminal nerve root compression, sham operation group, TN+HDACi group (TN plus selective histone deacetylase inhibitor injection into the TREZ), or TN+Veh group (TN plus vehicle injection into the TREZ). To measure the length of the central portion of the TREZ from the junction of the trigeminal nerve root entering the pons to the interface of the dome-shaped CNS-PNS transitional zone, immunofluorescent staining of glia and glial nuclei was performed using glial fibrillary acidic protein (GFAP) antibody and DAPI, respectively. To investigate the acetylation of histone H3 within the TREZ in a TN animal model group and a sham operation group, localization of histone H3K9, H3K18, and H3K27 acetylation was examined via immunohistochemical staining methods. RESULTS: Measurements of the CNS-PNS transitional zone in the TREZ revealed that the average length from the junction of the trigeminal nerve root connecting the pons to the glial fringe of the TREZ in the TN group was longer than that in the sham operation group (p < 0.05) and that the interface gradually migrated distally. Cells that stained positive for acetylated histone H3K9, H3K18, and H3K27 were distributed around both sides of the border of the CNS-PNS junction in the TREZ. The ratio of immunoreactive H3K9-, H3K18- and H3K27-positive cells in the TN group was obviously higher than that in the sham operation group on postoperative days 7, 14, 21, and 28 (p < 0.05). CONCLUSIONS: These results suggested that chronic compression of the trigeminal nerve root may be involved in the pathogenesis of TN in an animal model by influencing the plasticity of the CNS-PNS transitional zone and the level of histone acetylation in the TREZ.

The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor.

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin ß6. The 36 amino acid residues of DSP are sufficient to bind to integrin ß6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP (aa183-219) stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin ß6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP (aa183-219) in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin ß6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.

Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes.

Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription.

Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages.

miR-218 expression in osteosarcoma tissues and its effect on cell growth in osteosarcoma cells.

OBJECTIVE: To investigate the expression of miR-218 and its clinical significance in osteosarcoma tissues and explore its effect on proliferation and apoptosis in osteosarcoma cells. METHODS: miR-218 expression was detected in 76 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-218 was over-expressed by exogenous miR-218 plasmids in Saos-2 cells, and then BrdU cell proliferation assay and flow cytometry were used to determine cell proliferation and apoptosis. RESULTS: The expression of miR-218 in osteosarcoma tissues was significantly lower than those in normal tumor-adjacent tissues (t=8.735, P<0.001). MiR-218 expression in tumor tissues was significantly correlated with tumor size (χ(2)=5.380, P=0.020), clinical stage (χ(2)=6.692, P=0.010) and distant metastasis (χ(2)=4.180, P=0.041). MiR-218 was obviously over-expressed by exogenous miR-218 plasmids (t=19.42, P<0.001), and miR-218 overexpression significantly reduced cell proliferation (t=9.045, P<0.001) and induced apoptosis (t=12.38, P<0.001) in Saos-2 cells. CONCLUSIONS: The low-expression of miR-218 is correlated with the poor clinicopathological features in osteosarcoma. Moreover, miR-218 overexpression reduces cancer cell proliferation and induces apoptosis in Saos-2 cells, suggesting that miR-218 may play a key role in the progression of human osteosarcoma.

Role of immune dysfunction in pathogenesis of type 1 diabetes mellitus in children.

OBJECTIVES: To investigate the function of cytokines, chemokines, and regulatory T cells (Tregs) in the pathogenesis of type 1 diabetes mellitus (T1DM) in children. METHODS: A total of 35 children with T1DM and 30 healthy controls were enrolled in this study. Levels of serum cytokines (IL-1α, IL-6, IL-10, IL-12, and TNF-α) and chemokines (MIP-1α, MIP-1α and MCP-1) were detected by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMCs) were isolated and culture supernatant of phytohaemagglutinin (PHA)-stimulated PBMCs was subjected to ELISA for levels of cytokines (IL-1α, IL-6, IL-10, IL-12 and TNF-α) in T1DM and control group. Furthermore, flow cytometry was used to determine the percentage of Tregs in PBMCs of two groups. RESULTS: Levels of serum cytokines including IL-1α, IL-6, IL-10 and TNF-α as well as chemokines, such as MIP-1α and MIP-1α in children with T1DM children were significantly higher than those in healthy controls (P<0.05, respectively). PBMCs with PHA stimulation in T1DM group secreted more IL-1α and TNF-α (P<0.05, respectively), but less IL-10 (P<0.05), as compared with control group. Furthermore, the proportion of CD4(+), CD25(+), Foxp3(+), Tregs in PBMCs isolated from children with T1DM was obviously lower than those in healthy controls (P<0.05). CONCLUSIONS: Immune dysfunction, with upregulation of inflammatory factors such as IL-1α, IL-6, TNF-α and MIP-1α, downregulation of IL-10 and Tregs, plays an important role in the pathogenesis of T1DM in children.

Connections between EM2- and SP-containing terminals and GABAergic neurons in the mouse spinal dorsal horn.

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect in pain modulation. To investigate the potential interactions of EM2- and substance P (SP)-containing primary afferents and γ-amino butyric acid (GABA)-containing interneurons in lamina II in nociceptive transmission, connections between EM2- and SP-containing terminals and GABAergic neurons in the spinal dorsal horn were studied. Double-immunofluorescent labeling showed that approximately 62.3 % of EM2-immunoreactive neurons exhibited SP-immunostaining, and 76.9 % of SP-immunoreactive neurons demonstrated EM2-immunoreactivities in the dorsal root ganglion (DRG). Dense double-labeled EM2- and SP-immunoreactivities were mainly observed in lamina II of the lumbar dorsal horn. Furthermore, triple-immunofluorescent labeling results revealed that EM2 and SP double-labeled terminals overlapped with GABAergic neurons. Immuno-electron microscopy confirmed that the EM2- or SP-immunoreactive terminals formed synapses with GABA-immunoreactive dendrites in lamina II of the lumbar dorsal horn. During noxious information transmission induced by formalin plantar injection, GABAergic neurons expressing FOS in their nuclei were contacted with EM2- or SP-immunoreactive terminals. These results suggest that the interactions between EM2- and SP-containing terminals and GABAergic interneurons in the lamina II influence pain transmission and modulation in the spinal dorsal horn.