Hedgehog signaling regulates bone homeostasis through orchestrating osteoclast differentiation and osteoclast-osteoblast coupling.

Imbalance of bone homeostasis induces bone degenerative diseases such as osteoporosis. Hedgehog (Hh) signaling plays critical roles in regulating the development of limb and joint. However, its unique role in bone homeostasis remained largely unknown. Here, we found that canonical Hh signaling pathway was gradually augmented during osteoclast differentiation. Genetic inactivation of Hh signaling in osteoclasts, using Ctsk-Cre;Smof/f conditional knockout mice, disrupted both osteoclast formation and subsequent osteoclast-osteoblast coupling. Concordantly, either Hh signaling inhibitors or Smo/Gli2 knockdown stunted in vitro osteoclast formation. Mechanistically, Hh signaling positively regulated osteoclast differentiation via transactivation of Traf6 and stabilization of TRAF6 protein. Then, we identified connective tissue growth factor (CTGF) as an Hh-regulatory bone formation-stimulating factor derived from osteoclasts, whose loss played a causative role in osteopenia seen in CKO mice. In line with this, recombinant CTGF exerted mitigating effects against ovariectomy induced bone loss, supporting a potential extension of local rCTGF treatment to osteoporotic diseases. Collectively, our findings firstly demonstrate that Hh signaling, which dictates osteoclast differentiation and osteoclast-osteoblast coupling by regulating TRAF6 and CTGF, is crucial for maintaining bone homeostasis, shedding mechanistic and therapeutic insights into the realm of osteoporosis.

Chronic cauda equina syndrome decompression surgery recovery is very "bad"? Based on patient self-assessment.

BACKGROUND: Symptoms of cauda equina syndrome (CES) secondary to degenerative lumbar spine diseases are sometimes mild and tend to be ignored by patients, resulting in delayed treatment. In addition, the long-term efficacy of surgery is unclear. OBJECTIVE: To determine the predictive factors of CES and post-operative recovery in patients with symptoms lasting > 3 months. METHODS: From January 2011 to December 2020, data of 45 patients with CES secondary to lumbar disk herniation/lumbar spinal stenosis were collected from a single center. The patients had bladder, bowel or sexual dysfunction and decreased perineal sensation that lasted for > 3 months. A 2-year post-operative follow-up was conducted to evaluate recovery outcomes, which were measured by validated self-assessment questionnaires conducted by telephone and online. RESULTS: Overall, 45 CES patients (57.8% female; mean age, 56 years) were included. The duration of pre-operative CES symptoms was 79.6 weeks (range, 13-730 weeks). The incidence of saddle anesthesia before decompression was 71.1% (n = 32), bladder dysfunction 84.4% (n = 38), bowel dysfunction 62.2% (n = 28) and sexual dysfunction 64.4% (n = 29). The overall recovery rate of CES after a 2-year follow-up was 64.4%. The rates of the residual symptoms at the last follow-up were as follows: saddle anesthesia 22.2%, bladder dysfunction 33.3%, bowel dysfunction 24.4% and sexual dysfunction 48.9%. Pre-operative saddle anesthesia, overactive bladder and sexual dysfunction were risk factors for poor prognosis after decompression. CONCLUSION: CES patients with symptoms lasting > 3 months may recover after surgery. Sexual dysfunction has a high residual rate and should not be ignored during diagnosis and treatment.

SARS-CoV-2 ORF7a blocked autophagy flux by intervening in the fusion between autophagosome and lysosome to promote viral infection and pathogenesis.

The coronavirus disease 2019 (COVID-19) continues to pose a major threat to public health worldwide. Although many studies have clarified the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection process, the underlying mechanisms of viral invasion and immune evasion were still unclear. This study focused on SARS-CoV-2 ORF7a (open reading frame-7a), one of the essential open reading frames (ORFs) in infection and pathogenesis. First, by analyzing its physical and chemical characteristics, SARS-CoV-2 ORF7a is an unstable hydrophobic transmembrane protein. Then, the ORF7a transmembrane domain three-dimensional crystal structure model was predicted and verified. SARS-CoV-2 ORF7a localized in the endoplasmic reticulum and participated in the autophagy-lysosome pathway via interacting with p62. In addition, we elucidated the underlying molecular mechanisms by which ORF7a intercepted autophagic flux, promoted double membrane vesicle formation, and evaded host autophagy-lysosome degradation and antiviral innate immunity. This study demonstrated that ORF7a could be a therapeutic target, and Glecaprevir may be a potential drug against SARS-CoV-2 by targeting ORF7a. A comprehensive understanding of ORF7a's functions may contribute to developing novel therapies and clinical drugs against COVID-19.

Hsa-let-7f-1-3p targeting the circadian gene Bmal1 mediates intervertebral disc degeneration by regulating autophagy.

The intervertebral disc has an intrinsic circadian rhythm, elimination of which leads to stress in nucleus pulposus cells (NPCs), contributing to intervertebral disc degeneration (IDD). Disruption or deletion of Bmal1 (a core transcription factor) results in complete loss of circadian rhythms, make mice susceptibility to IDD. However, the underlying mechanism by which Bmal1 mediates IDD is remains enigmatic, and whether there are other upstream factors regulating Bmal1 in NPCs. In our study, we first found that the decrease of Bmal1 was significantly correlated with the grades of IDD. With gain- and loss-of-function, Bmal1 shown a protective effect on NPC viability and functions. Transcriptomic and proteomic landscape reveals the functional contributions of Bmal1, and phosphoproteomic analysis links to autophagy. Bioinformatics analysis identified that a novel miRNA hsa-let-7f-1-3p was directly target Bmal1 3'UTR and negatively correlated with NPC function. Finally, our animal model confirmed the protective role of Bmal1 in rat IDD and this effect could be attenuated by hsa-let-7f-1-3p. The hsa-let-7f-1-3p/Bmal1/autophagy axis provides a potential therapeutic strategy for the clinical treatment of IDD.

Effection of monoplanar pedicle screw on facet joint degeneration in thoracolumbar vertebral fractures.

BACKGROUND: This study aimed to compare the clinical outcomes and effect on instrument-related facet joints between fixed-axis pedicle screw (FAPS) and monoplanar pedicle screw (MPPS). METHODS: 816 pedicle screws of 204 patients with thoracolumbar vertebral fractures (TLVF) who underwent internal fixation surgery were analyzed in this retrospective study. All patients were divided into two groups (FAPS and MPPS). Preoperative, immediate postoperative, and 12-18-months postoperative CT and X-ray, and clinical data, including demographics, preoperative and immediate postoperative Visual Analogue Scale (VAS), blood loss (BL), operation time (OT) and hospital stay time (HST), were collected. Facet joint violation and degeneration grade were evaluated by CT according to Babu's criteria and Weishaupt's criteria respectively, and preoperative, immediate postoperative and 12-18-months postoperative anterior body compression index (ABCI) were measured by X-ray. RESULTS: Postoperative VAS of two groups was lower than preoperative VAS (p < 0.05). BL, OT, and HST were less in MPPS than FAPS, and the difference was statistically significant in BL and HST (p < 0.05) but no in OT (p > 0.05). Immediate postoperative and 12-18-months postoperative ABCI were significantly higher than preoperative (p < 0.05), and the difference of ABCI between immediate postoperative and 12-18-months postoperative were not significant in two groups (p > 0.05). Total violation rate (VR) was about 1.35% (11/816) and FAPS had a lower VR than MPPS, but no significant (p > 0.05). Weishaupt's criteria revealed that average class (AC) was 0.69 in FAPS and 0.67 in MPPS, and the distribution of degenerated facet joints in two groups did not differ preoperatively (p > 0.05). In 12-18 months postoperatively, AC was significantly higher in FAPS than in MPPS, and the distribution of degenerated facet joints in two groups was significantly different (p < 0.05). The comparison of cranial to caudal joints in two groups revealed that cranial joints had more severe degeneration than caudal joints. CONCLUSIONS: The findings suggested that both MPPS and FAPS were effective for patients with TLVF, but MPPS by percutaneous may be a better choice to avoid adjacent segment degeneration, especially the surgery-involved facet joints degeneration.

The value of Hounsfield units in predicting cage subsidence after transforaminal lumbar interbody fusion.

BACKGROUND: Cage subsidence may occur following transforaminal lumbar interbody fusion (TLIF) and lead to nonunion, foraminal height loss and other complications. Low bone quality may be a risk factor for cage subsidence. Assessing bone quality through Hounsfield units (HU) from computed tomography has been proposed in recent years. However, there is a lack of literature evaluating the correlation between HU and cage subsidence after TLIF. METHODS: Two hundred and seventy-nine patients suffering from lumbar degenerative diseases from April, 2016 to August, 2018 were enrolled. All underwent one-level TLIF with a minimum of 1-year follow-up. Cage subsidence was defined as > 2 mm loss of disc height at the fusion level. The participants were divided into 2 groups: cage subsidence group (CS) and non-cage subsidence group (non-CS). Bone quality was determined by HU, bone mineral density of lumbar (BMD-l) and femoral (BMD-f) from dual-emission X-ray absorptiometry (DXA). HU of each vertebra from L1 to L4 (e.g., HU1 for HU of L1) and mean value of the four vertebrae (HUm) were calculated. Visual analog scale (VAS) of back/leg pain and Oswestry disability index (ODI) were used to report clinical outcomes. RESULTS: Cage subsidence occurred in 82 (29.4%) cases at follow-ups. Mean age was 50.8 ± 9.0 years with a median follow-up of 18 months (range from 12 to 40 months). A total of 90.3% patients presented fusion with similar fusion rate between the two groups. ODI and VAS in leg were better in non-CS group at last follow-ups. Using receiver operating characteristic curves (ROCs) to predict cage subsidence, HUm provided a larger area under the curve (AUC) than BMD-l (Z = 3.83, P <  0.01) and BMD-f (Z = 2.01, P = 0.02). AUC for HU4 was larger than BMD-f and close to HUm (Z = 0.22, P = 0.481). CONCLUSIONS: Cage subsidence may indicate worse clinical outcomes. HU value could be a more effective predictor of lumbar cage subsidence compared with T-score of DXA after TLIF.

Clinical safety study of photobiomodulation in acute spinal cord injury by scattering fiber.

The study aimed to design a reliable and straightforward PBM method by implanting a medical scattering fiber above surgically exposed spinal cord in SCI patients. Moreover, the safety of this method was examined. Twelve patients with acute SCI (ASIA B) requiring posterior decompression were recruited. The medical scattering fiber was implanted above the spinal cord, and was continuously irradiated at 810 nm, 300 mW, 30 min/day, once per day for 7 days. The vital signs (temperature, blood pressure, respiratory rate, heart rate, and oxygen saturation), infection indicators (WBC, NEUT, hs-CRP, and PCT), photo-allergic reaction indicators (Eosinophil and Basophil), coagulation function indicators (PT, APTT, TT) and neurological stability indicators (ASIA sensory and motor scores) were recorded to evaluate the safety of PBM. Three months after surgery, 12 patients completed follow-up. In our study, direct PBM on SCI site did not cause clinically pathologic changes in vital signs of the patients. All patients had higher WBC, NEUT, and hs-CRP at day 3 during irradiation than those before surgery, and returned to normal at day 7. The changes in Eosinophil and Basophil that were closely associated with allergic reactions were within normal limits throughout the course of irradiation. The coagulation function (PT, APTT, and TT) of patients were also in the normal range. The ASIA sensory and motor scores of all patients had no changes throughout the irradiation process. However, in the follow-up, both ASIA sensory and motor scores of all patients had minor improvement than those in pre-irradiation, and 7 patients had adverse events, but they were not considered to be related to PBM. Our study might firstly employ direct PBM in the SCI by using scattered optical fibers. In a limited sample size, our study concluded that direct PBM at the site of SCI would not produce adverse effects within the appropriate irradiation parameters. The method is safe, feasible, and does not add additional trauma to the patient. Our preliminary study might provide a new methodology for the clinical PBM treatment of acute SCI.

Autogenous bone-guided induced membrane technique in closed/small-sized open high-energy fractures in benign inflammatory environment: a case series.

OBJECTIVE: Infection and nonunion are the two most challenging issues for high-energy fractures. This study aimed to explore the clinical effect of benign inflammation-cultivated bone growth activity in the treatment of closed/small-sized open and high-energy fractures. METHODS: This study is a case series of closed/small-sized open and high-energy fractures of the lower limbs treated at our hospital from April 2009 to February 2017. All patients underwent debridement and external fixation in the early stage, followed by internal fixation in the second stage. After the operation, fracture healing was monitored by X-ray, and early-stage knee function training was initiated. Also, bone grafting was performed to stimulate the healing reaction, eliminating the atrophic nonunion factors. RESULTS: The operation in all 75 cases was carried out after the inflammatory responses completely subsided, leading to secondary wound healing. Bony union appeared in 71 patients who did not suffer from any pain and could stand up and walk without any restriction. Among them, 68 patients could flex their knee > 100°, and three patients had knee flexion ranging from 80 to 100°. No infections occurred after the second operation. CONCLUSION: This two-stage treatment for high-energy fractures could avoid the damage caused by excessive inflammatory responses that occurred following early-stage one-time internal fixation. This method protected benign inflammatory-callus reactions induced by the primary injury and utilized the advantages of closed reduction in AO fixation with open reduction, thereby avoiding potential infection and nonunion caused by one-time fixation during the early stage.

Phenotypic characterization of Slc26a2 mutant mice reveals a multifactorial etiology of spondylolysis.

Confusion persists over pathogenesis of spondylolysis. To confirm pathogenicity of the previously identified causative mutation of spondylolysis and investigate the genetic etiology, we generate a new mouse line harboring D673V mutation in the Slc26a2 gene. D673V mutation induces delayed endochondral ossification characterized by transiently reduced chondrocyte proliferation in mice at the early postnatal stage. Adult D673V homozygotes exhibit dysplastic isthmus and reduced bone volume of the dorsal vertebra resembling the detached vertebral bony structure when spondylolysis occurs, including the postzygopophysis, vertebral arch, and spinous process, which causes biomechanical alterations around the isthmic region of L4-5 vertebrae indicated by finite element analysis. Consistently, partial ablation of Slc26a2 in vertebral skeletal cells using Col1a1-Cre; Slc26a2 fl/fl mouse line recapitulates a similar but worsened vertebral phenotype featured by lamellar isthmus. In addition, when reaching late adulthood, D673V homozygotes develop an evident bone-loss phenotype and show impaired osteogenesis. These findings support a multifactorial etiology, involving congenitally predisposed isthmic conditions, altered biomechanics, and age-dependent bone loss, which leads to SLC26A2-related spondylolysis.

Clinical efficacy and therapeutic value of delayed surgery in patients with symptomatic old thoracolumbar fractures.

BACKGROUND: To investigate the clinical efficacy and therapeutic value of posterior decompression reduction, bone grafting fusion, and internal fixation for treatment of symptomatic old thoracolumbar fractures. METHOD: Retrospective analysis was conducted for 14 patients (9 men, 5 women; average age 40.1 years) with old thoracolumbar fractures who underwent posterior operation. American Spinal Injury Association (ASIA) scores were used to evaluate neurologic function. Vertebral body height, Cobb angle in the sagittal plane, spinal canal volume ratio (%) and bone graft fusion were analyzed by radiography and computed tomography on different follow-up times. RESULTS: Mean follow-up was 27.1 months (23-36 months). Of three patients with ASIA grade A, 2 had improved postoperative urination and defecation, although no classification change. Preoperative ASIA score for eight patients with incomplete injury was grade B; four patients recovered to grade C at final follow-up. Preoperative ASIA score was C in three patients, increased to D in two patients and returned to normal E in one patient. Preoperative results showed average injured vertebra height loss rate decreased from 50.4 to 8.9%; average Cobb angle on the sagittal plane recovered from 39.6 to 6.9°; and the average spinal canal volume ratio recovered from 33.8 to 5.9%. Bony fusion was achieved; local lumbago and leg pain were relieved to some extent. No patients exhibited loosening of the fracture treated by internal fixation, pseudoarthrosis, or other related serious complications. CONCLUSION: Treatment of old thoracolumbar fractures by posterior decompression reduction, bone grafting fusion, and internal fixation can relieve spinal cord compression, improve neurologic function of some patients (ASIA grades B-C), effectively relieve pain, correct deformity, restore biomechanical stability, and significantly improve quality of life.

Time-restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury.

Delivery of multiple neurotrophic factors (NTFs), especially with time-restricted release kinetics, holds great potential for nerve repair. In this study, we utilized the tetracycline-regulatable Tet-On 3G system to control the expression of c-Jun, which is a common regulator of multiple NTFs in Schwann cells (SCs). In vitro, Tet-On/c-Jun-modified SCs showed a tightly controllable secretion of multiple NTFs, including glial cell line-derived NTF, nerve growth factor, brain-derived NTF, and artemin, by the addition or removal of doxycycline (Dox). When Tet-On/c-Jun-transduced SCs were grafted in vivo, the expression of NTFs could also be regulated by oral administration or removal of Dox. Fluoro-Gold retrograde tracing results indicated that a biphasic NTF expression scheme (Dox+3/-9, NTFs were up-regulated for 3 wk and declined to physiologic levels for another 9 wk) achieved more axonal regeneration than continuous up-regulation of NTFs (Dox+12) or no NTF induction (Dox-12). More importantly, the Dox+3/-9-group animals showed much better functional recovery than the animals in the Dox+12 and Dox-12 groups. Our findings, for the first time, demonstrated drug-controllable expression of multiple NTFs in nerve repair cells both in vitro and in vivo. These findings provide new hope for developing an optimal therapeutic alternative for nerve repair through the time-restricted release of multiple NTFs using Tet-On/c-Jun-modified SCs.-Huang, L., Xia, B., Shi, X., Gao, J., Yang, Y., Xu, F., Qi, F., Liang, C., Huang, J., Luo, Z. Time-restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury.

The Immune Privilege of the Intervertebral Disc: Implications for Intervertebral Disc Degeneration Treatment.

The intervertebral disc (IVD) is the largest avascular organ of the body. It is composed of three parts: the nucleus pulposus (NP), the annulus fibrosus (AF) and the cartilaginous endplate (CEP). The central NP is surrounded by the AF and sandwiched by the two CEPs ever since its formation. This unique structure isolates the NP from the immune system of the host. Additionally, molecular factors expressed in IVD have been shown inhibitive effect on immune cells and cytokines infiltration. Therefore, the IVD has been identified as an immune privilege organ. The steady state of immune privilege is fundamental to the homeostasis of the IVD. The AF and the CEP, along with the immunosuppressive molecular factors are defined as the blood-NP barrier (BNB), which establishes a strong barrier to isolate the NP from the host immune system. When the BNB is damaged, the auto-immune response of the NP occurs with various downstream cascade reactions. This effect plays an important role in the whole process of IVD degeneration and related complications, such as herniation, sciatica and spontaneous herniated NP regression. Taken together, an enhanced understanding of the immune privilege of the IVD could provide new targets for the treatment of symptomatic IVD disease. However, the underlying mechanism above is still not fully clarified. Accordingly, the current study will extensively review and discuss studies regarding the immune privilege of the IVD.

Dysplastic spondylolysis is caused by mutations in the diastrophic dysplasia sulfate transporter gene.

Spondylolysis is a fracture in part of the vertebra with a reported prevalence of about 3-6% in the general population. Genetic etiology of this disorder remains unknown. The present study was aimed at identifying genomic mutations in patients with dysplastic spondylolysis as well as the potential pathogenesis of the abnormalities. Whole-exome sequencing and functional analysis were performed for patients with spondylolysis. We identified a novel heterozygous mutation (c.2286A > T; p.D673V) in the sulfate transporter gene SLC26A2 in five affected subjects of a Chinese family. Two additional mutations (e.g., c.1922A > G; p.H641R and g.18654T > C in the intron 1) in the gene were identified by screening a cohort of 30 unrelated patients with the disease. In situ hybridization analysis showed that SLC26A2 is abundantly expressed in the lumbosacral spine of the mouse embryo at day 14.5. Sulfate uptake activities in CHO cells transfected with mutant SLC26A2 were dramatically reduced compared with the wild type, confirming the pathogenicity of the two missense mutations. Further analysis of the gene-disease network revealed a convergent pathogenic network for the development of lumbosacral spine. To our knowledge, our findings provide the first identification of autosomal dominant SLC26A2 mutations in patients with dysplastic spondylolysis, suggesting a new clinical entity in the pathogenesis of chondrodysplasia involving lumbosacral spine. The analysis of the gene-disease network may shed new light on the study of patients with dysplastic spondylolysis and spondylolisthesis as well as high-risk individuals who are asymptomatic.

Potential Mechanism of Neurite Outgrowth Enhanced by Electrical Stimulation: Involvement of MicroRNA-363-5p Targeting DCLK1 Expression in Rat.

Electrical stimulation (ES) promotes neurite outgrowth and nerve regeneration, but the underlying mechanisms remain undefined. In the present study, we investigated the role of micro RNAs (miRNAs) in ES-mediated neurite outgrowth. First, we performed microarray analyses to identify changes in the miRNAs profile of dorsal root ganglion neurons (DRGNs) following ES. The expression of 16 known miRNAs was altered by ES. Bioinformatics showed that the potential targets of these differentially expressed miRNAs were involved in neurite outgrowth. We focused on miRNA-363-5p (miR-363-5p), because its expression was consistently altered by ES in the present study. Silencing miR-363-5p promoted neurite outgrowth, while miR-363-5p mimic reduced neurite outgrowth. Downregulation of miR-363-5p indicated that double cortin-like kinase (DCLK) 1, a major microtubule-associated protein, was a direct target of miR-363-5p in DRGNs. Knockdown of DCLK1 recapitulated the beneficial effect of a miR-363-5p inhibitor on DRG neurite outgrowth. In conclusion, our data has indicated that miR-363-5p is involved in ES-promoted neurite outgrowth by targeting DCLK1. These findings provide new insights into the roles of miRNAs in ES-enhanced neurite outgrowth and regeneration.

Effects of Ginkgo Biloba Extract EGb-761 on Neuropathic Pain in Mice: Involvement of Opioid System.

Neuropathic pain is considered as one of the most difficult types of pain to manage with conventional analgesics. EGb-761 is extracted from leaves of Ginkgo biloba and has analgesia and anti-inflammatory properties. This study aimed to examine the effect of EGb-761 on chronic constriction injury (CCI)-induced neuropathic pain behaviors, including thermal hyperalgesia and mechanical allodynia, and to explore the possible mechanisms underlying this action. To this end, CCI mice were intraperitoneally injected with EGb-761 (10, 20, 40, and 80 mg/kg), and thermal hyperalgesia, mechanical allodynia, cytokines, and mu-opioid receptor expression were measured. Results showed that EGb-761 attenuated thermal hyperalgesia and mechanical allodynia dose-dependently and the best delivery time window was from day 7 to day 14 after CCI. Additionally, EGb-761 treatment significantly decreased pro-inflammatory cytokines and enhanced mu opioid receptor (MOR) expression in the sciatic nerve. Moreover, the opioid antagonist naloxone prevented the effect of EGb-761 on thermal hyperalgesia and mechanical allodynia but did not influence the effect of EGb-761 on inflammatory cytokines. In conclusion, this study suggests that the potential of EGb-761 as a new analgesic for neuropathic pain treatment, and opioid system may be involved in the EGb-761-induced attenuation of thermal hyperalgesia and mechanical allodynia. Copyright © 2016 John Wiley & Sons, Ltd.

The effects and mechanisms of clinorotation on proliferation and differentiation in bone marrow mesenchymal stem cells.

Data from human and rodent studies have demonstrated that microgravity induces observed bone loss in real spaceflight or simulated experiments. The decrease of bone formation and block of maturation may play important roles in bone loss induced by microgravity. The aim of this study was to investigate the changes of proliferation and differentiation in bone marrow mesenchymal stem cells (BMSCs) induced by simulated microgravity and the mechanisms underlying it. We report here that clinorotation, a simulated model of microgravity, decreased proliferation and differentiation in BMSCs after exposure to 48 h simulated microgravity. The inhibited proliferation are related with blocking the cell cycle in G2/M and enhancing the apoptosis. While alterations of the osteoblast differentiation due to the decreased SATB2 expression induced by simulated microgravity in BMSCs.

Inhibition of the spinal astrocytic JNK/MCP-1 pathway activation correlates with the analgesic effects of tanshinone IIA sulfonate in neuropathic pain.

BACKGROUND: Neuropathic pain (NP) continues to be challenging to treat due to lack of effective drugs. Accumulating evidence elucidated that glia-mediated inflammatory reactions play a pivotal role in the introduction and development of NP. Besides, activation of the c-Jun N-terminal kinase (JNK)/monocyte chemoattractant protein-1 (MCP-1) pathway in astrocytes has been reported to be critical for spinal astrocytic activation and neuropathic pain development after spinal nerve ligation (SNL). Tanshinone IIA, a major active component of a traditional Chinese drug, Danshen, possesses potent immuno-suppressive activities. The present study was undertaken to assess whether intraperitoneal administration of tanshinone IIA sulfonate (TIIAS) has analgesic effect on SNL-induced neuropathic pain and whether the inhibition of astrocytic activation and JNK/MCP-1 pathway is involved in the analgesic effect of TIIAS. METHODS: The effects of TIIAS on SNL-induced mechanical allodynia were assessed by behavioral testing. Immunofluorescence histochemical staining was used to detect changes of spinal astrocytes and spinal pJNK expression and localization. Immunofluorescence histochemistry and Western blot analysis were used to quantify the SNL-induced spinal pJNK expression after TIIAS administration. Enzyme-linked immunosorbent assay (ELISA) was used to detect the SNL-induced spinal expression of pro-inflammatory cytokines and MCP-1. RESULTS: Our results indicated that intraperitoneal TIIAS up-regulated the mechanical paw withdrawal threshold (PWT) of NP, while astrocytic activation was suppressed and accompanied by the down-regulation of IL-1ß and TNF-α expression, as well as JNK phosphorylation in the spinal dorsal horn. Additionally, the release of MCP-1 was dose dependently decreased. After co-treatment with TIIAS and JNK inhibitor (SP600125), no significant increases in mechanical PWT and MCP-1 expression were observed compared with the TIIAS-treated group. CONCLUSIONS: The present results suggest that the analgesic effects of TIIAS in neuropathic pain are mainly mediated by the down-regulation of SNL-induced astrocytic activation, which is via the inhibition of JNK/MCP-1 pathway.

[Transforming growth factor-ß1 induces bone marrow-derived mesenchymal stem cells to differentiate into cancer-associated fibroblasts].

OBJECTIVE: To investigate the effect of transforming growth factor-ß1 (TGF-ß1) on the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into cancer-associated fibroblasts(CAFs). METHODS: MSCs were cultured in α-MEM with recombinant human TGF-ß1 or in tumor-conditioned medium.The expression of CAFs markers were detected by immunofluorescence and quantitative RT-PCR. RESULTS: The qRT-PCR assay showed that the expression of CAFs markers FAP, ACTA, CAV, CCL5, CXCR4, FSP1, SDF-1 and vimentin were 9.92±2.16, 7.76±1.28, 3.04±0.95, 3.28±2.16, 2.13±0.71, 1.41±0.66, 2.25±0.86 and 1.38±0.56, respectively, significantly upregulated in the MSCs co-cultured with TGF-ß1 or TCM. The relative levels of FAP, ACTA, CAV, CCL5, CXCR4, FSP1, SDF-1 and vimentin mRNA in the TCM group were 7.52±1.76, 5.02±1.18, 1.98±1.19, 1.82±1.19, 2.95±0.86, 1.44±0.67, 2.08±0.74 and 1.47±0.55, respectively, indicating that MSCs can express CAFs phenotype.TGF beta signaling pathway inhibitor SB-431542 could inhibit the differentiation. Both immunofluorescence and Western blot confirmed the above results. CONCLUSIONS: TGF-ß1 induces differentiation of local MSCs to CAFs by upregulating the expression of pSmad3, so as to further promote the growth of cancer cells.

[Neurological complications of posterior vertebral column resection for severe rigid congenital spinal deformities].

OBJECTIVE: To analyze the risk factors of neurological complications of posterior vertebral column resection in the treatment of severe rigid congenital spinal deformities. METHODS: The clinical data of 88 patients with severe rigid congenital spinal deformities who underwent PVCR in Department Of Orthopaedics, Xijing Hospital, Fourth Military Medical University from June 2007 to November 2012 were collected. There were 39 males and 49 females at the average age of 16.9 years (range 6-46 years). To measure the Cobb angle and balance at preoperative, postoperative and follow up, and to record the operation report, neurological complications and at follow up. The relevant factors of neurological complications were analyzed by one-way analysis, including: age, Cobb angle, operation time, body mass index, pulmonary function, blood volume loss, resection level, number of vertebrae fixed, number of vertebrae resected, usage of cage or titanium mesh, preoperative neurologic function, the type of deformity and combination of spinal canal deformity, and further analyzed by multiariable Logistic regression analysis. RESULTS: The average follow up was 42 months (range 19 to 83 months). The number of resected vertebrae average 1.3 (range 1 to 3), operative time average 502.4 min (range 165.0 to 880.0 min), estimate blood loss average 2,238 ml (range 100 to 11,500 ml) for an average 69.3% blood volume loss (range 9% to 299%). The average preoperative major coronal curve of 93.6° corrected to 22.2°, at the final follow-up, the coronal curve was 22.2° with a correction of 76.8%. The average preoperative coronal imbalance (absolute value) was 2.5 cm decreasing to 1.3 cm at the final follow-up. The average preoperative major sagittal curve of 88.2° corrected to 28.7°, at the final follow-up, the sagittal curve was 29.2°, average decrease in kyphosis of 59.0°. The average preoperative sagittal imbalance (absolute value) was 3.1 cm decreasing to 1.2 cm at the final follow-up. There were 12 patients (13.6%) developed a neurological complications. High rate of neurological complications was occurred in patients with operative time greater than 480 min, pulmonary dysfunction, blood volume loss greater than 50%, T7-T99 osteotomy and preoperative neurologic compromise (P=0.046, 0.000, 0.000, 0.033, 0.043). CONCLUSIONS: Posterior vertebral column resection can achieve satisfactory efficacy in treatment of severe spinal deformities. Pulmonary dysfunction and blood volume loss greater than 50% were significant risk factors of neurological complications.

17ß-Estradiol inhibits ER stress-induced apoptosis through promotion of TFII-I-dependent Grp78 induction in osteoblasts.

Although many studies have suggested that estrogen prevents postmenopausal bone loss partially due to its anti-apoptosis effects in osteoblasts, the underlying mechanism has not been fully elucidated. In the present study, we found that 17ß-estradiol (17ß-E2), one of the primary estrogens, inhibited endoplasmic reticulum (ER) stress-induced apoptosis in MC3T3-E1 cells and primary osteoblasts. Interestingly, 17ß-E2-promoted Grp78 induction, but not CHOP induction in response to ER stress. We further confirmed that Grp78-specific siRNA reversed the inhibition of 17ß-E2 on ER stress-induced apoptosis by activating caspase-12 and caspase-3. Moreover, we found that 17ß-E2 markedly increased the phosphorylated TFII-I levels and nuclear localization of TFII-I in ER stress conditions. 17ß-E2 stimulated Grp78 promoter activity in a dose-dependent manner in the presence of TFII-I and enhanced the binding of TFII-I to the Grp78 promoter. In addition, 17ß-E2 notably increased phosphorylated ERK1/2 levels and Ras kinase activity in MC3T3-E1 cells. The ERK1/2 activity-specific inhibitor U0126 remarkably blocked 17ß-E2-induced TFII-I phosphorylation and Grp78 expression in response to ER stress. Together, 17ß-E2 protected MC3T3-E1 cells against ER stress-induced apoptosis by promoting Ras-ERK1/2-TFII-I signaling pathway-dependent Grp78 induction.