Sagittal imaging study of the lumbar spine with the short rod technique.

PURPOSE: The short rod technique (SRT) is a novel method for lumbar pedicle screw placement to reduce surgical trauma and avoid damage to the facet joint and articular surface. The core concept is to change the entry point and angle of the screw on the vertebrae at both ends in the sagittal plane to shorten the length of the longitudinal rods. The purpose of this study is to determine the sagittal screw angle (SSA) and its safe Maximum (MAX) value on each lumbar vertebra for the SRT and to observe the shortening effect on the longitudinal rods. METHODS: A total of 152 healthy adults were investigated by measuring the lumbar spine lateral view images. The SSA and MAX-SSA were measured with SRT as reference to the conventional placement technique method. The distance between the entry points of the proximal and distal vertebrae was measured to compare the changes in the length of the longitudinal rods using the two screw placement techniques. RESULTS: + SSA increased from L1 to L4, and -SSA increased from L2 to L5, in which the -SSA of L2, L3, and L4 were significantly greater than those of + SSA (P < 0.05). + MAX-SSA at L1-L4 was 23.26 ± 3.54°, 23.68 ± 3.37°, 24.12 ± 3.29°, and 24.26 ± 3.42°, respectively. -MAX-SSA at L2-L5 was 36.25 ± 3.26°, 38.26 ± 3.73°, 38.62 ± 3.63° and 37.33 ± 3.31°, respectively. Theoretical reductions by calculation for the 2-segment lumbar pedicles were: L1-2: 9 mm, L2-3: 9.29 mm, L3-4: 6.23 mm, and L4-5: 7.08 mm; And the 3-segment lumbar pedicles were: L1-3: 16.97 mm, L2-4: 16.73 mm, L3-5, and 18.24 mm, respectively. CONCLUSIONS: The application of the SRT to lumbar pedicles is a safe screw placement method that can significantly shorten the length of the used longitudinal rods.

Melatonin promotes osteoblastic differentiation and regulates PDGF/AKT signaling pathway.

Melatonin has been reported to participate in bone metabolism in recent studies. However, the underlying mechanism in melatonin-mediated osteoblastic differentiation remains largely unknown. The aim of this study is to investigate the role of melatonin in osteoblastic differentiation. In the present study, additional melatonin significantly promoted osteoblastic differentiation of MC3T3-E1 cells as evidenced by increased messenger RNA (mRNA) levels of osteogenic markers, alkaline phosphatase (ALP), collagen type I α1 chain, osteocalcin, and runt-related transcription factor 2 (Runx2). It was noteworthy that the expression level of platelet-derived growth factor subunit B (PDGFB) and content of its homodimer PDGF-BB were remarkably increased after melatonin administration. Moreover, the mRNA levels of phosphorylated PDGFRß (PDGF receptor ß) and Akt, a serine/threonine-specific protein kinase, were significantly upregulated in melatonin-treated MC3T3-E1 cells determined by a real-time polymerase chain reaction. Besides, by performing alizarin red staining, osteoblastic differentiation of MC3T3-E1 cells was conspicuously promoted by melatonin, which could be partially attenuated by crenolanib, a PDGFR inhibitor. Similarly, results from immunofluorescence and western blot assay showed that melatonin-induced upregulation of Runx2 and phosphorylated Akt was suppressed by crenolanib. Akt inhibition by MK-2206 also suppressed osteoblastic differentiation. Furthermore, by in vivo assay, additional melatonin promoted osteoblastic differentiation in mice with femoral fracture, and obvious callus formation was observed in melatonin-treated mice 5 weeks after fracture. Melatonin supplement also inhibited osteoclastic differentiation in mice. All statistical analysis was performed using GraphPad Prism and a P < 0.05 was deemed to be significant. To summarize, we demonstrate that melatonin promotes osteoblastic differentiation in MC3T3-E1 cells and enhances fracture healing in mouse femoral fracture model and regulates PDGF/AKT signaling pathway.

Melatonin Induces Osteoblastic Differentiation of Mesenchymal Stem Cells and Promotes Fracture Healing in a Rat Model of Femoral Fracture via Neuropeptide Y/Neuropeptide Y Receptor Y1 Signaling.

The function of melatonin (MLT) in promoting fracture healing has been demonstrated in previous studies. However, the molecular mechanism underlying therapeutic effects of MLT is not entirely clear. In this study, mesenchymal stem cells (MSCs) were isolated from rat bone marrow and identified by flow cytometry. We found that MLT treatment upregulated the neuropeptide Y (NPY) and NPY receptor Y1 (NPY1R) expression, and promoted the proliferation and migration of MSCs, which was suppressed by BIBP3226, an inhibitor of NPY1R. Moreover, the levels of NPY and NPY1R in MSCs undergoing osteoblastic differentiation were upregulated after MLT administration. MLT-induced osteoblastic differentiation of MSCs was suppressed by BIBP3226 treatment, as evidenced by decreased levels of alkaline phosphatase (ALP), collagen type I α1 chain, osteocalcin, and runt-related transcription factor 2, downregulated activity of ALP, as well as reduced calcium nodule formation. Furthermore, we demonstrated that MLT could promote fracture healing in a rat model of femoral fracture, which was accompanied by the elevated expression of NPY and NPY1R. The administration of BIBP3226 inhibited fracture healing mediated by MLT. To sum up, our results show that MLT promotes osteoblastic differentiation of MSCs and fracture healing by NPY/NPY1R signaling.

ARRB1 inhibits extracellular matrix degradation and apoptosis of nucleus pulposus cells by promoting autophagy and attenuates intervertebral disc degeneration.

OBJECTIVE: Intervertebral disc degeneration (IVDD) is the leading cause of lower back pain (LBP). ß-arrestin 1 (ARRB1) is a multifunctional protein that regulates numerous pathological processes. The aim of this study was to investigate the role of ARRB1 in IVDD. METHODS: The expression of ARRB1 in nucleus pulposus (NP) of rats with IVDD was assayed. Next, rat nucleus pulposus cells (NPCs) were infected with lentiviruses containing shArrb1 (LV-shArrb1) and overexpressing Arrb1 (LV-oeArrb1). The roles of Arrb1 in serum-deprived NPCs were investigated by measuring apoptosis, extracellular matrix degradation, and autophagic flux. For experiments in vivo, LV-oeArrb1 lentivirus was injected into the NP tissues of IVDD rats to evaluate the effects of Arrb1 overexpression on NP. RESULTS: In the NP tissues of IVDD rats, ARRB1 and cleaved caspase-3 expression increased, and the ratio of LC3II/LC3I protein expression was upregulated. Arrb1 knockdown aggravated extracellular matrix degradation, cellular apoptosis, and impairment of autophagic flux in rat NPCs under serum-deprived conditions, whereas Arrb1 overexpression significantly reversed these effects. ARRB1 interacted with Beclin 1, and Arrb1 knockdown suppressed the formation of the Beclin1-PIK3C3 core complex. The autophagy inhibitor 3-methyladenine (3-MA) offset the protective effects of Arrb1 overexpression in serum-deprived NPCs. Furthermore, Arrb1 overexpression inhibited apoptosis and extracellular matrix degradation, promoted autophagy in NP, and delayed the development of IVDD in rats. CONCLUSION: ARRB1 prevents extracellular matrix degradation and apoptosis of NPCs by upregulating autophagy and ameliorating IVDD progression, presenting an innovative strategy for the treatment of IVDD.

Induction of Axillary Bud Swelling of Hevea brasiliensis to Regenerate Plants through Somatic Embryogenesis and Analysis of Genetic Stability.

To overcome rubber tree (RT) tissue culture explant source limitations, the current study aimed to establish a new Hevea brasiliensis somatic embryogenesis (SE) system, laying the technical foundation for the establishment of an axillary-bud-based seedling regeneration system. In this study, in vitro plantlets of Hevea brasiliensis Chinese Academy of Tropical Agricultural Sciences 917 (CATAS 917) were used as the experimental materials. Firstly, the optimum conditions for axillary bud swelling were studied; then, the effects of phenology, the swelling time of axillary buds (ABs), and medium of embryogenic callus induction were studied. Plantlets were obtained through somatic embryogenesis. Flow cytometry, inter-simple sequence repeat (ISSR molecular marker) and chromosome karyotype analysis were used to study the genetic stability of regenerated plants along with budding seedlings (BSs) and secondary somatic embryo seedlings (SSESs) as the control. The results show that the rubber tree's phenology period was mature, and the axillary bud induction rate was the highest in the 2 mg/L 6-benzyladenine (6-BA) medium (up to 85.83%). Later, 3-day-old swelling axillary buds were used as explants for callogenesis and somatic embryogenesis. The callus induction rate was optimum in MH (Medium in Hevea) + 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) + 1.5 mg/L 1-naphthalene acetic acid (NAA) + 1.5 mg/L Kinetin (KT) + 70 g/L sucrose (56.55%). The regenerated plants were obtained after the 175-day culture of explants through callus induction, embryogenic callus induction, somatic embryo development, and plant regeneration. Compared with the secondary somatic embryo seedling control, axillary bud regeneration plants (ABRPs) were normal diploid plants at the cellular and molecular level, with a variation rate of 7.74%.

Timosaponin B-II alleviates osteoarthritis-related inflammation and extracellular matrix degradation through inhibition of mitogen-activated protein kinases and nuclear factor-κB pathways in vitro.

Osteoarthritis (OA), an inflammatory response in chondrocytes, leads to extracellular matrix (ECM) degradation and cartilage destruction. Timosaponin B-II (TB-II) is the main bioactive component of Rhizoma Anemarrhenae with reported antioxidant and anti-inflammatory effects. This study investigated the anti-OA function and mechanism of TB-II on IL-1ß-stimulated SW1353 cells and primary rat chondrocytes. We firstly screened the concentration of TB-II in SW1353 cells and primary rat chondrocytes using CCK-8 assay. Thereafter, SW1353 cells and chondrocytes were, respectively, pretreated with TB-II (20 and 40 µg/mL) and TB-II (10 and 30 µg/mL) for 24 h and then stimulated with interleukin 1ß (IL-1ß, 10 ng/mL) for another 24 hours. Results showed that TB-II suppressed the production of reactive oxygen species, the protein levels of inducible nitric oxide synthase and cyclooxygenase-2 in IL-1ß-stimulated SW1353 cells and chondrocytes. IL-1ß-induced high secretion levels of nitric oxide and prostaglandin 2, TNF-α, IL-6 and MCP-1 were down-regulated by TB-II treatment, indicating an anti-inflammatory effect of TB-II on OA in vitro condition. Moreover, TB-II weakened the mRNA and protein expression of (matrix metalloproteinase) MMPs including MMP-1, MMP-3, and MMP-13, indicating the protection of TB-II against ECM degradation. Mechanically, TB-II suppressed MAPKs and NF-κB pathways under IL-1ß stimulation evidenced by the down-regulated protein expression of p-ERK, p-p38, p-JNK, p-p65 and the reduced translocation of p65 subunit to the nucleus. The present study demonstrated that TB-II might become a novel therapeutic agent for OA treatment through repressing IL-1ß-stimulated inflammation, oxidative stress and ECM degradation via suppressing the MAPKs and NF-κB pathways.

Non-oxidative pentose phosphate pathway controls regulatory T cell function by integrating metabolism and epigenetics.

Regulatory T (Treg) cells are critical for maintaining immune homeostasis and preventing autoimmunity. Here, we show that the non-oxidative pentose phosphate pathway (PPP) regulates Treg function to prevent autoimmunity. Deletion of transketolase (TKT), an indispensable enzyme of non-oxidative PPP, in Treg cells causes a fatal autoimmune disease in mice, with impaired Treg suppressive capability despite regular Treg numbers and normal Foxp3 expression levels. Mechanistically, reduced glycolysis and enhanced oxidative stress induced by TKT deficiency triggers excessive fatty acid and amino acid catabolism, resulting in uncontrolled oxidative phosphorylation and impaired mitochondrial fitness. Reduced α-KG levels as a result of reductive TCA cycle activity leads to DNA hypermethylation, thereby limiting functional gene expression and suppressive activity of TKT-deficient Treg cells. We also find that TKT levels are frequently downregulated in Treg cells of people with autoimmune disorders. Our study identifies the non-oxidative PPP as an integrator of metabolic and epigenetic processes that control Treg function.

Role of Zip1 in the regulation of NPY expression by MLT to promote fracture healing in rats.

Our previous study documented that melatonin (MLT) induced the osteogenic differentiation of mesenchymal stem cells (MSCs) and promoted the healing of femoral fractures in rats via the neuropeptide Y (NPY)/neuropeptide Y1 receptor (NPY1R) signaling pathway. MLT treatment upregulated the expression of the zinc uptake transporter zinc transporter 1 (Zip1) in nerve cells. Prior research demonstrated that oral zinc upregulated NPY expression. MSCs were isolated from rat bone marrow and identified using flow cytometry in our study. The results showed that MLT treatment upregulated NPY and NPY1R levels in MSCs with osteogenic differentiation, which was accompanied by upregulated Zip1 expression. However, the MLT-induced osteogenic differentiation of MSCs was reversed after interference of Zip1 expression. It was confirmed by the decreased alkaline phosphatase (ALP) level; downregulated activities of type I collagen α1 chain (COL1A1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2) and ALP; and reduced mineralized nodule formation. MLT promoted fracture healing in rats with femoral fracture, which was accompanied by increased expression of NPY and NPY1R and significantly increased expression of Zip1. In contrast, the silencing of Zip1 expression reversed MLT-mediated fracture healing. In summary, Zip1 participated in the regulation of the NPY/NPY1R signaling pathway via MLT to promote the osteogenic differentiation of MSCs and fracture healing.

Transketolase Deficiency Protects the Liver from DNA Damage by Increasing Levels of Ribose 5-Phosphate and Nucleotides.

De novo nucleotide biosynthesis is essential for maintaining cellular nucleotide pools, the suppression of which leads to genome instability. The metabolic enzyme transketolase (TKT) in the nonoxidative branch of the pentose phosphate pathway (PPP) regulates ribose 5-phosphate (R5P) levels and de novo nucleotide biosynthesis. TKT is required for maintaining cell proliferation in human liver cancer cell lines, yet the role of TKT in liver injury and cancer initiation remains to be elucidated. In this study, we generated a liver-specific TKT knockout mouse strain by crossing TKTflox/flox mice with albumin-Cre mice. Loss of TKT in hepatocytes protected the liver from diethylnitrosamine (DEN)-induced DNA damage without altering DEN metabolism. DEN treatment of TKT-null liver increased levels of R5P and promoted de novo nucleotide synthesis. More importantly, supplementation of dNTPs in primary hepatocytes alleviated DEN-induced DNA damage, cell death, inflammatory response, and cell proliferation. Furthermore, DEN and high-fat diet (HFD)-induced liver carcinogenesis was reduced in TKTflox/floxAlb-Cre mice compared with control littermates. Mechanistically, loss of TKT in the liver increased apoptosis, reduced cell proliferation, decreased TNFα, IL6, and STAT3 levels, and alleviated DEN/HFD-induced hepatic steatosis and fibrosis. Together, our data identify a key role for TKT in promoting genome instability during liver injury and tumor initiation. SIGNIFICANCE: These findings identify transketolase as a novel metabolic target to maintain genome stability and reduce liver carcinogenesis.

Effects of ginsenoside Rb1 on oxidative stress injury in rat spinal cords by regulating the eNOS/Nrf2/HO-1 signaling pathway.

The present study aimed to investigate whether ginsenoside Rb1 (G-Rb1) attenuates spinal cord injury-associated oxidative stress in rats by regulating the endothelial nitric oxide synthase eNOS/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway. Sprague Dawley rats were randomly divided into the sham operation group (S group), spinal cord injury group (SCI group), G-Rb1 treatment group (G-Rb1 group) and SCI+G-Rb1+Inhibitor L-name group (L-name group). The posterior limb function was evaluated via the Basso, Beattie and Bresnahan scoring method. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione (GSH) in serum were measured by ELISA. The pathological changes in the spinal cord were observed by H&E staining. Reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect eNOS, phosphorylated (p)-eNOS, heat shock protein (HSP)90, Nrf2 and NAD(P)H quinone dehydrogenase 1 (Nqo1) at the mRNA and protein level. Immunohistochemistry was used to detect the expression of Nrf2 and p-eNOS. Compared with the S group, the scores of spinal cord function in the SCI group were significantly lower, and the levels of MDA were significantly increased, while the levels of SOD, CAT and GSH protein in spinal cord were significantly decreased (P<0.05). The spinal cord tissue exhibited hemorrhage, neuronal degeneration/necrosis, as well as mononuclear cell and lymphocyte infiltration. The eNOS, HSP90, Nrf2, Nqo1 and HO-1 mRNA levels were decreased (P<0.05). Compared with those in the SCI group, the spinal cord function score in the G-Rb1 group were significantly higher and the serum MDA content was significantly decreased, while the activity of SOD, CAT and GSH was significantly increased (P<0.05). The degeneration/necrosis of spinal cord neurons was attenuated, inflammatory cell infiltration was significantly reduced and the levels of eNOS, HSP90, Nrf2, Nqo1 and HO-1 were significantly upregulated (P<0.05). In the group that was administered the eNOS inhibitor L-name, the levels of eNOS, HSP90, Nrf2, Nqo1 and HO-1 were significantly decreased. In conclusion, G-Rb1 attenuates oxidative stress in injured spinal cords. The mechanism may at least in part involve the eNOS/Nrf2/HO-1 pathway.

Neuropeptide Y accelerates post-fracture bone healing by promoting osteogenesis of mesenchymal stem cells.

Fracture repair is a complex yet well orchestrated regenerative process involving numerous signaling and cell types including osteoblasts. Here we showed that NPY, a neurotransmitter with regulatory functions in bone homeostasis, may contribute to the post-fracture bone healing in patients with traumatic brain injury-fracture combined injuries. Our results suggested NPY levels were increased in patients with the combined injuries, accomplished by arising of bone healing markers, such as ALP, OC, PICP and ICTP, than in those with simple fractures, and NPY have direct actions on MSCs to promote their osteogenic differentiation. Our results provided clinical evidences for NPY participating in the bone healing process in a nonhypothalamic manner, most probably by directly promoting osteogenesis of mesenchymal stem cells.

Neuroprotective effect of allicin in a rat model of acute spinal cord injury.

AIMS: This study aims to investigate the effect of allicin on motor functions and histopathologic changes after spinal cord injury and the mechanism underlying its neuroprotective effects. MAIN METHODS: The motor function of rats was evaluated with the Basso, Beattie, and Bresna test. Histopathologic changes were evaluated by hematoxylin and eosin and Nissl staining. Spinal cord oxidative stress markers were determined by measuring glutathione and malondialdehyde content and superoxide dismutase activity using commercial kits. Inflammatory factors were determined by measuring tumor necrosis factor-α, interleukin-1ß and interleukin-6 using ELISA assay. Apoptosis was examined using TUNEL staining. The effect of allicin on Nrf2 protein levels and localization was assessed using immunofluorescence staining and Western blotting analysis. KEY FINDINGS: Results demonstrated that allicin accelerated the motor functional recovery and protected neuron damage against spinal cord injury (SCI). SCI-induced oxidative stress, inflammatory response and cell apoptosis in the spinal cord were also prevented by allicin. In addition, we observed that SCI increased Nrf2 nuclear expression, and allicin treatment further increased Nrf2 nuclear translocation in neurons and astrocytes. siRNA-mediated Nrf2 gene knockdown completely blocked the effect of allicin on spinal cord tissue. SIGNIFICANCE: Our finding suggests that allicin promotes the recovery of motor function after SCI in rats, and this effect may be related to its anti-oxidant, anti-inflammatory and anti-apoptotic effects. Allicin mediated Nrf2 nuclear translocation may be involved in the protective effect as well.

A Comparison of Anterior Cervical Corpectomy and Fusion Combined With Artificial Disc Replacement and Cage Fusion in Patients With Multilevel Cervical Spondylotic Myelopathy.

STUDY DESIGN: A retrospective study. OBJECTIVE: The aim of this study was to compare clinical and radiological outcomes of anterior cervical corpectomy and fusion (ACCF) combined with artificial disc replacement (C-ADR) and ACCF combined with anterior cervical discectomy and fusion (ACDF) in patients with consecutive 3-level cervical spondylotic myelopathy (CSM). SUMMARY OF BACKGROUND DATA: The optimal surgical strategy for multilevel CSM (MCSM) remains undefined. C-ADR maintains motion at the level of the surgical procedure and decreases strain on the adjacent segments. The clinical results of multilevel C-ADR have not yet been elucidated. ACCF combined with 1-level C-ADR for the treatment of consecutive 3-level CSM may be a reasonable alternative to 3-level fusion. METHODS: We retrospectively reviewed the histories of patients who underwent surgery for consecutive 3-level CSM between C3-4 and C6-7 from June 2007 to August 2011. A total of 42 patients were divided into 2 groups. Group A (n = 19) underwent ACCF combined with 1-level C-ADR; group B (n = 23) underwent ACCF combined with 1-level ACDF. We compared perioperative parameters, clinical parameters, and radiological parameters. RESULTS: There were no significant differences in the average age, sex ratio, the preoperative heights of the disc space or average blood loss between the 2 groups. Group A had longer operation times than group B (P < 0.05). During the follow-up period, group A showed a better Neck Dysfunction Index recovery (P < 0.05) at 24 months postoperatively, and less visual analogue scale scores at 12 and 24 months postoperatively (P < 0.05 and P < 0.001, respectively). Moreover, group A exhibited better C2-C7 range of motion recovery at 6, 12, and 24 months postoperatively (P < 0.05, respectively). CONCLUSION: Group A was superior to Group B in terms of better Neck Dysfunction Index recovery, less intermediate term pain, and better C2-C7 ROM recovery. ACCF hybrid 1-level C-ADR may be a suitable choice for the management of 3-level CSM in appropriate patients. LEVEL OF EVIDENCE: 3.