Luming Nong

Periodic Mechanical Stress Activates PKCδ-Dependent EGFR Mitogenic Signals in Rat Chondrocytes via PI3K-Akt and ERK1/2.

Background/Aims: The present study aimed to analyze the mechanisms by which periodic mechanical stress is translated into biochemical signals, and to verify the important role of signaling molecules including phosphatidylinositol-3-kinase (PI3K)-Akt, protein kinase C (PKC), and epidermal growth factor receptor (EGFR) in chondrocyte proliferation. The effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively in recent years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to periodic mechanical stress need further investigation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, GF109203X, Gö6976, rottlerin, and AG1478. They were maintained under static conditions or periodic mechanical stress for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. In the second step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, AG1478, and rottlerin. They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis. Results: Proliferation was inhibited by pretreatment with PKC or PKCδ inhibitor GF109203X or rottlerin and by short hairpin RNA (shRNA) targeted to PKCδ, but not by PKCα inhibitor Gö6976 in chondrocytes in response to periodic mechanical stress. Meantime, rottlerin and shRNA targeted to PKCδ also attenuated EGFR, Akt, and ERK1/2 activation. Furthermore, inhibiting EGFR activity by AG1478 and shRNA targeted to EGFR abrogated chondrocyte proliferation and phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK)1/2 subjected to periodic mechanical stress, while the phosphorylation site of PKCδ was not affected. In addition, pretreatment with the PI3K-Akt-selective inhibitor LY294002 and shRNA targeted to Akt reduced periodic mechanical stress-induced chondrocyte proliferation and phosphorylation of ERK1/2, while the phosphorylation levels of EGFR and PKCδ were not inhibited. Conclusion: These findings suggested that periodic mechanical stress promoted chondrocyte proliferation through PKCδ-EGFR-PI3K-Akt-ERK1/2. They provide a stronger viewpoint for further investigations into chondrocyte mechanobiology under periodic mechanical stress and the ways to improve the quality of tissue-engineered cartilage.

Application of lamina replantation with ARCH plate fixation in thoracic and lumbar intraspinal tumors

The aim of the present study was to investigate the clinical effects of lamina replantation with ARCH plate fixation on patients with thoracic and lumbar intraspinal tumors, following laminectomy. Thirteen patients with thoracic and lumbar intraspinal tumors underwent total lamina replantation with ARCH plate fixation and repair of the supraspinous ligaments, following laminectomy and tumor enucleation. To investigate the clinical effect of lamina replantation with ARCH plate fixation, pre- and postoperative visual analog scale (VAS), and Oswestry Disability Index (ODI) scores were determined, and pre- and postoperative X-ray and magnetic resonance imaging (MRI) examinations were conducted. Computed tomography (CT) examinations were also included in the follow-up. No complications were observed pre- or postoperatively. The VAS and ODI results 2 weeks following surgery and at the final follow-up examination demonstrated a significant improvement compared with the corresponding preoperative results. The X-ray examination results indicated a satisfactory internal fixation location, without any characteristics of a fracture, lumbar scoliosis, kyphosis or instability. Following the surgery, the CT and MRI examination results demonstrated that healing of the lamina bone and repair of the supraspinous ligament had occurred without tumor recurrence or spinal epidural scar recompression. Two of the 13 cases were lost to follow-up. The results indicated that in patients with thoracic and lumbar intraspinal tumors, lamina replantation with ARCH plate fixation following total laminectomy is effective and provides thoracolumbar stability. Furthermore, this has been identified to be an effective technique for preventing intraspinal scar proliferation.

An updated meta-analysis of the Fc receptor-like 3 –169T/C polymorphism and rheumatoid arthritis risk

Objectives: Published studies have shown conflicting results concerning the association between the –169T/C promoter polymorphism in the Fc receptor-like 3 (FCRL3) gene and rheumatoid arthritis (RA). In this study we conducted an up-to-date meta-analysis to examine the relationship. Method: We searched the PubMed database for all papers published up to 20 April 2012. Overall, 18 case–control studies with 12 620 cases and 12 613 controls were retrieved based on the search criteria for RA susceptibility related to the FCRL3 –169T/C polymorphism. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of this association. Publication bias was assessed using the Egger test. Results: We found that the FCRL3 –169T/C polymorphism increased the risk for RA overall in genetic models (allelic contrast: OR 1.09, 95% CI 1.03–1.14, p = 0.001; homozygote comparison: OR 1.20, 95% CI 1.08–1.34, p = 0.001; dominant genetic model: OR 1.03, 95% CI 1.01–1.05, p = 0.001). Stratified analysis by race also showed a significant positive association with Asians and Caucasians. Subgroup analysis of rheumatoid factor (RF) revealed a slightly positive relationship between the FCRL3 –169T/C polymorphism and RF-positive RA risk. No obvious evidence of publication bias was detected in the overall analysis. Conclusion: Our study indicates that the FCRL3 –169T/C polymorphism is significantly associated with increased RA risk.

Exploration about changes of IL-10, NF-κB and MMP-3 in a rat model of cervical spondylosis

Objectives To investigate the relationship and mechanism between IL‐10, NF‐&kgr;B and MMP‐3 in cervical degenerative disease induced by unbalanced dynamic and static forces in rats. Methods Sixty Sprague Dawley rats were randomized into test (n = 45) and control (n = 15) groups, which were randomly subdivided into three groups corresponding to one‐month, three‐month and six‐month post‐operation. Test group included 10, 15, 20 rats at corresponding postoperative stage and control group had five rats at each time point. By excising cervicodorsal muscles and ligaments of rats to establish unbalanced dynamic and static rat model in test group. The expression of IL‐10, NF‐&kgr;B and MMP‐3 in the intervertebral disc tissue samples of both test and control group rats were detected by immunohistochemistry at one‐month, three‐month and six‐month post‐operation. The results were analyzed and compared among groups. Results Compared with the control group, the positive expression of IL‐10 in test group was significantly higher at three‐month (P < 0.05). In the same model group, IL‐10 was highest at one‐month. Compared with the control group, NF‐kB in test group was higher at one‐month, three‐month and six‐month. In the same model group, NF‐kB was the highest at one‐month, followed by the time at three‐month and six‐month. And, compared with the control group, MMP‐3 was significantly higher in test group at one‐month (P < 0.05). Conclusion Cervical degeneration may accompanied with the changes of IL‐10, NF‐&kgr;B and MMP‐3. HighlightsWe successfully established the rat cervical model with non‐equilibrium of dynamic and static forces, histologic evidence of different degrees of cervical disc degeneration was seen in all test group at each time point postoperation.Cervical degeneration may accompanied with the changes of IL‐10, NF‐&kgr;B and MMP‐3.On the basis of our research, we speculate that some IL‐10 were produced in the process of IVD degeneration, which may activate NF‐&kgr;B signaling pathway.Activated NF‐&kgr;B drives expression of target genes that release of IL‐10 to activate the immune response in turn.IL‐10 suggests a dysregulation of aggrecan aseactivity in human degenerative IVD by damaging the balance between MMPs and TIMPs.

Lamina replacement with titanium plate fixation improves spinal stability after total lumbar laminectomy

Biomechanical experiments and strain analyses were performed to investigate the effects of lamina replacement surgery for intraspinal lesions on postoperative spinal stability. Eight specimens of thoracic and lumbar vertebrae (T12–L4) were collected from adult cadavers. Stepwise lumbar total laminectomy, and laminoplasty with lamina reduction and replacement was undertaken in combination with titanium-plate fixation to simulate the surgical setting. The effects of thoracic and lumbar vertebral strain, displacement, and rigidity on spinal stability were measured following both single and multiple segment laminectomy. Significant differences in mechanical indices of stability were seen between stepwise laminectomy of lumbar vertebrae and normal specimens (p < 0.05), between lamina replacement in combination with titanium-plate fixation and laminectomy (p < 0.05), and between single- and multiple-segment laminectomy (p < 0.05). Differences between laminoplasty with lamina replacement in combination with titanium-plate fixation and normal specimens need to be examined for further study. Lumbar laminectomy followed by reduction and replacement, in combination with titanium-plate fixation, was shown to be beneficial in terms of preserving spinal stability and maintaining biomechanical function and spinal loading capability.

Periodic Mechanical Stress Stimulates GIT1-Dependent Mitogenic Signals in Rat Chondrocytes Through ERK1/2 Activity

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

Periodic Mechanical Stress Stimulates Cav-1-Dependent IGF-1R Mitogenic Signals in Rat Chondrocytes Through ERK1/2

Background/Aims: The biological effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to mechanical stimuli remain to be determined. In the current study, we analyzed the mechanisms by which periodic mechanical stress is translated into biochemical signals and verified the key role of non-integrin mechanosensors including Caveolin-1 (Cav-1), and insulin-like growth factor-1 receptor (IGF-1R) in chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under static conditions or periodic mechanical stress for 0 h and 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to Cav-1 or IGF-1R or control scrambled shRNA. Moreover, they were pretreated with their selective inhibitors methyl β-cyclodextrin (MCD) or Linsitinib (OSI-906). They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of Cav-1 at Tyr14 and IGF-1R at Tyr1135/1136. Proliferation was inhibited by pretreatment with Cav-1 inhibitor MCD and by shRNA targeted to Cav-1 in chondrocytes in response to periodic mechanical stress. Meantime, MCD and shRNA targeted to Cav-1 also attenuated IGF-1R, and extracellular signal-regulated kinase (ERK)1/2 activation. In addition, inhibiting IGF-1R activity by Linsitinib and shRNA targeted to IGF-1R abrogated chondrocyte proliferation and phosphorylation level of ERK1/2 subjected to periodic mechanical stress, while the phosphorylation site of Cav-1 was not affected. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through Cav-1-IGF-1R-ERK1/2.

Periodic Mechanical Stress Induces Extracellular Matrix Expression and Migration of Rat Nucleus Pulposus Cells Through Src-GIT1-ERK1/2 Signaling Pathway

Background/Aims: Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. Methods: Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. Results: Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. Conclusion: In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.

Experience of operative treatment in 27 patients with intraspinal neurilemmoma

To investigate the clinical outcomes of posterior laminectomy on intraspinal neurilemmoma. Twenty-seven patients with intraspinal neurilemmoma admitted to the Second Peoples Hospital of Changzhou Affiliated to Nanjing Medical University from January 2010 to October 2015 were selected, and they received posterior laminectomy, and there were 5 patients whose nerve root and tumor were removed together because they could not be separated; 17 patients receiving total laminectomy or semi-laminectomy combined with facetectomy on the affected side were treated with spinal dynamic pedicle screw fixation or replantation of lamina or bone graft fusion and internal fixation. The patients were followed up regularly to observe the clinical outcomes after operation. The spinal stability and bone graft fusion were observed via X-ray film in review. Whether there was tumor recurrence was observed via MRI in review according to the individual condition. The operation time was 2.5–8 h (4.2 h on average). The intraoperative blood loss was 420–1,500 ml (760 ml on average). Tumors in 26 patients were totally resected, and tumor in 1 patient was partially resected due to tumor tissue invasion against the anterior sacral vascular plexus. All patients were pathologically confirmed as neurilemmoma after operation. Ten patients suffered from cerebrospinal fluid leakage in different degrees after operation. During the follow-up for 6–75 months after operation, clinical symptoms and neurological functions of all patients were significantly improved; there was 1 case of tumor residual and no recurrence. During the follow-up, the internal fixation in all patients receiving internal fixation of spine was stable with no vertebral instability. Posterior laminectomy can achieve satisfactory clinical outcomes in the treatment of intraspinal neurilemmoma. Internal fixation is needed to reestablish spinal stability during operation for those patients with great damage to spinal stability.

MicroRNA‑21 promotes neurite outgrowth by regulating PDCD4 in a rat model of spinal cord injury

Altered expression levels of microRNA-21 (miRNA-21) have been observed in a series of pathological processes, including cancer and central nervous system injury; however, the involvement of miRNA-21 in the molecular pathophysiology of spinal cord injury (SCI) has not been well documented. The present study examined the expression levels of miRNA-21 and its predicted target genes, programmed cell death 4 (PDCD4) and phosphatase and tensin homolog (PTEN), in rats using quantitative polymerase chain reaction and western blotting to further understand the role of miRNA-21 and the mechanisms underlying repair following SCI. The present study demonstrated that compared with uninjured spinal cords, miRNA-21 expression levels were significantly downregulated in injured spinal cords 4 and 8 h, and 1 day post-SCI, and were significantly upregulated after 3 and 7 days. Conversely, expression levels of PDCD4 and PTEN were significantly decreased at days 3 and 7 post-SCI compared with the control group. miRNA-21 overexpression in monolayer-cultured postnatal rat spinal cord neurons promoted neurite outgrowth and downregulated protein expression levels of PDCD4; however, PTEN protein expression levels were unaltered. To confirm that miRNA-21 directly targets PDCD4, a pRL-CMV luciferase reporter construct was used to detect miRNA-21 interactions with the PDCD4 3′-untranslated region. The results demonstrated that miRNA-21 decreased luciferase activity compared with a rat PDCD4 control reporter. The results of the present study suggested that increased miRNA-21 expression levels following SCI may promote the repair of injured spinal cords by inhibiting the expression of its target gene PDCD4.