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Objective@#To investigate the mechanisms of Pyrroloquinoline quinone (PQQ) against oxidative stress induced apoptosis in Schwann cells (SCs).@*Methods@#SCs were cultured in vitro, identified by S-100 immunofluorence staining. SCs were divided into control group, H2O2 induced group, H2O2 + PQQ treated group. CCK-8 assay was used to detect cell proliferation. Apoptosis was detected by flow cytometry with Annecin V-FITC/PI staining, mitochondrial transmembrane potential was detected by flow cytometry with JC-1 labeled staining, cytochrome C (CytC), Bax and Caspase-9 protein levels was detected by Western blot analysis.@*Results@#In this study, the S-100 positive cells were more than 95%, cell proliferation was decreased in H2O2 induced SCs, apoptotic rate was increased, mitochondrial transmembrane potential was decreased, CytC, Bax and Caspase-9 protein levels were increased. After PQQ added, cell proliferation was increased, apoptotic rate decreased, mitochondrial transmembrane potential increased, CytC, Bax and Caspase-9 protein levels decreased.@*Conclusions@#PQQ protects SCs from oxidative induced apoptosis by inhibiting mitochondrial signaling pathway.
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BACKGROUND:Lumbar fusion is a conventional effective measure to treat spondylolisthesis, spinal stenosis or with deformity. Bilateral pedicle screw fixation is recognized as the standard treatment for various spinal disorders, and has biomechanical and clinical advantages. OBJECTIVE:To evaluate the effects of bilateral pedicle screw fixation in the repair of lumbar disc herniation to restore disc height from the angle of imaging. METHODS: Clinical data of 42 patients with lumbar disc herniation were retrospectively analyzed. They al received bilateral pedicle screw fixation. Pain was evaluated before implantation, immediately and 1 month after implantation using Japanese Orthopaedic Association score of lower back pain and visual analog scale score. X-ray including anteriorposterior and lateral films of lumbar spine and MRI were used. CT was utilized to verify screw placement conditions and complications. RESULTS AND CONCLUSION:A total of 42 patients were folowed up for 3-6 months. Compared with pre-implantation, Japanese Orthopaedic Association score and visual analog scale score were significantly improved immediately after implantation (P 0.05). The height of intervertebral discs was significantly higher immediately and 1 month after implantation than pre-implantation (P < 0.01). The symptoms were lessened after fixation in al cases, and their qualities of life elevated. At 1 month, X-ray films and CT images revealed that no screw loosening, breakage or displacement occurred. The height of intervertebral discs was perfectly restored. No adverse events appeared in patients. These data indicate that bilateral pedicle screw fixation for lumbar intervertebral disc herniation can effectively restore the height of intervertebral discs, improve clinical symptoms and have biological and clinical superiority.
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BACKGROUND:It has been confirmed that carboxymethylated chitosan has an promoting effect on Schwann cel proliferation and secretion, but its impact on the cyclic adenosine monophosphate-mediated protein kinase A signaling pathway in schwann cel stil needs further study. OBJECTIVE:To investigate the effect of carboxymethylated chitosan on cyclic adenosine monophosphate/ protein kinase A signaling pathway in rat schwann cels. METHODS:The Schwann cels of the second generation neonatal rats were obtained and seeded in 6-wel plate at a concentration of 1×109/L. These Schwann cels were cultured and divided into four groups. The Schwann cels in the control group were cultured by adding PBS. The Schwann cels in the experimental groups were cultured by adding 50, 100 and 200 mg/L of carboxymethyl chitosan solution, respectively. After 24 hours, the concentration of cyclic adenosine monophosphate, protein kinase A activity and cyclic adenosine monophosphate response element binding protein mRNA expression were detected. RESULTS AND CONCLUSION:Compared with the control group, carboxymethyl chitosan increased cyclic adenosine monophosphate concentrations, the activity of protein kinase A and cyclic adenosine monophosphate response element binding protein mRNA expression within the Schwann cels in a dose-dependent manner (P < 0.05). These results demonstrate that carboxymethyl chitosan can increase the concentration of cyclic adenosine monophosphate within the Schwann cels and promote protein kinase A activity, thereby activating cyclic adenosine monophosphate/protein kinase A signaling pathway.
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Objective To investigate the protective effects of carboxymethylated chitosan (CMCS) on nitric oxide (NO) induced apoptosis in rat chondrocytes, and the probable roles and mechanisms of PI3K/Akt signaling pathway in these process.Methods Rat knee articular cartilage was used as the source of chondrocytes, the cells were identified by immunohistochemical staining against collagen type Ⅱ, odium nitroprusside (SNP, 3 mmol/L) was used to establish the apoptotic models of chondrocytes.Cells were divided into four groups: the control group, the SNP-induced group, the SNP+CMCS treated group, the SNP+CMCS+PI3K inhibitor Wortmannin treated group.Cell proliferation were assessed by cell proliferation assay kit (CCK-8), the apoptotic rate of chondrocytes was determined by FCM with Annexin V-FITC/PI double staining, the expression levels of MMP-13 and TIMP-1 mRNA were detected by real-time polymerase chain reaction (PCR) analysis, the expression of Akt and p-Akt protein levels was detected by Western blotting analysis.One-way analysis of variance (ANOVA) statistical analysis was used to calculate the data.Results Three mmol/L SNP can inhibit proliferation (0.221±0.023), and the proliferation was reduced by 70％ compared with the control group (0.736±0.032, F=8.203, P=0.021);and the induced apoptosis in cultured chondrocytes could be observed.The apoptotic rate was (68.8±5.2)％.Increased MMP-3 and decreased TIMP-1 mRNA expression were observed in SNP-induced cells.After adding CMCS to SNP-induced chondrocytes, the proliferation was increased while apoptotic rate was decreased, the apoptotic rate decreased to (14.7±2.3)％.CMCS could promote the activation of p-Akt in SNP-induced chondrocytes and restore SNP-induced MMP-13 and TIMP-1 mRNA expression.Conclusion CMCS could protect apoptosis in SNP induced chon-drocvtes via activation of PI3K/Akt pathwav.
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BACKGROUND:Carboxymethylated chitosan is shown to promote some kinds of cells proliferation, but its effects on proliferation of Schwann cells need further studies. OBJECTIVE:To investigate the effects of carboxymethylated chitosan on proliferation of Schwann cells and expression of nuclear factor-κB in cultured Schwann cells. METHODS:Schwann cells from Sprague-Dawley rats at logarithmic growth phase were seeded in 96-wel plates, and cultured respectively with PBS, 0, 10, 50, 100, 200, 500, 1 000 mg/L carboxymethyl chitosan for 24 hours. cellproliferation was detected using the cellcounting kit-8 assay. After trypsin digestion, Schwann cells from Sprague-Dawley rats at logarithmic growth phase were used to prepare cellsuspensions, which were seeded in 6-wel cellculture plates and cultured respectively with 50, 100 and 200 mg/L carboxymethyl chitosan and PBS for 24 hours. Then, 5-bromo-2-deoxyuridine, real-time PCR and western blot assay were performed. RESULTS AND CONCLUSION:cellcounting kit-8 and 5-bromo-2-deoxyuridine detection results showed that carboxymethyl chitosan at 50-1000 mg/L, especial y at 200-500 mg/L, could promote Schwann cellproliferation. Real-time PCR and western blot results showed 50-200 mg/L carboxymethyl chitosan could promote nuclear factorκB mRNA and protein expression in Schwann cells in a dose-dependent manner, suggesting carboxymethyl chitosan can promote Schwann cellproliferation and expression of nuclear factor-κB in Schwann cells cultured in vitro.
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BACKGROUND:Recently, non-fusion technology representing as artificial cervical disc replacement continues to improve. On the basis of reconstruction of disc structure and function of involved segments, cervical spine structure of surgery area segment is significantly close to dynamic and static load stress distribution required by natural physiological systems. It effects are apparent in protecting intervertebral facet joints of degenerated segment and structure and function of the cervical spine of adjacent segments and in maintaining cervical dynamic stability, which presented obvious methodological strengths compared with segmental fusion technology. OBJECTIVE:To evaluate the clinical outcomes of anterior cervical discectomy and fusion and Bryan artificial cervical disc replacement in the treatment of single-level cervical spondylotic myelopathy or radiculopathy. METHODS:A total of 43 middle and old age patients with single-level cervical spondylotic myelopathy or radiculopathy, who were treated from March 2010 to March 2012, were enrol ed in this study. They were randomly assigned to anterior cervical discectomy and fusion group (fusion group) and Bryan artificial cervical disc replacement group. Range-of-motion of cervical overal and adjacent intervertebral area near the intervertebral space was observed with radiography. During fol ow-up, postoperative recovery of neurological function was evaluated using Japanese Orthopaedic Association scale, visual analog scale and neck disability index. RESULTS AND CONCLUSION:None patients experienced complications of neurovascular injury during and after the surgery. Range-of-motion of postoperative overal cervical vertebra and adjacent joint was improved in the Bryan artificial cervical disc replacement group compared with the fusion group. Neurological function was apparently improved after surgery in each group. At 3 months after surgery, scores of Japanese Orthopaedic Association, visual analog scale and neck disability index were significantly improved in the Bryan artificial cervical disc replacement group compared with the fusion group (P<0.05). During final fol ow-up, there were significant differences in visual analog scale scores between the two groups. Japanese Orthopaedic Association scale score and neck disability index score were similar between the two groups. During fol ow-up, no prosthesis sinking, displacement or heterotopic ossification were detected. These data indicated that artificial cervical disc replacement could effectively keep the range of motion of cervical segments and protect disc degeneration of adjacent segment. Mid-term fol ow up obtained similar improvement of neurological function of fusion surgery. The moderate-term and short-term efficacies of non-fusion technology were better than fusion technology in the treatment of single-level cervical spondylopathy.
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<p><b>OBJECTIVE</b>To investigate the effects of Pyrroloquinoline quinine (PQQ) on hydrogen peroxide-induced apoptosis of Schwann cells (SCs) and its mechanism.</p><p><b>METHODS</b>SCs were isolated and cultured in vitro, and identified by S-100 immunofluorescence staining. The cultured SCs were divided into control group, hydrogen peroxide-treated group, hydrogen peroxide and PQQ treated groups. The intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) content was detected; the apoptotic rate of SCs induced by hydrogen peroxide was determined by flow cytometry assay. The Hoechst33342 staining was used to detect the nuclear fragmentation and apoptotic nuclear condensation of SCs; the Rhodamine123 staining was used to detect the changes of mitochondrial membrane potential in SCs, the Western blot analysis was used to detect the expression of Bcl-2 in hydrogen peroxide induced SCs.</p><p><b>RESULTS</b>The SOD activity was significantly decreased and MDA level was increased in H2O2 induced SCs (P < 0.05), after addition of PQQ, the SOD content increased and MDA content decreased (P < 0.05). Flow cytometry results showed that the early apoptotic rate was 58.8% in H2O2 induced SCs, which has significant difference compared with the control group (P < 0.05), after addition of 10, 50, 100 nmol/L PQQ, the apoptotic rates were reduced to 33.7%, 18.7%, 3.9% respectively, showing significantly different with injured group (P < 0.05). Hoechst 33342 staining showed that H2O2 induced SCs had typical morphological characteristics, such as uptake of nuclear chromatin, nuclear shrinkage, nuclear fragmentation phenomenon. The proportion of apoptotic cells after PQQ treatment reduced. Rhodamine staining results showed that the H2O2 induced mitochondrial membrane potential reduction in SCs, which was reversed by addition of PQQ. Western blot analysis showed that the expression of Bcl-2 was decreased in H2O2 induced SCs, while it increased significantly after addition of PQQ (P < 0.05).</p><p><b>CONCLUSION</b>PQQ has a protective effect on oxidative stress-induced apoptosis of SCs.</p>
Subject(s)
Humans , Apoptosis , Benzimidazoles , Cell Nucleus , DNA Fragmentation , Fluorescent Dyes , Hydrogen Peroxide , Pharmacology , Malondialdehyde , Metabolism , Oxidants , Pharmacology , Oxidative Stress , Pyrroles , Pharmacology , Quinine , Pharmacology , Quinolines , Pharmacology , Schwann Cells , Cell Biology , Superoxide Dismutase , MetabolismABSTRACT
Objective To study the effects of carboxymethylated chitosan (CMCS) to nitric oxide (NO)-induced apoptosis on rat chondrocytes,and explore p38MAPK signal transduction pathway in the process and its mechanism.Methods The rat articular cartilage cells were cultured in vitro,collagen type-2 (collagen-2) immunohistochemical staining was used to identify the cartilage cells.The model of chondrocyte apoptosis was built by different concentrations of sodium nitroprusside (SNP) induction.The cells were divided into the control group,the SNP treated group SNP+CMCS treated group,and the SNP+p38 MAPK inhibitor SB203580 treated group.The apoptotic rate of chondrocytes was calculated by FCM,apoptotic nuclei was identified by Hoechst33342 stain,the mitochondrial membrane potential changes was detected by Rhodamine123 (Rho123) stain,the expression of p38 and p-p38 were detected by Western blotting analysis.Results 1-3 mmol/L SNP could induce chondrocyte apoptosis,the apoptotic rate was increased with the SNP increasing,the most obvious apoptosis was occurred in 3 mmol/L SNP treated chondrocytes,which was 69.8％ (P＜0.05).SNP could increase the nuclear fragmentation of chondrocytes,the cells with nuclear fragmentation was significantly higher than that in the control group.SNP could reduce mitochondrial membrane potential in chondrocytes,which decreased significantly compared with the control group.SNP could increase the p-p38 expression in chondrocytes,which was 4.3 times compared to the control group.CMCS of different concentrations could reduce the apoptotic rate of SNP-induced chondrocytes,which was 51.0％,29.9％ and 15.2％,which was decreased significantly (P＜0.05) when compared with 3 mmol/L SNP induced group,CMCS decreased the cells number of SNP-induced nuclear fragmentation.CMCS increased the mitochondrial membrane potential in SNP-induced chondrocytes.CMCS reduced the expression levels of p-p38 in SNP-induced chondrocytes.Conclusion CMCS has protective effect on SNP-induced apoptosis of chondrocytes.This process is completed by inhibiting the activity of p38 MAPK signal pathway.
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BACKGROUND:Compared with traditional autogenous bone graft, composite, as the carrier of seed cells, possesses advantages of fewer traumas and no limitation of donor site in repairing bone defect.OBJECTIVE: To observe the ability of the composite of β-tricalcium phosphate artificial bone-hyaluronic acid-type I collagen (β-TCP/HA/COL-I), as induced bone marrow stromal cell (MSC) carrier, to repair rabbit radial defect, and the feasibility with the composite as bone substitute material.DESIGN: A randomized and controlled trial.SETTING: Department of Orthopaedics, Renmin HospitaL, Wuhan University.MATERIALS; The study was conducted in the Department of Orthopaedics Renmin Hospital, Wuhan University between September 2003 and July 2004. Thirty-one New Zealand big white rabbits, aged 6 months, with body mass of 1.5 to 2.0 kg were enrolled in this study. The rabbits were randomized into control group (n =4) and experimental group (n =27).METHODS : ①In vitro induction and culture of MSCs was performed on 31 white rabbits, and the alkaline phosphatase (ALP) positive ratio of induced MSCs was observed. The structure of β-TCP/HA/COL-I was observed under scanning electron microscope. ② A 2 cm radial defect was created through operation. Eight weeks later, composites of β-TCP/HA/COL-I/MSCs were implanted into one side of rabbits in the experimental group, and autogenous bone was implanted into the other side. Rabbits in the control group were untouched. ③All the animals in the experimental group were randomly sacrificed at postoperative 4,8 and 12 weeks, 6 rabbits at 4 and 8 weeks, 15 at 12 weeks; Animals in the control group were sacrificed at 12 weeks. Gross observation, X-ray photographing, haematoxylin-eosin (HE) dyeing, and assessment of inorganic ingredient were performed. Osteogenic area and biomechanical tests were performed at 12weeks. Repairing effects on bone defect in each group were compared.MAIN OUTCOME MEASURES: The ALP positive ratio of induced MSCs; The structure of composite ofβ-TCP/HA/COL-I;Gross observation; X-ray photographing; HE dyeing and assessment of inorganic ingredient; Osteogenic area and biomechanical tests.RESULTS: All the 31 rabbits entered the stage of result analysis. ① The ALP positive ratio of cells reached 75% after induction and culture. ② Scanning electron microscope observation showed that 3 kinds of materials with abundant cellular structure distributed evenly. ③ The osteogenic area at 12 weeks was (72.5±3.6)% and (76.7±6.2)% in the experimental group and autogenous group, respectively (P ＞ 0.05). ④The maximum bending moment was (521.0±61.1) and (554.3±53.3)N·mm in the experimental group and control group, respectively; The maximum displacement at point of application of force was (0.816±0.071)and (0.870±0.103)respectively, without significant difference (P ＞ 0.05). ⑤Inorganic ingredient in the composite was 75%, 57% and 42% at 4,8 and 12 weeks respectively, suggesting that the inorganic component in the material was gradually decomposed with the elongation of time. ⑥Results of gross observation,X-ray photographing, histopathological examination, biomechanical test showed that with the elongation of time, composite of β-TCP/HA/COL-I/MSCs could repair bone defect in the experimental group, while bone defect in the control group had not been repaired.CONCLUSTON: Composite of β-TCP/HA/COL-I /MSCs possesses the same effect on repairing bone defect as autogenous bone, so it may be used as autogenous bone graft substitute.
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BACKGROUND: Traumatic osteoarthritis (OA) resulted from the injury of joints and postoperation of joints is commonly observed. Intra-articular injection of sodium hyaluronate (Na-HA) has been considered as effective method for OA. OBJECTIVE: To observe the influence of intra-articular injection of NaHA on mRNA expression of inducible nitric oxide synthase (iNOS) in cartilage of traumatic OA induced by transection of anterior cruciate ligament. DESIGN: Randomized controlled animal experiment. SETTING: Department of Orthopaedics, Renmin Hospital, Wuhan Uni ersity. MATERIALS: The experiment was performed in Laboratory of Depart ment of Orthopaedics, Renmin Hospital, Wuhan University from April to December 2003, in which, 16 clean healthy flat-eared white rabbits, aged 5-6 months were employed. The rabbits were randomly divided into Na- HA injection group and saline control group with 8 rabbits in each group. Na-HA (2000, No 366095) was provided by Shanghai Jiahua Fine Biologi- cal Products Co. METHODS: ①OA model was established in rabbits of the two groups. Each rabbit was anesthetized intravenously with 1.0 mg/kg ketamine hy- drochloride and underwent unilateral anterior cruciate ligament transection. ②5 weeks after transection, Na-HA injection group rabbits received 0.3 mL of intra-articular 10 g/L Na-HA injection, once a week for 5 weeks. Ani mals in saline controlled group were treated with saline of the same vol ume. ③The rabbits were killed at week 10 after operation, general morphology and histopathological changes of articular cartilage degeneration of medial femoral condyle were evaluated (0 points as smooth articular surface with normal color and luster; 1 point as minimal fibrillation or a slight crevice and dark grey color of the surface; 2 points as erosion extending into superficial or middle layers of cartilage; 3 points as ulceration and erosion extending into the deep layers, and 4 points as denudation of cartilage, erosion extending to the sub-chondral bone). The mRNA expression of iNOS in cartilages was examined with reverse transcription-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURES: ①Observation of articular cartilage degeneration of medial femoral condyle, ②observation of articular cartilage degeneration of medial femoral condyle at light microscopic level, ③expression of iNOS in cartilages of each group. RESULTS: A total of 16 clean healthy rabbits entered the result analysis. ① Result of articular cartilage degeneration of medial femoral condyle: Pathological change of articular surface of femoral condyle was observed under anatomic microscope. Cartilage degradation in experimental group was significantly less severe than that in saline control group. ②Histological changes of articular cartilage degeneration of medial femoral condyle at light microscopic level: The Na-HA injection group showed cartilage changes: Membrane of cartilage presented denaturalization and abscission. Chondrocytes of superficial zone presented denaturalization, necrosis, turbulence and erosion. Animals treated with saline showed denaturalization, necrosis and disorder of chondrocytes, ulceration penetrating into the middle or deep zone of the cartilage. New hyperplasia of capillary vessels and fibroblasts were more obvious. Proliferation of fibrous tissue appeared at the bottom of ulcer. ③Expression of iNOS in cartilages of two group:The gene expression of iNOS in cartilage of Na-HA injection group was(1.09±0.18) and the expression of saline control group was (1.26±0.23). Nosignificant difference of iNOS expression was found between the two groups(P ＞ 0.05).CONCLUSION: Intra-articular injection of Na-HA has protective and repairing effect on cartilage with early OA and can significantly reduce the severity of cartilage degradation during early stage of traumatic OA. Intra-articular injection of Na-HA does not down-regulated iNOS expression in cartilage.
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@#ObjectiveTo observe the effects of chitosan injected intra-articular on cartilage degeneration of rabbits with osteoarthritis(OA).MethodsOA animal model was established by unilateral anterior cruciate ligament transaction(ACLT) in 18 rabbits.After operation,animals were randomly divided into the experimental group and control group with 9 rabbits in each group.The experimental group was treated with intra-articular injection of 0.3 ml of 2% chitosan every two weeks after operation.While,the control group was injected with 0.3 ml normal saline at the same time.All rabbits were killed at the 8th week.Cartilage degradation of femoral condyles was evaluated at macroscopic level and the changes of proteoglycan of articular cartilage matrix were detected by biochemical technique,and the expression of matrix metalloproteinases-1(MMP-1) was documented by immunohistochemistry.ResultsCartilage degradation in the control group was significantly more severe than that in the experimental group in macroscopic grading scale(P<0.05).The detection of proteoglycan showed that the quantity of glycosaminoglycans increased(P<0.01) and the chondroitin sulfate/keretan sulfate declined(P<0.05) in experimental group compared with that in the control group.Immunohistochemistry study showed that the expression of MMP-1 in the control group was significantly more severe than that in the experimental group(P<0.01).ConclusionChitosan can prevent cartilage degaradation and protect cartilage.
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Objective To improve the therapeutic efficacy of cervical spondylotic myelopathy (CSM) and reduce the complications by anterior cervical locking plate system (ACLPs).Methods 78 patients with CSM were treated with ACLPs.Thoughtful preparation and careful observation were given before and after the operation.Result All the cases obtained excellent results postoperatively according to the JOA score system except one dead.Conclusion ACLPs is a promising method for the treatment of CSM;careful preoperative preparation and postoperative observation can improve the efficacy and reduce the complications.
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Objective To investigate the therapeutic effect of two-stage delayed open reduction and internal fixation on Pilon fractures.Methods The clinical data of 36 patients(26 males and 10 females;aged 18-57 years with a mean of 38.5 years;9 patients suffered from open fractures and 27 with closed fractures;25 combined with fibular fractures) with monolateral Pilon fracture hospitalized in succession from Jan.2001 to Oct.2006 were analyzed retrospectively.According to the Ruedi-Allgower classification,there were 6 cases with type Ⅰ,21 with type Ⅱ and 9 with type Ⅲ fracture.All patients underwent temporary fixation with calcaneal traction or plaster slab for 7 to 14 days(mean 9.8d),and then they were treated with delayed surgery when the soft tissue injuries subsided.Ankle functions were evaluated to assess the clinical outcomes,and complications were documented for further analysis.Results Follow-up was performed for 19 to 34 months(in mean of 28 months).Based on the Mazur classification,the ankle function in 13 cases was excellent,17 as good,4 as fair and 2 as poor,with a satisfactory rate of 83.3%.Postoperative complications included 2 cases of superficial incision infections,2 of skin slough and 19 of articular degeneration.Conclusion Two-stage delayed open reduction and internal fixation is a reasonable and effective procedure in treating Pilon fractures with advantages of few complications and satisfactory ankle function.
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Objective To investigate the effect of Saussurea involucrata Injection(SII)on counteracting adjuvant-induced arthritis and its immunoregulation function,thus to supply the pharmacological evidences for the clinical treatment of arthritis.Methods Adjuvant arthritis was induced by plantar injection of Freundi' s complete adjuvant.MTT method was used to detect T and B lymphocytes proliferation,and sheep red blood cell immunization was used for hemolysin determination.Results(1)Rats right posterior metatarsus which was injected adjuvant was swollen from the second day to the 22nd day(the primary injury),and left posterior metatarsus not receiving adjuvant injection was swollen from the sixth day to the 20 th day(the secondary lesion).The differences of swelling degree were significant between SII group and NS group.(2)Consecutive intramuscular injection of SII 0.2,0.4,0.8 mL? kg-1? d-1 for 22 days suppressed the primary injury and the secondary lesion of adjuvant arthritis in rats,relieved swelling significantly from the sixth day(P