stilbenes and Intervertebral-Disc-Degeneration

To investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.. Ten patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the. Immunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group (. RES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.. 探讨白藜芦醇（resveratrol，RES）对退变髓核细胞（nucleus pulposus cells，NPC）细胞外基质（extracellular matrix，ECM）表达的调控作用及其相关分子机制。.. 选取临床上接受椎间盘摘除术的 10 例患者，其中 5 例为年轻脊柱爆裂骨折患者，作为对照组；余 5 例为老年腰椎间盘突出症患者，作为退变组。取两组患者髓核组织，免疫组织化学染色比较 β-catenin 的原位表达，Western blot 检测 β-catenin、Ⅱ型胶原和聚集蛋白聚糖（Aggrecan）蛋白表达。取退变组髓核组织分离、培养 NPC，分别用 IL-1β 单独（B 组）或联合 RES（C 组）刺激第 3 代 NPC，并设未刺激细胞作为空白对照组（A 组），Western blot 检测Ⅱ型胶原和 Aggrecan 蛋白表达。进一步采用小干扰 RNA（small interfering RNA，siRNA）靶向沉默 SIRT1 和 β-catenin 后，采用 Western blot、实时荧光定量 PCR 检测 β-catenin、SIRT1 蛋白和基因表达。用完全培养基（1 组）、IL-1β（2 组）、RES+IL-1β（3 组）、SIRT1-siRNA+RES+IL-1β（4 组）刺激第 3 代 NPC 培养 24 h 后，细胞免疫荧光染色检测 β-catenin 核转位情况；用完全培养基（Ⅰ组）、IL-1β（Ⅱ组）、IL-1β+β-catenin-siRNA（Ⅲ组）、IL-1β+RES（Ⅳ组）、IL-1β+RES+SIRT1-siRNA（Ⅴ组）刺激第 3 代 NPC 培养 24 h 后，采用 Western blot 检测Ⅱ型胶原和 Aggrecan 蛋白表达。.. 免疫组织化学染色及 Western blot 检测示，与对照组比较，退变组髓核组织中 β-catenin 阳性表达的细胞比例显著升高（. RES 可以通过抑制 Wnt/β-catenin 信号通路维持 NPC ECM 的表达，为椎间盘退行性疾病的治疗提供了新思路。.

Topics: Aggrecans; beta Catenin; Cell Count; Cells, Cultured; Collagen Type II; Diskectomy; Extracellular Matrix; Gene Expression; Humans; Interleukin-1beta; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Nucleus Pulposus; Resveratrol; Sirtuin 1; Stilbenes

Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain. Polydatin (PD) has been shown to exert multiple pharmacological effects on different diseases; here, we test the therapeutic potential of PD for IVDD. In in-vitro experiments, we confirmed PD is nontoxic to nucleus pulposus cells (NPCs) under the concentration of 400 μmol/L. Furthermore, PD was able to decrease the level of senescence in TNF-α-treated NPCs, as indicated by β-gal staining as well as senescence markers p53 and p16 expression. In the aspect of extracellular matrix (ECM), PD not only reduced metalloproteinase 3 (MMP-3), metalloproteinase 13 (MMP-13) and a disintegrin-like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) expression, but also increased aggrecan and collagen II levels. Mitochondrion is closely related to cellular senescence and ECM homeostasis; mechanistically, we found PD may rescue TNF-α-induced mitochondrial dysfunction, and it may also promote Nrf2 expression and activity. Silencing Nrf2 partly abolished the protective effects of PD on mitochondrial homeostasis, senescence and ECM homeostasis in TNF-α-treated NPCs. Correspondingly, PD ameliorated IVDD in rat model by promoting Nrf2 activity, preserving ECM and inhibiting senescence in nucleus pulposus cells. To sum up, our study suggests that PD exerts protective effects in NPCs against IVDD and reveals the underlying mechanism of PD on Nrf2 activation in NPCs.

Topics: ADAMTS4 Protein; Aggrecans; Animals; Cells, Cultured; Cellular Senescence; Collagen; Disease Models, Animal; Extracellular Matrix; Glucosides; Humans; Intervertebral Disc; Intervertebral Disc Degeneration; Low Back Pain; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; NF-E2-Related Factor 2; Nucleus Pulposus; Rats; Stilbenes; Tumor Necrosis Factor-alpha

Intervertebral disc degeneration (IVDD) is closely related with aging, whereas mitochondrial damage is a common feature of aging that results in cell apoptosis. Resveratrol (RES) is a natural antioxidant that protects against mitochondrial dysfunction in various cells. This study aimed to investigate the protective role of RES against mitochondrial dysfunction and human nucleus pulposus cell (NPC) apoptosis. We found that mitochondrial dysfunction and NPC apoptosis could be induced under oxidative stress by 100 μmol/l of H

Topics: Animals; Antioxidants; Cells, Cultured; Dose-Response Relationship, Drug; Female; Humans; Intervertebral Disc Degeneration; Male; Mitochondria; Nucleus Pulposus; Rabbits; Resveratrol; Stilbenes; Treatment Outcome

This study was done to evaluate whether injections of resveratrol, a natural compound found in the skin of grapes, had anabolic effects on degenerated intervertebral discs in a rabbit model. Two non-continuous lumbar discs were punctured in rabbits to induce disc degeneration. Four weeks and 6 weeks after puncture, the rabbits were treated by injections with dimethylsulfoxide (DMSO) or resveratrol. At 4, 8, and 16 weeks after initial injection, rabbits were sacrificed and the spine was extracted for magnetic resonance image (MRI), mRNA expression, and histological staining. Resveratrol treatment resulted in stronger signal intensity in T2-weighted images. MRI grade showed significantly lower in the resveratrol group than the DMSO group (P = 0.039). In the resveratrol group, aggrecan gene expression was significantly increased than that in the DMSO group at 16 weeks after injection (P = 0.027). MMP-13 mRNA levels in the resveratrol group were significantly decreased than those in the DMSO group at 8 and 16 weeks (P = 0.006 and P = 0.048, respectively). In hematoxylin and eosin stain, resveratrol-treated discs showed the features of regeneration. Histologic grade revealed improvement in resveratrol-treated discs, compared with DMSO-treated discs (P = 0.024). These anabolic effects on degenerated discs indicate that resveratrol is a promising candidate for treatment of degenerative disc disease.

Topics: Aggrecans; Anabolic Agents; Animals; Disease Models, Animal; Drug Administration Schedule; Intervertebral Disc Degeneration; Magnetic Resonance Imaging; Matrix Metalloproteinase 13; Rabbits; Radiography; Resveratrol; RNA, Messenger; Spine; Stilbenes

To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere.. Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression.. The cultured cells were identified to be NPCs. Morphological observation, senescence-associated P-galactosidase (SA-P-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P < 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P < 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P < 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P < 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P < 0.05).. Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.

Topics: Adult; Aggrecans; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chondrocytes; Extracellular Matrix; Female; Gene Expression Regulation; Humans; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Phenotype; Resveratrol; RNA, Messenger; Sirtuin 1; Stilbenes; Tissue Engineering

Many studies have demonstrated that SIRT1, an NAD(+)-dependent deacetylase, reduces apoptosis in several different cells. However, the role of SIRT1 in apoptosis of disc nucleus pulposus (NP) cells remains unclear. The present study was performed to determine whether degenerative human NP would express SIRT1, and to investigate the role of SIRT1 in NP cells apoptosis. The expression of SIRT1 in disc NP of patients (>55 years) with lumbar disc degenerative disease (DDD) and the disc NP of patients (<25 years) with lumbar vertebra fracture (LVF) was assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and Western blot analysis. The results showed that SIRT1 mRNA and protein levels were greater in LVF disc NP than those in DDD disc NP. Degenerative human NP cells were treated in culture with activator or inhibitor of SIRT1, resveratrol or nicotinamide, or SIRT1 small interfering RNA (siRNA), and cell apoptosis was quantified via flow cytometry. The rate of apoptosis was far fewer in resveratrol-treated NP cells than in SIRT1 siRNA-transfected or nicotinamide-treated NP cells. After SIRT1 siRNA was transfected, NP cells decreased phosphorylation of Akt, while resveratrol phosphorylated Akt. Treatment with LY294002 or Akt siRNA increased the rate of apoptosis. Our results suggested that SIRT1 plays a critical role in survival of degenerative human NP cells through the Akt anti-apoptotic signaling pathway.

Topics: Adult; Aged; Apoptosis; Blotting, Western; Cells, Cultured; Enzyme Inhibitors; Female; Gene Expression Regulation; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Intervertebral Disc Degeneration; Lumbar Vertebrae; Male; Middle Aged; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Resveratrol; Ribonucleotide Reductases; RNA, Messenger; Signal Transduction; Sirtuin 1; Stilbenes; Young Adult