This study aims to investigate the effects of Bushen Huoxue Decoction regulating adipose-derived stem cells(ADSCs)-exosomes(Exos) on the apoptosis of intervertebral disc nucleus pulposus cells(NPCs) and extracellular signal-regulated kinase(ERK) signaling pathway. Tert-butyl hydrogen peroxide(TBHP)-induced NPCs were divided into control, model, drug-containing serum, blank Exos, normal serum Exos, and drug-containing serum Exos groups. Cell viability and proliferation were examined by the CCK-8 assay and EdU staining, respectively. The cell cycle and apoptosis were evaluated by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin(IL)-1β, tumor necrosis factor(TNF)-α, and IL-6. The mRNA levels of aggrecan, collagen type Ⅱ alpha 1 chain(COL2A1), and ERK were determined by qRT-PCR, and the protein levels of aggrecan, COL2A1, and p-ERK were determined by Western blot. The results showed that compared with the model group, the treatments with drug-containing serum, blank Exos, and normal serum Exos enhanced the viability and proliferation of NPCs, decreased the proportion of cells in the G_0/G_1 phase, increased the proportion of cells in the S phase, reduced apoptosis, lowered the levels of IL-1β, TNF-α, and IL-6, up-regulated the mRNA and protein levels of aggrecan and COL2A1, and down-regulated the mRNA level of ERK and the protein level of p-ERK. Compared with the drug-containing serum, blank Exos, and normal serum Exos groups, the treatment with drug-containing serum Exos enhanced the viability and proliferation of NPCs, decreased the proportion of cells in the G_0/G_1 phase, increased the cells in the S phase, reduced apoptosis, lowered the levels of IL-1β, TNF-α, and IL-6, up-regulated the mRNA and protein levels of aggrecan and COL2A1, and down-regulated the mRNA level of ERK and the protein level of p-ERK. The results confirmed that the Exos secreted by ADSCs after treatment with Bushen Huoxue Decoction-containing serum promoted the proliferation of degenerated NPCs, inhibited apoptosis and the expression of inflammatory mediators, and promoted the production of proteoglycans and collagen, thus delaying the progression of intervertebral disc degeneration, the mechanism of which was related to the regulation of the ERK signaling pathway.
Intervertebral Disc Degeneration/drug therapy* ; Apoptosis/drug effects* ; Nucleus Pulposus/cytology* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Rats ; MAP Kinase Signaling System/drug effects* ; Rats, Sprague-Dawley ; Collagen Type II/metabolism* ; Humans ; Cell Proliferation/drug effects* ; Stem Cells/metabolism* ; Aggrecans/metabolism* ; Cell Survival/drug effects* ; Male ; Cells, Cultured ; Tumor Necrosis Factor-alpha/metabolism*

Intervertebral disc degeneration is considered as a primary cause of clinical low back pain, however the molecular mechanism is not clear yet. Recently, researches on the molecular basis of intervertebral disc degeneration have become a hotspot. The special structure and biomechanics properties of the disc contribute to its propensity toward degeneration. Intervertebral disc degeneration is associated with the changes of the cytological behavior,including the increase in cell death and the degradation of extracellular matrix. However, the mechanism of cell death including cell apoptosis and autophagy in intervertebral disc degeneration remains unclear. Further study on the molecular mechanism of intervertebral disc degeneration is the foundation of improving and treating the intervertebral disc degeneration in the future. Although some progresses are made in the aspect of biological study, the biological environment of intervertebral disc itself is still a challenge for the development of biological treatment. This article is to review the latest advance on the biological characteristics of normal intervertebral disc and the cell death in the process of the intervertebral disc degeneration.
Animals ; Apoptosis ; Cell Death ; Extracellular Matrix ; metabolism ; Humans ; Intervertebral Disc ; cytology ; metabolism ; Intervertebral Disc Degeneration ; metabolism ; physiopathology

The effect of amygdalin joint hydroxysafflor yellow A (HSYA) on the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and the possible mechanism were studied and explored. Chondrocytes were obtained from endplate of one-month SD rat intervertebral discs and cultured primary endplate chondrocytes. After identification, they were divided into normal group, induced group, amygdalin group, HSYA group and combined group. CCK-8 kit was adopted to detect the proliferation of the endplate chondrocytes. FCM was measured to detect the apoptosis. Real-time PCR method was adopted to observe the mRNA expression of Aggrecan, Col 2 alpha1, Col 10 alpha1, MMP-13 and the inflammatory cytokines IL-1beta. The protein expression of Col II, Col X was tested through immunofluorescence. Compared with the normal group, the proliferation of the endplate chondrocytes decreased while the apoptosis increased (P < 0.05). With down regulation of the mRNA expressions of Aggrecan, Col 2 alpha1 and up regulation of the mRNA expressions of Col 10 alpha1, MMP-13, IL-1beta (P < 0.05), the protein expression of Col II decreased while the protein expression of Col X increased. Compared with the induced group, amygdalin group, HSYA group, the combined group could inhibit the apoptosis and promote the proliferation (P < 0.05). They could increase the mRNA expressions of Aggrecan and Col 2 alpha1 while decrease the mRNA expressions of Col 10 alpha1, MMP-13 and IL-1beta (P < 0.05). They could also enhance the protein expression of Col II while reduce the protein expression of Col X. The effect of the combined group was significantly better than that of amygdalin and HSYA. Amygdalin joint HSYA could inhibit the degeneration of the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and better than the single use of amygdalin or HSYA.
Amygdalin ; pharmacology ; Animals ; Apoptosis ; Cells, Cultured ; Chalcone ; analogs & derivatives ; pharmacology ; Chondrocytes ; drug effects ; Collagen ; metabolism ; Drug Synergism ; Interleukin-1beta ; Intervertebral Disc ; cytology ; Quinones ; pharmacology ; Rats

BACKGROUNDRecent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs). However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.

METHODSTissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).

RESULTSMore than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.

CONCLUSIONSNon-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

Adult ; Antigens, CD ; metabolism ; CD24 Antigen ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Endoglin ; Female ; Flow Cytometry ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin beta1 ; metabolism ; Intervertebral Disc ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, Cell Surface ; metabolism ; Young Adult

OBJECTIVETo evaluate the biological effect on the synthesis of the extracellular matrix (ECM) in the cultivation of adult degenerative nucleus pulposus cells using the stiring microcarrier system in vitro.

METHODSThirty-four specimens were collected after intervertebral fusion operations of the patients with intervertebral disc herniation diseases from September 2005 to May 2009. The specimens were then randomly allocated into 2 groups for in vitro cultivation: monolayer culture group and microcarrier culture group. On the exponential phase, SP-ABC immunohistochemical staining and Western blot quantitative analysis were conducted in the two groups to detect the collagen type I and II. Proteoglycan contents of two groups in different growth phases were detected with (35)S-sulfate incorporation assay.

RESULTThe expressions of collagen type I and II in microcarrier culture group were significantly higher than those in monolayer culture group: SP-ABC immunohistochemical staining (collagen type I: 32.5 ± 4.4 vs. 15.2 ± 1.2, t = 2.871, P < 0.01; collagen type II: 43.6 ± 4.1 vs. 23.1 ± 2.2, t = 2.375, P < 0.05); Western blot quantitative analysis (collagen type I: 0.62 ± 0.08 vs. 0.50 ± 0.06, t = 3.327, P < 0.01; collagen type II: 1.46 ± 0.08 vs. 0.86 ± 0.04, t = 2.453, P < 0.05). Nucleus pulposus cells cultivated in stiring microcarrier system showed significantly increased proteoglycan synthesis than monolayer culture group does on both exponential phase and stationary phase (exponential phase: 34 821 ± 312 vs. 21 046 ± 673, t = 2.134, P < 0.05; stationary phase: 45 134 ± 175 vs. 32 193 ± 713, t = 2.801, P < 0.01).

CONCLUSIONSThe expression of collagen type I, II and proteoglycan of adult degenerative nucleus pulposus cells are positive regulated by the stiring microcarrier system, which can be used in the mass amplification of the adult degenerative nucleus pulposus cells.

Adult ; Aged ; Cell Culture Techniques ; Collagen ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Humans ; Intervertebral Disc ; cytology ; Male ; Middle Aged ; Proteoglycans ; metabolism ; Random Allocation ; Young Adult

Intervertebral disc (IVD) degeneration is implicated as a major cause of low back pain. The alternated phenotypes, reduced cell survival, decreased metabolic activity, loss of matrix production and dystrophic mineralization of nucleus pulposus (NP) cells may be key contributors to progressive IVD degeneration. IVD is the largest avascular structure in the body, characterized by low oxygen tension in vivo. Hypoxia-inducible factor (HIF) is a master transcription factor that is induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. This review summarizes relevant studies concerning the involvement of HIF in the regulation of biological behaviors of NP cells. We describe current data on the expression of HIF in NP cells and further discuss the various roles that HIF plays in the regulation of the phenotype, survival, metabolism, matrix production and dystrophic mineralization of NP cells. Here, we conclude that HIF may be a promising target for the prevention and treatment of IVD degeneration.
Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; Cell Survival ; Extracellular Matrix/metabolism ; Humans ; Hypoxia-Inducible Factor 1/genetics/*metabolism ; Intervertebral Disc/*cytology/metabolism ; Intervertebral Disc Degeneration/*metabolism/*pathology

BACKGROUNDIntervertebral disc degeneration is the main cause of low back pain. The purpose of this study was to explore potential methods for reversing the degeneration of lumbar intervertebral discs by transplantation of gene-modified nucleus pulposus cells into rabbit degenerative lumbar intervertebral discs after transfecting rabbit nucleus pulposus cells with adeno-associated virus 2 (AAV2)-mediated connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinases 1 (TIMP1) genes in vitro.

METHODSComputer tomography (CT)-guided percutaneous annulus fibrosus injury was performed to build degenerative lumbar intervertebral disc models in 60 New Zealand white rabbits. rAAV2-CTGF-IRES-TIMP1-transfected rabbit nucleus pulposus cells were transplanted into degenerative lumbar intervertebral discs (transplantation group), phosphate-buffered saline (PBS) was injected into degenerative lumbar intervertebral discs (degeneration control group) and normal lumbar intervertebral discs served as a blank control group. After 6, 10 and 14 weeks, the disc height index (DHI) and signal intensity in intervertebral discs were observed by X-ray and magnetic resonance imaging (MRI) analysis. The expression of CTGF and TIMP1 in nucleus pulposus tissue was determined by Western blotting analysis, the synthesis efficiency of proteoglycan was determined by a (35)S-sulfate incorporation assay, and the mRNA expression of type II collagen and proteoglycan was detected by RT-PCR.

RESULTSMRI confirmed that degenerative intervertebral discs appeared two weeks after percutaneous puncture. Transgenic nucleus pulposus cell transplantation could retard the rapid deterioration of the DHI. MRI indicated that degenerative intervertebral discs were relieved in the transplantation group compared with the degeneration control group. The expression of collagen II mRNA and proteoglycan mRNA was significantly higher in the transplantation group and the blank control group compared with the degeneration control group (P < 0.05).

CONCLUSIONSCT-guided percutaneous puncture can successfully build rabbit degenerative intervertebral disc models. Both CTGF and TIMP1-transfected cell transplantation helps to maintain disc height, and promotes the biosynthesis of type II collagen and proteoglycan in intervertebral discs, reversing the degeneration of intervertebral discs.

Animals ; Blotting, Western ; Cell Transplantation ; methods ; Cells, Cultured ; Collagen Type II ; genetics ; Connective Tissue Growth Factor ; genetics ; metabolism ; Dependovirus ; genetics ; Intervertebral Disc ; cytology ; diagnostic imaging ; metabolism ; Intervertebral Disc Degeneration ; diagnostic imaging ; metabolism ; therapy ; Magnetic Resonance Imaging ; Rabbits ; Radiography ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism

OBJECTIVE: To evaluate the effect of biglycan on the signaling of cytokines (epidermal growth factor, osteogenic protein-1, and interleukin-1) in bovine intervertebral disc cells. METHODS: Nucleoplasty (NP) and annulus fibrosus (AF) cells of the intervertebral disc tissues were isolated from the tails of young adult bovine. First, the cells were treated in 3 ways: Biglycan alone, cytokines alone (epidermal growth factor, osteogenic protein-1, or interleukin-1), and biglycan combined with cytonkines. Western blot was used to observe the singling of biglycan and cytokines in bovine intervertebral disc cells, and to identify the effect of biglycan on cytokines mentioned above. RESULTS: Biglycan upregulated the signaling (3- 4 folds) with the optimal effect at 10 min and 20 μmol/L both in the AF cells and NP cells. Epidermal growth factor, osteogenic protein-1, or interleukin-1 also upregulated the protein expression in the extracellular matrix of intervertebral disc cells. When combined different biglycan concentrations with epidermal growth factor, osteogenic protein-1, or interleukin-1 to treat the intervertebral disc cells, the concentration of biglycan rose, whereas the cytokine signal decreased both in the bovine AF and NP cells (P<0.01). There was no significant difference between the AF and NP cells. CONCLUSION Biglycan can adhere to the intervertebral disc cells to activate the extracellular signal-regulated kinase (ERK) pathway and this effect is time and concentration dependent. Byglycan can decrease not only the anabolism effect of epidermal growth factor and osteogenic protein-1, but also the catabolism effect of interleukin-1. This regulatory role of biglycan may be very important to maintain the metabolism balance. Biglycan may be good for the repair of intervertebral disc.
Animals ; Biglycan ; physiology ; Bone Morphogenetic Protein 7 ; metabolism ; physiology ; Cattle ; Cells, Cultured ; Cytokines ; metabolism ; physiology ; Epidermal Growth Factor ; metabolism ; physiology ; Interleukin-1 ; metabolism ; physiology ; Intervertebral Disc ; cytology ; metabolism ; Signal Transduction

BACKGROUNDLow back pain has emerged as a widespread disease often caused by intervertebral disc degeneration. This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1 (TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type II and proteoglycan levels. The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.

METHODSRhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion, cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOI) of 10(6). The expression of collagen type II and proteoglycan was measured using RT-PCR and Western blotting. The synthetic rate of proteoglycan was measured using (35)S incorporation.

RESULTSRhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected. Compared to the control, CTGF promoted the synthesis of collagen type II and proteoglycan. TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type II. Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type II.

CONCLUSIONSSingle gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan. CTGF expression can also enhance collagen type II protein synthesis. Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type II to levels greater than transduction of a single gene alone. Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.

Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type II ; biosynthesis ; Connective Tissue Growth Factor ; genetics ; metabolism ; Intervertebral Disc ; cytology ; Macaca mulatta ; Proteoglycans ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transduction, Genetic

BACKGROUNDThe most significant biological change in intervertebral disc degeneration is the decrease of chondrocyte specific gene and protein expression of Sox9 and collagen type II. Interleukin-1 (IL-1) is not expressed in the normal intervertebral disc tissue but increases in the degenerated intervertebral disc tissue. This suggests that IL-1 may play a role in regulation of the expression of Sox9 and collagen type II.

METHODSHuman intervertebral disc cells were isolated and cultured. Sox9 and collagen type II expression during treatment with IL-1, with or without the nuclear factor-kappaB (NF-kappaB) activity inhibitor curcumin, were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the activity of the NF-kappaB signaling pathway was detected by the electrophoretic mobility shift assay (EMSA).

RESULTSIL-1 lowered the mRNA level and protein expression of Sox9 and collagen type II in the cultured intervertebral disc cells in a dose dependent manner (P < 0.05), and this effect was attenuated by curcumin. Curcumin alone had no effect on Sox9 and collagen type II expression (P > 0.05). IL-1 at concentrations of 0.1 ng/ml, 1 ng/ml and 10 ng/ml could stimulate the activity of NF-kappaB in the intervertebral disc cells in a dose dependent manner (P < 0.05) that was inhibited by curcumin.

CONCLUSIONSWe demonstrated the previously unknown function of IL-1 in inhibiting Sox9 and collagen type II via NF-kappaB in the intervertebral disc cells. This inhibition can be attenuated by curcumin, which is an effective NF-kappaB activity inhibitor.

Adult ; Cells, Cultured ; Collagen Type II ; genetics ; metabolism ; Curcumin ; pharmacology ; Electrophoretic Mobility Shift Assay ; Gene Expression ; drug effects ; Humans ; Immunoblotting ; Interleukin-1 ; pharmacology ; Intervertebral Disc ; cytology ; Male ; NF-kappa B ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; genetics ; metabolism