Nucleus Pulposus Cells Of Intervertebral Disc Research Articles

Role of Autophagy and Pyroptosis in Intervertebral Disc Degeneration.

Intervertebral disc degeneration is a chronic degenerative disease caused by the interaction of genetic and environmental factors, mainly manifested as lower back pain. At present, the diagnosis of intervertebral disc degeneration mainly relies on imaging. However, early intervertebral disc degeneration is usually insidious, and there is currently a lack of relevant clinical biomarkers that can reliably reflect early disease progression. Pyroptosis is a regulatory form of cell death triggered by the activation of inflammatory bodies and caspase, which can induce the formation of plasma membrane pores and cell swelling or lysis. Previous studies have shown that during the progression of intervertebral disc degeneration, sustained activation of inflammasomes leads to nuclear cell pyroptosis, which can occur in the early stages of intervertebral disc degeneration. Moreover, intervertebral disc nucleus pulposus cells adapt to the external environment through autophagy and maintain cellular homeostasis and studying the mechanism of autophagy in IDD and intervening in its pathological and physiological processes can provide new ideas for the clinical treatment of IDD. This review analyzes the effects of pyroptosis and autophagy on IDD by reviewing relevant literature in recent years, in order to explore the relationship between pyroptosis, autophagy and IDD.

Effect of electroacupuncture at "Jiaji" (EX-B2) on senescence of nucleus pulposus cells of degenerated lumbar intervertebral disc in rabbits.

To observe the effect of electroacupuncture (EA) at "Jiaji"(EX-B2) on body mass, motor function, expression of caveolin-1 (Cav-1) in nucleus pulposus cells and annulus fibrosus tissue, telomerase activi-ty, relative telomere length and different cell cycle ratio of nucleus pulposus cells in rabbits with intervertebral disc degeneration(IVDD), so as to investigate its mechanism underlying delaying senescence of the degenerated lumbar intervertebral disc nucleus pulposus cells. Twenty-five male New Zealand rabbits with mature bones were divided into control, sham operation, model, EA, and acupuncture groups, with 5 rabbits in each group. The IVDD model was established by inserting kirschner wires to the vertebral bone surface between the lumbar (L)4 and L5 vertebrae, followed by applying continuous axial pressure for 28 d. EA (2 Hz/15 Hz, 1-2 mA) or acupuncture (only insertion of acupuncture needles into bilateral EX-B2, but without electrical stimulation) was applied to bilateral EX-B2 for 20 min, once daily, 6 times a week for 4 weeks. The hindlimb locomotor function (locomotor score) was assessed by using Faden's and colleagues' methods. The general conditions of rabbits in each group were observed, and their body weight changes were measured every week. Nucleus pulposus cells were isolated using enzyme digestion method. After the treatment, the Cav-1 positive cell counts in nucleus pulposus cells and annulus fibrosus tissues were detected by immunohistochemistry, and the telomerase activity of nucleus pulposus cells was detected by PCR-ELISA. The relative telomere length of nucleus pulposus cells was measured by real-time quantitative polymerase chain reaction (real-time qPCR), and the cell cycle of nucleus pulposus was detected by flow cytometry. Compared with the sham operation group, the body mass from 4 to 11 week, locomotor score at 4, 7 and 11 week, telomerase activity, relative telomere length and the proportion of cells in G2/M phase of nucleus pulposus cells were significantly decreased (P<0.01), while Cav-1 positive cell counts of nucleus pulposus and annulus fibrosus tissue, and the proportion of nucleus pulposus cells in the G0/G1 phase considerably increased (P<0.01) in the model group. Relevant to the model group, the EA group rather than the acupuncture group had an increase in the body mass from 8 to 11 week, locomotor score at 11 week, telomerase activity, relative telomere length of nucleus pulposus cells, and the proportion of nucleus pulposus cells in G2/M phase (P<0.01), and a decrease in the Cav-1 positive cell counts of nucleus pulposus and annulus fibrosus tissue and the proportion of cells in G0/G1 phase (P<0.01). No significant differences were found between the model and acupuncture groups in all the indexes mentioned above. EA at EX-B2 has a bene-ficial effect in improving motor function in rabbits with IVDD, which may be related to its functions in reducing the expression of Cav-1 in nucleus pulposus cells and annulus fibrosus, improving cycle arrest, enhancing the telomerase activity and the relative telomere length of nucleus pulposus cells, delaying the senescence of nucleus pulposus cells of the degenerated lumbar intervertebral discs.

Retracted: Effects of Fluid Shear Stress on Human Intervertebral Disc Nucleus Pulposus Cells Based on Label-Free Quantitative Proteomics.

[This retracts the article DOI: 10.1155/2022/3860898.].

Polyphyllin I suppressed the apoptosis of intervertebral disc nucleus pulposus cells induced by IL-1β by miR-503-5p/Bcl-2 axis

BackgroundThere are no studies that have shown the role and underlying mechanism of Polyphyllin I (PPI)-mediated anti-apoptosis activity in nucleus pulposus cells (NPCs). The research aimed to evaluate the effects of PPI in interleukin (IL)-1β-induced NPCs apoptosis in vitro.MethodsCell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and cell apoptosis was evaluated by double-stained flow cytometry (FITC Annexin V/PI). The expression of miR-503-5p was quantified by real-time quantitative PCR (qRT-PCR), and the expression of Bcl-2, Bax, and cleaved caspase-3 was quantified by Western blot. Dual-luciferase reporter gene assay was used to detect the targeting relationship between miR-503-5p and Bcl-2.ResultsPPI at 40 μg·mL−1 markedly promoted the viability of NPCs (P < 0.01). Also, PPI inhibited apoptosis and reduction in proliferative activity induced by IL-1β in the NPCs (P < 0.001, 0.01). PPI treatment significantly inhibited the expression of apoptosis-related protein Bax, cleaved caspase-3 (P < 0.05, 0.01), and enhanced the level of anti-apoptotic protein Bcl-2 (P < 0.01). The proliferative activity of NPCs was significantly decreased and the apoptosis rate of NPCs was increased under IL-1β treatment (P < 0.01, 0.001). Moreover, miR-503-5p was highly expressed in IL-1β-induced NPCs (P < 0.001). Furthermore, the effect of PPI on NPCs viability and apoptosis in IL-1β treatment was dramatically reversed by the overexpression of miR-503-5p (P < 0.01, 0.01). The targeted binding of miR-503-5p to the 3'UTR of Bcl-2 mRNA was confirmed by dual-luciferase reporter gene assays (P < 0.05). In further experiments, compared with miR-503-5p mimics, the effects of PPI on IL-1β-induced NPCs viability and apoptosis were greatly reversed by the co-overexpression of miR-503-5p and Bcl-2 (P < 0.05, 0.05).ConclusionPPI suppressed the apoptosis of intervertebral disk (IVD) NPCs induced by IL-1β via miR-503-5p/Bcl-2 molecular axis.

Effects of Fluid Shear Stress on Human Intervertebral Disc Nucleus Pulposus Cells Based on Label-Free Quantitative Proteomics.

Objective To explore the possible mechanism of fluid shear stress on human nucleus pulposus cells based on label-free proteomics technology. Methods The human nucleus pulposus cell line was purchased and subcultured in vitro. The Flexcell STR-4000 multiflow field cell fluid shear stress loading culture system was used to apply continuous laminar fluid shear stress (12 dyne/cm2, 45 mins) to the monolayer adherent cells. Those without mechanical loading were used as the control group, and those subjected to fluid shear loading were used as the experimental group. Differential protein expression was identified using mass spectrometry identification technology, and bioinformatics analysis was performed using Gene Ontology GO (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes KEGG (Kyoto Encyclopedia of Genes and Genomes). Results The proteomics results of the experimental group and the control group showed that the total number of mass spectra was 638653, the number of matched mass spectra was 170110, the total number of identified peptides was 32050, the specific peptide was 30564, and the total number of identified proteins was 4745. Comparing the two groups, 47 proteins were significantly differentially expressed, namely, 25 upregulated proteins and 22 downregulated proteins. Bioinformatics analysis showed that significantly different proteins were mainly manifested in cellular process, biological regulation, metabolic process, binding, catalytic activity, cellular components (cell part), organelle part (organelle part), and other molecular biological functions. Conclusion Using proteomics technology to screen human nucleus pulposus cells after fluid shear stress loading, the differential protein expression provides a basis for further exploration of the mechanism of mechanical factors on nucleus pulposus.

Tanshinone IIA and Astragaloside IV Inhibit miR-223/JAK2/STAT1 Signalling Pathway to Alleviate Lipopolysaccharide-Induced Damage in Nucleus Pulposus Cells.

Astragaloside IV (AS IV) and tanshinone (TS IIA) are the main natural components of Salvia miltiorrhiza and Radix Astragali, respectively. The amalgam of TS IIA and AS IV has potential therapeutic value in many inflammation-related diseases. However, the aftereffect of TS IIA and AS IV for lumbar disc herniation is not clear. Although the function of miR-223 in the inflammation-related JAK/STAT pathway is unknown, it is particularly expressed in human degenerative nucleus pulposus cells. This study has investigated the efficacy of the combined application of TS IIA and AS IV in the treatment of intervertebral disc nucleus pulposus cells (NP cells) injured by lipopolysaccharide (LPS). After miR-223 inhibitor imitated NP cells, the state of the JAK family and STAT family was recognized by Western blotting (Western blot, WB) and reverse transcriptase quantitative polymerase chain reaction (qPCR). The shRNA lentivirus interference vector targeting the STAT family was constructed, and the NP cell line stably interfering with the STAT gene was established after transfection. The expression of TNF-α, IL-6, MMP-9, MMP-3, caspase-1, and caspase-3 was detected by lipopolysaccharide (WTNP cells), control virus NP cells, STAT downregulation NP cells, enzyme-linked immunosorbent assay (ELISA), Western blot, and qPCR, respectively. The cell survival rate was detected by flow cytometry and TUNEL staining reverse transcriptase-polymerase chain reaction (qPCR). NP cells were treated with TS IIA and AS IV which had been made into different concentrations, and then, the expression of miR-223, p-STAT1, and p-JAK families was detected by WB Western blotting and qPCR. MiR-223 selectively acts on JAK2/STAT1 pathway, increases the expression of TNF-α, IL-6, MMP-9, MMP-3, caspase3-1, and caspase-3, and induces apoptosis, which can be eliminated by silencing STAT1. TS IIA combined with AS IV could inhibit the expression of miR-223, p-STAT1, and p-JAK2 in NP cells, and they showed a dose-dependent tendency to p-STAT1 and p-JAK2. This study shows that miR-223 promotes the inflammatory response and induces cell injury of NP cells by acting on the JAK2/STAT1 pathway, and the combination of TS IIA and AS IV may protect NP cells by downregulating miR-223 and inhibiting the expression of JAK2 and STAT1.

MicroRNA‑25 protects nucleus pulposus cells against apoptosis via targeting SUMO2 in intervertebral disc degeneration.

It has been reported that microRNA (miRNA/miR)‑25 is downregulated in patients with intervertebral disc degeneration (IVDD). However, the potential role of miR‑25 in IVDD remains unclear. Therefore, the present study aimed to investigate the effects of miR‑25 on human intervertebral disc nucleus pulposus cells (NPCs). The expression levels of miR‑25 and those of small ubiquitin‑related modifier2(SUMO2) were determined in human nucleus pulposus (NP) tissues by reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analyses. Subsequently, the potential interaction between miR‑25 and SUMO2 was validated via dual‑luciferase reporter assay and RNA pull‑down assay with biotinylated miRNA. The effects of miR‑25 on NPC proliferation and apoptosis were evaluated using Cell Counting Kit‑8 assay, 5‑ethynyl‑2'‑deoxyuridine incorporation assay, and flow cytometry. The results showed that miR‑25 was downregulated in patients with IVDD. In addition, miR‑25 increased the proliferation of NPCs and inhibited their apoptosis. Furthermore, the current study verified that miR‑25 could directly target SUMO2 and regulate its expression via the p53 signaling pathway. Additionally, the effects of miR‑25 on NPCs were abrogated following SUMO2 overexpression. Overall, the results of the present study demonstrated that miR‑25 could promote the proliferation and inhibit the apoptosis of NPCs via targeting SUMO2, suggesting that miR‑25 may be a potential target in the treatment of IVDD.

Efficacy of Yishen Huoxue Tongluo() medicated serum on apoptosis of nucleus pulposus cells of human intervertebral disc under overload static pressure

To explore the efficacy of Yishen Huoxue Tongluo() decoction containing serum on the apoptosis of human disc nucleus pulposus cells under the overload static pressure stress and its related mechanism. Human nucleus pulposus cells were divided into three groups. The blank group had no intervention. The model group and traditional Chinese medicine serum intervention group were treated with 3 MPa compressive stress in vitro for 2, 4 and 6 hours. The changes and differences of morphology, growth status and ultrastructure of intervertebral disc nucleus pulposus cells were observed. The apoptosis rate of nucleus pulposus cells and nuclear factor kappa-B p65 (NF-κB p65), SRY-related high mobility group box 9 (SOX9), C/EBP-homologous protein (CHOP), matrix metalloprotein-13 (MMP-13) and corresponding gene expression were detected. At the same time, compared with the blank group, the nucleus pulposus cells in the model group were smaller in volume, less in cytoplasm and worse in growth; the nucleus pulposus cells in the traditional Chinese medicine serum intervention group were slightly larger in volume, more complete in morphology, richer in cytoplasm and better in growth. Under the same action time, the ultrastructure of nucleus pulposus cells in blank group was complete, and the structures of primary and secondary processes were not broken;and the ultrastructure of model group and traditional Chinese medicine serum intervention group were damaged, the main and secondary processes were broken to varying degrees, and there was no significant difference between the two groups. At the same time, the apoptosis rate of nucleus pulposus cells in model group was higher than that in blank group, while the apoptosis rate of nucleus pulposus cells in the traditional Chinese medicine serum intervention group was lower than that inmodel group, the difference was statistically significant (P<0.05); with the increase of action time, there was no significant difference in the apoptosis rate of nucleus pulposus cells between blank group and traditional Chinese medicine serum intervention group, and the apoptosis rate of nucleus pulposus cells in model group was increased. Compared with model group, the expression of NF-κB p65, CHOP, MMP-13 were decreased and SOX9 was increased in traditional Chinese medicine serum intervention group at the same time (P<0.05);with the increase of action time, the expression of NF-κB p65, CHOP and MMP-13 were increased, and the expression of SOX9 was decreased in blank group and model group(P<0.05), and the expression level of model group was higher than that of blank group(P<0.05). Overall observation by gene expression, under the same action time, the relative quantifications of NF-κB p65, CHOP and MMP-13 in traditional Chinese medicine serum intervention group were lower than that in model group, while SOX9 was increased (P<0.05);compared with model group, the relative quantifications of NF-κB p65, CHOP and MMP-13 in blank group were decreased(P<0.05), and the relative quantification of SOX9 was increased(P<0.05);with the increase of action time, the relative quantifications of NF-κB p65, CHOP and MMP-13 of nucleus pulposus cells in blank group and model group were increased and SOX9 was decreased(P<0.05). Yishen Huoxue Tongluo() decoction can reduce the apoptosis of nucleus pulposus cells under overload and static pressure, and has the effect of delaying the degeneration of nucleus pulposus cells. Its mechanism may be related to the decrease of CHOP, MMP-13 expression and the increase of SOX9 expression by inhibiting NF-κB p65 signal pathway of nucleus pulposus cells.

Decorin inhibits nucleus pulposus apoptosis by matrix-induced autophagy via the mTOR pathway.

Decorin (Dcn) is a member of the class I small leucine-rich proteoglycans, whose expression in the nucleus pulposus (NP) of intervertebral discs (IVDs) has been shown to increase with aging in humans and sheep. Dcn induces autophagy in endothelial cells; however, its precise role in NP and IVD degeneration during aging is not well understood. We addressed this question in the present study by treating rat nucleus pulposus cells (NPCs) with different concentrations of Dcn. The Western blot analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling assay results showed that Dcn treatment induced autophagy and decreased apoptosis caused by interleukin (IL)-1β application. This effect was dependent on the protein kinase B/mechanistic target of rapamycin (mTOR)/p70 S6 kinase signaling. Dcn treatment also decreased the expression of matrix metalloproteinase-3 and -13 and decreased the IL-1β-induced attenuation of collagen type II and aggrecan levels. The role of Dcn in stimulating autophagy was further supported by the fact that the observed effects were abrogated by knocking down autophagy-related protein 7 with Atg7 small interfering RNA. Thus, Dcn protects NPCs in IVDs from IL-1β-induced apoptosis and degeneration by promoting autophagy through mTOR signaling.

MiR‑222 induces apoptosis in human intervertebral disc nucleus pulposus cells by targeting Bcl‑2.

Intervertebral disc degeneration (IDD) is characterized by abnormal induction of apoptosis in intervertebral disc nucleus pulposus (NP) cells. Previous studies indicated that miR-222 was upregulated in patients with rheumatoid arthritis. However, the effects of miR-222 in IDD remain unclear. The present study aimed to demonstrate the role of miR-222 in NP cells. The levels of miR-222 in patients with IDD were measured by reverse transcription-quantitative PCR. Cell Counting Kit-8 and western blotting assays were used to detect cell proliferation and apoptosis-associated protein levels, respectively. In addition, luciferase reporter assays were performed to validate the predicted target genes of miR-222. miR-222 was significantly upregulated in patients with IDD. Overexpression of miR-222 inhibited cell proliferation and induced cell apoptosis. Moreover, overexpression of miR-222 resulted in an upregulation in the levels of Bax and cleaved caspase 3, and a downregulation in the levels of Bcl-2 in NP cells. The luciferase reporter assays demonstrated that Bcl-2 is a target of miR-222. Furthermore, overexpression of miR-222 increased the levels of cytochrome c, apoptotic protease activating factor-1 and cleaved caspase 9 in NP cells. Conversely, downregulation of miR-222 could promote the proliferation of NP cells. The present data demonstrated that miR-222 induced apoptosis in NP cells by directly targeting Bcl-2. Therefore, miR-222 may act as a potential therapeutic target for the treatment of IDD.

Knockdown of miR-222 inhibits inflammation and the apoptosis of LPS-stimulated human intervertebral disc nucleus pulposus cells.

It has been demonstrated that miR-222 is upregulated in human intervertebral disc (IVD) degeneration tissues; however, the underlying mechanisms remain unclear. In this study, we aimed to elucidate the mechanisms of action of miR-222 in IVD tissues. Nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) to simulate IVD degeneration. The expression level of miR-222 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in cells and tissues. Cell apoptosis was analyzed by flow cytometry. Additionally, western blot analysis was used to determine the levels of Toll-like receptor 4 (TLR4), Iκβ-alpha (IκBα) and p65. Interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) and IL-6 protein expression levels were determined by enzyme-linked immunosorbent assay (ELISA). The target gene of miR-222 was determined by TargetScan7.2 and dual luciferase reporter gene analysis. Western blot analysis and RT-qPCR were used to determine the mRNA and protein levels of tissue inhibitor of metalloproteinase 3 (TIMP3). The mRNA expression level of miR-222 was found to be increased in IVD tissues and in LPS-stimulated cells, and its expression was positively associated with the clinical MRI grade. In vitro, apoptosis was promoted/inhibited by miR-222 mimics/inhibitors. Transfection with miR-222 mimics/inhibitors significantly increased/decreased the production of TNF-α, IL-1β and IL-6 and suppressed/enhanced collagen II and aggrecan expression. The protein levels of TLR4, p-IκBα and p-p65 were upregulated/downregulated by transfection with the mimics/inhibitors. In addition, it was demonstrated that TIMP3 was a direct target gene of miR-222, and was negatively regulated by miR-222 in NP cells. The silencing of TIMP3 reversed the inhibitory effects of miR-222 inhibitor on cell apoptosis, which was induced by LPS. Thus, on the whole, the findings of this study demonstrate that miR-222 functions as a promoter of IVD development, partly via the regulation of TIMP3.

Effect of Matrix Metalloproteinase 3 Gene Silencing and SRY-Related High Mobility Group-Box Gene 9 Gene Overexpression on Human Degeneration Nucleus Pulposus Cells In Vitro

This study aims to detect the biological effects of matrix metalloproteinase 3 (MMP3) and SRY-related high mobility group-box gene 9 (SOX9) gene regulation in the human intervertebral disc degeneration of nucleus pulposus cells mediated by lentiviral vectors in vitro. The culture and development of human degenerated intervertebral disc nucleus pulposus cells was performed. The cell morphology and structure were observed, as identified by hematoxylin and eosin (H&amp;E) staining. The experiment was divided into five groups: blank control group, SOX9 gene overexpression group, MMP3 gene silencing group, SOX9 gene overexpression+MMP3 gene silencing group, and blank vector group. RT-PCR and western blot were performed to detect the influence of MMP3 gene silencing and SOX9 gene overexpression on degenerated human intervertebral disc nucleus pulposus cells, include the secretion of polysaccharide and expression of type II collagen, at the mRNA and protein level. The originally generated degenerated human intervertebral disc nucleus pulposus cells had the same cellular morphology. The MTT assay revealed that the blank group and blank vector group had no statistical significance in cell proliferation. The MMP3 gene silencing and SOX9 gene overexpression could promote the proliferation of degenerated human intervertebral disc nucleus pulposus cells, when compared with the blank control group and blank vector group, and this more obvious in the MMP3 gene silencing group and SOX9 gene overexpression group. RT-PCR and western blot revealed that MMP3 gene silencing and SOX9 gene overexpression can promote cells to secrete polysaccharide and type-II collagen (P &lt; 0.05). MMP3 gene silencing and SOX9 gene overexpression can promote the proliferation of degenerated human intervertebral disc nucleus pulposus cells, and their ability to secrete polysaccharide and type-II collagen.

Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide

Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide

The mechanism of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc by Wnt/β-catenin signaling pathway

Objective To study the effects of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc. Methods Nucleus pulposus cells were identified via the toluidine blue and safranin O. Next, P3 cells were cultured with naringin 0 μg/ml (control group), naringin group (10, 20, 40, 80, 100 μg/ml). After 48 hours, the best concentration of naringin was selected by cell counting kit-8 (CCK-8) and flow cytometry. β-catenin, Cyclin D1, c-Myc gene expression was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The expression of B cell lymphoma/leukemia-2 (bcl-2), bcl-2 associated X protein (bax), Caspase-3 protein, type Ⅱ collagen and proteoglycan was determined by enzyme linked immunosorbent assay (ELISA). Western blotting detected of type Ⅱ collagen and proteoglycan expression. Results The results of cells staining were positive. CCK-8 and flow cytometry were used to determine the optimum concentration of naringin (20 μg/ml) to inhibit the apoptosis of nucleus pulposus cells. In the human degeneration nucleus pulposus cells, the expression of β-catenin gene was overexpressed and the expression of Cyclin D1 and c-Myc gene was down regulated. 20g/ml naringin could down regulate the expression of β-catenin gene (P=0.000), and up regulate the expression of Cyclin D1 and c-Myc gene (P=0.000, 0.001). The naringin (20 μg/ml) could obviously regulate up the expression of bcl-2, type Ⅱ collagen and proteoglycan protein (P=0.001, 0.000, 0.023), and regulate down the expression of bax and Caspase-3 protein, (P=0.001, 0.001). Western blotting showed that 20 μg/ml naringin could promote the expression of type Ⅱ collagen and proteoglycan. Conclusion Naringin may inhibit the apoptosis of nucleus pulposus cells by regulating the Wnt/β-catenin pathway and the mitochondrial apoptosis pathway. Key words: Naringin; Human nucleus pulpous; Apoptosis; Intervertebral disc; Degeneration

MiR-532 downregulation of the Wnt/β-catenin signaling via targeting Bcl-9 and induced human intervertebral disc nucleus pulposus cells apoptosis

ObjectiveIntervertebral disc degeneration was characterized with aberrant intervertebral disc nucleus pulposus cells apoptosis. MiR-532 was reported to be up-regulated in the patients with intervertebral disc degeneration. However, the role of miR-532 in intervertebral disc degeneration remains unclear. Thus, current study aim to investigate the effects of miR-532 on human intervertebral disc nucleus pulposus cells. MethodsQuantitative PCR was firstly used to evaluate the level of miR-532 expression in intervertebral disc nucleus pulposus cells. Then, luciferase reporter assays was used to confirm the target genes of miR-532. The effects of miR-532 on cell proliferation were assayed using EdU transfection. Next, apoptosis level was examined by flow cytometry utilizing Annexin V-FITC/PI double staining, and the protein expression in cells was detected with Western blotting. Finally, The expression of β-catenin protein was demonstrated by fluorescence confocal microscopy and the interaction between β-catenin and Bcl-9 was detected by co-immunoprecipitation. ResultsMiR-532 was significantly upregulated in patient with intervertebral disc degeneration. In addition, intervertebral disc nucleus pulposus cells apoptosis was significantly increased in miR-532 mimics treated group. Moreover, Bcl-9 was confirmed to be a direct target of miR-532 via luciferase reporter assays and western blot assays. ConclusionMiR-532 downregulation of the Wnt/β-catenin signaling via targeting Bcl-9 and induced human intervertebral disc nucleus pulposus cells apoptosis.

Protective effect of p53 on the viability of intervertebral disc nucleus pulposus cells under low glucose condition

P53 is a famous cancer suppressor and plays key roles in metabolism. Intervertebral disc (IVD) is the largest avascular cartilaginous structure in humans and its degeneration is a common cause of spine diseases initiated from damaged nucleus pulposus (NP) cells. The potential cause of disc degeneration has been attributed to aging, genetic factors, mechanical factors and nutrition. In this study, we found that p53 decreased and leaked to the cytoplasm in NP cells as the glucose level decreases, in contrast to cancer cells in which p53 increases and concentrates to the nuclei. Comparing with in p53 knockdown NP cells, relative high p53 expression in normal control NP cells inhibited autophagy and the pentose phosphate pathway. Furthermore, the expression of Sox 9 and type II collagen were higher in p53 normal control than p53 knockdown NP cells. Based on these results, we believe that relative high p53 facilitates NP cell viability and integrity.

The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells

Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

The expression and role of protease nexin-1 in intervertebral disc cells nucleus pulposus

Objective To investigate the expression of Protease nexin-1 (PN-1) in Intervertebral Disc nucleus pulposus (NP) cells and the related signal pathways. Methods Human primary NP cells were isolated and cultured, and divided into control group, interleukine-1β (IL-1β), TNF-α group. Histological examinations were undertaken to observe the morphologic changes of NP cells during the process of differentiation at day 3, 7, 14 and 21. MMPs expression in NP cells were accessed by Western blot. Then, NP cells were cultured in medium containing 10 ng/ml IL-1β and different concenration of PN-1 (0, 10, 50, 100 ng/ml) for 24h. Protein were extracted, and The expression level of Interleukin-1 receptor-associated kinase 1 (IRAK1) , p38 mitogen-activated protein kinase (p38 MAP Ki-nase), phosphos-P38 MAP Kinase (P-P38) wereanalyzed by using Western blot. Results Histologically, under induction with IL-1β, both morphological and biological properties of NP cells were changed, cell proliferation became slowly, cell density was sparse within disordered arrangement, and obvious cell necrosis appeared. The results also showed the degenerative process of NP cells, MMPs production increased and Agg and Col II protein decreased significantly. PN-1 administration could inhibit the activation of MMP3,MMP9 and MMP13 induced by IL-1β, and enhance the expression of AGG and Col2. IL-1β administration could stimulate Nuclear factor κB (NF-κB) signal pathway through the up-regulated expression of IRAK1 and P-P38, while PN-1 could inhibit the inductive effects by suppress the expression of IRAK1 and the phosphorylation of P-38, thereby, inhibit the degeneration of NP cells. Conclusion PN-1 is involved during the degeneration of NP cells. PN-1 administration could antagonize IL-1β-induced proteases and inhibit the activation of MMPs, and potentially preserve NP cells, and reverse its degeneration via IRAK1/NF-κB signaling pathway. Key words: Intervertebral disc degeneration; Interleukin-1 Receptor-Associated Kinases; NF-kappa B

SOX9 directly Regulates CTGF/CCN2 Transcription in Growth Plate Chondrocytes and in Nucleus Pulposus Cells of Intervertebral Disc.

Several lines of evidence indicate that connective tissue growth factor (CTGF/CCN2) stimulates chondrocyte proliferation and maturation. Given the fact that SOX9 is essential for several steps of the chondrocyte differentiation pathway, we asked whether Ctgf (Ccn2) is the direct target gene of SOX9. We found that Ctgf mRNA was down-regulated in primary sternal chondrocytes from Sox9flox/flox mice infected with Ad-CMV-Cre. We performed ChIP-on-chip assay using anti-SOX9 antibody, covering the Ctgf gene from 15 kb upstream of its 5′-end to 10 kb downstream of its 3′-end to determine SOX9 interaction site. One high-affinity interaction site was identified in the Ctgf proximal promoter by ChIP-on-chip assay. An important SOX9 regulatory element was found to be located in −70/−64 region of the Ctgf promoter. We found the same site for SOX9 binding to the Ctgf promoter in nucleus pulposus (NP) cells. The loss of Sox9 in growth plate chondrocytes in knee joint and in NP cells in intervertebral disc led to the decrease in CTGF expression. We suggest that Ctgf is the direct target gene of SOX9 in chondrocytes and NP cells. Our study establishes a strong link between two regulatory molecules that have a major role in cartilaginous tissues.

The effect of capacitively coupled (CC) electrical stimulation on human disc nucleus pulposus cells and the relationship between CC and BMP-7.

An in vitro study using human intervertebral disc nucleus pulposus cells to evaluate the effects of CC stimulation on disc-matrix macromolecule production. Nucleus pulposus cells were cultured in alginate beads and treated with CC stimulation. The effect of BMP on CC stimulation of the cells was evaluated by applying a BMP blocker (noggin) or by applying additional BMP-7 to the culture. The mRNA levels of the disc extracellular matrix genes (collagen I, II, aggrecan) and BMPs were measured by real-time PCR. The protein levels of aggrecan, collagen II, and BMPs were determined by ELISAs and Western blots. Sulfated glycosaminoglycan (sGAG) content was assayed using the DMMB method. (1) CC stimulation upregulates the production of the disc-matrix macromolecular components: sGAG, aggrecan and collagen II; (2) CC stimulation increases the ratio of mRNA expression levels of collagen II to collagen I; (3) CC stimulation induces the expression of endogenous BMP-4 and BMP-7; (4) inhibition of BMP activity (using noggin) reduces CC-mediated upregulation of aggrecan and collagen II; (5) CC and BMP-7 act in synergy to increase the upregulation of disc-matrix macromolecules. CC stimulation upregulates the production of the intervertebral disc-matrix macromolecules aggrecan, collagen II, and sGAG by a mechanism involving BMPs. CC stimulation acts in synergy with BMP-7 to increase the upregulation of these disc-matrix macromolecules.