Matrix protein gene expression in intervertebral disc cells subjected to altered osmolarity

Physiologic loading of the intervertebral disc may lead to changes in the osmotic pressure experienced by the resident cells. In this study, changes in gene expression levels for extracellular matrix and cytoskeletal proteins were quantified in disc cells subjected to hypo-osmotic (255 mOsm) or hyper-osmotic conditions (450 mOsm), relative to iso-osmotic conditions (293 mOsm). Important differences were observed in osmolarity and between cells of different regions, corresponding to the transition zone and nucleus pulposus. Under hypo-osmotic conditions, gene expressions for aggrecan and type II collagen were up-regulated in the transition zone, but not in the nucleus pulposus cells. Genes for the small proteoglycans, biglycan, and decorin, but not lumican, were up-regulated in transition zone cells following incubation in either hypo- or hyper-osmotic media. The same genes were down-regulated in nucleus pulposus cells under either hypo- or hyper-osmotic conditions. Differences in the response to altered osmolarity between cells of the intervertebral disc may relate to their different cytoskeletal structures or embryological origins.     

周期性机械应力对髓核细胞增殖和细胞外基质表达的影响

目的：探讨周期性机械应力对髓核细胞增殖和细胞外基质表达的影响。方法：对兔髓核细胞进行体外细胞培养,对细胞施加周期性机械应力（0.25Mpa,0.1Hz）。实验分为2组,不加压组和加压组,不加压组置于单纯旋转式生物反应器内,加压组每天置于周期性机械应力场内2小时。分别于3天,7天检测细胞数目以及聚集蛋白聚糖（aggrecan）和Ⅱ型胶原的基因表达。结果：髓核细胞的增殖和聚集蛋白聚糖、Ⅱ型胶原基因的表达水平与周期性压力密切相关,在周期性机械应力刺激下髓核细胞增殖明显,细胞外基质的分泌增加,组织工程髓核细胞的活性显著提高。结论：周期性机械应力能够显著促进髓核细胞增殖,同时上调聚集蛋白聚糖、Ⅱ型胶原的基因的表达。     

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert‐butyl hydroperoxide (TBHP)‐treated nucleus pulposus cells of SD rats in vitro as well as in a puncture‐induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen‐II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts‐5. Additionally, autophagy blockade using 3‐MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine‐treated rats had an accentuated T2‐weighted signal and a diminished histological degenerative grade than vehicle‐treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.     

Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells

In this experiment, the cross-talk betweenNotch and the NF-κB signaling pathway was examined to reveal the mechanism of slowing down the type II collagen (ColII) and aggrecan degeneration affected by inflammatory cytokines. The expression levels of ColII and aggrecan in the intervertebral disc were observed through immunohistochemistry and hematoxylin-eosin staining+alcian blue staining, respectively. The expression levels of ColII, aggrecan, Runx2, and NF-κB in the nuclei of human nucleus pulposus cells (hNPCs) in each group, as well as the phosphorylation and acetylation levels of p65, were examined through Western blot analysis. The 293T cells were transfected with a plasmid containing the overexpressed relative domain of Notch1 intracellular domain (NICD1), and immunoprecipitation (IP) was performed to observe the combination of NICD1 and p65. HNPCs were transfected with a lentiviral-contained overexpression lacking the ANK region of NICD1, and IP was performed to observe the combination of NICD1 and p65. The expression of ColII and aggrecan in the intervertebral disc culture increased when γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-Sphenylglycine t-butyl ester (DAPT) was added to the disc culture medium. Western blot revealed that DAPT inhibited p65 phosphorylation and acetylation, and the p65 and p50 levels in the nucleus decreased. NICD1 was found to be combined with p65 in contrast to the reverse consequences after ANK domain deletion in hNPCs. In nucleus pulposus cells, the combination of p65 and the ANK domain of NICD1 is a critical procedure for the degeneration related to the NF-κB signaling pathway activation induced by IL-1β and TNF-α.     

Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.     

Leptin Downregulates Aggrecan through the p38-ADAMST Pathway in Human Nucleus Pulposus Cells

The mechanistic basis of obesity-associated intervertebral disc degeneration (IDD) is unclear. Aberrant expression of aggrecan and its degrading enzymes ADAMTS-4 and ADAMTS-5 is implicated in the development of IDD. Here, we investigated the effect of leptin, a hormone with increased circulating levels in obesity, on the expression of aggrecan and ADAMTSs in primary human nucleus pulposus (NP) cells. Real-time PCR and Western blots showed that leptin increased the mRNA and protein expression of ADAMTS-4 and ADAMTS-5 and reduced the level of aggrecan in NP cells, accompanied by a prominent induction of p38 phosphorylation. Treatment of NP cells with SB203580 (a p38 inhibitor) abolished the regulation of aggrecan and ADAMTSs by leptin. Knockdown of ADAMTS-4 and ADAMTS-5 by siRNAs also attenuated the degradation of aggrecan in leptin-stimulated NP cells. To conclude, we demonstrated that leptin induces p38 to upregulate ADAMTSs and thereby promoting aggrecan degradation in human NP cells. These results provide a novel mechanistic insight into the molecular pathogenesis of obesity-associated IDD.