ABSTRACT
Intervertebral disc degeneration (IDD) refers to the biomechanical and structural changes of intervertebral disc tissues due to the effects of a variety of factors. Theses physical or chemical factors lead to the rupture of the annulus fibrous, protrusion of the nucleus pulposus tissue, compression of the spinal cord and nerve roots and causing the patient's back and leg pain ultimately. Degeneration of intervertebral disc is a common condition in clinical practice, which affects working ability and daily living quality of patients seriously. Due to the change of living habits, the population with IDD tend to be younger recently. The etiology, pathogenesis and diagnosis and interventions of IDD have always been hot topics in spinal surgery. Thus, animal models of IDD close to the human body has a of great clinical significance for exploring the etiology, pathological mechanism and non-surgical treatment of IDD. At present, the establishment of IDD model mainly includes two following aspects, in vitro model and in vivo model. There are two main in vitro models, cell culture and tissue or organ culture. There are seven kinds of in vivo models, which can be divided into two categories, namely spontaneous and induced model. Among them, the spontaneous degeneration model is also regarded as age-related degeneration, while the induced model refers to the construction of the animal model of IDD by injuring the structure of the intervertebral disc, changing the biomechanical structure of the vertebral body, development spinal instability caused by surgery or constructing nerve root compression and gene knockout. Although there are many methods of animal modeling and literature reports, each method has its own advantages and disadvantages. The advantages and disadvantages should be weighed when choosing the animal models.
ABSTRACT
Low back pain is becoming an important factor affecting people's quality of life, while the age of its onset is getting younger and younger, and the social and economic losses caused by low back pain are huge every year. Intervertebral disc degeneration (IDD) is an important cause of low back pain. Due to multiple factors, biomechanical and structural changes occur in intervertebral disc tissue, including rupture of annulus fibrosus, protrusion of nucleus pulposus, which cause compression of spinal cord and nerve root, and lower back pain. Micro-RNA (miRNA) is a kind of single-stranded non-coding small molecule RNA, with 18-24 nucleotides in length, which exists widely in eukaryotes. As one of the important regulatory molecules of gene expression, it has been proved to play a key role in the initiation and progression of many diseases, and it may also play an important role in intervertebral disc degeneration. At present, the clinical treatment for IDD is mainly surgical treatment to alleviate clinical symptoms. Even if surgical treatment can achieve good results, it will bring great physical trauma and economic burden to patients. The role of miRNA in IDD is one of the hotspots in the current academic research. Studies have shown that miRNA has abnormal expression patterns in degenerative intervertebral disc tissues and participates in a variety of pathological processes of IDD. At present, some miRNAs have been proved to be related to a variety of pathological processes in IDD, including nucleus pulposus cell apoptosis and proliferation, extracellular matrix degradation, autophagy, inflammation and cartilage endplate degeneration. The comparative study of gene chip showed that there were significant differences in the expression of some miRNAs between degenerative and normal nucleus pulposus cells. These differentially expressed miRNAs may be involved in the process of nucleus pulposus cell degeneration by regulating their respective upstream or downstream pathways. Most of the regulatory pathways are crossed and parallel, thus constructing a huge miRNA regulatory network. Understanding the target genes and mechanisms of miRNA in the pathogenic process can provide an important reference for the origin, development and prognosis of IDD. In this article, the important role of miRNA in IDD and the potential significance of clinical treatment are reviewed. With the in-depth study of miRNA and the molecular biological mechanism can provide new ideas for the diagnosis and treatment of IDD, which is likely to become a new strategy for biological treatment of IDD.
ABSTRACT
Objective:Resorption can occur after lumbar disc herniation, and Thymic stromal lymphopoietin (TSLP) is considered to be a key factor mediating reabsorption. Studies have found that under the action of inflammatory factors like TNF-α, IL-6, etc,the expression of TSLP in intervertebral disc tissue was increased, and then mononuclear macrophage chemokine-1 (MCP-1) was induced to induce infiltration of macrophage to promote reabsorption. To determine the mechanism, we design the experiment to explore the mechanism of IL-6-mediated JAK/STAT pathway and TGF-β/Smad pathway to promote the reabsorption of intervertebral disc by regulating the expression of TSLP.Methods:Rat bone marrow mesenchymal stem cells (MSC) and rat nucleus pulposus cells (NPC) were cultured in vitro, treating the cells withrat IL-6 recombinant protein, STAT3inhibitor and Smad2/3 inhibitorrespectively, and use real-time quantitative PCR (qRT-PCR) technology to detect the expression of JAK1, STAT3, Smad2, TSLP mRNA under different conditions; Western blot to detect the expression of TSLP, Smad2 and phosphate STAT3 protein; using enzyme-linked immunosorbent assay (Elisa) to detect the expression of TGF-β in two rat cells under the treatment of IL-6.Results:After stimulation ofIL-6 (10 ng/ml or 100 ng/ml) the expression of JAK1in MSC (10 ng/ml: 5.13±1.21; 100 ng/ml: 5.23±0.35; control group: 0.97±0.03), STAT3 (10 ng/ml: 6.50±0.38; 100 ng/ml: 6.74±0.61; control group: 0.87±0.19) was significantly increased, and TSLP also showed high expression in MSC (10 ng/ml: 4.26±0.38; 100 ng/ml: 5.05±0.46; control group: 1.04±0.04).The expression of STAT3 (10 ng/ml: 2.91±0.08; control group: 1.12±0.11), TSLP (10 ng/ml: 7.32±0.37; control group: 1.03±0.03) in NPC also increased. After stimulation of IL-6, the expression of Smad2 increasing in MSC was observed (10 ng/ml: 15.92±0.62; 100 ng/ml: 20.28±0.58; control group: 0.96±0.08), and increased expression of Smad2 in NPC (10 ng/ml: 5.01±0.17; control group: 0.96±0.03). The expression of TSLP in MSC decreased after adding STAT3 inhibitor (BP group, BP group: 0.17±0.01; control group: 0.90±0.09), the expression of TSLP also decreased in NPC (BP group: 0.42±0.11; control group: 0.90±0.11). After adding Smad2/3 inhibitor (SB group), the expression of TSLP in MSC decreased (SB group: 0.33±0.01; control group: 1.02±0.02), and the expression of TSLP in NPC also decreased (SB group: 0.40±0.04; control group: 0.99±0.01).Conclusion:IL-6 up-regulates TSLP expression via the JAK1/STAT3 signaling pathway and promotes prominent intervertebral disc reabsorption. At the same time, IL-6 can activate the TGF-β/Smad2/3 pathway and up-regulate the expression of TSLP, which play a synergistic role in the reabsorption process.
ABSTRACT
Low back pain is becoming an important factor that affects people's quality of life today, and the social losses caused by lowback pain are hugeevery year. Lumbar disc herniation (LDH) is one of the main diseases that cause low back pain. The mechanism of lumbar disc herniation in the biomedical science is still controversial. Inflammatory factor is a cytokine secreted by tissue cells and involved in mediating the inflammatory response. Studies have shown that some factors stimulated by the extrusive nucleus pulposus, like inflammatory factors, degeneration-related genes and downstream expression products, can cause the degeneration of intervertebral disc. IL-1, IL-6, TNF-α, MMPs, and TGF-β have become the hot topicin disc degeneration. Signaling pathway is the main pathway for inflammatory factors to participate in the regulation of various biochemical reactions in cells. The inflammatory factors interact with different proteins to activate or inhibit different pathways, thereby achieving regulation of the cell cycle, regulates gene expression, induces immune inflammatory response, and apoptosis. Research on the role of various inflammatory factors in the body and related molecular signaling pathways will help us understand the mechanism of LDH. Most of the experimental studies only focus on the influence of a certain cytokine or single pathway on intervertebral disc degeneration, but different inflammatory factors and their signaling pathways often crosstalk with each other through special channels, forming a complex and precise signal transduction regulation network jointly regulates various physiological or pathological processes in the body, and the occurrence of disease is often accompanied by multiple factors. Studying the effect of a single signal network on the disease cannot fully explain the cause of the disease and related clinical manifestations. Therefore, clarifying the role of various inflammatory factors in IDD and exploring and analyzing the ways in which each factor regulates each other will provide ideas for understanding the mechanism of lumbar degeneration and exploring new methods for preventing and treating LDH in the future.
ABSTRACT
Objective The aim of current study is to determine the effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells by investigating the apoptotic activity and variation of intracellular phosphorylated protein kinase B (p-Akt),X-linkedinhibitor of apoptosis protein (XIAP),cysteinyl aspartate specific proteinase-3 (caspase-3),with the treatment of thymic stromal lymphopoietin.Methods Mouse lumbar nucleus pulposus cells were cultured and identified under a fluorescence microscope.Second or third passage cells maintained in monolayers were used for the following experiments.The groups were divided randomly into normal group,TNF-α treated group,TSLP treated group,TSLP+LY94002 treated group and TSLP+Embelin treated group.As a control,normal group was treated with PBS.TNF-α treated group was treated with 500 ng/ml TNF-αt as a positive control.TSLP treated group was treated with 10 ng/ml rhTSLP.TSLP+LY94002 treated group and TSLP+ Embelin treated group were treated with 10 ng/ml TSLP with the pretreatment of different pathway inhibitors for 30 ain in different corresponding experiments,for which 10 μ mol LY294002 or 50 LY294002 responding experimentsreatment of different pathway inhibitors formouse nucleus pulposus cells was detected by FACS.The expression levels of the intracellular p-Akt,XIAP,caspase-3 were investigated by Western blot analysis.Results As the culture cell type Ⅱ collagen staining was positive observed by fluorescence microscopy,we confirmed that the cuhured cells were nucleus pulposus cells.In comparison with negative control,the levels of p-Akt,XIAP in TSLP treated group were elevated (t=9.510,P=0.001;t=8.851,P=0.001).Thecaspase-3 activity were slightly enhanced and the rate of cells apoptosis was no significance.Compared with TSLP treated group,downregulated level of pAkt and XIAPand upregulatedcaspase-3 activity in TSLP+LY294002 treated group were observed (t=8.798,P=0.001;t=7.032,P=0.002;t=5.908,P=0.004).Upregulated caspase-3 activity were also observed in TSLP+ Embelin treated group (t=7.990,P=0.001).Furthermore,significant increased apoptotic cell rate was observed in TSLP+LY294002 or TSLP+Embelin treated groups (t=21.268,P=0.001;t=21.279,P=0.001).Conclusion TSLP may have a potential anti-apoptotic effect on mouse NP cells via upregulating XIAP in PI3K/Akt signaling pathway to restrain the activation of caspase-3.