Circ-STC2 promotes the ferroptosis of nucleus pulposus cells via targeting miR-486-3p/TFR2 axis.

BACKGROUND: Low back pain (LBP) has become the second leading cause of disability worldwide, which has brought great economic burden to people. It is generally believed that intervertebral disc degeneration (IDD) is the main cause of LBP. This study aimed to explore the role of circ-STC2 in the pathogenesis of IDD. METHODS: Nucleus pulposus cells (NPCs) were treated with T-Butyl Hydrogen Peroxide (TBHP) to establish IDD model in vitro. RT-qPCR was performed to detect mRNA expressions. The cell viability was detected with CCK-8 assay. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+ and glutathione (GSH) of NPCs were measured by corresponding kits. The protein expressions were determined by western blot. Dual-luciferase reporter and RNA pull-down assays were conducted to verify the relationship between circ-STC2 or transferrin recepto 2 (TFR2) and miR-486-3p. RESULTS: Circ-STC2 and TFR2 expressions were up-regulated in IDD tissues, and miR-486-3p expression was down-regulated. Knockdown of circ-STC2 promoted the cell viability and inhibited the ferroptosis of the NPCs. The GSH levels, and glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) protein expressions were increased, the LDH, MDA and Fe2+ levels and achaete-scute complexlike 4 (ASCL4) protein expressions were decreased after circ-STC2 knockdown. Knockdown of miR-486-3p abrogated the si-circ-STC2 effects and overexpression of TFR2 reversed the miR-486-3p mimic effects. CONCLUSIONS: Circ-STC2 inhibits the cell viability, induced the ferroptosis of the TBHP treated NPCs via targeting miR-486-3p/TFR2 axis.

Pathomechanism of the IVDs Degeneration and the Role of Neurotrophic Factors and Concentration of Selected Elements in Genesis of Low Back Pain.

Degenerative disc disease of the lumbosacral spine is a very common medical problem. An episode of sciatica occurs at least once in the life of 60-90% of the human population. A phenomenon that is closely related to the process of lowering the pH of the extracellular matrix degenerating the intervertebral disc (IVD) is the precipitation of calcium salts, especially pyrophosphate dehydrate and hydroxyapatite. In such an altered environment of the IVD, we can observe an increased influx of monocytes, macrophages, T-lymphocytes, as well as non-immunocompetent cells, which are a source of cytokines, e.g., tumor necrosis alpha (TNF-α), interleukin- (IL-1ß, IL-8). The above-mentioned mediators of an inflammatory condition contribute to an increase in the expression of Brain-Derived Neurotrophic Factor (BDNF) and Glial cell Derived Neurotrophic Factor (GDNF) in mast cells and chondrocytes, as well as to the descending transport of these mediators along the nerve endings. In the process of degeneration of the IVD as a result of repeated and even slight injuries, there is damage to the connections of the endplate of the vertebral bodies with the IVD, which results in an impairment of the penetration of nutritional substances and water into the disc. As a consequence, there is an overexpression of the brain-derived neurotrophic factor GDNF, as well as neuromodulin (GAP-43) in the mast cells and chondrocytes of the IVDs, while descending transport of these mediators along the nerve fibers is also observed.

Development of a rat model with lumbar vertebral endplate lesion.

PURPOSE: Vertebral endplate lesion (EPL) caused by severe disc degeneration is associated with low back pain. However, there is no suitable animal model to elucidate the pathophysiology of EPL. This study aimed to develop a rat model of EPL and evaluate rat behavior and imaging and histological findings. METHODS: The L4-5 intervertebral discs of Sprague-Dawley rats were transperitoneally removed, except for the outer annulus fibrosus and cartilage endplate, in the EPL group. The L4-5 discs were not removed and simply exposed in the sham group. Changes around the vertebral endplate on magnetic resonance imaging (MRI) and computed tomography (CT) were evaluated. Additionally, pain-related behavioral and histological assessments were performed. RESULTS: In the EPL group, a low-signal area around the vertebral endplate was observed on T1-weighted and T2-weighted fat-saturated MRI at 8 weeks or later after surgery. In the same group, CT showed osteosclerosis around the vertebral endplate at 12 weeks after surgery. The sham group did not show abnormal imaging features on the MRI and CT. Behavioral evaluation showed that the EPL group had a significantly longer grooming time than the sham group. Conversely, the 12-week postoperative locomotion time and the 1- and 12-week postoperative standing times were significantly shorter in the EPL group than in the sham group. Histological evaluation showed a high degree of vertebral endplate degeneration and an increased number of osteoclasts and proportion of nerve fibers expressing calcitonin gene-related peptide in the EPL group compared to those in the sham group. CONCLUSION: Our rat EPL model showed pain-related behavioral patterns and an increased expression of pain-related neuropeptide. This model could contribute to the study of the pathophysiology of EPL and will help in the treatment of low back pain in the future.

Neuroimmune signatures in chronic low back pain subtypes.

We recently showed that patients with different chronic pain conditions (such as chronic low back pain, fibromyalgia, migraine and Gulf War illness) demonstrated elevated brain and/or spinal cord levels of the glial marker 18-kDa translocator protein (TSPO), which suggests that neuroinflammation might be a pervasive phenomenon observable across multiple aetiologically heterogeneous pain disorders. Interestingly, the spatial distribution of this neuroinflammatory signal appears to exhibit a degree of disease specificity (e.g. with respect to the involvement of the primary somatosensory cortex), suggesting that different pain conditions may exhibit distinct 'neuroinflammatory signatures'. To explore this hypothesis further, we tested whether neuroinflammatory signal can characterize putative aetiological subtypes of chronic low back pain patients based on clinical presentation. Specifically, we explored neuroinflammation in patients whose chronic low back pain either did or did not radiate to the leg (i.e. 'radicular' versus 'axial' back pain). Fifty-four patients with chronic low back pain, 26 with axial back pain [43.7 ± 16.6 years old (mean ± SD)] and 28 with radicular back pain (48.3 ± 13.2 years old), underwent PET/MRI with 11C-PBR28, a second-generation radioligand for TSPO. 11C-PBR28 signal was quantified using standardized uptake values ratio (validated against volume of distribution ratio; n = 23). Functional MRI data were collected simultaneously to the 11C-PBR28 data (i) to functionally localize the primary somatosensory cortex back and leg subregions; and (ii) to perform functional connectivity analyses (in order to investigate possible neurophysiological correlations of the neuroinflammatory signal). PET and functional MRI measures were compared across groups, cross-correlated with one another and with the severity of 'fibromyalgianess' (i.e. the degree of pain centralization, or 'nociplastic pain'). Furthermore, statistical mediation models were used to explore possible causal relationships between these three variables. For the primary somatosensory cortex representation of back/leg, 11C-PBR28 PET signal and functional connectivity to the thalamus were: (i) higher in radicular compared to axial back pain patients; (ii) positively correlated with each other; (iii) positively correlated with fibromyalgianess scores, across groups; and finally (iv) fibromyalgianess mediated the association between 11C-PBR28 PET signal and primary somatosensory cortex-thalamus connectivity across groups. Our findings support the existence of 'neuroinflammatory signatures' that are accompanied by neurophysiological changes and correlate with clinical presentation (in particular, with the degree of nociplastic pain) in chronic pain patients. These signatures may contribute to the subtyping of distinct pain syndromes and also provide information about interindividual variability in neuroimmune brain signals, within diagnostic groups, that could eventually serve as targets for mechanism-based precision medicine approaches.

Oxygen-Ozone Therapy in the Rehabilitation Field: State of the Art on Mechanisms of Action, Safety and Effectiveness in Patients with Musculoskeletal Disorders.

In recent years, the interest in oxygen-ozone (O2O3) therapy application has considerably increased in the field of rehabilitation. Despite its widespread use in common clinical practice, the biochemical effects of O2O3 are still far from being understood, although its chemical properties seem to play a pivotal role in exerting its positive effects on different pathological conditions. Indeed, the effectiveness of O2O3 therapy might be partly due to the moderate oxidative stress produced by O3 interactions with biological components. O2O3 therapy is widely used as an adjuvant therapeutic option in several pathological conditions characterized by chronic inflammatory processes and immune over-activation, and most musculoskeletal disorders share these pathophysiological processes. The present comprehensive review depicts the state-of-the-art on the mechanisms of action, safety and effectiveness of O2O3 therapy in the complex scenario of the management of musculoskeletal disorders. Taken together, our findings suggest that O2O3 therapy seems to reduce pain and improve functioning in patients affected by low back pain and knee osteoarthritis, as reported by several studies in the literature. However, to date, further studies are warranted to clearly investigate the therapeutic effects of this promising therapy on other musculoskeletal disorders in the field of rehabilitation.

The potential role and trend of HIF­1α in intervertebral disc degeneration: Friend or foe? (Review).

Lower back pain (LBP) is one of the most common reasons for seeking medical advice in orthopedic clinics. Increasingly, research has shown that symptomatic intervertebral disc degeneration (IDD) is mostly related to LBP. This review first outlines the research and findings of studies into IDD, from the physiological structure of the intervertebral disc (IVD) to various pathological cascades. The vicious cycles of IDD are re­described in relation to the analysis of the relationship among the pathological mechanisms involved in IDD. Interestingly, a 'chief molecule' was found, hypoxia­inducible factor­1α (HIF­1α), that may regulate all other mechanisms involved in IDD. When the vicious cycle is established, the low oxygen tension activates the expression of HIF­1α, which subsequently enters into the hypoxia­induced HIF pathways. The HIF pathways are dichotomized as friend and foe pathways according to the oxygen tension of the IVD microenvironment. Combined with clinical outcomes and previous research, the trend of IDD development has been predicted in this paper. Lastly, an early precautionary diagnosis and treatment method is proposed whereby nucleus pulposus tissue for biopsy can be obtained through IVD puncture guided by B­ultrasound when the patient is showing symptoms but MRI imaging shows negative results. The assessment criteria for biopsy and the feasibility, superiority and challenges of this approach have been discussed. Overall, it is clear that HIF­1α is an indispensable reference indicator for the accurate diagnosis and treatment of IDD.

Resveratrol-enhanced SIRT1-mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors.

Low back pain (LBP) is a major clinical problem that lacks effective treatments. The sensory innervation in porous vertebral endplates and anxiety contributes to spinal hyperalgesia. We hypothesized that SIRT1 activator resveratrol alleviates LBP and anxiety via promotion of osteogenesis in the porous endplates. The hyperalgesia and anxiety-related behaviors; sensory innervation, inflammation and porosity of endplates; and osteogenic/osteoclastic factors expression were measured following resveratrol treatment after lumbar spine instability (LSI) surgery. To explore whether resveratrol promotes endplates osteogenesis and thus alleviates LBP through activation of SIRT1 in the osteoprogenitor cells of endplates, SIRT1OSX-/- mice were employed. Additionally, the levels of inflammation markers, phosphorylation of cAMP response element-binding protein (pCREB), and brain-derived neurotrophic factor (BDNF) in hippocampus were evaluated. After 4 or 8 weeks LSI surgery, the mice suffered from hyperalgesia and anxiety, which were efficiently attenuated by resveratrol at 8 weeks. Resveratrol treatment-enhanced osteogenesis and decreased endplates porosities accompanied with the reduction of TNFα, IL-1ß, and COX2 levels and CGRP+ nerve fibers innervation in porous endplates. Resveratrol-mediated endplates osteogenesis, decreased endplates porosities, and analgesic and antianxiety effects were abrogated in SIRT1OSX-/- mice. Furthermore, resveratrol relieved inflammation and increased pCREB and BDNF expression in the hippocampus after 8 weeks, which alleviate anxiety-related behaviors. This study provides that resveratrol-mediated porous endplates osteogenesis via the activation of SIRT1 markedly blocked sensory innervation and inflammation in endplates, therefore, alleviating LSI surgery-induced LBP and hippocampus-related anxiety.

The role of structure and function changes of sensory nervous system in intervertebral disc-related low back pain.

Low back pain (LBP) is a common musculoskeletal symptom, which can be developed in multiple clinical diseases. It is widely recognized that intervertebral disc (IVD) degeneration (IVDD) is one of the leading causes of LBP. However, the pathogenesis of IVD-related LBP is still controversial, and the treatment means are also insufficient to date. In recent decades, the role of structure and function changes of sensory nervous system in the induction and the maintenance of LBP is drawing more and more attention. With the progress of IVDD, IVD cell exhaustion and extracellular matrix degradation result in IVD structural damage, while neovascularization, innervation and inflammatory activation further deteriorate the microenvironment of IVD. New nerve ingrowth into degenerated IVD amplifies the impacts of IVD-derived nociceptive molecules on sensory endings. Moreover, IVDD is usually accompanied with disc herniation, which could injure and inflame affected nerves. Under mechanical and pro-inflammatory stimulation, the pain-transmitting pathway exhibits a sensitized function state and ultimately leads to LBP. Hence, relevant pathogenic factors, such as neurotrophins, ion channels, inflammatory factors, etc., are supposed to serve as promising therapeutic targets for LBP. The purpose of this review is to comprehensively summarize the current evidence on 1) the pathological changes of sensory nervous system during IVDD and their association with LBP, and 2) potential therapeutic strategies for LBP targeting relevant pathogenic factors.

Icariin Ameliorates Lower Back Pain in Rats via Suppressing the Secretion of Cytokine-Induced Neutrophil Chemoatractant-1.

PURPOSE: To investigate whether icariin (ICA), a well-known medicine extracted from the stem and leaf of Epimedium brevicornum Maxim, had analgesic effect on lower back pain (LBP) in rats. METHODS: In a puncture-induced LBP rat model, the severity of LBP was quantified using the paw/foot withdrawal threshold method after intragastric administration of ICA at a dosage of 50 mg/kg/d or 100 mg/kg/d. The pain-related peptides of substance P (SP) and calcitonin gene-related peptide (CGRP) were also measured in intervertebral disc (IVD) tissue using RT-PCR after ICA treatment. In addition, the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) in IVD was quantified using RT-PCR and ELISA examination. RESULTS: ICA treatment resulted in a significant amelioration of mechanical allodynia in a dose-response manner, and the analgesic effect could last for two weeks even during the washout period. More importantly, the mechanism of analgesic pharmacological effect in ICA was to suppress the upregulated CINC-1, the homolog of IL-8 in rats, which is a crucial proalgesic factor contributing to LBP, in IVDs. CONCLUSION: ICA is a novel herbal extract to relieve LBP, and it may be a promising alternative pain killer in the future.

Effects of Glucose Deprivation on ATP and Proteoglycan Production of Intervertebral Disc Cells under Hypoxia.

As the most common cause of low back pain, the cascade of intervertebral disc (IVD) degeneration is initiated by the disappearance of notochordal cells and progressive loss of proteoglycan (PG). Limited nutrient supply in the avascular disc environment restricts the production of ATP which is an essential energy source for cell survival and function such as PG biosynthesis. The objective of this study was to examine ATP level and PG production of porcine IVD cells under prolonged exposure to hypoxia with physiological glucose concentrations. The results showed notochordal NP and AF cells responded differently to changes of oxygen and glucose. Metabolic activities (including PG production) of IVD cells are restricted under the in-vivo nutrient conditions while NP notochordal cells are likely to be more vulnerable to reduced nutrition supply. Moreover, provision of energy, together or not with genetic regulation, may govern PG production in the IVD under restricted nutrient supply. Therefore, maintaining essential levels of nutrients may reduce the loss of notochordal cells and PG in the IVD. This study provides a new insight into the metabolism of IVD cells under nutrient deprivation and the information for developing treatment strategies for disc degeneration.

Endocannabinoids release after Osteopathic Manipulative Treatment. A brief review.

OBJECTIVES: Since 70's, scientific research has analyzed how many acute and chronic issues can affect body systems. In case of depression, chronic pain and overtraining, centrals and peripherals systems act to manage and maintain body adaptations. The aim of this study is to evaluate if the osteopathic treatment can increase the release of Cannabinoid receptor (CB) and promote the linkage with their receptors. CONTENT: Documents research is based on PubMed and Google Scholar databases. Keywords used were "osteopathic treatment", "manual therapy", "endocannabinoid", "beta endorphin (BE)", and " CB1" "massage". From 70 articles collected (published in the last 10 years) 52 were excluded as non-relevant to the study aim. SUMMARY: The Key points have been the similar results found by different authors during different treatment periods and with different doses. From 22 articles examined, 13 have established positive effects on CB increasing post osteopathic treatment, three articles have indicated the most targeted tissues in which the substances are most expressed, two articles indicate how physical activities produce antalgic effects by increasing CB's values. OUTLOOK: As a result of this review, osteopathic manipulation treatment seems to be a valid and effective instrument for the treatment of a series of pathologies such as chronic low back pain, fibromyalgia, spinal cord lesions, myofascial graft point, migraine, GI tract dysfunctions, and depression.

Low back pain in athletes can be controlled with acupuncture by a catecholaminergic pathway: clinical trial.

BACKGROUND: Activation of the sympathetic nervous system attenuates inflammation via catecholamines. Recent evidence has shown that electroacupuncture (EA) activates neuronal networks involved in the release of dopamine and norepinephrine that control systemic inflammation. In muscle, catecholamines are related to cyclic adenosine monophosphate (cAMP). This signaling molecule has been implicated in recovery from sustained contractile activity, which may induce muscular pain, such as that which occurs during low back pain (LBP). OBJECTIVE: Our aim was to evaluate the effects of EA used for the control of LBP on the activation of the sympathetic nervous system in a randomized controlled clinical trial in athletes. METHODS: Two groups of athletes with acute or chronic low back pain were studied. EA, sham EA and pharmacological treatment (diclofenac sodium) were evaluated. The outcome measures included a pain score represented by a visual analogue scale (VAS) and serum levels of catecholamines quantified by enzyme-linked immunosorbent assay. In addition, blood was collected into chilled heparin tubes, placed in 96-well cell culture plates and incubated with an equal volume of Roswell Park Memorial Institute (RPMI) medium, with lipopolysaccharide (LPS) alone or with catecholamines. Tumor necrosis factor (TNF)-α levels in the supernatants were analyzed. RESULTS: The results indicated that the initial pain ratings did not differ between the groups analyzed. EA induced epinephrine secretion but not norepinephrine or dopamine secretion. Although EA and pharmacological treatment did not differ in terms of pain relief, in vitro epinephrine and norepinephrine reduced TNF-α production in response to LPS stimuli. CONCLUSION: EA activates the sympathetic nervous system and induces the release of epinephrine, which could ameliorate inflammation and protect muscular tissue in addition to relieving pain.

Identification of significant gene biomarkers of low back pain caused by changes in the osmotic pressure of nucleus pulposus cells.

The incidence of intervertebral disc (IVD) degeneration disease, caused by changes in the osmotic pressure of nucleus pulposus (NP) cells, increases with age. In general, low back pain is associated with IVD degeneration. However, the mechanism and molecular target of low back pain have not been elucidated, and there are no data suggesting specific biomarkers of low back pain. Therefore, the research aims to identify and verify the significant gene biomarkers of low back pain. The differentially expressed genes (DEGs) were screened in the Gene Expression Omnibus (GEO) database, and the identification and analysis of significant gene biomarkers were also performed with various bioinformatics programs. A total of 120 patients with low back pain were recruited. Before surgery, the degree of pain was measured by the numeric rating scale (NRS), which enables comparison of the pain scores from individuals. After surgery, IVD tissues were obtained, and NP cells were isolated. The NP cells were cultured in two various osmotic media, including iso-osmotic media (293 mOsm/kg H2O) to account for the morbid environment of NP cells in IVD degeneration disease and hyper-osmotic media (450 mOsm/kg H2O) to account for the normal condition of NP cells in healthy individuals. The relative mRNA expression levels of CCL5, OPRL1, CXCL13, and SST were measured by quantitative real-time PCR in the in vitro analysis of the osmotic pressure experiments. Finally, correlation analysis and a neural network module were employed to explore the linkage between significant gene biomarkers and pain. A total of 371 DEGs were identified, including 128 downregulated genes and 243 upregulated genes. Furthermore, the four genes (CCL5, OPRL1, SST, and CXCL13) were identified as significant gene biomarkers of low back pain (P < 0.001) based on univariate linear regression, and CCL5 (odds ratio, 34.667; P = 0.003) and OPRL1 (odds ratio, 19.875; P < 0.001) were significantly related to low back pain through multivariate logistic regression. The expression of CCL5 and OPRL1 might be correlated with low back pain in patients with IVD degeneration disease caused by changes in the osmotic pressure of NP cells.

Biopsy samples from the erector spinae of persons with nonspecific chronic low back pain display a decrease in glycolytic muscle fibers.

BACKGROUND CONTEXT: Low back pain (LBP) in Western Europe was classified as having the highest disability and overall burden among 291 studied conditions. For an extensive period of time, evidence related to morphological changes (eg, atrophy and fat infiltration) of the paraspinal muscles in persons with LBP has accumulated. Despite this evidence, there is limited knowledge on muscle fiber type composition of these muscles, and their relation to LBP. PURPOSE: The aim of the study is to investigate differences in muscle fiber type composition between persons with nonspecific chronic low back pain (NSCLBP) and healthy controls for the lumbar erector spinae (ES) and multifidus (MF) muscle. STUDY DESIGN AND SETTING: A cross-sectional study took place in the REVAL Rehabilitation Research Center, Hasselt University, Diepenbeek, Belgium. PATIENT SAMPLE: Twenty persons with NSCLBP (age: 44.5±7.42) and 18 healthy controls (age: 39.89±7.90) participated in this study. OUTCOME MEASURES: The primary outcome measure was paraspinal muscle fiber type composition. Secondary outcomes consisted of physiologic measures (maximal aerobic capacity and back muscle strength) and functional measures (activity level). METHODS: Biopsy samples were taken from the lumbar ES and MF muscle at the L4 spinal level. These samples were stained using immunofluorescent antibodies against myosin heavy chains. In each sample, number and size (CSA) of type I, I/IIa, IIa, IIa/x, and IIx muscle fibers were quantified. From these data the relative cross-sectional fiber areas (RCSA) were calculated. To analyze differences in fiber type composition between healthy persons and persons with NSCLBP, a repeated measurements analysis of variance was used. Secondary outcome measures were analyzed using a Student's t test, and Wilcoxon test. This study was supported by the research fund of Hasselt University without potential conflict of interest. RESULTS: There were no significant differences between both groups regarding anthropometric data. There were no significant between group differences for CSA in the ES. Persons with NSCLBP displayed a nonsignificant (p=.0978) increase in the number of type I muscle fibers, and a significant decrease (p=.0019) in the number of type IIx muscle fibers in the ES muscle. Persons with NSCLBP also displayed a trend toward a higher (p=.0596) RCSA for type I fibers and a significantly lower RCSA for type IIx fibers (p=.0411). There were no significant between group differences within the MF. Regarding the secondary outcome measures, there was a significant between group difference in activity level (p=.0004) and isokinetic back muscle strength (p=.0342). CONCLUSIONS: This is the first study to examine muscle fiber type characteristics in both the ES and MF muscle of persons with NSCLBP. Based on muscle fiber characteristics, the paraspinal muscles of persons with NSCLBP seems to display a larger oxidative potential based on an increase of the number type I fibers at the expense of type IIx glycolytic fibers.

BMP-3 Promotes Matrix Production in Co-cultured Stem Cells and Disc Cells from Low Back Pain Patients.

Low back pain is one of the most common disorders and believed to be due to intervertebral disc degeneration. Transplantation of human mesenchymal stem cells (hMSCs) is suggested as potential treatment option. Bone morphogenetic growth factor 3 (BMP-3) promotes chondrogenesis and is proven effective in enhancing chondrogenesis in hMSCs pretreated with interleukin-1 beta (IL-1ß) in hydrogel model. Three-dimensional co-cultures of hMSCs and disc cells (DCs) have previously been demonstrated to result in increased proteoglycan production. The aim was to study the effects of BMP-3 on hMSCs, DCs, as well as hMSCs and DCs in co-culture in a pellet system, both as single treatment and after pretreatment of IL-1ß. Cell pellet cultures with hMSCs, DCs, and co-culture (1:1 ratio) were performed and stimulated with BMP-3 at 1 or 10 ng/mL concentrations. For pretreatment (PRE-T), cell pellets were first stimulated with IL-1ß, for 24 h, and then BMP-3. The pellets were harvested on day 7, 14, and 28. Results demonstrated that BMP-3 stimulation at 10 ng/mL promoted cell viability, proteoglycan accumulation, as well as chondrogenesis in all pellet groups compared to 1 ng/mL. Cellular proliferation and chondrogenic differentiation of hMSCs were best promoted by PRE-T at 10 ng/mL, whereas BMP-3 best enhanced chondrogenesis in DC and co-culture pellets at the same concentration. Impact Statement Current therapies for low back pain include pain modulation and surgery, which do not tackle the underlying cellular mechanisms of the degenerated intervertebral discs (IVDs). To develop an understanding of the degeneration process and to further reverse its course, the effects of growth factor and cytokine on the native cells of the IVDs were investigated, revealing the potency of bone morphogenetic growth factor 3 on disc cells (DCs) and combined culture of mesenchymal stem cells and DCs. These results may impact future strategies in development of cell therapies that could directly influence the IVD degeneration process, which might alter the treatment models of today.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1ß, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1ß by suppressing the increase in IL-1ß, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1ß-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1ß to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Interleukin-8 as a therapeutic target for chronic low back pain: Upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model.

BACKGROUND: Low back pain (LBP) is the leading global cause of disability and is associated with intervertebral disc degeneration (DD) in some individuals. However, many adults have DD without LBP. Understanding why DD is painful in some and not others may unmask novel therapies for chronic LBP. The objectives of this study were to a) identify factors in human cerebrospinal fluid (CSF) associated with chronic LBP and b) examine their therapeutic utility in a proof-of-concept pre-clinical study. METHODS: Pain-free human subjects without DD, pain-free human subjects with DD, and patients with chronic LBP linked to DD were recruited and lumbar MRIs, pain and disability levels were obtained. CSF was collected and analyzed by multiplex cytokine assay. Interleukin-8 (IL-8) expression was confirmed by ELISA in CSF and in intervertebral discs. The SPARC-null mouse model of progressive, age-dependent DD and chronic LBP was used for pre-clinical validation. Male SPARC-null and control mice received systemic Reparixin, a CXCR1/2 (receptors for IL-8 and murine analogues) inhibitor, for 8 weeks. Behavioral signs of axial discomfort and radiating pain were assessed. Following completion of the study, discs were excised and cultured, and conditioned media was evaluated with a protein array. FINDINGS: IL-8 was elevated in CSF of chronic LBP patients with DD compared to pain-free subjects with or without DD. Chronic inhibition with reparixin alleviated low back pain behaviors and attenuated disc inflammation in SPARC-null mice. INTERPRETATION: These studies suggest that the IL-8 signaling pathway is a viable therapy for chronic LBP. FUND: Supported by NIH, MMF, CIHR and FRQS.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Plasma N-glycome composition associates with chronic low back pain.

BACKGROUND: Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease. METHODS: Plasma samples of 1128 CLBP patients and 760 healthy controls were collected in clinical centers in Italy, Belgium and Croatia and used for N-glycosylation profiling by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) after N-glycans release, fluorescent labeling and clean-up. Observed N-glycosylation profiles have been compared with a cohort of 126 patients with acute inflammation that underwent abdominal surgery. RESULTS: We have found a statistically significant increase in the relative amount of high-branched (tri-antennary and tetra-antennary) N-glycan structures on CLBP patients' plasma glycoproteins compared to healthy controls. Furthermore, relative amounts of disialylated and trisialylated glycan structures were increased, while high-mannose and glycans containing bisecting N-acetylglucosamine decreased in CLBP. CONCLUSIONS: Observed changes in CLBP on the plasma N-glycome level are consistent with N-glycosylation changes usually seen in chronic inflammation. GENERAL SIGNIFICANCE: To our knowledge, this is a first large clinical study on CLBP patients and plasma N-glycome providing a new glycomics perspective on potential disease pathology.

Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.

Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 µg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.