DUSP-1 Induced by PGE2 and PGE1 Attenuates IL-1β-Activated MAPK Signaling,Leading to Suppression of NGF Expression in Human Intervertebral Disc Cells

The molecular mechanism of discogenic low back pain (LBP) involves nonphysiological nerve invasion into a degenerated intervertebral disc (IVD), induced by nerve growth factor (NGF). Selective cyclooxygenase (COX)-2 inhibitors are mainly used in the treatment of LBP, and act by suppressing the inflammatory mediator prostaglandin E₂ (PGE₂), which is induced by inflammatory stimuli, such as interleukin-1β (IL-1β). However, in our previous in vitro study using cultured human IVD cells, we demonstrated that the induction of NGF by IL-1β is augmented by a selective COX-2 inhibitor, and that PGE₂ and PGE₁ suppress NGF expression. Therefore, in this study, to elucidate the mechanism of NGF suppression by PGE₂ and PGE₁, we focused on mitogen-activated protein kinases (MAPKs) and its phosphatase, dual-specificity phosphatase (DUSP)-1. IL-1β-induced NGF expression was altered in human IVD cells by MAPK pathway inhibitors. PGE₂ and PGE₁ enhanced IL-1β-induced DUSP-1 expression, and suppressed the phosphorylation of MAPKs in human IVD cells. In DUSP-1 knockdown cells established using small interfering RNA, IL-1β-induced phosphorylation of MAPKs was enhanced and prolonged, and NGF expression was significantly enhanced. These results suggest that PGE₂ and PGE₁ suppress IL-1β-induced NGF expression by suppression of the MAPK signaling pathway, accompanied by increased DUSP-1 expression.     

Should Degenerated Intervertebral Discs of Patients with Modic Type 1 Changes Be Treated with Mesenchymal Stem Cells?

Low back pain (LBP) has been among the leading causes of disability for the past 30 years. This highlights the need for improvement in LBP management. Many clinical trials focus on developing treatments against degenerative disc disease (DDD). The multifactorial etiology of DDD and associated risk factors lead to a heterogeneous patient population. It comes as no surprise that the outcomes of clinical trials on intradiscal mesenchymal stem cell (MSC) injections for patients with DDD are inconsistent. Intradiscal MSC injections have demonstrated substantial pain relief and significant disability-related improvements, yet they have failed to regenerate the intervertebral disc (IVD). Increasing evidence suggests that the positive outcomes in clinical trials might be attributed to the immunomodulatory potential of MSCs rather than to their regenerative properties. Therefore, patient stratification for inflammatory DDD phenotypes may (i) better serve the mechanisms of action of MSCs and (ii) increase the treatment effect. Modic type 1 changes—pathologic inflammatory, fibrotic changes in the vertebral bone marrow—are frequently observed adjacent to degenerated IVDs in chronic LBP patients and represent a clinically distinct subpopulation of patients with DDD. This review discusses whether degenerated IVDs of patients with Modic type 1 changes should be treated with an intradiscal MSC injection.     

Genetic Therapy for Intervertebral Disc Degeneration

Intervertebral disc (IVD) degeneration can cause chronic lower back pain (LBP), leading to disability. Despite significant advances in the treatment of discogenic LBP, the limitations of current treatments have sparked interest in biological approaches, including growth factor and stem cell injection, as new treatment options for patients with chronic LBP due to IVD degeneration (IVDD). Gene therapy represents exciting new possibilities for IVDD treatment, but treatment is still in its infancy. Literature searches were conducted using PubMed and Google Scholar to provide an overview of the principles and current state of gene therapy for IVDD. Gene transfer to degenerated disc cells in vitro and in animal models is reviewed. In addition, this review describes the use of gene silencing by RNA interference (RNAi) and gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR) system, as well as the mammalian target of rapamycin (mTOR) signaling in vitro and in animal models. Significant technological advances in recent years have opened the door to a new generation of intradiscal gene therapy for the treatment of chronic discogenic LBP.     

Effects of Neuropeptides and Mechanical Loading on Bone Cell Resorption in Vitro

Neuropeptides such as vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are present in nerve fibers of bone tissues and have been suggested to potentially regulate bone remodeling. Oscillatory fluid flow (OFF)-induced shear stress is a potent signal in mechanotransduction that is capable of regulating both anabolic and catabolic bone remodeling. However, the interaction between neuropeptides and mechanical induction in bone remodeling is poorly understood. In this study, we attempted to quantify the effects of combined neuropeptides and mechanical stimuli on mRNA and protein expression related to bone resorption. Neuropeptides (VIP or CGRP) and/or OFF-induced shear stress were applied to MC3T3-E1 pre-osteoblastic cells and changes in receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) mRNA and protein levels were quantified. Neuropeptides and OFF-induced shear stress similarly decreased RANKL and increased OPG levels compared to control. Changes were not further enhanced with combined neuropeptides and OFF-induced shear stress. These results suggest that neuropeptides CGRP and VIP have an important role in suppressing bone resorptive activities through RANKL/OPG pathway, similar to mechanical loading.     

In Situ Cell Signalling of the Hippo-YAP/TAZ Pathway in Reaction to Complex Dynamic Loading in an Intervertebral Disc Organ Culture

Recently, a dysregulation of the Hippo-YAP/TAZ pathway has been correlated with intervertebral disc (IVD) degeneration (IDD), as it plays a key role in cell survival, tissue regeneration, and mechanical stress. We aimed to investigate the influence of different mechanical loading regimes, i.e., under compression and torsion, on the induction and progression of IDD and its association with the Hippo-YAP/TAZ pathway. Therefore, bovine IVDs were assigned to one of four different static or complex dynamic loading regimes: (i) static, (ii) “low-stress”, (iii) “intermediate-stress”, and (iv) “high-stress” regime using a bioreactor. After one week of loading, a significant loss of relative IVD height was observed in the intermediate- and high-stress regimes. Furthermore, the high-stress regime showed a significantly lower cell viability and a significant decrease in glycosaminoglycan content in the tissue. Finally, the mechanosensitive gene CILP was significantly downregulated overall, and the Hippo-pathway gene MST1 was significantly upregulated in the high-stress regime. This study demonstrates that excessive torsion combined with compression leads to key features of IDD. However, the results indicated no clear correlation between the degree of IDD and a subsequent inactivation of the Hippo-YAP/TAZ pathway as a means of regenerating the IVD.     

NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia—a locus for craniofacial sensory nerves—were used to examine how NGF affects the Ca²⁺-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca²⁺-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca²⁺-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181–197 and 198–206) to support CAP-evoked Ca²⁺-dependent exocytosis at different intensities of stimulation.     

The Application of Mesenchymal Stromal Cells and Their Homing Capabilities to Regenerate the Intervertebral Disc

Chronic low back pain (LBP) remains a challenging condition to treat, and especially to cure. If conservative treatment approaches fail, the current “gold standard” for intervertebral disc degeneration (IDD)-provoked back pain is spinal fusion. However, due to its invasive and destructive nature, the focus of orthopedic research related to the intervertebral disc (IVD) has shifted more towards cell-based therapeutic approaches. They aim to reduce or even reverse the degenerative cascade by mimicking the human body’s physiological healing system. The implementation of progenitor and/or stem cells and, in particular, the delivery of mesenchymal stromal cells (MSCs) has revealed significant potential to cure the degenerated/injured IVD. Over the past decade, many research groups have invested efforts to find ways to utilize these cells as efficiently and sustainably as possible. This narrative literature review presents a summary of achievements made with the application of MSCs for the regeneration of the IVD in recent years, including their preclinical and clinical applications. Moreover, this review presents state-of-the-art strategies on how the homing capabilities of MSCs can be utilized to repair damaged or degenerated IVDs, as well as their current limitations and future perspectives.     

Lycium barbarum Polysaccharides and Capsaicin Inhibit Oxidative Stress,Inflammatory Responses,and Pain Signaling in Rats with Dextran Sulfate Sodium-Induced Colitis

Ulcerative colitis (UC) is an inflammatory disease with chronic relapsing symptoms. This study investigated the effects of Lycium barbarum polysaccharides (LBP) and capsaicin (CAP) in dextran sulfate sodium (DSS)-induced UC rats. Rats were divided into normal, DSS-induced UC, and UC treated with 100 mg LBP/kg bw, 12 mg CAP/kg bw, or 50 mg LBP/kg bw and 6 mg CAP/kg bw. Rats were fed LBP or CAP orally by gavage for 4 weeks, and UC model was established by feeding 5% DSS in drinking water for 6 days during week 3. Oral CAP and mixture significantly reduced disease activity index. Oral LBP significantly decreased serum malondialdehyde, interleukin (IL)-6, colonic tumor necrosis factor (TNF)-α levels, and protein expression of transient receptor potential cation channel V1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1), but increased serum catalase activity. Oral CAP significantly suppressed serum IL-6, colonic TRPV1 and TRPA1 protein expression, but elevated IL-10 levels, serum superoxide dismutase and catalase activities. The mixture of LBP and CAP significantly reduced serum IL-6, colonic TNF-α and TRPA1 protein. In conclusion, administration of LBP and/or CAP attenuate DSS-induced UC symptoms through inhibiting oxidative stress, proinflammatory cytokines, and protein expression of TRPV1 and TRPA1.     

Pressure Loading Induces DNA Damage in Human Hepatocyte Line L02 Cells via the ERK1/2–Dicer Signaling Pathway

Alteration of liver tissue mechanical microenvironment is proven to be a key factor for causing hepatocyte injury and even triggering the occurrence of hepatocellular carcinoma; however, the underlying mechanisms involved are not fully understood. In this study, using a customized, pressure-loading device, we assess the effect of pressure loading on DNA damage in human hepatocytes. We show that pressure loading leads to DNA damage and S-phase arresting in the cell cycle, and activates the DNA damage response in hepatocytes. Meanwhile, pressure loading upregulates Dicer expression, and its silencing exacerbates pressure-induced DNA damage. Moreover, pressure loading also activates ERK1/2 signaling molecules. Blockage of ERK1/2 signaling inhibits pressure-upregulated Dicer expression and exacerbates DNA damage by suppressing DNA damage response in hepatocytes. Our findings demonstrate that compressive stress loading induces hepatocyte DNA damage through the ERK1/2–Dicer signaling pathway, which provides evidence for a better understanding of the link between the altered mechanical environment and liver diseases.     

A Single Injection of NTG-101 Reduces the Expression of Pain-Related Neurotrophins in a Canine Model of Degenerative Disc Disease

Background: Tissue sources of pain emanating from degenerative discs remains incompletely understood. Canine intervertebral discs (IVDs) were needle puncture injured, 4-weeks later injected with either phosphate-buffered saline (PBS) or NTG-101, harvested after an additional fourteen weeks and then histologically evaluated for the expression of NGFr, BDNF, TrkB and CALCRL proteins. Quantification was performed using the HALO automated cell-counting scoring platform. Immunohistochemical analysis was also performed on human IVD tissue samples obtained from spinal surgery. Immunohistochemical analysis and quantification of neurotrophins and neuropeptides was performed using an in vivo canine model of degenerative disc disease and human degenerative disc tissue sections. Discs injected with NTG-101 showed significantly lower levels of Nerve Growth Factor receptor (NGFr/TrkA, p = 0.0001), BDNF (p = 0.009), TrkB (p = 0.002) and CALCRL (p = 0.008) relative to PBS injections. Human IVD tissue obtained from spinal surgery due to painful DDD show robust expression of NGFr, BDNF, TrkB and CALCRL proteins. A single intradiscal injection of NTG-101 significantly inhibits the expression of NGFr, BDNF, TrkB and CALCRL proteins in degenerative canine IVDs. These results strongly suggest that NTG-101 inhibits the development of neurotrophins that are strongly associated with painful degenerative disc disease and may have profound effects upon the management of patients living with discogenic pain.     

Recent Advances in Managing Spinal Intervertebral Discs Degeneration

Low back pain (LBP) represents a frequent and debilitating condition affecting a large part of the global population and posing a worldwide health and economic burden. The major cause of LBP is intervertebral disc degeneration (IDD), a complex disease that can further aggravate and give rise to severe spine problems. As most of the current treatments for IDD either only alleviate the associated symptoms or expose patients to the risk of intraoperative and postoperative complications, there is a pressing need to develop better therapeutic strategies. In this respect, the present paper first describes the pathogenesis and etiology of IDD to set the framework for what has to be combated to restore the normal state of intervertebral discs (IVDs), then further elaborates on the recent advances in managing IDD. Specifically, there are reviewed bioactive compounds and growth factors that have shown promising potential against underlying factors of IDD, cell-based therapies for IVD regeneration, biomimetic artificial IVDs, and several other emerging IDD therapeutic options (e.g., exosomes, RNA approaches, and artificial intelligence).     

Mechanical Stretch-Induced NLRP3 Inflammasome Expression on Human Annulus Fibrosus Cells Modulated by Endoplasmic Reticulum Stress

Excessive mechanical loading is a major cause of spinal degeneration, typically originating from a tear in the annulus fibrosus (AF). Endoplasmic reticulum (ER) stress and NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome have been implicated in the pathogenesis of intervertebral disc (IVD) degeneration. However, the causal relationship between the mechanical stretching of AF cells and the NLRP3 inflammasome response associated with ER stress remains scarce. To elucidate the pathogenesis and regulatory mechanisms of mechanical stretch-induced IVD degeneration, human AF cell lines were subjected to different degrees of cyclic stretching to simulate daily spinal movements. Our results indicated that 15% high cyclic stretch (HCS) induced the expression of NLRP3 and interleukin-1 beta (IL-1β) and was also responsible for the increased expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2) and reactive oxygen species (ROS) in human AF cells. In addition, HCS increased the expression of glucose-regulated protein 78 (GRP78), an ER stress chaperone, which was neutralized with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. In addition, HCS was found to induce thioredoxin-interacting protein (TXNIP) expression and NLRP3 inflammasome activation, which can be suppressed by si-NOX2 or the NOX2 inhibitor GSK2795039. Consequently, HCS upregulated ER stress and ROS production, leading to increased NLRP3 and IL-1β expression in human AF cells, and may further accelerate IVD degeneration.     

Systemic Inflammation and the Breakdown of Intestinal Homeostasis Are Key Events in Chronic Spinal Cord Injury Patients

Our aim was to investigate the subset distribution and function of circulating monocytes, proinflammatory cytokine levels, gut barrier damage, and bacterial translocation in chronic spinal cord injury (SCI) patients. Thus, 56 SCI patients and 28 healthy donors were studied. The levels of circulating CD14^+highCD16⁻, CD14^+highCD16⁺, and CD14^+lowCD16⁺ monocytes, membrane TLR2, TLR4, and TLR9, phagocytic activity, ROS generation, and intracytoplasmic TNF-α, IL-1, IL-6, and IL-10 after lipopolysaccharide (LPS) stimulation were analyzed by polychromatic flow cytometry. Serum TNF-α, IL-1, IL-6 and IL-10 levels were measured by Luminex and LPS-binding protein (LBP), intestinal fatty acid-binding protein (I-FABP) and zonulin by ELISA. SCI patients had normal monocyte counts and subset distribution. CD14^+highCD16⁻ and CD14^+highCD16⁺ monocytes exhibited decreased TLR4, normal TLR2 and increased TLR9 expression. CD14^+highCD16⁻ monocytes had increased LPS-induced TNF-α but normal IL-1, IL-6, and IL-10 production. Monocytes exhibited defective phagocytosis but normal ROS production. Patients had enhanced serum TNF-α and IL-6 levels, normal IL-1 and IL-10 levels, and increased circulating LBP, I-FABP, and zonulin levels. Chronic SCI patients displayed impaired circulating monocyte function. These patients exhibited a systemic proinflammatory state characterized by enhanced serum TNF-α and IL-6 levels. These patients also had increased bacterial translocation and gut barrier damage.     

Comparative Metabolic Study of Two Contrasting Chinese Cabbage Genotypes under Mild and Severe Drought Stress

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important leafy vegetable crop cultivated worldwide. Drought is one of the most important limiting factors for the growth, production and quality of Chinese cabbage due to its weak drought tolerance. In order to deepen the understanding of drought stress response in Chinese cabbage, metabolomics studies were conducted in drought−tolerant (DT) and drought−susceptible (DS) genotypes of Chinese cabbage under water deficit−simulated mild and severe drought stress conditions. A total of 777 metabolites were detected, wherein 90 of them were proposed as the drought−responsive metabolites in Chinese cabbage, with abscisic acid (ABA), serine, choline alfoscerate, and sphingosine as potential representative drought stress biomarkers. We also found that drought−tolerant and drought−susceptible genotypes showed differential metabolic accumulation patterns with contrasting drought response mechanisms. Notably, constitutively high levels of ABA and glutathione were detected in drought−tolerant genotype in all tested and control conditions. In addition, proline, sucrose, γ−aminobutyric acid, and glutathione were also found to be highly correlated to drought tolerance. This study is the first metabolomic study on how Chinese cabbage responds to drought stress, and could provide insights on how to develop and cultivate new drought−resistant varieties.     

Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice

The effect of a cellular prion protein (PrP^c) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP^c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp^−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na⁺/K⁺ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp^−/− vs. WT control group, without a substantial change in the Na⁺/K⁺ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp^−/− vs. WT mice. The present study demonstrates that a lack of PrP^c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp^−/− vs. WT mice. Our findings provide evidence that PrP^c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.