Identification of key eRNAs for intervertebral disc degeneration by integrated multinomial bioinformatics analysis.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a common degenerative condition leading to abnormal stress distribution under load, causing intervertebral stenosis, facet joint degeneration, and foraminal stenosis. Very little is known about the molecular mechanism of eRNAs in IVDD. METHODS: Gene expression profiles of 38 annulus disc samples composed of 27 less degenerated discs (LDs) and 11 more degenerated discs (MDs) were retrieved from the GEO database. Then, differentially expressed enhancer RNAs (DEeRNAs), differentially expressed target genes (DETGs), and differentially expressed transcription factors (DETFs), hallmark of cancer signalling pathways according to GSVA; the types and quantity of immune cells according to CIBERSORT; and immune gene sets according to ssGSEA were analysed to construct an IVDD-related eRNA network. Then, multidimensional validation was performed to explore the interactions among DEeRNAs, DETFs and DEGs in space. RESULTS: A total of 53 components, 14 DETGs, 15 DEeRNAs, 3 DETFs, 5 immune cells, 9 hallmarks, and 7 immune gene sets, were selected to construct the regulatory network. After validation by online multidimensional databases, 21 interactive DEeRNA-DEG-DETF axes related to IVDD exacerbation were identified, among which the C1S-CTNNB1-CHD4 axis was the most significant. CONCLUSION: Based upon the results of our study, we theorize that the C1S-CTNNB1-CHD4 axis plays a vital role in IVDD exacerbation. Specifically, C1S recruits CTNNB1 and upregulates the expression of CHD4 in IVDD, and subsequently, CHD4 suppresses glycolysis and activates oxidative phosphorylation, thus generating insoluble collagen fibre deposits and leading to the progression of IVDD. Overall, these DEeRNAs could comprise promising therapeutic targets for IVDD due to their high tissue specificity.

Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis.

The purpose of this study was to investigate the initiation of autophagy activation and apoptosis in nucleus pulposus cells under temporary compression (TC) and sustained compression (SC) to identify ideal research approaches in intervertebral disc degeneration. Various techniques were used: radiography (X-ray), magnetic resonance imaging (MRI), transmission electron microscope (TEM), H&E staining, Masson's trichrome staining, immunohistochemistry (IHC) (LC3, beclin-1, and cleaved caspase-3), and real-time polymerase chain reaction (RT-qPCR) for autophagy-related (beclin-1, LC3, and P62) and apoptosis-related (caspase-3 and PARP) gene expression analysis. X-ray and MRI revealed varying degrees of disc degeneration, ranging from moderate to severe in both groups. The severity was directly linked to compression duration, with SC resulting in notably severe central NP cell degeneration. Surprisingly, TC also caused similar, though less severe, degeneration. Elevated expression of LC3 and beclin-1 was identified after 6 weeks, but it notably declined after 12 weeks. Central NP cells in both groups exhibited increased expression of cleaved caspase-3 that was positively correlated with the duration of SC. TC showed fewer apoptotic markers compared to SC. LC3, beclin-1, and P62 mRNA expression peaked after 6 weeks and declined after 12 weeks in both groups. Cleaved caspase-3 and PARP expression peaked in SC, positively correlating with longer compression duration, while TC showed lower levels of apoptosis gene expression. Furthermore, TEM results revealed different events of the autophagic degradation process after 2 weeks of compression. TCmay be ideal for studying early triggered autophagy-mediated degeneration, while SC may be ideal for studying late or slower-triggered apoptosis-mediated degeneration.

The long non-coding RNA KLF3-AS1/miR-10a-3p/ZBTB20 axis improves the degenerative changes in human nucleus pulposus cells.

Excessive apoptosis of intervertebral disc cells, namely nucleus pulposus (NP) cells, results in decreased cell density and extracellular matrix (ECM) catabolism, hence leading to intervertebral disc degeneration (IVDD). As a cell model in the present study, a commercially available human NP cell line was utilized. Long noncoding RNAs and microRNAs may regulate the proliferation or apoptosis of human NP cells, hence exerting a significant influence on the occurrence of IVDD. KLF3-AS1 was discovered to be abnormally downregulated in IVDD tissues. Overexpression of KLF3-AS1 enhanced NP cell viability, prevented cell apoptosis, boosted ECM synthesis, and lowered MMP-13 and ADAMTS4 levels. ZBTB20 and KLF3-AS1 were co-expressed in IVDD; ZBTB20 overexpression had similar effects on NP cells, ECM production, and MMP-13 and ADAMTS4 levels as KLF3-AS1 overexpression. miR-10a-3p may target KLF3-AS1 and ZBTB20 and inhibit the expression of ZBTB20. Inhibition of miR-10a-3p enhanced NP cell viability, reduced apoptosis, and enhanced ECM synthesis. KLF3-AS1 overexpression increased ZBTB20 expression, whereas miR-10a-3p overexpression decreased ZBTB20 expression; miR-10a-3p overexpression reduced the effects of KLF3-AS1 on ZBTB20. Overexpression of miR-10a-3p consistently decreased the effects of KLF3-AS1 overexpression on NP cell survival, apoptosis, and ECM synthesis. In conclusion, KLF3-AS1 overexpression may ameliorate degenerative NP cell alterations through the miR-10a-3p/ZBTB20 axis.

ISO 15189 is a sufficient instrument to guarantee high-quality manufacture of laboratory developed tests for in-house-use conform requirements of the European In-Vitro-Diagnostics Regulation.

The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.

Strontium Ranelate Ameliorates Intervertebral Disc Degeneration via Regulating TGF-ß1/NF-κB Axis.

Intervertebral disc degeneration (IVDD) is a prevalent and debilitating condition characterized by chronic back pain and reduced quality of life. Strontium ranelate (SRR) is a compound traditionally used for treating osteoporosis via activating TGF-ß1 signaling pathway. Recent studies have proved the anti-inflammatory effect of SRR on chondrocytes. Although the exact mechanism of IVDD remains unclear, accumulating evidences have emphasized the involvement of multifactorial pathogenesis including inflammation, oxidative stress damage, and etc. However, the biological effect of SRR on IVDD and its molecular mechanism has not been investigated. Firstly, this study proved the decreased expression of Transforming Growth Factor-beta 1(TGF-ß1) in degenerated human intervertebral disc tissues. Subsequently, we confirmed for the first time that SRR could promote cell proliferation, mitigate inflammation and oxidative stress in human nucleus pulposus cells in vitro via increasing the expression of TGF-ß1 and suppressing the Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB) pathway. The molecular docking result proved the interaction between SRR and TGF-ß1 protein. To further verify this interaction, gain- and loss- of function experiments were conducted. We discovered that both TGF-ß1 knockdown and overexpression influenced the activation of the NF-κB pathway. Taken together, SRR could mitigate IL-1ß induced-cell dysfunction in human nucleus pulposus cells by regulating TGF-ß1/NF-κB axis in vitro. Finally, the in vivo therapeutic effect of SRR on IVDD was confirmed. Our findings may contribute to the understanding of the complex interplay between inflammation and degenerative processes in the intervertebral disc and provide valuable insights into the development of targeted treatment-based therapeutics for IVDD.

Role of Necroptosis in Intervertebral Disc Degeneration.

Apoptosis has historically been considered the primary form of programmed cell death (PCD) and is responsible for regulating cellular processes during development, homeostasis, and disease. Conversely, necrosis was considered uncontrolled and unregulated. However, recent evidence has unveiled the significance of necroptosis, a regulated form of necrosis, as an important mechanism of PCD alongside apoptosis. The activation of necroptosis leads to cellular membrane disruption, inflammation, and vascularization. This process is crucial in various pathological conditions, including intervertebral disc degeneration (IVDD), neurodegeneration, inflammatory diseases, multiple cancers, and kidney injury. In recent years, extensive research efforts have shed light on the molecular regulation of the necroptotic pathway. Various stimuli trigger necroptosis, and its regulation involves the activation of specific proteins such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and the mixed lineage kinase domain-like (MLKL) pseudokinase. Understanding the intricate mechanisms governing necroptosis holds great promise for developing novel therapeutic interventions targeting necroptosis-associated IVDD. The objective of this review is to contribute to the growing body of scientific knowledge in this area by providing a comprehensive overview of necroptosis and its association with IVDD. Ultimately, these understandings will allow the development of innovative drugs that can modulate the necroptotic pathway, offering new therapeutic avenues for individuals suffering from necroptosis.

Rac1 regulates nucleus pulposus cell degeneration by activating the Wnt/ß-catenin signaling pathway and promotes the progression of intervertebral disc degeneration.

Nucleus pulposus cell (NPC) dysfunction is considered as an important event related to intervertebral disc degeneration (IVDD). In the present study, tandem mass spectrometry (TMT) was used to detect total protein expression of nucleus pulposus (NP) in patients with IVDD and healthy controls. Bioinformatic analysis was performed to identify differentially expressed proteins that may be involved in the degeneration of NP. The results show that Rac1 may be a key protein involved in the degeneration of NP via Wnt/ß-catenin pathway activation. We investigated the influence of Rac1 on IVDD degeneration and associated mechanisms. Rac1 expression increased in interleukin (IL)-1ß-stimulated human NPCs, consistent with the results of TMT. The Rac1 inhibitor NSC23766 alleviated the degeneration of NPCs in vitro. Furthermore, Rac1 activated Wnt/ß-catenin signaling, and the inhibition of this pathway significantly ameliorated the Rac1-mediated degenerative phenotype. NSC23766 exerted protective effects on IVDD in a puncture rat model. Taken together, these data suggest that Rac1 inhibition can delay NPC degeneration, probably through the regulation of the Wnt/ß-catenin pathway. This study has the potential to advance understanding of the mechanism of occurrence of degenerative NP tissues and to provide novel strategies for slowing IVDD progression.

Interleukin-18 Inhibition Protects Against Intervertebral Disc Degeneration via the Inactivation of Caspase-3/9 Dependent Apoptotic Pathways.

BACKGROUND: The present study was designed to identify and understand the potential effectiveness of therapeutic target in intervertebral disc degeneration (IVDD) and its regulation mechanism. METHODS: The role and mechanism of interleukin-18 (IL-18) in the disease were investigated. The IVDD degenerative nucleus pulposus (NP) tissues from the human and mouse models were used.A total of three groups of Male BALB/c mice were randomly made i.e control, IVDD, and IVDD+Ad-shIL-18 groups. After Ad-shIL-18 transfection, the expression of ECM synthesis related protein Aggrecan (ACAN) and Collagen II, apoptotic effector Caspases (Caspase-3, 8, 9, 12 and Cleaved-Caspase 3, 8, 9, 12), pro-apoptotic gene Bax and anti-apoptotic factors Bcl-2 in NP cells of the human were evaluated. RESULTS: The results of our study revealed that the mRNA and protein expression levels of IL-18 were notably increased in the NP tissues of IVDD patients and mice models. In the IVDD mice model, Ad-sh-IL-18 treatment reversed the IVDD progression. The levels of Aggrecan and Collagen II, contributing to ECM degradation in NP cells, were also significantly increased. Additionally, Ad-sh-IL-18 could inhibit the NP cell's apoptosis via regulating the caspase-3/9 pathway. CONCLUSION: The IL-18 knockdown via the caspase-3/9 pathway, might reduce the NP cell's death as well as the imbalance between catabolism and anabolism of ECM in IVDD.

Evaluation of the analgesic effect of fentanyl-ketamine and fentanyl-lidocaine constant rate infusions in isoflurane-anesthetized dogs undergoing thoracolumbar hemilaminectomy.

OBJECTIVE: To evaluate anesthetic conditions and postoperative analgesia with the use of intraoperative constant rate infusions (CRIs) of fentanyl-lidocaine or fentanyl-ketamine in dogs undergoing thoracolumbar hemilaminectomy. STUDY DESIGN: Prospective, randomized, blinded, clinical study. ANIMALS: A total of 32 client-owned dogs. METHODS: Dogs were premedicated with fentanyl (5 µg kg-1) administered intravenously (IV), anesthesia was induced with IV alfaxalone and maintained with isoflurane. Fentanyl (0.083 µg kg-1 minute-1) was infused IV with either ketamine (0.5 mg kg-1; then 40 µg kg-1 minute-1; group KF) or lidocaine (2 mg kg-1; then 200 µg kg-1 minute-1; group LF) assigned randomly. Heart rate, noninvasive arterial pressures, respiratory rate, esophageal temperature, end-tidal partial pressure of carbon dioxide and isoflurane concentration were recorded throughout anesthesia. Maintenance of anesthesia, recovery and postoperative pain (Glasgow Composite Pain Scale) were scored. Cardiopulmonary data were analyzed using a two-way anova with repeated measures, demographics of the two groups with a t test, and scores with Mann-Whitney U test, with p < 0.05. RESULTS: All dogs recovered from anesthesia without complications. No significant difference was found between groups for cardiopulmonary variables, total anesthesia time, sedation score and requirement for postoperative sedation or for rescue analgesia. Anesthetic maintenance score was of lower quality in KF than in LF [median (interquartile range): 0 (0-0.5) versus 0 (0-0); p = 0.032)], but still considered ideal. Recovery score was higher and indicative of less sedation in LF than in KF [1 (1-1.5) versus 0.5 (0-1); p < 0.0001]. Pain score was higher in KF than in LF [2 (1-3) versus 1 (1-2); p = 0.0009]. CONCLUSIONS AND CLINICAL RELEVANCE: Both CRIs of KF and LF provided adequate anesthetic conditions in dogs undergoing thoracolumbar hemilaminectomy. Based on requirement for rescue analgesia, postoperative analgesia was adequate in both groups.

Mesenchymal stem cells-derived exosomes ameliorate intervertebral disc degeneration through inhibiting pyroptosis.

Mesenchymal stem cell (MSCs)-based therapies have shown a promised result for intervertebral disc degeneration (IVDD) treatment. However, its molecular mechanisms remain unclear. Exosomes involve cell-cell communication via transference of its contents among different cells, and the present potential effect on cell death regulation. This study aimed to investigate the role of MSCs-derived exosomes on IVDD formation. Here, we first found the NLRP3-mediated nucleus pulposus cell (NP cell) pyroptosis was activated in the IVDD mice model and lipopolysaccharide (LPS)-induced model. However, MSCs treatment could inhibit NP cell pyroptosis in vitro. We then isolated MSCs-derived exosomes by differential centrifugation and identified the characteristics. Secondly, we investigated the function of MSCs-derived exosomes on LPS-induced NP cell pyroptosis. Finally, we presented evidence that MSCs-derived exosomal miR-410 was a crucial regulator of pyroptosis. Results showed that MSCs-derived exosomes play an anti-pyroptosis role by suppressing the NLRP3 pathway. Moreover, it suggested that this effect was attributed to miR-410, which was derived from MSCs-exosomes and could directly bind to NLRP3mRNA. In conclusion, for the first time, we demonstrated that MSCs-exosome treatment may inhibit pyroptosis and could be a promising therapeutic strategy for IVDD.

Mfn2 is involved in intervertebral disc degeneration through autophagy modulation.

OBJECTIVE: To explore whether Mitofusin 2 (Mfn2) is implicated in the pathogenesis of intervertebral disc degeneration (IVDD). METHODS: We detected the protein content of Mfn2 in degenerated human nucleus pulposus (NP) tissues and investigated the effects of Mfn2 knockdown and Mfn2 overexpression on rat nucleus pulposus cells (NPCs) under oxidative stress by using a range of biological techniques. Afterwards, we confirmed the effects of Mfn2 overexpression on NPCs in vivo and further evaluated the therapeutic action of adenovirus (AV)-Mfn2 injection in a rodent IVDD model. RESULTS: Mfn2 expression was decreased in human NP tissues during IVDD. Mfn2 knockdown aggravated the impairment of autophagic flux, mitochondrial dysfunction and cellular apoptosis in rat NPCs after Tert-Butyl hydroperoxide (TBHP) treatment, while Mfn2 overexpression significantly reversed these alterations. Besides, Mfn2 overexpression promoted an ROS (reactive oxygen species)-dependent mitophagy via PINK1 (PTEN-induced putative kinase 1)/Parkin pathway in TBHP-treated NPCs. Inhibition of autophagy with chloroquine (CQ) disordered the protective effects of Mfn2 overexpression on NPCs. Furthermore, Mfn2 overexpression in discs by AV-Mfn2 injection ameliorated the development of IVDD in rats. CONCLUSION: Mfn2 repression is deeply involved in the pathogenesis of IVDD with its impairment on autophagy, leading to the aggravation of mitochondrial dysfunction and apoptotic cell death, which ought to be a promising therapeutic target for IVDD.

BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.

Diabetes mellitus may lead to intervertebral disc degeneration (IVDD). Matrix metalloproteinase-13 (MMP-13) is one of the major catabolic factors in extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and contributes to diabetic IVDD. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is implicated in chronic inflammation. Here, we report that the expression of BRD4 and MMP-13 was elevated in diabetic nucleus pulposus tissues as well as in advanced glycation end products (AGEs)-treated NPCs; also, the regulatory effect of BRD4 on MMP-13 was studied. We found that MMP-13 was regulated by MAPK and NF-κB signaling as well as autophagy in AGEs-treated NPCs. Next, we explored the role of BRD4 in regulation of MAPK, NF-κB signaling, and autophagy. The results showed that BRD4 is the upstream regulator of all of these 3 factors, and inhibition of BRD4 may suppress MAPK and NF-κB signaling while activating autophagy in AGEs-treated NPCs. Finally, we demonstrated that BRD4 inhibition may suppress MMP-13 expression in diabetic NPCs in vitro as well as in vivo; meanwhile, it may preserve ECM in diabetic rats. Our study demonstrates that inhibition of BRD4 may suppress MAPK and NF-κB signaling and activate autophagy to suppress MMP-13 expression in diabetic IVDD, and diabetic IVDD may be compromised by BRD4 inhibitors.-Wang, J., Hu, J., Chen, X., Huang, C., Lin, J., Shao, Z., Gu, M., Wu, Y., Tian, N., Gao, W., Zhou, Y., Wang, X., Zhang, X. BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.

Adrenoceptor Expression during Intervertebral Disc Degeneration.

Healthy and degenerating intervertebral discs (IVDs) are innervated by sympathetic nerves, however, adrenoceptor (AR) expression and functionality have never been investigated systematically. Therefore, AR gene expression was analyzed in both tissue and isolated cells from degenerated human IVDs. Furthermore, human IVD samples and spine sections of wildtype mice (WT) and of a mouse line that develops spontaneous IVD degeneration (IVDD, in SM/J mice) were stained for ARs and extracellular matrix (ECM) components. In IVD homogenates and cells α1a-, α1b-, α2a-, α2b-, α2c-, ß1-, and ß2-AR genes were expressed. In human sections, ß2-AR was detectable, and its localization parallels with ECM alterations. Similarly, in IVDs of WT mice, only ß2-AR was expressed, and in IVDs of SM/J mice, ß2AR expression was stronger accompanied by increased collagen II, collagen XII, decorin as well as decreased cartilage oligomeric matrix protein expression. In addition, norepinephrine stimulation of isolated human IVD cells induced intracellular signaling via ERK1/2 and PKA. For the first time, the existence and functionality of ARs were demonstrated in IVD tissue samples, suggesting that the sympathicus might play a role in IVDD. Further studies will address relevant cellular mechanisms and thereby help to develop novel therapeutic options for IVDD.

Oxidative damage induces apoptosis and promotes calcification in disc cartilage endplate cell through ROS/MAPK/NF-κB pathway: Implications for disc degeneration.

Cartilage endplate (CEP) cell calcification and apoptosis play a vital role in the intervertebral disc degeneration (IVDD). Oxidative stress is a key factor in inducing programmed cell death and cartilage calcification. However, the cell death and calcification of cartilage endplate cells under oxidative stress have never been described. The present study investigated the apoptosis and calcification in the cartilage endplate cell under oxidative stress induced by H2O2 to understand the underlying mechanism of IVDD. The cartilage endplate cells isolated from human lumbar discs were subjected to different concentrations of H2O2 for various time periods. The cell viability was determined by CCK-8 assay, whereas Western blot, immunofluorescence, and Alcian blue, Alizarin red, and Von Kossa staining evaluated the apoptosis and calcification. The level of mitochondria-specific reactive oxygen species (ROS) was quantified with an oxygen radical-sensitive probe-MitoSOX. The potential signaling pathways were investigated by Western blot after the addition of N-acetyl-l-cysteine (NAC). We found that the oxidative stress induced by H2O2 increased the apoptosis and subsequently the calcification in the cartilage endplate cells through the ROS/p38/ERK/p65 pathway. The apoptosis and the calcification of the cartilage endplate cells induced by H2O2 can be abolished by NAC. These results suggested that regulating the apoptosis and the calcification in the cartilage endplate cells under oxidative stress should be advantageous for the survival of cells and might delay the process of disc degeneration.

Association Between Natural Killer Cell Activity and Colorectal Cancer in High-Risk Subjects Undergoing Colonoscopy.

BACKGROUND & AIMS: Low activity of natural killer (NK) cells has been associated with increased risk of cancer and has been reported in patients with colorectal cancer (CRC). Activity of NK cells can be measured in a small volume of whole blood by a commercially available test. We investigated whether this test could be used to identify patients with CRC, using findings from colonoscopy as a reference standard. METHODS: We performed an open-label, prospective, cross-sectional study of 872 high-risk subjects (more than 40 years old) screened for CRC by colonoscopy at a university hospital in Montreal, Canada from October 2014 through January 2016. Blood samples were collected on the day of colonoscopy, prior to the procedure. The test involves stimulation of whole blood with cytokine that induces NK cells to secrete interferon gamma (IFNG), which is quantified by an ELISA. Tissue samples were taken from lesions during the colonoscopy and analyzed histologically; subjects were classified as having no evidence of disease, adenomatous polyps of less than 10 mm, of 10 mm or more, or CRC. We used the non-parametric Mann-Whitney test to compare NK cell activity between subjects with no evidence of CRC and subjects found to have CRC. Receiver operating characteristic curve analysis was used to assess the ability of the test to identify individuals with CRC. The primary objective was to determine the difference in NK cell activity between subjects with vs without CRC. The secondary objective was the test performance, based on receiver operating characteristic analysis, and cut-off value that most accurately identified individuals with CRC. RESULTS: We found a significant difference in NK cell activity between the 23 subjects with CRC (based on pathology analysis) and the 849 subjects without CRC: subjects found to have CRC by colonoscopy had a median level of 86.0 pg IFNG/mL (inter-quartile range, 43.3-151.0 pg IFNG/mL), whereas subjects without CRC had a median level of 298.1 pg IFNG/mL (inter-quartile range, 100.4-920.2 pg IFNG/mL) (P = .0002). The cut-off value that most accurately identified subjects with CRC was 181 pg/mL. The NK cell activity test identified subjects with CRC with 87.0% sensitivity, 60.8% specificity, a positive predictive value of 5.7%, and a negative predictive value of 99.4%. The odds ratio for detection of CRC in subjects with low NK cell activity vs subjects with higher NK cell activity was 10.3 (95% CI, 3.03-34.9). CONCLUSIONS: Using colonoscopy as the reference standard, a test for NK cell activity in whole blood samples identified patients with CRC with 87.0% sensitivity and a negative predictive value of 99.4%. Subjects with low NK cell activity had a 10-fold higher risk of CRC compared with subjects with high NK cell activity. This test might be used in clinical practice to assess patients for risk of CRC. Clinicaltrials.gov number: NCT02291198.

Inhibition of MAGL attenuates Intervertebral Disc Degeneration by Delaying nucleus pulposus senescence through STING.

Intervertebral disc degeneration (IVDD) stands as the primary cause of low back pain (LBP). A significant contributor to IVDD is nucleus pulposus cell (NPC) senescence. However, the precise mechanisms underlying NPC senescence remain unclear. Monoacylglycerol lipase (MAGL) serves as the primary enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), breaking down monoglycerides into glycerol and fatty acids. It plays a crucial role in various pathological processes, including pain, inflammation, and oxidative stress. In this study, we utilized a lipopolysaccharide (LPS)-induced NPC senescence model and a rat acupuncture-induced IVDD model to investigate the role of MAGL in IVDD both in vitro and in vivo. Initially, our results showed that MAGL expression was increased 2.41-fold and 1.52-fold within NP tissues from IVDD patients and rats induced with acupuncture, respectively. This increase in MAGL expression was accompanied by elevated expression of p16INK4α. Following this, it was noted that the suppression of MAGL resulted in a notable decrease in the quantity of SA-ß-gal-positive cells and hindered the manifestation of p16INK4α and the inflammatory factor IL-1ß in NPCs. MAGL inhibition promotes type II collagen (Col-2) expression and inhibits matrix metalloproteinase 13 (MMP13), thereby restoring the balance of extracellular matrix (ECM) metabolism both in vitro and in vivo. A significant role for STING has also been demonstrated in the regulation of NPC senescence by MAGL. The expression of the STING protein was reduced by 57% upon the inhibition of MAGL. STING activation can replicate the effects of MAGL and substantially increase LPS-induced inflammation while accelerating the senescence of NPCs. These results strongly indicate that the inhibition of MAGL can significantly suppress nucleus pulposus senescence via its interaction with STING, consequently restoring the balance of ECM metabolism. This insight provides new perspectives for potential treatments for IVDD.

Fisetin suppresses ferroptosis through Nrf2 and attenuates intervertebral disc degeneration in rats.

Low back pain, primarily caused by intervertebral disc degeneration (IVDD), lacks effective pharmacological treatments. Oxidative stress has been identified as a significant contributor to IVDD. This study aims to establish an in vitro model of IVDD induced by oxidative stress and identify potential therapeutic agents and their underlying mechanisms. By screening the natural product library, fisetin emerged as the most promising compound in suppressing cell death induced by oxidative stress in nucleus pulposus cells (NPCs). Furthermore, our investigation revealed that the cell death induced by oxidative stress was predominantly associated with ferroptosis, and fisetin demonstrated the ability to inhibit ferroptosis in NPCs. Mechanistic exploration suggested that the impact of fisetin on ferroptosis may be mediated through the Nrf2/HO-1 (Nuclear factor erythroid 2-related factor 2/heme oxygenase-1) axis. Notably, the in vivo study demonstrated that fisetin could alleviate IVDD in rats. These findings highlight fisetin as a potential therapeutic option for IVDD and implicate the involvement of the Nrf2/HO-1 pathway in its mechanism of action.

Mast Cells and Their Related Gene HK-1 are Closely Associated with Discogenic Low Back Pain: A Bioinformatics and Clinical Sample Study.

Background: Low back pain (LBP) is primarily caused by intervertebral disc degeneration (IVDD). Immune cells penetrating nucleus pulposus (NP) tissues may play an important role in generating IVDD and LBP. Methods: The clinical data from 100 cases of IVDD patients was initially analyzed retrospectively. Subsequently, peripheral blood and NP tissues from 41 IVDD patients were gathered for a validated investigation. Among them, ribosome-removed-RNA sequencing (RNA-seq) was performed on 10 cases of NP tissues of specific classifications (VAS 3 and Pfirrmann 3 were used as the controls, while patients with VAS 6 and Pfirrmann 5 were used as the experimental group). Differentially expressed genes (DEGs) were identified for the subsequent bioinformatics analysis. Further methods to confirm the underlying cause of discogenic LBP included mast cell immunohistochemistry (IHC), 12 cytokine detection, Western blot (WB), and real-time polymerase chain reaction (RT-PCR). Results: Discogenic LBP and IVDD severity are strongly associated, and immunological cell infiltration has been demonstrated to be a significant factor in LBP by bioanalytical research. Tryptase-positive mast cells were found to be significantly more abundant in the VAS 6 NP tissues of IVDD patients than in the VAS 3 NP tissues. It was initially demonstrated that IVDD and LBP were significantly impacted by hemokinin-1 (HK-1), the mast cell-related gene. Furthermore, blood levels of interleukin 12 p70 (IL-12P70) are noticeably elevated and strongly correlated with HK-1, indicating that HK-1 may be involved in the regulation of mast cell activity and IL-12P70 production. Conclusion: The severity of LBP was observed to be positively correlated with the IVDD Pfirrmann grading. Further research indicates that patients with IVDD may experience persistent low back pain due to HK-1 activation of mast cells and the release of the cytokine IL12P70. This work will offer new insights into the diagnosis and treatment of discogenic LBP.

High glucose enhances fibrosis in human annulus fibrosus cells by activating mTOR, PKCδ, and NF-κB signaling pathways.

Low back pain stands as a significant factor in disability, largely resulting from intervertebral disc degeneration (IVDD). High glucose (HG) levels have been implicated in the pathogenesis of IVDD. However, the detailed mechanism of HG in IVDD is largely unknown. Our clinical results revealed that fibrosis markers such as CTGF, Col1a1, ATF4, and EIF2 are highly expressed in advanced-stage IVDD patients. Stimulation of human annulus fibrosus cells (HAFCs) with HG, but not mannitol, promotes fibrosis protein production. Ingenuity Pathway Analysis in the GSE database found that the mTOR, PKCδ, and NF-κB pathways were significantly changed during IVDD. The mTOR, PKCδ, and NF-κB inhibitors or siRNAs all abolished HG-induced fibrosis protein production. In addition, treatment of HAFCs with HG enhances the activation of mTOR, PKCδ, and NF-κB pathways. Thus, HG facilitates fibrosis in IVDD through mTOR, PKCδ, and NF-κB pathways. These results underscore the critical role of HG as a fibrotic factor in the progression of IVDD.

The Survival of Human Intervertebral Disc Nucleus Pulposus Cells under Oxidative Stress Relies on the Autophagy Triggered by Delphinidin.

Delphinidin (Delp), a natural antioxidant, has shown promise in treating age-related ailments such as osteoarthritis (OA). This study investigates the impact of delphinidin on intervertebral disc degeneration (IVDD) using human nucleus pulposus cells (hNPCs) subjected to hydrogen peroxide. Various molecular and cellular assays were employed to assess senescence, extracellular matrix (ECM) degradation markers, and the activation of AMPK and autophagy pathways. Initially, oxidative stress (OS)-induced hNPCs exhibited notably elevated levels of senescence markers like p53 and p21, which were mitigated by Delp treatment. Additionally, Delp attenuated IVDD characteristics including apoptosis and ECM degradation markers in OS-induced senescence (OSIS) hNPCs by downregulating MMP-13 and ADAMTS-5 while upregulating COL2A1 and aggrecans. Furthermore, Delp reversed the increased ROS production and reduced autophagy activation observed in OSIS hNPCs. Interestingly, the ability of Delp to regulate cellular senescence and ECM balance in OSIS hNPCs was hindered by autophagy inhibition using CQ. Remarkably, Delp upregulated SIRT1 and phosphorylated AMPK expression while downregulating mTOR phosphorylation in the presence of AICAR (AMPK activator), and this effect was reversed by Compound C, AMPK inhibitor. In summary, our findings suggest that Delp can safeguard hNPCs from oxidative stress by promoting autophagy through the SIRT1/AMPK/mTOR pathway.