Human Nucleus Research Articles

Simultaneous quantification of cefuroxime and clindamycin in human lumbar anulus fibrosus, nucleus pulposus and serum via UPLC-MS/MS

BackgroundThis study aimed to develop a sensitive, accurate method for simultaneously quantifying cefuroxime and clindamycin in human serum, lumbar anulus fibrosus and nucleus pulposus. MethodsCefuroxime and clindamycin were quantified using ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode on a triple-quadrupole AB Qtrap 5500 system in positive ion mode. Internal standards were D3-cefuroxime and D3,13C-clindamycin. Samples were pretreated by precipitating total protein. ResultsThe method showed high sensitivity and good linearity over broad calibration ranges from 100 to 100 000 ng/mL for cefuroxime and 10 to 10 000 ng/mL for clindamycin in serum, and from 10 to 10 000 ng/mL for cefuroxime and 1 to 1 000 ng/mL for clindamycin in lumbar nucleus pulposus. In all sample types, correlation coefficients were greater than 0.99, intra- and inter-day precision (relative standard deviation) was less than 15%, and accuracy (relative error) was within 14% for both analytes. This method was effective at quantifying penetration of cefuroxime and clindamycin in patients undergoing oblique lumbar interbody fusion surgery. ConclusionsA very sensitive, specific method for simultaneous detection of cefuroxime and clindamycin has been developed for human lumbar anulus fibrosus, nucleus pulposus and serum samples.

Cell Type-Specific Predictive Models Perform Prioritization of Genes and Gene Sets Associated With Autism.

Bulk transcriptomic analyses of autism spectrum disorder (ASD) have revealed dysregulated pathways, while the brain cell type-specific molecular pathology of ASD still needs to be studied. Machine learning-based studies can be conducted for ASD, prioritizing high-confidence gene candidates and promoting the design of effective interventions. Using human brain nucleus gene expression of ASD and controls, we construct cell type-specific predictive models for ASD based on individual genes and gene sets, respectively, to screen cell type-specific ASD-associated genes and gene sets. These two kinds of predictive models can predict the diagnosis of a nucleus with known cell type. Then, we construct a multi-label predictive model for predicting the cell type and diagnosis of a nucleus at the same time. Our findings suggest that layer 2/3 and layer 4 excitatory neurons, layer 5/6 cortico-cortical projection neurons, parvalbumin interneurons, and protoplasmic astrocytes are preferentially affected in ASD. The functions of genes with predictive power for ASD are different and the top important genes are distinct across different cells, highlighting the cell-type heterogeneity of ASD. The constructed predictive models can promote the diagnosis of ASD, and the prioritized cell type-specific ASD-associated genes and gene sets may be used as potential biomarkers of ASD.

In Vivo Study for Clinical Application of Dental Stem Cell Therapy Incorporated with Dental Titanium Implants.

The aim of this study was to investigate the behavior of dental-derived human mesenchymal stem cells (d-hMSCs) in response to differently surface-treated implants and to evaluate the effect of d-hMSCs on local osteogenesis around an implant in vivo. d-hMSCs derived from alveolar bone were established and cultured on machined, sandblasted and acid-etched (SLA)-treated titanium discs with and without osteogenic induction medium. Their morphological and osteogenic potential was assessed by scanning electron microscopy (SEM) and real-time polymerase chain reaction (RT-PCR) via mixing of 5 × 106 of d-hMSCs with 1 mL of Metrigel and 20 μL of gel-cell mixture, which was dispensed into the defect followed by the placement of customized mini-implants (machined, SLA-treated implants) in New Zealand white rabbits. Following healing periods of 2 weeks and 12 weeks, the obtained samples in each group were analyzed radiographically, histomorphometrically and immunohistochemically. The quantitative change in osteogenic differentiation of d-hMSCs was identified according to the type of surface treatment. Radiographic analysis revealed that an increase in new bone formation was statistically significant in the d-hMSCs group. Histomorphometric analysis was in accordance with radiographic analysis, showing the significantly increased new bone formation in the d-hMSCs group regardless of time of sacrifice. Human nuclei A was identified near the area where d-hMSCs were implanted but the level of expression was found to be decreased as time passed. Within the limitations of the present study, in this animal model, the transplantation of d-hMSCs enhanced the new bone formation around an implant and the survival and function of the stem cells was experimentally proven up to 12 weeks post-sacrifice.

Elevated lymphotoxin-\u03b1 (TNF\u03b2) is associated with intervertebral disc degeneration

BackgroundIntervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFβ, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB.ResultsThe levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 μg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels.ConclusionsThis study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.

Inhibition of LRRK2 restores parkin-mediated mitophagy and attenuates intervertebral disc degeneration

To elucidate the role of LRRK2 in intervertebral disc degeneration (IDD) as well as its mitophagy regulation mechanism. The expression of LRRK2 in human degenerative nucleus pulposus tissues as well as in oxidative stress-induced rat nucleus pulposus cells (NPCs) was detected by western blot. LRRK2 was knocked down in NPCs by lentivirus (LV)-shLRRK2 transfection; apoptosis and mitophagy were assessed by western blot, TUNEL assay, immunofluorescence staining and mitophagy detection assay in LRRK2-deficient NPCs under oxidative stress. After knockdown of Parkin in NPCs with siRNA transfection, apoptosis and mitophagy were further assessed. In puncture-induced rat IDD model, X-ray, MRI, hematoxylin-eosin (HE) and Safranin O-Fast green (SO) staining were performed to evaluate the therapeutic effects of LV-shLRRK2 on IDD. We found that the expression of LRRK2 was increased in degenerative NPCs both in vivo and in vitro. LRRK2 deficiency significantly suppressed oxidative stress-induced mitochondria-dependent apoptosis in NPCs; meanwhile, mitophagy was promoted. However, these effects were abolished by the mitophagy inhibitor, suggesting the effect of LRRK2 on apoptosis in NPCs is mitophagy-dependent. Furthermore, Parkin knockdown study showed that LRRK2 deficiency activated mitophagy by recruiting Parkin. In vivo study demonstrated that LRRK2 inhibition ameliorated IDD in rats. The results revealed that LRRK2 is involved in the pathogenesis of IDD, while knockdown of LRRK2 inhibits oxidative stress-induced apoptosis through mitophagy. Thus, inhibition of LRRK2 may be a promising therapeutic strategy for IDD.

Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

BackgroundMicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment.MethodsNP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined.ResultsBioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO.ConclusionMir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.

An analytical method for the identification of cell type-specific disease gene modules

BackgroundGenome-wide association studies have identified genetic variants associated with the risk of brain-related diseases, such as neurological and psychiatric disorders, while the causal variants and the specific vulnerable cell types are often needed to be studied. Many disease-associated genes are expressed in multiple cell types of human brains, while the pathologic variants affect primarily specific cell types. We hypothesize a model in which what determines the manifestation of a disease in a cell type is the presence of disease module comprised of disease-associated genes, instead of individual genes. Therefore, it is essential to identify the presence/absence of disease gene modules in cells.MethodsTo characterize the cell type-specificity of brain-related diseases, we construct human brain cell type-specific gene interaction networks integrating human brain nucleus gene expression data with a referenced tissue-specific gene interaction network. Then from the cell type-specific gene interaction networks, we identify significant cell type-specific disease gene modules by performing statistical tests.ResultsBetween neurons and glia cells, the constructed cell type-specific gene networks and their gene functions are distinct. Then we identify cell type-specific disease gene modules associated with autism spectrum disorder and find that different gene modules are formed and distinct gene functions may be dysregulated in different cells. We also study the similarity and dissimilarity in cell type-specific disease gene modules among autism spectrum disorder, schizophrenia and bipolar disorder. The functions of neurons-specific disease gene modules are associated with synapse for all three diseases, while those in glia cells are different. To facilitate the use of our method, we develop an R package, CtsDGM, for the identification of cell type-specific disease gene modules.ConclusionsThe results support our hypothesis that a disease manifests itself in a cell type through forming a statistically significant disease gene module. The identification of cell type-specific disease gene modules can promote the development of more targeted biomarkers and treatments for the disease. Our method can be applied for depicting the cell type heterogeneity of a given disease, and also for studying the similarity and dissimilarity between different disorders, providing new insights into the molecular mechanisms underlying the pathogenesis and progression of diseases.

Angiotensin II Type 1 Receptor Antagonist Losartan Inhibits TNF-α-Induced Inflammation and Degeneration Processes in Human Nucleus Pulposus Cells

Our recent study detected the expression of a tissue renin–angiotensin system (tRAS) in human intervertebral discs (IVDs). The present study sought to investigate the impact of the angiotensin II receptor type 1 (AGTR1) antagonist losartan on human nucleus pulposus (NP) cell inflammation and degeneration induced by tumor necrosis factor-α (TNF-α). Human NP cells (4 donors; Pfirrmann grade 2–3; 30–37-years–old; male) were isolated and expanded. TNF-α (10 ng/mL) was used to induce inflammation and degeneration. We examined the impact of losartan supplementation and measured gene expression of tRAS, anabolic, catabolic, and inflammatory markers in NP cells after 24 and 72 h of exposure. T0070907, a PPAR gamma antagonist, was applied to examine the regulatory pathway of losartan. Losartan (1 mM) significantly impaired the TNF-α-induced increase of pro-inflammatory (nitric oxide and TNF-α), catabolic (matrix metalloproteinases), and tRAS (AGTR1a and angiotensin-converting enzyme) markers. Further, losartan maintained the NP cell phenotype by upregulating aggrecan and downregulating collagen type I expression. In summary, losartan showed anti-inflammatory, anti-catabolic, and positive phenotype-modulating effects on human NP cells. These results indicate that tRAS signaling plays an important role in IVD degeneration, and tRAS modulation with losartan could represent a novel therapeutic approach.

Chapter 31 - The intermediate nucleus in humans: Cytoarchitecture, chemoarchitecture, and relation to sleep, sex, and Alzheimer disease

The intermediate nucleus of Brockhaus (INH), also known as the interstitial nucleus of the anterior hypothalamus-1 of Allen and Gorski (INAH-1), the sexually dimorphic nucleus of Swaab and colleagues (SDN), and the ventrolateral preoptic nucleus of Saper and colleagues (VLPO), is a cluster of largely galanin-expressing neurons in the lateral preoptic area, at the level of the crossing of the anterior commissure and dorsal to the supraoptic nucleus. The number of Nissl-stained neurons in the INH has been reported to be larger in men than women and to decrease with aging, although these findings have been controversial, in part because of differences in patient populations and methods used to assess the nucleus. However, recent studies have confirmed that the number of galanin-immunoreactive INH neurons is larger in men than women and decreases with age and have reported further loss with Alzheimer disease. The galanin-immunoreactive VLPO neurons have been thought to drive sleep behavior in many species, and their numbers in older humans correlate with the amount of consolidated sleep they experience. Sleep differences between men and women, during aging, and with Alzheimer disease may also depend upon the integrity of this nucleus.

Chapter 22 - Monogenic human obesity syndromes

Neural circuits in the hypothalamus play a key role in the regulation of human energy homeostasis. A critical circuit involves leptin-responsive neurons in the hypothalamic arcuate nucleus (the infundibular nucleus in humans) expressing the appetite-suppressing neuropeptide proopiomelanocortin (POMC) and the appetite-stimulating Agouti-related peptide. In the fed state, the POMC-derived melanocortin peptide α-melanocyte-stimulating hormone stimulates melanocortin-4 receptors (MC4Rs) expressed on second-order neurons in the paraventricular nucleus of the hypothalamus (PVN). Agonism of MC4R leads to reduced food intake and increased energy expenditure. Disruption of this hypothalamic circuit by inherited mutations in the genes encoding leptin, the leptin receptor, POMC, and MC4R can lead to severe obesity in humans. The characterization of these and closely related genetic obesity syndromes has informed our understanding of the neural pathways by which leptin regulates energy balance, neuroendocrine function, and the autonomic nervous system. A broader understanding of these neural and molecular mechanisms has paved the way for effective mechanism-based therapies for patients whose severe obesity is driven by disruption of these pathways.

Chapter 25 - Imaging of the human subthalamic nucleus

The human subthalamic nucleus (STN) is a small lens shaped iron rich nucleus, which has gained substantial interest as a target for deep brain stimulation surgery for a variety of movement disorders. The internal anatomy of the human STN has not been fully elucidated, and an intensive debate, discussing the level of overlap between putative limbic, associative, and motor zones within the STN is still ongoing. In this chapter, we have summarized anatomical information obtained using different neuroimaging modalities focusing on the anatomy of the STN. Additionally, we have highlighted a number of major challenges faced when using magnetic resonance imaging (MRI) approaches for the visualization of small iron rich deep brain structures such as the STN. In vivo MRI and postmortem microscopy efforts provide valuable complementary information on the internal structure of the STN, although the results are not always fully aligned. Finally, we provide an outlook on future efforts that could contribute to the development of an integrative research approach that will help with the reconciliation of seemingly divergent results across research approaches.

Augmenting postgraduate ophthalmology residency training during the COVID-19 pandemic

Dear Editor, The coronavirus pandemic resulted in an unsurmountable loss to human life worldwide and created unprecedented challenges for healthcare systems including ophthalmology. The postgraduate medical and surgical training was not far from this negative influence.[1] We read an interesting article by Sarkar et al.[2] regarding surgical simulator-assisted postgraduate training and we must congratulate the authors highlighting the impact of virtual reality simulators (Eyesi, VRmagic, Mannheim, Germany). Although surgical training forms the core of ophthalmology postgraduate training but as a trainee resident, other skills also deserve equal attention for a holistic training program.[3] Here, we want to add a few important points for maximizing resident training output when the patient flow is less and surgical training is at a halt. The residents can practice anterior segment slit-lamp examination and retinoscopy on dummy eyeballs. They can use a slit-lamp and dummy eyeball holders for practicing oblique illumination, retro, and diffuse illumination. In addition, they can also master how to identify the myopic and hypermetropic reflex using a retinoscope on dummy eyeballs. The 3D printed or dummy eyeballs can be used for practicing indirect ophthalmology with the retinal image printed within.[4] They can even practice scleral depression using an earbud or a scleral depressor. The dummy eyeballs can be employed for practicing manual keratometry by identifying mires on the eyeball. In addition, they can have surgical video discussions where they can exchange scientific ideas. The wet lab can be utilized for practicing and tying sutures on a thermocol or a lemon. This initial technique can be taught by a senior, and later, scoring can be done based on the performance of the trainee. The goat’s eyeballs or leftover cadaveric eyeballs can be used to practice scleral tunnel incision, side port incision, tunnel sutures, rhexis in goat’s eyeball, trench, and quadrant emulsification in goat’s eye, and anterior vitrectomy. One innovative technique we follow is that the goat’s eyeballs can be hardened by baking them in the microwave so that the nucleus becomes hard and gives a real-time feel of trenching and emulsification. In addition, the goat’s nucleus can be replaced with a human nucleus and soap pellet to give a real-time feel of phacoemulsification. We believe that this training module can be implemented across different centers for maximizing medical and surgical output when the pandemic is at its peak.[5] Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Digital images of multipolar neurons from the human dentate nucleus: topologic and morphometric analysis accompanied with the classification by cluster analysis

Digital images of multipolar neurons from the human dentate nucleus: topologic and morphometric analysis accompanied with the classification by cluster analysis

Long non-coding RNA MT1DP interacts with miR-365 and induces apoptosis of nucleus pulposus cells by repressing NRF-2-induced anti-oxidation in lumbar disc herniation.

BackgroundThis study investigated the effects of the long non-coding (lnc) RNA MT1DP on the apoptosis of nucleus pulposus (NP) cells. The interactions between MT1DP and the microRNA miR-365, and its effects on the anti-oxidant activity of nuclear factor erythroid 2-related factor 2 (NRF-2) were investigated in lumbar disc herniation (LDH).MethodsHuman degenerative intervertebral disc NP tissues were obtained from 10 patients with LDH who underwent lumbar spine surgery. Normal intervertebral disc NP tissues were obtained from 10 patients with lumbar vertebrae fractures and used as negative controls (NCs).ResultsThe gene expressions of MT1DP and miR-365 in human degenerative disc NP tissues and nucleus pulposus cells (NPCs) were significantly increased, while the level of NRF-2 was significantly decreased. Overexpression of MT1DP and miR-365 (MT1DP + miR-365) and inhibition of NRF-2 suppressed NP cell viability and induced apoptosis. MT1DP + miR-365 caused inflammation in NP cells by damaging the mitochondrial membrane. The combination of lnc-MT1DP and miR-365 reduced cell mitochondrial function and led to a decrease in the ability of cells to elimination reactive oxygen species (ROS).ConclusionsThe combination of lnc-MT1DP and miR-365 damaged the cell mitochondrial membrane, reduced mitochondrial function and the ability to eliminate ROS, increased cell apoptosis, and caused LDH.

Scutellarin Protects Against Mitochondrial ROS-Dependent NLRP3 Inflammasome Activation to Attenuate Intervertebral Disc Degeneration

Background: Intervertebral disc degeneration (IVDD) is a predominant reason for disc herniation, which is widespread all over the world. It is known that inflammatory responses, mitochondrial dysfunction and extracellular matrix degradation are involved in IVDD. Scutellarin, an active ingredient extracted from Erigeron breviscapus (Vaniot) Ha, Hand-Mazz, is reported to exhibit a therapeutic potential in several degenerative diseases through suppressing inflammation and regulating metabolism. However, whether scutellarin can improve IVDD remains unknown. Methods: Human primary nucleus pulposus cells (HNPCs) were cultured and stimulated with TNF-α, in the presence or absence of scutellarin. Furthermore, a rat needle puncture model was established and scutellarin was injected into the IVD to verify its protective function against IVDD. Moreover, activation of the NF-κB signaling pathway, MAPK signaling pathway and NLRP3 inflammasome was analyzed both in vitro and in vivo. Findings: Scutellarin attenuated the inflammatory reaction and retained the production of major IVD components both in vitro and in vivo. Mechanically, scutellarin reduced reactive oxygen species amount, alleviate d mitochondrial damage and decreased the expression levels of apoptosis-related biomarkers in stimulation of TNF-α. In addition, scutellarin antagonized the activation of NF-κB signaling pathway and p38 signaling pathway, and suppressed activity of NLRP3 inflammasome mediated by TNF-α. Interpretation: This study reveals that scutellarin protects against degeneration of nucleus pulposus cells, which might shed light on treatment of IVDD in the future. Funding: This study was financially supported by grants from the National Natural Science Foundation of China (81572191, 82073437, 82072478). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University.

2D Images of Astrocytes in the Human Principal Olivary Nucleus: Monofractal Analysis of the Morphology

Introduction: Current knowledge postulated glia as active participants in various metabolic processes within nervous tissue. The most numerous glial cells were astrocytes, and qualitative analysis divided them into two types based on their anatomical locations: fibrous and protoplasmic. The main goal of this research was to examine the morphological difference between types, analyzing four features of the image. The secondary objective of this research was to explore their morphology through maturation and aging. Materials and Methods: The material originated from bilateral sections of the human principal olivary nucleus, without disorders in the central nervous system. The brains were taken from 30 human cadavers (35 - 90 years) and cut into samples corresponding to dimensions of the principal olivary nucleus. A light microscope digitized the histological preparations. The selection of 294 images was analyzed by monofractal parameters derived from the box-counting. These parameters quantified four image properties (size, shape, complexity and homogeneity) of the glial body or whole glial cell. Results: The first results showed that images of two types of astrocytes were significantly different (p < 0.05 and higher) in all properties of whole cells. The second results examined the differences between three age groups in both types of astrocytes. The differences between groups were more evident for protoplasmic than fibrous (nine vs. three parameters). Conclusions: The main limitation of this study lies in the fact that the quantification was performed only by fractal analysis techniques. Nevertheless, a detailed monofractal analysis of astrocytes was performed for the first time. Thus, although this study can be seen as an improvement of the previous qualitative results, future research will provide the complete procedure of the image analysis.

Lumican silencing alleviates tumor necrosis factor-α-induced nucleus pulposus cell inflammation and senescence by inhibiting apoptosis signal regulating kinase 1/p38 signaling pathway via inactivating Fas ligand expression

ABSTRACT A recent study has reported that lumican (LUM) is expressed at a high level in the nucleus pulposus specimens from herniated lumbar disc, without description of the specific mechanism. This study was designed to investigate the function and mechanism of LUM in intervertebral disc degeneration (IDD). In this study, human nucleus pulposus cells (hNPCs) cells were challenged with tumor necrosis factor (TNF)-α to establish the IDD in vitro model. After LUM silencing, cell viability was detected using CCK-8 kit, and the expression of inflammatory factors was evaluated using RT-qPCR and ELISA. Flow cytometry and β-galactosidase staining were used to determine cell cycle and cell senescence. The expression of cycle and senescence-related proteins was evaluated with western blotting. Then, Fas ligand (FasL) was overexpressed and proteins in apoptosis signal regulating kinase 1 (ASK1)/p38 signaling were tested. Finally, GS-4997, an inhibitor of ASK1, was used to explore the regulatory effects of LUM on ASK1/p38 signaling in TNF-α-induced hNPCs. Results indicated that LUM expression was upregulated in TNF-α-challenged hNPCs. LUM gene interference mitigated TNF-α-induced inflammatory response, cell cycle arrest, and senescence of hNPCs. It was then found that LUM silencing could inhibit ASK1/p38 signaling in TNF-α-treated hNPCs, which was reversed by FasL overexpression. Additionally, ASK1/p38 participated in the mediation by LUM of TNF-α-induced inflammation, cell cycle arrest, and senescence of hNPCs. To conclude, interference with LUM effectively mitigated TNF-α-induced inflammatory response, cell cycle arrest, and cell senescence. Further experiments showed the involvement of ASK1/p38 pathway in LUM-mediated NP cell phenotypes through FasL.

CGRP Regulates Nucleus Pulposus Cell Apoptosis and Inflammation via the MAPK/NF-κB Signaling Pathways during Intervertebral Disc Degeneration.

Chronic low back pain (CLBP) has been proved to be the dominating cause of disability in patients with lumbar degenerative diseases. Of the various etiological factors, intervertebral disc degeneration (IVDD) has been the dominating cause. In the past few decades, the role and changes of nerve systems, especially the peripheral sensory fibers and their neurotransmitters, in the induction and progression of IVDD have attracted growing concerns. The expression of many neuropeptides, such as SP, NPY, and CGRP, in the nociceptive pathways is increased during the progression of IVDD and responsible for the discogenic pain. Here, the role of CGRP in the progression of IVDD was firstly investigated both in vitro and in vivo. Firstly, we confirmed that human degenerated intervertebral disc tissue exhibited elevated expression of CGRP and its receptor. Secondly, in vitro experiments suggested that CGRP could inhibit the proliferation and induce apoptosis in human nucleus pulposus (NP) cells, as well as promote inflammation and degenerated phenotypes through activating NF-κB and MAPK signaling pathways. Thirdly, CGRP receptor antagonist, Rimegepant, can ameliorate the adverse effects of CGRP imposed on NP cells, which were confirmed in vitro and in vivo. Our results will bring about a brand-new insight into the roles of neuromodulation in IVDD and related therapeutic attempts.

Ulinastatin Ameliorates IL-1β-Induced Cell Dysfunction in Human Nucleus Pulposus Cells via Nrf2/NF-κB Pathway.

Low back pain (LBP) has been a wide public health concern worldwide. Among the pathogenic factors, intervertebral disc degeneration (IDD) has been one of the primary contributors to LBP. IDD correlates closely with inflammatory response and oxidative stress, involving a variety of inflammation-related cytokines, such as interleukin 1 beta (IL-1β), which could result in local inflammatory environment. Ulinastatin (UTI) is a kind of acidic protein extracted from human urine, which inhibits the release of tumor necrosis factor alpha (TNF-α) and other inflammatory factors to protect organs from inflammatory damage. However, whether this protective effect of UTI on human nucleus pulposus (NP) exists, and how UTI affects the biological behaviors of human NP cells during IDD remain elusive. In this current study, we revealed that UTI could improve the viability of NP cells and promote the proliferation of NP cells. Additionally, UTI could protect human NP cells via ameliorating IL-1β-induced apoptosis, inflammatory response, oxidative stress, and extracellular matrix (ECM) degradation. Molecular mechanism analysis suggested that the protective effect from UTI on IL-1β-treated NP cells were through activating nuclear factor- (erythroid-derived 2-) like 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway and the suppression of NF-κB signaling pathway. Therefore, UTI may be a promising therapeutic medicine to ameliorate IDD.

FAM134B-Mediated ER-phagy Upregulation Attenuates AGEs-Induced Apoptosis and Senescence in Human Nucleus Pulposus Cells.

Previous studies have established the pathogenic role of advanced glycation end products (AGEs) accumulation in intervertebral disc degeneration (IDD). Emerging evidence indicates that ER-phagy serves as a crucial cellular adaptive mechanism during stress conditions. This study is aimed at investigating the role of FAM134B-mediated ER-phagy in human nucleus pulposus (NP) cells upon AGEs treatment and exploring its regulatory mechanisms. We observed that AGEs treatment resulted in significantly increased apoptosis, senescence, and ROS accumulation in human NP cells; meanwhile, the enhanced apoptosis and senescence by AGEs treatment could be partially alleviated with the classic ROS scavenger NAC administration. Furthermore, we confirmed that FAM134B-mediated ER-phagy was activated under AGEs stimulation via ROS pathway. Importantly, it was also found that FAM134B overexpression could efficiently relieve intracellular ROS accumulation, apoptosis, and senescence upon AGEs treatment; conversely, FAM134B knockdown markedly resulted in opposite effects. In conclusion, our data demonstrate that FAM134B-mediated ER-phagy plays a vital role in AGEs-induced apoptosis and senescence through modulating cellular ROS accumulation, and targeting FAM134B-mediated ER-phagy could be a promising therapeutic strategy for IDD treatment.