Palmitic acid induces lipid droplet accumulation and senescence in nucleus pulposus cells via ER-stress pathway.

Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder affecting millions of adults worldwide, but a poor understanding of its pathogenesis has limited the effectiveness of therapy. In the current study, we integrated untargeted LC/MS metabolomics and magnetic resonance spectroscopy data to investigate metabolic profile alterations during IDD. Combined with validation via a large-cohort analysis, we found excessive lipid droplet accumulation in the nucleus pulposus cells of advanced-stage IDD samples. We also found abnormal palmitic acid (PA) accumulation in IDD nucleus pulposus cells, and PA exposure resulted in lipid droplet accumulation and cell senescence in an endoplasmic reticulum stress-dependent manner. Complementary transcriptome and proteome profiles enabled us to identify solute carrier transporter (SLC) 43A3 involvement in the regulation of the intracellular PA level. SLC43A3 was expressed at low levels and negatively correlated with intracellular lipid content in IDD nucleus pulposus cells. Overexpression of SLC43A3 significantly alleviated PA-induced endoplasmic reticulum stress, lipid droplet accumulation and cell senescence by inhibiting PA uptake. This work provides novel integration analysis-based insight into the metabolic profile alterations in IDD and further reveals new therapeutic targets for IDD treatment.

D-mannose alleviates intervertebral disc degeneration through glutamine metabolism.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a multifaceted condition characterized by heterogeneity, wherein the balance between catabolism and anabolism in the extracellular matrix of nucleus pulposus (NP) cells plays a central role. Presently, the available treatments primarily focus on relieving symptoms associated with IVDD without offering an effective cure targeting its underlying pathophysiological processes. D-mannose (referred to as mannose) has demonstrated anti-catabolic properties in various diseases. Nevertheless, its therapeutic potential in IVDD has yet to be explored. METHODS: The study began with optimizing the mannose concentration for restoring NP cells. Transcriptomic analyses were employed to identify the mediators influenced by mannose, with the thioredoxin-interacting protein (Txnip) gene showing the most significant differences. Subsequently, small interfering RNA (siRNA) technology was used to demonstrate that Txnip is the key gene through which mannose exerts its effects. Techniques such as colocalization analysis, molecular docking, and overexpression assays further confirmed the direct regulatory relationship between mannose and TXNIP. To elucidate the mechanism of action of mannose, metabolomics techniques were employed to pinpoint glutamine as a core metabolite affected by mannose. Next, various methods, including integrated omics data and the Gene Expression Omnibus (GEO) database, were used to validate the one-way pathway through which TXNIP regulates glutamine. Finally, the therapeutic effect of mannose on IVDD was validated, elucidating the mechanistic role of TXNIP in glutamine metabolism in both intradiscal and orally treated rats. RESULTS: In both in vivo and in vitro experiments, it was discovered that mannose has potent efficacy in alleviating IVDD by inhibiting catabolism. From a mechanistic standpoint, it was shown that mannose exerts its anti-catabolic effects by directly targeting the transcription factor max-like protein X-interacting protein (MondoA), resulting in the upregulation of TXNIP. This upregulation, in turn, inhibits glutamine metabolism, ultimately accomplishing its anti-catabolic effects by suppressing the mitogen-activated protein kinase (MAPK) pathway. More importantly, in vivo experiments have further demonstrated that compared with intradiscal injections, oral administration of mannose at safe concentrations can achieve effective therapeutic outcomes. CONCLUSIONS: In summary, through integrated multiomics analysis, including both in vivo and in vitro experiments, this study demonstrated that mannose primarily exerts its anti-catabolic effects on IVDD through the TXNIP-glutamine axis. These findings provide strong evidence supporting the potential of the use of mannose in clinical applications for alleviating IVDD. Compared to existing clinically invasive or pain-relieving therapies for IVDD, the oral administration of mannose has characteristics that are more advantageous for clinical IVDD treatment.

Genkwanin alleviates intervertebral disc degeneration via regulating ITGA2/PI3K/AKT pathway and inhibiting apoptosis and senescence.

Intervertebral disc degeneration (IVDD) is a progressive degenerative disease influenced by various factors. Genkwanin, a known anti-inflammatory flavonoid, has not been explored for its potential in IVDD management. This study aims to investigate the effects and mechanisms of genkwanin on IVDD. In vitro, cell experiments revealed that genkwanin dose-dependently inhibited Interleukin-1ß-induced expression levels of inflammatory factors (Interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2) and degradation metabolic protein (matrix metalloproteinase-13). Concurrently, genkwanin upregulated the expression of synthetic metabolism genes (type II collagen, aggrecan). Moreover, genkwanin effectively reduced the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways. Transcriptome sequencing analysis identified integrin α2 (ITGA2) as a potential target of genkwanin, and silencing ITGA2 reversed the activation of PI3K/AKT pathway induced by Interleukin-1ß. Furthermore, genkwanin alleviated Interleukin-1ß-induced senescence and apoptosis in nucleus pulposus cells. In vivo animal experiments demonstrated that genkwanin mitigated the progression of IVDD in the rat model through imaging and histological examinations. In conclusion, This study suggest that genkwanin inhibits inflammation in nucleus pulposus cells, promotes extracellular matrix remodeling, suppresses cellular senescence and apoptosis, through the ITGA2/PI3K/AKT, NF-κB and MAPK signaling pathways. These findings indicate that genkwanin may be a promising therapeutic candidate for IVDD.

Injectable, self-healing hydrogels based on gelatin, quaternized chitosan, and laponite as localized celecoxib delivery system for nucleus pulpous repair.

Utilization of injectable hydrogels stands as a paradigm of minimally invasive intervention in the context of intervertebral disc degeneration treatment. Restoration of nucleus pulposus (NP) function exerts a profound influence in alleviating back pain. This study introduces an innovative class of injectable shear-thinning hydrogels, founded on quaternized chitosan (QCS), gelatin (GEL), and laponite (LAP) with the capacity for sustained release of the anti-inflammatory drug, celecoxib (CLX). First, synthesis of Magnesium-Aluminum-Layered double hydroxide (LDH) was achieved through a co-precipitation methodology, as a carrier for celecoxib and a source of Mg ions. Intercalation of celecoxib within LDH layers (LDH-CLX) was verified through a battery of analytical techniques, including FTIR, XRD, SEM, EDAX, TGA and UV-visible spectroscopy confirmed a drug loading efficiency of 39.22 ± 0.09 % within LDH. Then, LDH-CLX was loaded in the optimal GEL-QCS-LAP hydrogel under physiological conditions. Release behavior (15 days profile), mechanical properties, swelling ratio, and degradation rate of the resulting composite were evaluated. A G* of 15-47 kPa was recorded for the hydrogel at 22-40 °C, indicating gel stability in this temperature range. Self-healing properties and injectability of the composite were proved by rheological measurements. Also, ex vivo injection into intervertebral disc of sheep, evidenced in situ forming and NP cavity filling behavior of the hydrogel. Support of GEL-QCS-LAP/LDH-CLX (containing mg2+ ions) for viability and proliferation (from ~94 % on day 1 to ~134 % on day 7) of NP cells proved using MTT assay, DAPI and Live/Dead assays. The hydrogel could significantly upregulate secretion of glycosaminoglycan (GAG, from 4.68 ± 0.1 to 27.54 ± 1.0 µg/ml), when LHD-CLX3% was loaded. We conclude that presence of mg2+ ion and celecoxib in the hydrogel can lead to creation of a suitable environment that encourages GAG secretion. In conclusion, the formulated hydrogel holds promise as a minimally invasive candidate for degenerative disc repair.

Ephrin B2 (EFNB2) potentially protects against intervertebral disc degeneration through inhibiting nucleus pulposus cell apoptosis.

Nucleus pulposus (NP) cell apoptosis is a significant indication of accelerated intervertebral disc degeneration; however, the precise mechanism is unelucidated as of yet. Ephrin B2 (EFNB2), the only gene down-regulated in the three degraded intervertebral disc tissue microarray groups (GSE70362, GSE147383 and GSE56081), was screened for examination in this study. Subsequently, EFNB2 was verified to be down-regulated in degraded NP tissue samples. Interleukin-1 (IL-1ß) treatment of NP cells to simulate the IDD environment indicated that IL-1ß treatment decreased EFNB2 expression. In degenerative NP cells stimulated by IL-1ß, EFNB2 knockdown significantly increased the rate of apoptosis as well as the apoptosis-related molecules cleaved-caspase-3 and the Bax to Bcl-2 ratio. EFNB2 was found to promote AKT, PI3K, and mTOR phosphorylation; the PI3K/AKT signaling role was investigated using the PI3K inhibitor LY294002. EFNB2 overexpression significantly increased PI3K/AKT pathway activity in IL-1ß-stimulated NP cells than the normal control. Moreover, EFNB2 partially alleviated NP cell apoptosis induced by IL-1ß, reduced the cleaved-cas3 level, and decreased the Bax/Bcl-2 ratio after the addition of the inhibitor LY294002. Additionally, EFNB2 overexpression inhibited the ERK1/2 phosphorylation; the effects of EFNB2 overexpression on ERK1/2 phosphorylation, degenerative NP cell viability, and cell apoptosis were partially reversed by ERK signaling activator Ceramide C6. EFNB2 comprehensively inhibited the apoptosis of NP cells by activating the PI3K/AKT signaling and inhibiting the ERK signaling, obviating the exacerbation of IDD. EFNB2 could be a potential target to protect against degenerative disc changes.

Catalytic Nanodots-Driven Pyroptosis Suppression in Nucleus Pulposus for Antioxidant Intervention of Intervertebral Disc Degeneration.

Low back pain resulting from intervertebral disc degeneration (IVDD) is a prevalent global concern; however, its underlying mechanism remains elusive. Single-cell sequencing analyses revealed the critical involvement of pyroptosis in IVDD. Considering the involvement of reactive oxygen species (ROS) as the primary instigator of pyroptosis and the lack of an efficient intervention approach, this study developed carbonized Mn-containing nanodots (MCDs) as ROS-scavenging catalytic biomaterials to suppress pyroptosis of nucleus pulposus (NP) cells to efficiently alleviate IVDD. Catalytic MCDs have superior efficacy in scavenging intracellular ROS and rescuing homeostasis in the NP microenvironment compared with N-acetylcysteine, a classical antioxidant. The data validates that pyroptosis plays a vital role in mediating the protective effects of catalytic MCDs against oxidative stress. Systematic in vivo assessments substantiate the effectiveness of MCDs in rescuing a puncture-induced IVDD rat model, further demonstrating their ability to suppress pyroptosis. This study highlights the potential of antioxidant catalytic nanomedicine as a pyroptosis inhibitor and mechanistically unveils an efficient strategy for the treatment of IVDD.

Does oseltamivir protect human chondrocyte and nucleus pulposus cells from degeneration by inhibiting senescence and proinflammation mediated by the NLRP3 inflammasome and NF-κB?

OBJECTIVE: Recent drug design studies suggest that inflammation is among the most important factors in the development of both intervertebral disc (IVD) degeneration (IVDD) and osteoarthritis (OA) due to cartilage damage. This study aimed to investigate whether the anti-inflammatory drug oseltamivir has a toxic effect on IVD and cartilage tissue cells. It assessed what effect oseltamivir has on hypoxia-inducible factor (HIF)-1 alpha (HIF1α), which plays an important role in anabolic pathways in IVD and cartilage tissue. In addition, the study analyzed whether oseltamivir could inhibit the release of inflammatory interleukin-1 beta (IL-1ß) via the nuclear factor kappa-B (NF-κB) signaling pathway by activating the nucleotide-binding oligomerization domain and leucine-rich repeat protein-3 (NLRP3) inflammasome. MATERIALS AND METHODS: Human lumbar IVD (n = 8) tissues were isolated for annulus fibrosus (AF) and nucleus pulposus (NP) primary cell cultures, and human tibial and femoral cartilage tissues (n = 8) were isolated for primary chondrocyte cultures. Untreated groups served as the control and oseltamivir-treated groups as the study sample. Cell viability and cytotoxicity were evaluated at 0, 24, 48, and 72 h in all groups for changes in HIF-1α, IL-1ß, NF-κB, and the NLRP3-inflammasome protein expressions using Western blotting. The α significance value was < 0.05. RESULTS: In the oseltamivir-treated groups, cell proliferation decreased in both AF/NP cell and chondrocyte cultures obtained from IVD cartilage tissues. After Western blotting analysis, changes were observed in the protein expressions of HIF-1α, IL-1ß, NF-κB, and the NLRP3 inflammasome in both AF/NP cells and chondrocytes. The results were statistically significant (p < 0.05). CONCLUSIONS: Oseltamivir treatment may be a promising regenerative strategy to manage IVDD and osteoarthritic cartilage tissues.

Naringin Inhibits Apoptosis Induced by Cyclic Stretch in Rat Annular Cells and Partially Attenuates Disc Degeneration by Inhibiting the ROS/NF-κB Pathway.

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo. Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κB signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF-κB signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

Naringin protects human nucleus pulposus cells against TNF-α-induced inflammation, oxidative stress, and loss of cellular homeostasis by enhancing autophagic flux via AMPK/SIRT1 activation.

Activation of the proinflammatory-associated cytokine, tumor necrosis factor-α (TNF-α), in nucleus pulposus (NP) cells is essential for the pathogenesis of intervertebral disc degeneration (IDD). Restoring autophagic flux has been shown to effectively protect against IDD and is a potential target for treatment. The goal of this study was to explore particular autophagic signalings responsible for the protective effects of naringin, a known autophagy activator, on human NP cells. The results showed that significantly increased autophagic flux was observed in NP cells treated with naringin, with pronounced decreases in the inflammatory response and oxidative stress, which rescued the disturbed cellular homeostasis induced by TNF-α activation. Autophagic flux inhibition was detectable in NP cells cotreated with 3-methyladenine (3-MA, an autophagy inhibitor), partially offsetting naringin-induced beneficial effects. Naringin promoted the expressions of autophagy-associated markers via SIRT1 (silent information regulator-1) activation by AMPK (AMP-activated protein kinase) phosphorylation. Either AMPK inhibition by BML-275 or SIRT1 silencing partially counteracted naringin-induced autophagic flux enhancement. These findings indicate that naringin boosts autophagic flux through SIRT1 upregulation via AMPK activation, thus protecting NP cells against inflammatory response, oxidative stress, and impaired cellular homeostasis. Naringin can be a promising inducer of restoration autophagic flux restoration for IDD.

Cyanidin attenuates the apoptosis of rat nucleus pulposus cells and the degeneration of intervertebral disc via the JAK2/STAT3 signal pathway in vitro and in vivo.

CONTEXT: Cyanidin has been shown to have therapeutic potential in osteoarthritis. However, it is unclear whether cyanidin prevents the progression of intervertebral disc degeneration (IVDD). OBJECTIVE: This study evaluates the effects of cyanidin on IVDD in vitro and in vivo. MATERIALS AND METHODS: Nucleus pulposus cells (NPCs) isolated from lumbar IVD of 4-week-old male Sprague-Dawley (SD) rats were exposed to 20 ng/mL IL-1ß, and then treated with different doses (0-120 µM) of cyanidin for 24 h. SD rats were classified into three groups (n = 8) and treated as follows: control (normal saline), IVDD (vehicle), IVDD + cyanidin (50 mg/kg). Cyanidin was administered intraperitoneally for 8 weeks. RESULTS: The IC50 of cyanidin for NPCs was 94.78 µM, and cyanidin had no toxicity at concentrations up to 500 mg/kg in SD rats. Cyanidin inhibited the apoptosis of NPCs induced by IL-1ß (12.73 ± 0.61% vs. 18.54 ± 0.60%), promoted collagen II (0.82-fold) and aggrecan (0.81-fold) expression, while reducing MMP-13 (1.02-fold) and ADAMTS-5 (1.40-fold) expression. Cyanidin increased the formation of autophagosomes in IL-1ß-induced NPCs, and promoted LC3II/LC3I (0.83-fold) and beclin-1 (0.85-fold) expression, which could be reversed by chloroquine. Cyanidin inhibited the phosphorylation of JAK2 (0.47-fold) and STAT3 (0.53-fold) in IL-1ß-induced NPCs. The effects of cyanidin could be enhanced by AG490. Furthermore, cyanidin mitigated disc degeneration in IVDD rats in vivo. DISCUSSION AND CONCLUSIONS: Cyanidin improved the function of NPCs in IVDD by regulating the JAK2/STAT3 pathway, which may provide a novel alternative strategy for IVDD. The mechanism of cyanidin improving IVDD still needs further work for in-depth investigation.

GSK-3ß inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway.

BACKGROUND: Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this process remain unclear. Increasing evidence indicates that GSK-3ß is related to cell apoptosis induced by oxidative stress. In this study, we explored whether GSK-3ß inhibition protects human NP cell against apoptosis under oxidative stress. METHODS AND RESULTS: Immunofluorescence staining was used to show the expression of GSK-3ß in human NP cells (NPCs). Flow cytometry, mitochondrial staining and western blot (WB) were used to detect apoptosis of treated NPCs, changes of mitochondrial membrane potential and the expression of mitochondrial apoptosis-related proteins using GSK-3ß specific inhibitor SB216763. Co-Immunoprecipitation (Co-IP) was used to demonstrate the interaction between GSK-3ß and Bcl-2. We delineated the protective effect of GSK-3ß specific inhibitor SB216763 on human NPCs apoptosis induced by oxidative stress in vitro. Further, we showed SB216763 exert the protective effect by preservation of the mitochondrial membrane potential and inhibition of caspase 3/7 activity during oxidative injury. The detailed mechanism underlying the antiapoptotic effect of GSK-3ß inhibition was also studied by analyzing mitochondrial apoptosis pathway in vitro. CONCLUSIONS: We concluded that the GSK-3ß inhibitor SB216763 protected mitochondrial membrane potential to delay nucleus pulposus cell apoptosis by inhibiting the interaction between GSK-3ß and Bcl-2 and subsequently reducing cytochrome c(Cyto-C) release and caspase-3 activation. Together, inhibition of GSK-3ß using SB216763 in NPCs may be a favorable therapeutic strategy to slow intervertebral disc degeneration.

The protective effects of dezocine on interleukin-1ß-induced inflammation, oxidative stress and apoptosis of human nucleus pulposus cells and the possible mechanisms.

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. We aimed to investigate the role of dezocine (DEZ) in the development of IDD. Human nucleus pulposus cells (HNPCs) induced by interleukin (IL)-1ß was used as a cellular model of IDD. After treatment with DEZ, HNPCs viability was evaluated with a CCK-8 assay. Then, the levels of inflammatory factors, including IL-6 and tumor necrosis factor-α (TNF-α), and oxidative stress-related markers, including reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH), were tested by RT-qPCR or kits. TUNEL staining was employed to detect cell apoptosis and Western blot was used to determine the expression of proteins related to inflammation, oxidative stress, apoptosis, endoplasmic reticulum stress (ERS) and MAPK signaling. Afterward, PMA, a MAPK signaling pathway agonist, was adopted for exploring the regulatory effects of DEZ on MAPK pathway. Results indicated that DEZ enhanced cell viability of HNPCs after IL-1ß exposure. DEZ alleviated the inflammation and oxidative stress, evidenced by decreased levels of IL-6, TNF-α, ROS, MDA, p-NF-κB p65, NF-κB p65 in nucleus, cox-2 and increased levels of NF-κB p65 in cytoplasm, GSH, SOD1 and SOD2. Moreover, DEZ notably inhibited IL-1ß-induced apoptosis of HNPCs. Furthermore, DEZ suppressed the levels of ERS-related proteins. The levels of related proteins in MAPK signaling including p-P38 and p-ERK1/2 were remarkably reduced after DEZ administration. By contrast, PMA crippled the impacts of DEZ on inflammation, oxidative stress and apoptosis of HNPCs induced by IL-1ß. Collectively, DEZ ameliorates IL-1ß-induced HNPCs injury via inhibiting MAPK signaling.

Hyaluronic acid-based interpenetrating network hydrogel as a cell carrier for nucleus pulposus repair.

Hyaluronic acid (HA) is a key component of the intervertebral disc (IVD) that is widely investigated as an IVD biomaterial. One persisting challenge is introducing materials capable of supporting cell encapsulation and function, yet with sufficient mechanical stability. In this study, a hybrid interpenetrating polymer network (IPN) was produced as a non-covalent hydrogel, based on a covalently cross-linked HA (HA-BDDE) and HA-poly(N-isopropylacrylamide) (HA-pNIPAM). The hybrid IPN was investigated for its physicochemical properties, with histology and gene expression analysis to determine matrix deposition in vitro and in an ex vivo model. The IPN hydrogel displayed cohesiveness for at least one week and rheological properties resembling native nucleus pulposus (NP) tissue. When implanted in an ex vivo IVD organ culture model, the IPN supported cell viability, phenotype expression of encapsulated NP cells and IVD matrix production over four weeks under physiological loading. Overall, our results indicate the therapeutic potential of this HA-based IPN hydrogel for IVD regeneration.

Dimethyl Fumarate Ameliorates Nucleus Pulposus Cell Dysfunction through Activating the Nrf2/HO-1 Pathway in Intervertebral Disc Degeneration.

BACKGROUND: Oxidative stress, inflammation, and nucleus pulposus cells (NPCs) apoptosis are involved in pathogenesis of intervertebral disc (IVD) degeneration (IVDD). Dimethyl fumarate (DMF) has been found to effectively depress oxidative stress and inflammation via the Nrf2 pathway. Hence, this project was designed to explore the underlying mechanisms of how DMF protects NPCs from damage by LPS challenge. METHODS AND RESULTS: CCK8 assay and flow cytometry of apoptosis indicated that DMF treatment attenuated LPS-induced NPC damage. Western blot analysis demonstrated that DMF enhanced the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in LPS-challenged NPCs. DMF treatment significantly decreased the accumulation of ROS, downregulated inflammatory cytokines (p-NF-κB, IL-1ß, and TNF-α), and ER stress-associated apoptosis proteins (Bip, calpain-1, caspase-12, caspase-3, and Bax) in LPS-challenged NPCs. The level of antiapoptotic protein Bcl-2 was promoted by DMF treatment in LPS-challenged NPCs. Glutathione (GSH) assay showed that DMF treatment improved reduced to oxidized glutathione ratio in LPS-challenged NPCs. Furthermore, the results of western blot analysis indicated that in LPS-challenged NPCs, DMF treatment ameliorated the elevated levels of matrix degradation enzymes (MMP-13, aggrecanase 1) and type I collagen and the reduced levels of matrix composition (type II collagen and ACAN). However, Nrf2 knockdown abolished these protective effects of DMF. CONCLUSION: Our data suggested that treatment with DMF mitigated LPS-induced oxidative stress, inflammation, and ER stress-associated apoptosis in NPCs via the Nrf2/HO-1 signaling pathway, thus reliving LPS-induced dysfunction of NPCs, which offered a novel potential pharmacological treatment strategy for IVDD.

Melatonin Suppresses Apoptosis of Nucleus Pulposus Cells through Inhibiting Autophagy via the PI3K/Akt Pathway in a High-Glucose Culture.

Diabetes mellitus- (DM-) associated hyperglycemia promotes apoptosis of disc nucleus pulposus (NP) cells, which is a contributor to intervertebral disc degeneration (IDD). Melatonin is able to protect against cell apoptosis. However, its effects on apoptosis of NP cell in a high-glucose culture remain unclear. The purpose of the present study was to investigate the effects and molecular mechanism of melatonin on NP cell apoptosis in a high-glucose culture. NP cells were cultured in the baseline medium supplemented with a high-glucose concentration (0.2 M) for 3 days. The control cells were only cultured in the baseline medium. Additionally, the pharmaceutical inhibitor LY294002 was added along with the culture medium to investigate the possible role of the PI3K/Akt pathway. Apoptosis, autophagy, and activity of the PI3K/Akt pathway of NP cells among these groups were evaluated. Compared with the control NP cells, high glucose significantly increased cell apoptosis ratio and caspase-3/caspase-9 activity and decreased mRNA expression of Bcl-2, whereas it increased mRNA or protein expression of Bax, caspase-3, cleaved caspase-3, cleaved PARP, and autophagy-related molecules (Atg3, Atg5, Beclin-1, and LC3-II) and decreased protein expression of p-Akt compared with the control cells. Additionally, melatonin partly inhibited the effects of high glucose on those parameters of cell apoptosis, autophagy, and activation of PI3K/Akt. In conclusion, melatonin attenuates apoptosis of NP cells through inhibiting the excessive autophagy via the PI3K/Akt pathway in a high-glucose culture. This study provides new theoretical basis of the protective effects of melatonin against disc degeneration in a DM patient.

Tanshinone IIA and Astragaloside IV Inhibit miR-223/JAK2/STAT1 Signalling Pathway to Alleviate Lipopolysaccharide-Induced Damage in Nucleus Pulposus Cells.

Astragaloside IV (AS IV) and tanshinone (TS IIA) are the main natural components of Salvia miltiorrhiza and Radix Astragali, respectively. The amalgam of TS IIA and AS IV has potential therapeutic value in many inflammation-related diseases. However, the aftereffect of TS IIA and AS IV for lumbar disc herniation is not clear. Although the function of miR-223 in the inflammation-related JAK/STAT pathway is unknown, it is particularly expressed in human degenerative nucleus pulposus cells. This study has investigated the efficacy of the combined application of TS IIA and AS IV in the treatment of intervertebral disc nucleus pulposus cells (NP cells) injured by lipopolysaccharide (LPS). After miR-223 inhibitor imitated NP cells, the state of the JAK family and STAT family was recognized by Western blotting (Western blot, WB) and reverse transcriptase quantitative polymerase chain reaction (qPCR). The shRNA lentivirus interference vector targeting the STAT family was constructed, and the NP cell line stably interfering with the STAT gene was established after transfection. The expression of TNF-α, IL-6, MMP-9, MMP-3, caspase-1, and caspase-3 was detected by lipopolysaccharide (WTNP cells), control virus NP cells, STAT downregulation NP cells, enzyme-linked immunosorbent assay (ELISA), Western blot, and qPCR, respectively. The cell survival rate was detected by flow cytometry and TUNEL staining reverse transcriptase-polymerase chain reaction (qPCR). NP cells were treated with TS IIA and AS IV which had been made into different concentrations, and then, the expression of miR-223, p-STAT1, and p-JAK families was detected by WB Western blotting and qPCR. MiR-223 selectively acts on JAK2/STAT1 pathway, increases the expression of TNF-α, IL-6, MMP-9, MMP-3, caspase3-1, and caspase-3, and induces apoptosis, which can be eliminated by silencing STAT1. TS IIA combined with AS IV could inhibit the expression of miR-223, p-STAT1, and p-JAK2 in NP cells, and they showed a dose-dependent tendency to p-STAT1 and p-JAK2. This study shows that miR-223 promotes the inflammatory response and induces cell injury of NP cells by acting on the JAK2/STAT1 pathway, and the combination of TS IIA and AS IV may protect NP cells by downregulating miR-223 and inhibiting the expression of JAK2 and STAT1.

Notoginsenoside R1 suppresses inflammatory response and the pyroptosis of nucleus pulposus cells via inactivating NF-κB/NLRP3 pathways.

Intervertebral disc degeneration (IVDD) is the main cause of low back pain. Notoginsenoside R1 (NR1) is widely applied in the treatment of bone disorders, including IVDD. The present study aimed to investigate the effects of NR1 on the development of IVDD and the potential mechanisms. AF puncture was performed to establish IVDD rat model. Histology changes were analyzed by hematoxylin and eosin (H&E) staining. mRNA expressions were determined using qRT-PCR. Protein expressions were detected with western blot. Cellular functions were detected by MTT, EdU, flow cytometry, and TUNEL assays. The results showed that NR1 suppressed AF puncture induced IVDD, restored intervertebral disc (IVD) function, and suppressed mechanical hyperalgesia and thermal hyperalgesia. Moreover, NR1 promoted the release of extracellular matrix (ECM) in vivo and in vitro, and decreased the mRNA expressions of proinflammation cytokines. Additionally, NR1 inactivated NF-κB/NLRP3 pathways, improved cellular functions of nucleus pulposus cells (NPCs), and suppressed cell pyroptosis, which was reversed by NLRP3 activation. Taken together, NR1 may protect against IVDD via suppressing NF-κB/NLRP3 pathways. This may provide a novel therapy for IVDD.

The effect of Nrf2 activators tBHQ and 4-octyl itaconate on the nucleus pulposus cell degeneration.

OBJECTIVE: The present study aimed to investigate the impact of two Nrf2 agonists, tBHQ and 4-Octyl Itaconate, on nucleus pulposus (NP) degeneration and explore the underlying mechanism. PATIENTS AND METHODS: We isolated the NP cells from the disc tissue of disc herniation patients. NP cells were pretreated with an adequate dose of tBHQ, Itaconate, or the mixture of them, and then subjected to the Lipopolysaccharides (LPS) stimulation to induce degeneration. Besides, the Nrf2 gene silenced NP cells were also used as a comparison. Moreover, the LPS-treated NP cells were also cultured in the mix of tBHQ and Itaconate to determine whether the agonists affected reverse degeneration. RESULTS: LPS treatment suppressed Nrf2 expression and induced the NP cell degeneration with a decrease of cell viability and collagen II expression, an increase of reactive oxygen species (ROS) production, inflammatory cytokine accumulation (IL-1ß, TNF-α), and apoptosis (Caspase3, Caspase8). However, tBHQ or Itaconate pretreated NP cells contained a higher level of Nrf2 protein and alleviated the negative effect caused by LPS, which was abolished with the silencing of Nrf2. Additionally, tBHQ showed a better ability to suppress ROS than Itaconate. Meanwhile, Itaconate inhibited a higher amount of IL-1ß and TNF-α than tBHQ. Interestingly, when NP cells were pretreated with both tBHQ and Itaconate, the result indicated an excellent anti-ROS and anti-inflammatory peculiarity. Furthermore, when NP cells suffered from LPS first and then treated with the agonist, the anti-ROS and anti-inflammatory effects remained. However, the cell viability, collagen II, and apoptotic degree were not improved. CONCLUSIONS: Both tBHQ and Itaconate effectively prevent NP cells from degeneration through anti-ROS and anti-inflammation, and the combined use of them may have better effects. But in comparison, their impact on reversing NP cell degeneration has yet to be proven.

Injectable exosome-functionalized extracellular matrix hydrogel for metabolism balance and pyroptosis regulation in intervertebral disc degeneration.

Exosome therapy is a promising therapeutic approach for intervertebral disc degeneration (IVDD) and achieves its therapeutic effects by regulating metabolic disorders, the microenvironment and cell homeostasis with the sustained release of microRNAs, proteins, and transcription factors. However, the rapid clearance and disruption of exosomes are the two major challenges for the application of exosome therapy in IVDD. Herein, a thermosensitive acellular extracellular matrix (ECM) hydrogel coupled with adipose-derived mesenchymal stem cell (ADSC) exosomes (dECM@exo) that inherits the superior properties of nucleus pulposus tissue and ADSCs was fabricated to ameliorate IVDD. This thermosensitive dECM@exo hydrogel system can provide not only in situ gelation to replenish ECM leakage in nucleus pulposus cells (NPCs) but also an environment for the growth of NPCs. In addition, sustained release of ADSC-derived exosomes from this system regulates matrix synthesis and degradation by regulating matrix metalloproteinases (MMPs) and inhibits pyroptosis by mitigating the inflammatory response in vitro. Animal results demonstrated that the dECM@exo hydrogel system maintained early IVD microenvironment homeostasis and ameliorated IVDD. This functional system can serve as a powerful platform for IVD drug delivery and biotherapy and an alternative therapy for IVDD.

Ozone as Modulator of Resorption and Inflammatory Response in Extruded Nucleus Pulposus Herniation. Revising Concepts.

Ozone therapy has been used to treat disc herniation for more than four decades. There are several papers describing results and mechanism of action. However, it is very important to define the characteristics of extruded disc herniation. Although ozone therapy showed excellent results in the majority of spinal diseases, it is not yet fully accepted within the medical community. Perhaps it is partly due to the fact that, sometimes, indications are not appropriately made. The objective of our work is to explain the mechanisms of action of ozone therapy on the extruded disc herniation. Indeed, these mechanisms are quite different from those exerted by ozone on the protruded disc herniation and on the degenerative disc disease because the inflammatory response is very different between the various cases. Extruded disc herniation occurs when the nucleus squeezes through a weakness or tear in the annulus. Host immune system considers the nucleus material to be a foreign invader, which triggers an immune response and inflammation. We think ozone therapy modulates this immune response, activating macrophages, which produce phagocytosis of extruded nucleus pulposus. Ozone would also facilitate the passage from the M1 to M2 phase of macrophages, going from an inflammatory phase to a reparative phase. Further studies are needed to verify the switch of macrophages.