In vivo therapy of osteosarcoma using anion transporters-based supramolecular drugs.

BACKGROUND: Osteosarcoma represents a serious clinical challenge due to its widespread genomic alterations, tendency for drug resistance and distant metastasis. New treatment methods are urgently needed to address those treatment difficulties in osteosarcoma to improve patient prognoses. In recent years, small-molecule based anion transporter have emerged as innovative and promising therapeutic compound with various biomedical applications. However, due to a lack of efficient delivery methods, using ion transporters as therapeutic drugs in vivo remains a major challenge. RESULT: Herein, we developed self-assembled supramolecular drugs based on small-molecule anion transporters, which exhibited potent therapeutic effect towards osteosarcoma both in vitro and in vivo. The anion transporters can disrupt intracellular ion homeostasis, inhibit proliferation, migration, epithelial-mesenchymal transition process, and lead to osteosarcoma cell death. RNA sequencing, western blot and flow cytometry indicated reprogramming of HOS cells and induced cell death through multiple pathways. These pathways included activation of endoplasmic reticulum stress, autophagy, apoptosis and cell cycle arrest, which avoided the development of drug resistance in osteosarcoma cells. Functionalized with osteosarcoma targeting peptide, the assembled supramolecular drug showed excellent targeted anticancer therapy against subcutaneous xenograft tumor and lung metastasis models. Besides good tumor targeting capability and anti-drug resistance, the efficacy of the assembly was also attributed to its ability to regulate the tumor immune microenvironment in vivo. CONCLUSIONS: In summary, we have demonstrated for the first time that small-molecule anion transporters are capable of killing osteosarcoma cells through multiple pathways. The assemblies, OTP-BP-L, show excellent targeting and therapeutic effect towards osteosarcoma tumors. Furthermore, the supramolecular drug shows a strong ability to regulate the tumor immune microenvironment in vivo. This work not only demonstrated the biomedical value of small-molecule anion transporters in vivo, but also provided an innovative approach for the treatment of osteosarcoma.

Methyl 3,4-dihydroxybenzoate inhibits RANKL-induced osteoclastogenesis via Nrf2 signaling in vitro and suppresses LPS-induced osteolysis and ovariectomy-induced osteoporosis in vivo.

Osteoporosis deteriorates bone mass and biomechanical strength and is life-threatening to the elderly. In this study, we show that methyl 3,4-dihydroxybenzoate (MDHB), an antioxidant small-molecule compound extracted from natural plants, inhibits receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro. Furthermore, MDHB attenuates the activation of mitogen-activated protein kinase (MAPK) and NF-κB pathways by reducing the levels of reactive oxygen species (ROS), which leads to downregulated protein expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1). We also confirm that MDHB upregulates the protein expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), an important transcription factor involved in ROS regulation, by inhibiting the ubiquitination-mediated proteasomal degradation of Nrf2. Next, animal experiments show that MDHB has an effective therapeutic effect on lipopolysaccharide (LPS)- and ovariectomized (OVX)-induced bone loss in mice. Our study demonstrates that MDHB can upregulate Nrf2 and suppress excessive osteoclast activity in mice to treat osteoporosis.

Damage to the human lumbar cartilage endplate and its clinical implications.

The cartilaginous endplate (CEP) is a thin layer of hyaline cartilage, and plays an important role in the diffusion of nutrients into the intervertebral discs. Its damage may seriously affect the disc degeneration, and result in low back pain (LBP). However, the structural features of damaged CEPs have not been well characterized, and this hinders our understanding of the etiology of disc degeneration and pain. To present the structural features of micro-damaged CEPs in patients with disc degeneration and LBP that might even be regarded as an initial factor for disc degeneration, we performed a histological study of micro-damaged CEPs harvested from human lumbar intervertebral discs and analyzed its clinical implications. Human lumbar CEPs were excised from 35 patients (mean age 60.91 years) who had disc degeneration and LBP. Control tissue was obtained from 15 patients (mean age 54.67 years) with lumbar vertebral burst fractures. LBP and disability were assessed clinically, and all patients underwent anterior vertebral body fusion surgery. CEPs together with some adjacent nucleus pulposus (NP) were sectioned at 4 µm, and stained using H&E, Safranin O/Fast Green, and Alcian Blue. Immunostaining and PCR were used to identify various markers of degeneration, innervation, and inflammation. Histology demonstrated physical micro-damage in 14/35 CEPs from the disc degeneration group. Six major types of damage could be distinguished: fissure, traumatic nodes, vascular mimicry, incorporation of NP tissue within the CEP, incorporation of bone within the CEP, and incorporation of NP and bone within the CEP. Pain and disability scores (ODI: p = 0.0190; JOA: p = 0.0205; JOABPEQ: p = 0.0034) were significantly higher in those with micro-damaged CEPs (N = 14) than in those with non-damaged CEPs (N = 21). CEP damage was significantly associated with elevated MMP3 (p = 0.043), MMP13 (p = 0.0191), ADAMTS5 (p = 0.0253), TNF-α (p = 0.0011), and Substance P (p = 0.0028), and with reduced Sox9 (p = 0.0212), aggrecan (p = 0.0127), and type II collagen (p = 0.0139). In conclusion, we presented a new classification of human lumbar micro-damaged CEPs. Furthermore, we verify disc degeneration, innervation, and discogenic pain in micro-damaged CEPs.

Preparation and Characterization of Ultrafine Nitroguanidine with Different Aspect Ratios.

In order to improve the mechanical properties and thermodynamic stability of the long needle-like raw material Nitroguanidine (NQ), it was pulverized by a mechanical pulverization method and dried by a spray drying method, and three ultrafine NQ samples with different aspect ratios (2.26, 1.87, and 1.25) were prepared. The samples were tested and characterized by scanning electron microscopy, digital imaging particle size analysis, X-ray diffraction, and differential scanning calorimetry (DSC), and the impact sensitivity and bulk density of the raw NQ and ultrafine NQ samples were also tested. The results show that ultrafine NQ has the smallest particle size D50 of 9.18 µm, with uniform particle size distribution, unchanged crystal structure, and no introduction of impurities, and the impact sensitivity was the same as that of the feedstock NQ, which was 0. In addition, with the decreasing aspect ratio of the NQ particles, their apparent activation energy increased, and the energy required to be absorbed in the thermal decomposition increased; their thermal stability was also better.

S1P regulates intervertebral disc aging by mediating endoplasmic reticulum - mitochondrial calcium ion homeostasis.

Mechanistically, S1P deficiency impeded COP II-mediated transport vesicles formation, which leads to proteins retention in the endoplasmic reticulum (ER) and subsequently ER distension. ER distension increased the contact between the ER and mitochondria, disrupting ER-to-mitochondria calcium flow, resulting in mitochondrial dysfunction and energy metabolism disturbance. Finally, using 2-APB to inhibit calcium ion channels and the senolytic drug dasatinib and quercetin (D + Q) partially rescued the aging and degenerative phenotypes caused by S1P deficiency. In conclusion, our findings suggest that S1P is a critical factor in causing IVDD in the process of aging and highlight the potential of targeting S1P as a therapeutic approach for age-related IVDD.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches.

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.

Advanced fabrication approach for innovative triple base propellants with enhanced continuous fracture resistance.

This paper initially contrasts the solvent-based and solventless molding processes, subsequently optimizing a sustainable and efficient solventless molding route for both STP and SLTP. Key physicochemical parameters such as extrusion rate, residual volatile solvents, moisture content, and apparent density of both propellant types are meticulously compared. Furthermore, the orientation of crystal particles and the structure of the matrix-bound interface are analyzed. Comprehensive examination of triaxial progressive failure phenomena-including static thermal mechanical responses, quasi-static structural deformation, and dynamic structural damage-is conducted, leading to the formulation of a damage mechanism and model. Subsequently, a structural mechanics model for nitroguanidine micrometer rod-reinforced triple base propellants is established, quantitatively evaluating the influence of nitroguanidine crystal arrangement angles on the structural strength of both propellant types. This study furnishes a theoretical foundation for specialized internal structural and mechanical behaviors through theoretical computations.

The deubiquitinase UCHL1 negatively controls osteoclastogenesis by regulating TAZ/NFATC1 signalling.

The ubiquitin‒proteasome system (UPS) plays a key role in maintaining protein homeostasis and bone remodelling. However, the role of deubiquitinating enzymes (DUBs) in bone resorption is still not well defined. Here, we identified the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) as a negative regulator of osteoclastogenesis by using the GEO database, proteomic analysis, and RNAi. Osteoclast-specific UCHL1 conditional knockout mice exhibited a severe osteoporosis phenotype in an ovariectomized model. Mechanistically, UCHL1 deubiquitinated and stabilized the transcriptional coactivator with PDZ-binding motif (TAZ) at the K46 residue, thereby inhibiting osteoclastogenesis. The TAZ protein underwent K48-linked polyubiquitination, which was degraded by UCHL1. As a substrate of UCHL1, TAZ regulates NFATC1 through a nontranscriptional coactivator function by competing with calcineurin A (CNA) for binding to NFATC1, which inhibits NFATC1 dephosphorylation and nuclear transport to impede osteoclastogenesis. Moreover, overexpression of UCHL1 locally alleviated acute and chronic bone loss. These findings suggest that activating UCHL1 may serve as a novel therapeutic approach targeting bone loss in various bone pathological states.

A Dual-Targeted Metal-Organic Framework Based Nanoplatform for the Treatment of Rheumatoid Arthritis by Restoring the Macrophage Niche.

Inflammatory infiltration and bone destruction are important pathological features of rheumatoid arthritis (RA), which originate from the disturbed niche of macrophages. Here, we identified a niche-disrupting process in RA: due to overactivation of complement, the barrier function of VSIg4+ lining macrophages is disrupted and mediates inflammatory infiltration within the joint, thereby activating excessive osteoclastogenesis and bone resorption. However, complement antagonists have poor biological applications due to superphysiologic dose requirements and inadequate effects on bone resorption. Therefore, we developed a dual-targeted therapeutic nanoplatform based on the MOF framework to achieve bone-targeted delivery of the complement inhibitor CRIg-CD59 and pH-responsive sustained release. The surface-mineralized zoledronic acid (ZA) of ZIF8@CRIg-CD59@HA@ZA targets the skeletal acidic microenvironment in RA, and the sustained release of CRIg-CD59 can recognize and prevent the complement membrane attack complex (MAC) from forming on the surface of healthy cells. Importantly, ZA can inhibit osteoclast-mediated bone resorption, and CRIg-CD59 can promote the repair of the VSIg4+ lining macrophage barrier to achieve sequential niche remodeling. This combination therapy is expected to treat RA by reversing the core pathological process, circumventing the pitfalls of traditional therapy.

Destabilization of macrophage migration inhibitory factor by 4-IPP reduces NF-κB/P-TEFb complex-mediated c-Myb transcription to suppress osteosarcoma tumourigenesis.

BACKGROUND: As an inflammatory factor and oncogenic driver protein, the pleiotropic cytokine macrophage migration inhibitory factor (MIF) plays a crucial role in the osteosarcoma microenvironment. Although 4-iodo-6-phenylpyrimidine (4-IPP) can inactivate MIF biological functions, its anti-osteosarcoma effect and molecular mechanisms have not been investigated. In this study, we identified the MIF inhibitor 4-IPP as a specific double-effector drug for osteosarcoma with both anti-tumour and anti-osteoclastogenic functions. METHODS: The anti-cancer effects of 4-IPP were evaluated by wound healing assay, cell cycle analysis, colony formation assay, CCK-8 assay, apoptosis analysis, and Transwell migration/invasion assays. Through the application of a luciferase reporter, chromatin immunoprecipitation assays, and immunofluorescence and coimmunoprecipitation analyses, the transcriptional regulation of the NF-κB/P-TEFb complex on c-Myb- and STUB1-mediated proteasome-dependent MIF protein degradation was confirmed. The effect of 4-IPP on tumour growth and metastasis was assessed using an HOS-derived tail vein metastasis model and subcutaneous and orthotopic xenograft tumour models. RESULTS: In vitro, 4-IPP significantly reduced the proliferation and metastasis of osteosarcoma cells by suppressing the NF-κB pathway. 4-IPP hindered the binding between MIF and CD74 as well as p65. Moreover, 4-IPP inhibited MIF to interrupt the formation of downstream NF-κB/P-TEFb complexes, leading to the down-regulation of c-Myb transcription. Interestingly, the implementation of 4-IPP can mediate small molecule-induced MIF protein proteasomal degradation via the STUB1 E3 ligand. However, 4-IPP still interrupted MIF-mediated communication between osteosarcoma cells and osteoclasts, thus promoting osteoclastogenesis. Remarkably, 4-IPP strongly reduced HOS-derived xenograft osteosarcoma tumourigenesis and metastasis in an in vivo mouse model. CONCLUSIONS: Our findings demonstrate that the small molecule 4-IPP targeting the MIF protein exerts an anti-osteosarcoma effect by simultaneously inactivating the biological functions of MIF and promoting its proteasomal degradation. Direct destabilization of the MIF protein with 4-IPP may be a promising therapeutic strategy for treating osteosarcoma.

Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2.

Sigma-1 receptor (Sigmar1) is a specific chaperone located in the mitochondria-associated endoplasmic reticulum membrane (MAM) and plays a role in several physiological processes. However, the role of Sigmar1 in bone homeostasis remains unknown. Here, we show that mice lacking Sigmar1 exhibited severe osteoporosis in an ovariectomized model. In contrast, overexpression of Sigmar1 locally alleviated the osteoporosis phenotype. Treatment with Sigmar1 agonists impaired both human and mice osteoclast formation in vitro. Mechanistically, SERCA2 was identified to interact with Sigmar1 based on the immunoprecipitation-mass spectrum (IP-MS) and co-immunoprecipitation (co-IP) assays, and Q615 of SERCA2 was confirmed to be the critical residue for their binding. Furthermore, Sigmar1 promoted SERCA2 degradation through Hrd1/Sel1L-dependent ER-associated degradation (ERAD). Ubiquitination of SERCA2 at K460 and K541 was responsible for its proteasomal degradation. Consequently, inhibition of SERCA2 impeded Sigmar1 deficiency enhanced osteoclastogenesis. Moreover, we found that dimemorfan, an FDA-approved Sigmar1 agonist, effectively rescued bone mass in various established bone-loss models. In conclusion, Sigmar1 is a negative regulator of osteoclastogenesis, and activation of Sigmar1 by dimemorfan may be a potential treatment for osteoporosis in clinical practice.

Urolithin A Promotes Angiogenesis and Tissue Regeneration in a Full-Thickness Cutaneous Wound Model.

The treatment of chronic wound is an important topic of current clinical issue. Neovascularization plays a crucial role in skin wound healing by delivering fresh nutrients and oxygen to the wound area. The aim of this study was to investigate the mechanisms of urolithin A (UA) in angiogenesis during wound healing. The results of in vitro experiments showed that treatment with UA (5-20 µM) promoted the proliferation, migration, and angiogenic capacity of HUVECs. Furthermore, we investigated the effect of UA in vivo using a full-thickness skin wound model. Subsequently, we found that UA promoted the regeneration of new blood vessels, which is consistent with the results of accelerated angiogenesis in vitro experiments. After UA treatment, the blood vessels in the wound are rapidly formed, and the deposition and remodeling process of the collagen matrix is also accelerated, which ultimately promotes the effective wound healing. Mechanistic studies have shown that UA promotes angiogenesis by inhibiting the PI3K/AKT pathway. Our study provides evidence that UA can promote angiogenesis and skin regeneration in chronic wounds, especially ischemic wounds.

Application of Supercritical CO2 Foaming Technology for Waste Double-Base Propellants.

This paper used a supercritical CO2 batch foaming process to treat a waste SP double-base propellant, which is a type of double-base propellants containing 58.6% nitrocellulose, 40.0% nitroglycerin, 0.8% centralite, and 0.5% vaseline, to solve a problem of poor stability of industrial explosives directly prepared by the propellant. Experiments show that this process can produce dense pores inside the SP double-base propellant. With the increase of the pressure of supercritical CO2, the number of pores inside the foamed SP double-base propellant increased, and these pores served as hotspots in the detonation reaction. An increased number of hotspots improved the detonation stability of the perfusion explosive. During the explosion, the energy of the perfusion explosive with the foamed SP double-base propellant was released more completely, so the shock wave energy and bubble energy of the explosive gradually increased with the increase of pressure. Therefore, the supercritical CO2 foaming process can promote the treatment technology of waste double-base propellants and can optimize the detonation performance of perfusion explosives by increasing the pressure of supercritical CO2.

Site-1 protease controls osteoclastogenesis by mediating LC3 transcription.

Site-1 protease (S1P) is a Golgi-located protein that activates unique membrane-bound latent transcription factors, and it plays an indispensable role in endoplasmic reticulum stress, lipid metabolism, inflammatory response and lysosome function. A patient with S1P mutation exhibits severe skeletal dysplasia with kyphoscoliosis, dysmorphic facial features and pectus carinatum. However, whether S1P regulates bone remodeling by affecting osteoclastogenesis remains elusive. Here, we show that S1P is indeed a positive regulator of osteoclastogenesis. S1P ablation in mice led to significant osteosclerosis compared with wild-type littermates. Mechanistically, S1P showed upregulated during osteoclastogenesis and was identified as a direct target of miR-9-5p. S1P deletion in bone marrow monocytes (BMMs) inhibited ATF6 and SREBP2 maturation, which subsequently impeded CHOP/SREBP2-complex-induced LC3 expression and autophagy flux. Consistently, transfection of LC3 adenovirus evidently rescued osteoclastogenesis in S1P-deficient BMMs. We then identified the interaction regions between CHOP and SREBP2 by Co-immunoprecipitation (Co-IP) and molecular docking. Furthermore, S1P deletion or inhibitor efficaciously rescued ovariectomized (OVX)- and LPS-induced bone loss in vivo. Collectively, we showed that S1P regulates osteoclast differentiation in a LC3 dependent manner and so is a potential therapy target for osteoporosis.

EIF4A3-induced circular RNA PRKAR1B promotes osteosarcoma progression by miR-361-3p-mediated induction of FZD4 expression.

Emerging evidence indicates that circRNAs are broadly expressed in osteosarcoma (OS) cells and play a crucial role in OS progression. Recently, cancer-specific circRNA circPRKAR1B has been identified by high-throughput sequencing and is recorded in publicly available databases. Nevertheless, the detailed functions and underlying mechanisms of circPRKAR1B in OS remains poorly understood. By functional experiments, we found that circPRKAR1B enhanced OS cell proliferation, migration, and promotes OS epithelial-mesenchymal transition (EMT). Mechanistic investigations suggested that circPRKAR1B promotes OS progression through sponging miR-361-3p to modulate the expression of FZD4. Subsequently, we identified that EIF4A3 promoted cirPRKAR1B formation through binding to the downstream target of circPRKAR1B on PRKAR1B mRNA. Further rescue study revealed that overexpression of the Wnt signalling could impair the onco-suppressor activities of the silencing of circPRKAR1B. Interestingly, further experiments indicated that circPRKAR1B is involved in the sensitivity of chemoresistance in OS. On the whole, our results demonstrated that circPRKAR1B exerted oncogenic roles in OS and suggested the circPRKAR1B/miR-361-3p/FZD4 axis plays an important role in OS progression and might be a potential therapeutic target.

Lubricin expression in the lumbar endplate and its association with Modic changes.

OBJECTIVE: To explore the expression of lubricin in the lumbar endplate and its association with Modic changes (MCs). METHODS: Human endplate specimens harvested from patients undergoing surgery for thoracolumbar spine fractures â€‹or lumbar interbody fusion were divided into two groups: MCs group and normal group. Lubricin expression was examined by immunohistochemistry, and differences between the groups were analysed using quantitative polymerase chain reaction (qPCR). Lubricin expression and differences between endplates with MCs and normal endplates were confirmed using a rabbit model. In a final experiment, rabbit endplate chondrocytes were cocultured with Propionibacteria acnes (P. acnes) supernatant, and the expression of lubricin and endplate degeneration related genes were evaluated. In addition, the expression of matrix metalloproteinase 1(MMP-1), A disintegrin-like and metalloproteinase with thrombospondin type 5 motif (ADAMTS5) and inflammatory factors (Interleukin- 1ß (IL-1ß) and Interleukin-6 (IL-6)) were evaluated after lubricin overexpression. RESULTS: Lubricin was found in human lumbar endplates and its expression was lower in the MCs group compared to the normal group. In the rabbit model, lubricin was also found in the endplate. In rabbits injected with P. acnes (the MCs group), lubricin expression of endplate decreased compared to the normal group. In the culture of rabbit endplate chondrocytes with P. acnes supernatant, the expression of lubricin, aggrecan, sox9 and collagen type-II decreased significantly, while that of MMP-1 and ADAMTS5 increased significantly. Moreover, lubricin overexpression could downregulate the expression of MMP-1, ADAMTS5 and inflammatory factors (IL-1ß and IL-6) compared to negative control. CONCLUSION: Lubricin is present in the lumbar endplate where it may have an anti-inflammatory role. P. acnes infection inhibits lubricin expression by cartilage endplate cells and this may facilitate the progression of MCs and endplate degeneration. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Lubricin may have an anti-inflammatory role. P. acnes infection inhibits lubricin expression by cartilage endplate cells and this may facilitate the progression of MCs and endplate degeneration.

The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs.

Calcipressin-1, also known as regulator of calcineurin 1 (RCAN1), can specifically bind calcineurin at or near the calcineurin A catalytic domain and downregulate calcineurin activity. However, whether RCAN1 affects the hypoxic intervertebral disc (IVD) phenotype through the calcineurin/NFAT signaling pathway remains unclear. First, we confirmed the characteristics of the degenerative nucleus pulposus (NP) by H&E, safranin O/fast green and Alcian blue staining, and detected increased RCAN1 levels in the degenerative NP by immunohistochemistry. Then, we demonstrated that the protein level of RCAN1.4 was higher than that of RCAN1.1 and progressively elevated from the control group to the Pfirrmann grade V group. In vitro, both hypoxia (1% O2) and overexpression of HIF-1α reduced the protein level of RCAN1.4 in rat NP cells in a dose- and time-dependent manner. We further found that miRNA-124, through a nondegradative pathway (without the proteasome or lysosome), suppressed the expression of RCAN1.4. As expected, calcineurin in NP cells was activated and primarily promoted nuclear translocation of NFATc1 under hypoxia or RCAN1.4 siRNA transfection. Furthermore, SOX9, type II collagen and MMP13 were elevated under hypoxia, RCAN1.4 siRNA transfection or NFATc1 overexpression. Using chromatin immunoprecipitation (ChIP) and a luciferase reporter assay (with mutation), we clarified that NFATc1 increasingly bound the SOX9 promotor region (bp -367~-357). Interaction of HIF-1α and NFATc1 promoted MMP13 transcription. Finally, we found that FK506 reversed hypoxia-induced activation of the calcineurin/NFAT signaling pathway in NP cells and an ex vivo model. Together, these findings show that the RCAN1.4-calcineurin/NFAT signaling pathway has a vital role in the hypoxic phenotype of NP cells. RCAN1.4 might be a therapeutic target for degenerative disc diseases.

Homocysteine induces oxidative stress and ferroptosis of nucleus pulposus via enhancing methylation of GPX4.

Homocysteine (Hcy) is an amino acid involved in gene methylation. Plasma concentration of Hcy is elevated in the pathological condition hyperhomocysteinemia (HHcy), which increases the risk of disorders of the vascular, nervous and musculoskeletal systems, including chondrocyte dysfunction. The present study aimed to explore the role of Hcy in intervertebral disc degeneration (IVDD), using a range of techniques. A clinical epidemiological study showed that HHcy is an independent risk factor for human IVDD. Cell culture using rat nucleus pulposus cells showed that Hcy promotes a degenerative cell phenotype (involving increased oxidative stress and cell death by ferroptosis) which is mediated by upregulated methylation of GPX4. An in-vivo mouse 'puncture' model of IVDD showed that folic acid (which is used to treat HHcy in humans) reduced the ability of diet-induced HHcy to promote IVDD. We conclude that Hcy upregulates oxidative stress and ferroptosis in the nucleus pulposus via enhancing GPX4 methylation, and is a new contributing factor in IVDD.

Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol.

Ethanol is one of the most commonly abused psychotropic substances with deleterious effects on the central nervous system. Ethanol exposure during development results in the loss of neurons in brain regions and when exposed to ethanol cultured cells undergo apoptosis. To date no information is available on whether abnormally high AChE activity is characteristic of apoptosis in animals exposed to ethanol. The aims of the present study were to determine whether induction of AChE activity is associated with ethanol-induced apoptosis and to explore the mechanism of enhanced AChE activity induced by ethanol. For this purpose, in vitro and in vivo experiments were performed. AChE activity was quantified by spectrophotometry and apoptosis by flow cytometer in SH-SY5Y cells exposed to ethanol. The results showed that cells treated with 500mM ethanol for 24h had a 9-fold increase in apoptotic cells and a 6-fold increase in AChE activity compared with controls. Mice exposed acutely to 200µl of 20% ethanol daily on days 1-4 had elevated AChE activity in plasma on days 3-7. On day 4, plasma AChE activity was 2.4-fold higher than pretreatment activity. More apoptotic cells were found in the brains of treated mice compared to controls. Cells in brain sections that were positive in the TUNEL assay stained for AChE activity. In conclusion, AChE activity and apoptosis were induced in SH-SY5Y cells and mice treated with ethanol, which may indicate that increased AChE may related to apoptosis induced by ethanol. Unusually high AChE activity may be an effect marker of exposure to ethanol. The relationship between AChE and apoptosis might represent a novel mechanism of ethanol-associated neuronal injury.