NFATc1-mediated expression of SLC7A11 drives sensitivity to TXNRD1 inhibitors in osteoclast precursors.

Excess osteoclast activity is found in many bone metabolic diseases, and inhibiting osteoclast differentiation has proven to be an effective strategy. Here, we revealed that osteoclast precursors (pre-OCs) were more susceptible to thioredoxin reductase 1 (TXNRD1) inhibitors than bone marrow-derived monocytes (BMDMs) during receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis. Mechanistically, we found that nuclear factor of activated T-cells 1 (NFATc1) upregulated solute carrier family 7 member 11 (SLC7A11) expression through transcriptional regulation during RANKL-induced osteoclastogenesis. During TXNRD1 inhibition, the rate of intracellular disulfide reduction is significantly reduced. Increased cystine transport leads to increased cystine accumulation, which leads to increased cellular disulfide stress and disulfidptosis. We further demonstrated that SLC7A11 inhibitors and treatments that prevent disulphide accumulation could rescue this type of cell death, but not the ferroptosis inhibitors (DFO, Ferro-1), the ROS scavengers (Trolox, Tempol), the apoptosis inhibitor (Z-VAD), the necroptosis inhibitor (Nec-1), or the autophagy inhibitor (CQ). An in vivo study indicated that TXNRD1 inhibitors increased bone cystine content, reduced the number of osteoclasts, and alleviated bone loss in an ovariectomized (OVX) mouse model. Together, our findings demonstrate that NFATc1-mediated upregulation of SLC7A11 induces targetable metabolic sensitivity to TXNRD1 inhibitors during osteoclast differentiation. Moreover, we innovatively suggest that TXNRD1 inhibitors, a classic drug for osteoclast-related diseases, selectively kill pre-OCs by inducing intracellular cystine accumulation and subsequent disulfidptosis.

A double-edged sword with a therapeutic target: iron and ferroptosis in immune regulation.

Cellular activities such as DNA synthesis, adenosine triphosphate production, and mitochondrial respiration are affected by iron metabolism. Disturbance of iron homeostasis usually leads to damage in cells and organs in the context of iron overload or deficiency. Thus, iron, a key regulator in nutritional immunity, was shown to be critical in innate and adaptive immunity. Unlike apoptosis, ferroptosis, a feature of iron-dependent lipid peroxidation, is thought to be associated with immune regulation because of its immunogenic nature. In this review, we summarize the role of iron and ferroptosis in immune regulation and discuss their therapeutic potential in the treatment of arthropathies like osteoarthritis and rheumatoid arthritis.

Adipose-Derived Mesenchymal Stem Cells Reprogram M1 Macrophage Metabolism via PHD2/HIF-1α Pathway in Colitis Mice.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease worldwide. Infiltration of pro-inflammatory macrophages (M1 macrophages) contributes to the occurrence of bowel inflammation. Transplantation of mesenchymal stem cells (MSCs) is a promising therapeutic strategy for UC, but the exact mechanism remains unknow yet. Here, we treated DSS-induced colitis mice with adipose-derived mesenchymal stem cells (ADMSCs) and revealed that ADMSCs alleviated colon inflammation by reducing the infiltration of M1 macrophages. Moreover, ADMSCs exerted this therapeutic effect by inhibiting succinate accumulation, increasing PHD2 to prevent M1 macrophages from overexpressing HIF-1α and thereby reprogramming the glycolytic pathway of M1 macrophages. Meanwhile, the succinate secreted by M1 macrophages triggered ADMSCs to secrete PGE2 in return, which could also shift macrophages from M1 phenotype to M2. Our work demonstrated an immunomodulatory effect of ADMSCs and provided a novel perspective on UC therapy.

Iron Metabolism and Immune Regulation.

Iron is a critical element for living cells in terrestrial life. Although iron metabolism is strictly controlled in the body, disturbance of iron homeostasis under certain type of condition leads to innate and adaptive immune response. In innate immunity, iron regulates macrophage polarizations, neutrophils recruitment, and NK cells activity. In adaptive immunity, iron had an effect on the activation and differentiation of Th1, Th2, and Th17 and CTL, and antibody response in B cells. In this review, we focused on iron and immune regulation and listed the specific role of iron in macrophage polarization, T-cell activation, and B-cells antibody response. In addition, correlations between iron and several diseases such as cancer and aging degenerative diseases and some therapeutic strategies targeting those diseases are also discussed.

Gut microbiota modulates osteoclast glutathione synthesis and mitochondrial biogenesis in mice subjected to ovariectomy.

OBJECTIVES: Osteoporosis is a common bone disease in the elderly mainly regulated by osteoblasts (OBs) and osteoclasts (OCs). The gut microbiota has been recognized as an important factor in many physiological and pathological processes in the host. Thus, we hypothesize that the gut microbiota is necessary for postmenopausal osteoporosis and that germ-free (GF) mice are protected from osteoporosis. MATERIAL AND METHODS: Osteoporosis models were established by performing ovariectomy (OVX) in mice. Bone mass was measured by micro-CT, and gut microbiota were assessed by 16s rDNA sequencing. Reactive oxygen species (ROS) were detected by dihydroethidium (DHE) staining in vivo and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in vitro. RESULTS: Firmicutes and Bacteroidetes in the intestine are pivotal in OC differentiation, and the Firmicutes/Bacteroidetes ratio (F/B ratio) is a specific indicator of osteoporosis. Furthermore, we found that Firmicutes and Bacteroidetes affect the de novo synthesis of glutathione (GSH) by regulating its key enzyme glutamate-cysteine ligase catalytic subunit (Gclc) and inhibiting mitochondrial biogenesis and ROS accumulation via the cAMP response element-binding (CREB) pathway. In addition, supplementing OVX mice with the probiotic Lactobacillus salivarius LI01 from the Firmicutes phylum prevented osteoporosis. CONCLUSIONS: Our results reveal that GSH plays a vital role in OVX-induced bone loss, and probiotics that affect GSH metabolism are potential therapeutic targets for overcoming osteoporosis.

Iron Regulates the Warburg Effect and Ferroptosis in Colorectal Cancer.

Iron promotes the proliferation of cancer cells, but it also contributes to cell death. Here we explored whether iron could promote the Warburg effect of colorectal cancer (CRC) cells and suppress sensitivity to ferroptosis by inducing reactive oxygen species (ROS) and regulating nuclear factor erythroid 2-related factor 2 (NRF2). In this study, cell proliferation abilities were measured by CCK-8, EdU incorporation, and colony formation assays. Seahorse XF96 respirometry assays were used to detect the Warburg effect and the level of ROS was assess by DCFH-DA fluorescent probes. Results showed that iron exposure promoted the Warburg effect of CRC cells by inducing ROS and activating NRF2 both in vivo and in vitro. In addition, iron exposure also induced ferroptosis in CRC cells, but at the same time its inhibitory proteins SLC7A11 and GPX4 were also upregulated, indicating an enhanced resistance to ferroptosis. Our results revealed that iron can effectively promote tumorigenesis. Meanwhile, iron elimination or a low-iron diet might be valid therapeutic approaches for CRC.

Hypoxia inhibits RANKL-induced ferritinophagy and protects osteoclasts from ferroptosis.

Ferroptosis is a new form of regulated cell death. Several studies have demonstrated that ferroptosis was involved in multiple diseases. However, the precise role of ferroptosis in osteoporosis remains unclear. Here, we demonstrated that ferroptosis was involved in osteoclasts over the course of RANKL-induced differentiation, and it was induced by iron-starvation response and ferrintinophagy. Mechanistically, under normoxia but not hypoxia, ferroptosis could be induced due to iron-starvation response (increased transferrin receptor 1, decreased ferritin) followed by RANKL stimulation, and this was attributed to the down-regulation of aconitase activity. We further investigated intracellular iron homeostasis and found that ferritinophagy, a process initiated by FTH-NCOA4 complex autophagosome degradation, was activated followed by RANKL stimulation under normoxia. Interestingly, these processes could not be observed under hypoxia. Moreover, we demonstrated that HIF-1α contributed to the decrease of ferritinophagy and autophagy flux under hypoxia. Additionally, HIF-1α impair autophagy flux via inhibition of autophagosome formation under hypoxia in BMDMs. In vivo study, we indicated that HIF-1α specific inhibitor 2ME2 prevent OVX bone loss. In conclusion, our study comprehensively investigated the role of ferroptosis in osteoclasts in vitro and in vivo, and innovatively suggested that targeting HIF-1α and ferritin thus inducing ferroptosis in osteoclasts could be an alternative in treatment of osteoporosis.

Schisandrin A restrains osteoclastogenesis by inhibiting reactive oxygen species and activating Nrf2 signalling.

OBJECTIVES: Intracellular reactive oxygen species (ROS) induced by receptor activator of NF-kB ligand (RANKL) has been proven to be a critical factor in the development of osteoclasts. This study aimed to prove that schisandrin A (Sch), a novel anti-oxidant compound, is able to suppress osteoclastogenesis and prevent bone loss in ovariectomized (OVX) mice by suppressing ROS via nuclear factor erythroid 2-related factor (Nrf2). MATERIAL AND METHODS: Micro-CT was used to detect bone formation. The effects of Sch on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS) were measured by dihydroethidium (DHE) staining in vivo and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in vitro. Immunofluorescence staining was used to detect the expression of Nrf2 in vivo. siRNA was used to evaluate the effect of Nrf2 in osteoclastogenesis. RESULTS: Sch suppresses RANKL-induced ROS production by regulating nuclear factor erythroid 2-related factor (Nrf2) in vitro and vivo. Mechanistically, Sch enhances the expression of Nrf2 by regulating the degradation of Nrf2. Further, Sch suppresses phosphorylation of P65 and its nuclear translocation, as well as the degradation of IκBα. Collectively, our findings reveal that Sch protects against OVX-induced bone loss by suppressing ROS via Nrf2. CONCLUSIONS: Our results showed the potential of anti-oxidant compound schisandrin A in the treatment of osteoporosis, highlighting Nrf2 as a novel promising target in osteoclast-related disease.

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL-induced osteoclastogenesis.

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL-induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F-actin ring formation and tartrate-resistant acid phosphatase (TRAP) staining in dose-dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK-related trigger RANKL by phosphorylation JNK/ERK/p38-MAPK, IκBα/p65-NF-κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K-AKT-NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL-induced RANK-TRAF6 association and RANKL-related gene and protein markers such as NFATc1, Cathepsin K, MMP-9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast-related diseases such as osteoporosis.

Mitochondrion-mediated iron accumulation promotes carcinogenesis and Warburg effect through reactive oxygen species in osteosarcoma.

BACKGROUND: Iron metabolism disorder is closely associated with several malignant tumors, however the mechanisms underlying iron and the carcinogenesis in osteosarcoma are not yet well understood. METHODS: Cell proliferation ability of osteosarcoma cell lines was measured by CCK-8, EdU incorporation and colony formation assays. Cell cycle analysis was detected by flow cytometry. The carcinogenesis of osteosarcoma was measured by soft-agar formation, trans-well and Wound healing-scratch assay. Warburg effect was detected by Seahorse respirometry assays. Reactive oxygen species (ROS) level was measured by Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes. Western blotting was used to measure the expression of mitoferrin 1 (SLC25A37) and mitoferrin 2 (SLC25A28). Iron level in vitro and vivo was detected by iron assay kit. RNAi stable cell lines was generated using shRNA. RESULTS: Iron promoted proliferation, carcinogenesis and Warburg effect of osteosarcoma cells. Iron-induced reactive oxygen species (ROS) played an important role in these processes. Iron accumulated more in mitochondrion than in cytoplasm, suggesting mitochondrion-mediated iron accumulation was involved in the development of osteosarcoma. Moreover, iron upregulated the expression of mitoferrin 1 (SLC25A37) and mitoferrin 2 (SLC25A28). Knock-down of mitoferrin 1 (SLC25A37) and mitoferrin 2 (SLC25A28) decreased the production of ROS. In addition, iron increased the expression of Warburg key enzymes HK2 and Glut1, and affected AMPK/mTORC1 signaling axis. CONCLUSIONS: Mitochondrion-mediated iron accumulation promotes carcinogenesis and Warburg effect of osteosarcoma cells. Meanwhile, iron deprivation might be a novel effective strategy in the treatment of osteosarcoma.

The fiber metabolite butyrate reduces gp130 by targeting TRAF5 in colorectal cancer cells.

BACKGROUND: Dietary fiber is effective for colorectal cancer (CRC) treatment. Interleukin-6 (IL-6) and its adaptors are potential targets for CRC therapy. Butyrate, a metabolite of dietary fiber, is a new, highly safe type of targeted drug. METHODS: In this study, Cell Counting Kit-8 cell viability and wound healing assays, western blot analysis, immunofluorescence staining, and xenograft tumor mouse models were used to evaluate the anticancer effect of butyrate and its possible mechanism in vivo and in vitro. RESULTS: Dietary fiber and sodium butyrate (NaB) decreased CRC burden by decreasing IL-6 receptor gp130 and blocking IL-6/JAK2/STAT3 axis activation in vitro and in vivo. Furthermore, NaB reduced the gp130 protein level by regulating its degradation rate via targeting TRAF5. CONCLUSIONS: The fiber metabolite butyrate inhibits CRC development by reducing gp130 via TRAF5.

Finite element analysis of dual small plate fixation and single plate fixation for treatment of midshaft clavicle fractures.

BACKGROUND: Midshaft clavicle fractures are one of the most familiar fractures. And, dual small plate fixation has been reported as can minimize hardware-related complications. However, the biomechanical properties of the dual small plate fixation have not yet been thoroughly evaluated. Here, we report the results of a finite element analysis of the biomechanical properties of midshaft clavicle fractures treated with dual small plating and superior and anteroinferior single plate fixation. METHODS: A three-dimensional (3D) finite element model of the midshaft clavicle fractures was created, whose 4-mm transverse fracture gap, having an angle < 30 degree and devoid of overlapping triangles, was simulated between the fractured segments of the middle-shaft of the clavicle. The equivalent von Mises stress and displacement of the model was used as the output measures for analysis. RESULTS: No significant differences were found between dual plating, superior or anteroinferior single plating in cantilever bending, axial compression, and axial torsion. Dual plating with a smaller plate-screw construct is biomechanically eligible to compare with superior and anteroinferior single plate fixation using larger plate-screw constructs. CONCLUSIONS: This study demonstrated that larger plate-screw constructs for the treatment of simple are placed clavicular fractures; however, weight-bearing and exorbitant shoulder activity should be avoided after the operation. Therefore, dual plating may provide a viable option for fixing midshaft clavicle fractures and, thus, may be preferred for patients who need early activity.

Juglanin Inhibits Osteoclastogenesis in Ovariectomized Mice via the Suppression of NF-κB Signaling Pathways.

Bone metabolism is a physiological process that involves both osteoblasts and osteoclasts. Pathological changes of osteoclasts are commonly seen in osteoporosis diseases. Juglanin is a natural compound, reported to have an inhibitory effect on inflammation, oxidative stress and cancer progression. The purpose of this study is to explore the role that Juglanin plays on the osteoclast functions and underlying signaling pathways. In vitro study demonstrated that Juglanin had negative influence on osteoclastic differentiation by suppressing the transcription activity of osteoclastogenesis-related genes and proteins. To determine the underlying mechanism, Western blot was employed to show that Juglanin could significantly have negative effect on the phosphorylation of P50, P65, I-κB， ultimately suppressing the expression and transcriptional activity of nuclear factor of activated T cells (NFATc1). In vivo Juglanin treatment attenuate bone reducing in mice with removed ovary through suppressing osteoclast functioning. Taken together, our study demonstrated that in the molecular mechanism, JUG inhibited the expression of receptor activator of nuclear factor-κ B ligand (RANKL) induced NF - κ B signaling pathway, thus may play a vital part in preventing postmenopausal osteoporosis.

Tetrandrine Prevents Bone Loss in Ovariectomized Mice by Inhibiting RANKL-Induced Osteoclastogenesis.

Postmenopausal osteoporosis (PMOP) is a metabolic bone disease characterized by decreased bone density and strength due to the imbalance between osteogenesis and osteoclastogenesis. Postmenopausal estrogen withdrawal increases proinflammatory cytokines and increases the serum level of Receptor activator of NF-kB ligand (RANKL)/Osteoprotegerin (OPG), which then leads to the overactivation of osteoclastogenesis. Tetrandrine, a bis-benzylisoquinoline alkaloid, has been widely used in the treatment of rheumatoid arthritis clinically in China. Here, we demonstrate that tetrandrine significantly prevented ovariectomy-induced bone loss and inhibited RANKL-induced osteoclastogenesis. In vivo, we found that intraperitoneal injection of tetrandrine (30 mg/kg) every other day markedly reduced bone loss in ovariectomized mice and the serum levels of TRAcp5b, TNF-a, IL-6, CTX-I, and RANKL/OPG were significantly decreased. In vitro, we found that tetrandrine significantly inhibited osteoclast differentiation in bone marrow monocytes (BMMs) and RAW264.7 cells according to the results of osteoclastogenesis-related gene expression, tartrate-resistant acid phosphatase (TRAP) staining and actin-ring formation as well as bone resorption assay. Mechanistically, tetrandrine inhibited RANKL-induced osteoclastogenesis by suppressing NF-kB, Ca2+, PI3K/AKT, and MAPKs signaling pathways. Taken together, our findings suggest that tetrandrine suppresses osteoclastogenesis through modulation of multiple pathways and has potential value as a therapeutic agent for PMOP, especially for those suffering from RA and PMOP at the same time.

[Expression characteristics of Piezo1 protein in stress models of human degenerative chondrocytes].

OBJECTIVE: To explore the expression characteristics of new mechanosensitive ion channel Piezo1 protein in stress models of human degenerative chondrocytes. METHODS: The stress stimulation model of human degenerative chondrocytes in vitro was constructed. Multi-channel cell stretch stress loading system FX-4000T was used to treat chondrocytes. According to the results of pre-test, the loading frequency of 0.5 Hz and the cell elongation of 20% were loaded. According to cell processing time, it was divided into 0 h, 2 h, 12 h, 24 h and 48 h mechanical stress group. The RT-PCR and Western-blot were used to test the expression of the Piezo1, also the Laser scanning confocal microscope (LSCM) was used to test the intensity of the fluorescence of the Piezo1. RESULTS: (1)The result of the RT-PCR showed that the expression of the Piezo1 in the 2 h group was higher than the 0 h group(F=13.917, q=0.037 1, P<0.05). The expression of the piezo1 in the 24 h group was the highest. While the expression of the piezo1 in the 48 h group was lower than the expression of the piezo1 in the 24 h group(F=13.917, q=0.049 5, P<0.05). (2)The result of the Western-blot showed that the 2 h group was higher than the 0 h group(F=19.341, q=0.037 1, P<0.05). The expression of the 24 h had the highest expression which was higher than the 48 h group(F=19.341, q=0.017 7, P<0.05). (3)The Piezo1 protein was extensively expressed in the cytoplasm and nucleus of the nucleus pulposus cells. And with the increase of stress processing time, the fluorescence intensity of the protein also increased. CONCLUSIONS: In human degeneration cartilage cells, the new mechanio sensitive ion channel Piezo1 protein has a trace expression. After loading periodic mechanical tensile force, the expression of Piezo1 protein increases with time dependence.

Interferonregulatoryfactor-8(IRF-8) regulates the expression of matrix metalloproteinase-13 (MMP-13) in chondrocytes.

Low levels of inflammation-induced expression of matrix metalloproteinase (MMP) play a crucial role in articular cartilage matrix destruction in osteoarthritis (OA) patients. Interferon regulatory factor-8 (IRF-8), an important member in the IRF family, plays a key role in regulating the inflammation-related signaling pathway. The aim of this study is to investigate the physiological roles of IRF-8 in the pathological progression of OA. We found that IRF-8 was expressed in human primary chondrocytes. Interestingly, the expression of IRF-8 was upregulated in OA chondrocytes. In addition, IRF-8 was increased in response to interleukin-1ß (IL-1ß) treatment, mediated by the Janus kinase 2 (JAK2) pathway. Overexpression of IRF-8 in human chondrocytes by transduction with lentiviral-IRF-8 exacerbated IL-1ß-induced expression of matrix metalloproteinase-13 (MMP-13) in human chondrocytes. In contrast, knockdown of IRF-8 inhibited IL-1ß-induced expression of MMP-13. Importantly, IRF-8 could bind to the promoter of MMP-13 and stimulate its activity. Additionally, overexpression of IRF-8 exacerbated IL-1ß-induced degradation of type II collagen. However, silencing IRF-8 abrogated the degradation of type II collagen. Taken together, our findings identified a novel function of IRF-8 in regulating articular cartilage matrix destruction by promoting the expression of MMP-13.

Applying Electrospun Gelatin/Poly(lactic acid-co-glycolic acid) Bilayered Nanofibers to Fabrication of Meniscal Tissue Engineering Scaffold.

The menisci are fibrocartilaginous tissues composed primarily of an interlacing network of collagen fibers with nanoscale diameter. Electrospinning is a suitable process of producing nanoscale fibers that mimic collagen fibers. In this study, a bilayered scaffold (group B), which consists of a gelatin nanofiber mesh and a PLGA nanofiber mesh, has been fabricated through an electrospinning method. At the same time, we electrospun pure PLGA fibrous mesh (group A) and gelatin/PLGA composite fibrous mesh (group C) as control groups. In order to compare all scaffold morphologies, the scaffolds were imaged by SEM and some parameters were measured and analyzed as following: Diameters of fibrils are from the smallest of less than average 0.14 µm for group C to the biggest of nearly average 0.38 µm for group B. The scaffolds pore diameters are from average 4.9 µm for group A to average 11.2 µm for group B. Porosity rates show that the group B has the highest porosity rate at about 91%. The scaffolds' properties were compared and analyzed, including hydrophilicity property (water contact angle) and mechanical properties (tensile strength). The results of water contact angle showed the group B is the most hydrophil among the groups. The results of tensile strength showed the tensile strength of group C is the weakest among the groups. All the results showed significant differences between the groups. Finally, in vitro, the meniscal cells derived from New Zealand white rabbits menisci were seeded in the scaffolds. We observed the cells proliferation behavior in the scaffolds. All above demonstrates that a bi-layered gelatin/PLGA scaffold reveals not only concurrent effects of mechanics and cytocompatibility in a fibrous context, but also a promising scaffold for future meniscal repair strategies.