Vitamin C promotes ACE2 degradation and protects against SARS-CoV-2 infection.

ACE2 is a major receptor for cellular entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2 binding, strategies to flexibly and sufficiently reduce ACE2 levels for the prevention of SARS-CoV-2 infection have not been explored. Here, we reveal vitamin C (VitC) administration as a potent strategy to prevent SARS-CoV-2 infection. VitC reduces ACE2 protein levels in a dose-dependent manner, while even a partial reduction in ACE2 levels can greatly inhibit SARS-CoV-2 infection. Further studies reveal that USP50 is a crucial regulator of ACE2 levels. VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination of ACE2 at Lys788 and subsequent degradation of ACE2 without affecting its transcriptional expression. Importantly, VitC administration reduces host ACE2 levels and greatly blocks SARS-CoV-2 infection in mice. This study reveals that ACE2 protein levels are down-regulated by an essential nutrient, VitC, thereby enhancing protection against infection of SARS-CoV-2 and its variants.

XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity.

XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.

Evaluation of Novel Anticaries Adhesive in a Secondary Caries Animal Model.

We investigated the anticaries properties of an adhesive containing dimethylaminododecyl methacrylate (DMADDM) in vivo via a secondary caries animal model. Cavities were prepared in the maxillary first molars of Wistar rats. DMADDM-containing adhesives were applied on one side and commercial adhesives on the opposite side as a control. After a 3-week feeding period to induce secondary caries, the molars were harvested for the evaluation of the secondary caries. Lesion depth (LD) and mineral loss (ML) were measured via a micro-CT method, and a modified Keyes scoring method yielded scores for the caries lesions. Statistical analysis was divided into 2 parts: a correlation analysis between 2 evaluations with one-way ANOVA and a least-significant differences (LSD) test, and an evaluation of anticaries adhesives with a paired samples t test. The results showed that: (1) secondary caries was successfully produced in rats; (2) there was a correlation between the modified Keyes scoring method and micro-CT in the evaluation of the secondary caries; (3) the adhesive containing DMADDM significantly reduced both LD and ML (according to micro-CT), and also lowered the scores (based on the modified Keyes scoring method). This suggests that the novel DMADDM adhesive could perform an anticaries function in vivo via the secondary caries animal model which was also developed and testified in research. Secondary caries is one of the major reasons leading to the failure of caries restoration treatment. As a solution, anticaries adhesives perform well in biofilm inhibition in vitro. However, the lack of secondary caries animal models limits the evaluation of anticaries adhesives in vivo.

diABZI and poly(I:C) inhibit osteoclastic bone resorption by inducing IRF7 and IFIT3.

Type I interferons (IFN-I) are pleiotropic factors endowed with multiple activities that play important roles in innate and adaptive immunity. Although many studies indicate IFN-I inducers exert favorable effects on broad-spectrum antivirus, immunomodulation, and anti-tumor by inducing endogenous IFN-I and IFN-stimulated genes (ISGs), their function in bone homeostasis still needs further exploration. Here, our study demonstrates two distinct IFN-I inducers, diABZI and poly(I:C), as potential therapeutics to alleviate osteolysis and osteoporosis. Firstly, IFN-I inducers suppress the genes that control osteoclast (OC) differentiation and activity in vitro. Moreover, diABZI alleviates bone loss in Ti particle-induced osteolysis and ovariectomized (OVX)-induced osteoporosis in vivo by inhibiting OC differentiation and function. In addition, the inhibitory effects of IFN-I inducers on OC differentiation are not observed in macrophages derived from Ifnar1-/- mice, which indicate that the suppressive effect of IFN-I inducers on OC is IFNAR-dependent. Mechanistically, RNAi-mediated silencing of IRF7 and IFIT3 in OC precursors impair the suppressive effect of the IFN-I inducers on OC differentiation. Taken together, these results demonstrate that IFN-I inducers play a protective role in bone turnover by limiting osteoclastogenesis and bone resorption through the induction of OC-specific mediators via the IFN-ß signaling pathway.

XAF1 promotes osteoclast apoptosis by antagonizing the XIAP-caspase axis.

Background: Over-activated osteoclast (OC) is a major cause of diseases related to bone loss and bone metabolism. Both bone resorption inhibition and apoptosis induction of osteoclast are crucial in treating these diseases. X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is an important interferon-stimulated and apoptotic gene. However, how XAF1 regulates bone formation and remodeling is unknown. Methods: We generate global and chimeric Xaf1 knockout mouse models and utilize these models to explore the function and mechanism of XAF1 in regulating bone formation and remodeling in vivo and in vitro. Results: We show that XAF1 depletion enhances osteoclast generation in vitro. XAF1 knockout increases osteoclast number and bone resorption, thereby exacerbating bone loss in both OVX and osteolysis models. Activation of XAF1 with BV6 (a potent XIAP inhibitor) suppresses osteoclast formation. Mechanistically, XAF1 deletion decreases osteoclast apoptosis by facilitating the interaction between XIAP and caspase-3/7. Conclusions: Our data illustrates an essential role of XAF1 in controlling osteoclastogenesis in both osteoporosis and osteolysis mouse models and highlights its underlying mechanism, indicating a potential role in clinical treatment.The translational potential of this article: The translation potential of this article is that we first indicated that osteoclast apoptosis induced by XAF1 contribute to the progression of osteoporosis and osteolysis, which provides a novel strategy in the prevention of osteoporosis and osteolysis.

Bio-clickable mussel-inspired peptides improve titanium-based material osseointegration synergistically with immunopolarization-regulation.

Upon the osteoporotic condition, sluggish osteogenesis, excessive bone resorption, and chronic inflammation make the osseointegration of bioinert titanium (Ti) implants with surrounding bone tissues difficult, often lead to prosthesis loosening, bone collapse, and implant failure. In this study, we firstly designed clickable mussel-inspired peptides (DOPA-N3) and grafted them onto the surfaces of Ti materials through robust catechol-TiO2 coordinative interactions. Then, two dibenzylcyclooctyne (DBCO)-capped bioactive peptides RGD and BMP-2 bioactive domain (BMP-2) were clicked onto the DOPA-N3-coated Ti material surfaces via bio-orthogonal reaction. We characterized the surface morphology and biocompatibility of the Ti substrates and optimized the osteogenic capacity of Ti surfaces through adjusting the ideal ratios of BMP-2/RGD at 3:1. In vitro, the dual-functionalized Ti substrates exhibited excellent promotion on adhesion and osteogenesis of mesenchymal stem cells (MSCs), and conspicuous immunopolarization-regulation to shift macrophages to alternative (M2) phenotypes and inhibit inflammation, as well as enhancement of osseointegration and mechanical stability in osteoporotic rats. In summary, our biomimetic surface modification strategy by bio-orthogonal reaction provided a convenient and feasible method to resolve the bioinertia and clinical complications of Ti-based implants, which was conducive to the long-term success of Ti implants, especially in the osteoporotic or inflammatory conditions.

Bioclickable Mussel-Derived Peptides With Immunoregulation for Osseointegration of PEEK.

Polyether ether ketone (PEEK)-based biomaterials have been widely used in the field of spine and joint surgery. However, lack of biological activity limits their further clinical application. In this study, we synthesized a bioclickable mussel-derived peptide Azide-DOPA4 as a PEEK surface coating modifier and further combined bone morphogenetic protein 2 functional peptides (BMP2p) with a dibenzylcyclooctyne (DBCO) motif through bio-orthogonal reactions to obtain DOPA4@BMP2p-PEEK. As expected, more BMP2p can be conjugated on PEEK after Azide-DOPA4 coating. The surface roughness and hydrophilicity of DOPA4@BMP2p-PEEK were obviously increased. Then, we optimized the osteogenic capacity of PEEK substrates. In vitro, compared with the BMP2p-coating PEEK material, DOPA4@BMP2p-PEEK showed significantly higher osteogenic induction capability of rat bone marrow mesenchymal stem cells. In vivo, we constructed a rat calvarial bone defect model and implanted PEEK materials with a differently modified surface. Micro-computed tomography scanning displayed that the DOPA4@BMP2p-PEEK implant group had significantly higher new bone volume and bone mineral density than the BMP2p-PEEK group. Histological staining of hard tissue further confirmed that the DOPA4@BMP2p-PEEK group revealed a better osseointegrative effect than the BMP2p-PEEK group. More importantly, we also found that DOPA4@BMP2p coating has a synergistic effect with induced Foxp3+ regulatory T (iTreg) cells to promote osteogenesis. In summary, with an easy-to-perform, two-step surface bioengineering approach, the DOPA4@BMP2p-PEEK material reported here displayed excellent biocompatibility and osteogenic functions. It will, moreover, offer insights to engineering surfaces of orthopedic implants.

Oridonin promotes osteogenesis through Wnt/ß-catenin pathway and inhibits RANKL-induced osteoclastogenesis in vitro.

AIMS: To study the molecular mechanism of oridonin (ORI) on osteoblast differentiation and osteoclast formation in vitro. MAIN METHODS: Rat bone marrow mesenchymal stem cells (BMSCs) were treated with different concentrations of ORI in osteogenic medium (OM). CCK-8 assay and were used to detect the effect on BMSCs viability. Alizarin red staining and ALP activity were used to illuminate the effect of ORI on osteogenic differentiation. Expressions of osteogenic differentiation related genes were detected by real-time quantitative PCR (qRT-PCR), and expressions of osteogenic related proteins were detected by Western blot (WB) and immunofluorescence. Similarly, bone marrow mononuclear cells (BMMs) were treated with different concentrations of ORI. CCK-8 assay and Live/Dead staining were used to detect the effect of ORI on BMMs activity. TRAP staining was used to detect its effect on osteoclast differentiation. Expressions of osteoclast-related genes were detected by qRT-PCR, and expressions of osteoclast-related proteins were detected by WB and immunofluorescence. KEY FINDINGS: (1) ORI (2 µM) promoted the ALP activity of BMSCs differentiation into osteoblasts and increased the number of calcium nodules. (2) ORI stimulated the expressions of wnt1, ß-catenin and Runx2, but with no significantly effect on p-GSK-3ß and GSK-3ß. (3) ORI promoted the expression of OPG and inhibited the expression of RANKL. (4) ORI directly/indirectly inhibited the osteoclast formation and expressions of osteoclast-related genes TRAP, NFATc1 and c-Fos. SIGNIFICANCE: ORI may promote BMSCs differentiate into osteoblasts through the Wnt/ß-catenin signaling pathway. At the same time, it may also inhibit the formation of osteoclasts mediated by RANKL.

Characterization and immunogenicity of bone marrow-derived mesenchymal stem cells under osteoporotic conditions.

Mesenchymal stem cells (MSCs) are characterized by their multilineage potential and low immunogenicity. However, the properties of MSCs under pathological conditions are unclear. The current study investigated the differentiation potential and immunological characteristics of bone marrow-derived MSCs from ovariectomized-osteoporotic rats (OP-BMSCs). Although the expression of cell morphology- and stemness-related surface markers was similar between OP-BMSCs and BMSCs from healthy rats (H-BMSCs), the proliferation rate was significantly decreased compared with that of H-BMSCs. Regarding multilineage potential, osteogenesis and chondrogenesis abilities of OP-BMSCs decreased, but the adipogenesis ability was significantly enhanced compared with that of H-BMSCs. As expected, decreased osteogenesis following osteogenic induction resulted in reduced expression of ß-catenin, osteocalcin, and runt-related transcription factor 2 in OP-BMSCs. Remarkably, the expression of the co-stimulatory proteins CD40 and CD80 was significantly higher, whereas the expression of the negative co-stimulatory molecule programmed cell death ligand 1 was significantly lower in the OP-BMSCs than that in H-BMSCs. Consequently, H-BMSCs inhibited the proliferation and secretion of inflammatory cytokines from anti-CD3 antibody-activated T cells, whereas OP-BMSCs did not. These results indicate that decreased osteogenesis and increased immunogenicity of OP-BMSCs contribute to bone loss in osteoporosis.

Haem oxygenase-1 induction prevents glucocorticoid-induced osteoblast apoptosis through activation of extracellular signal-regulated kinase1/2 signalling pathway.

BACKGROUND: High-dose glucocorticoid (GC) therapy always causes osteoporosis partly by inducing osteoblast apoptosis. However, the underlying mechanisms of GC-induced apoptosis remain elusive. Haem oxygenase-1 (HO-1) is a cytoprotective protein that rescues cells from H2O2 or high glucose-induced apoptosis. In bone metabolism, HO-1 also participates in osteoclast and osteoblast differentiation. OBJECTIVE: The present study aimed to investigate the protective role of HO-1 against GC-induced osteoblast apoptosis and to elucidate the underlying mechanism. METHODS: Mouse osteoblastic MC3T3-E1 cells were treated with dexamethasone (Dex) for 24 h in the presence or absence of cobalt (III) protoporphyrin IX chloride (CoPP, an inducer of HO-1). In some experiments, U0126 was added to the culture 1 h before CoPP treatment. The induction of apoptosis was determined by flow cytometry. Cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. The expression levels of Bax and bcl-2 were measured by real-time polymerase chain reaction and Western blot. HO-1, extracellular signal-regulated kinase (ERK)-1/2 and pERK1/2 protein levels were measured by Western blot analysis. RESULTS: Dex promoted apoptosis and inhibited cell viability in MC3T3-E1 cells. In addition, Dex significantly increased Bax expression and reduced Bcl-2 expression. The expression of HO-1 was also reduced after Dex treatment. HO-1 induction by CoPP significantly attenuated Dex-induced apoptosis as evidenced by Annexin V/PI staining. The mRNA expression level of antiapoptotic gene Bcl-2 was also increased after CoPP treatment. Moreover, CoPP treatment increased the phosphorylation of ERK1/2. U0126, an inhibitor of ERK activation, significantly abrogated the protective effects of CoPP. CONCLUSION: Our results demonstrate that HO-1 induction by CoPP can attenuate Dex-induced apoptosis of mouse osteoblastic MC3T3-E1 cells. The antiapoptotic effect of HO-1 induction may be correlated with the activation of ERK1/2 signalling pathway. The translational potential of this article: HO-1 induction by CoPP can prevent GC-induced osteoblast apoptosis. Our findings will highlight the therapeutic potential of HO-1 induction in GC-induced osteoporosis.

Mussel-Inspired Peptide Coatings on Titanium Implant to Improve Osseointegration in Osteoporotic Condition.

Osteoporosis greatly impairs in vivo implant osseointegration because of poor osteogenesis in osteoporotic conditions and the low bioactivity of implants, such as titanium-based biomaterials. Various surface engineering strategies, including unstable physical absorption or complex chemical conjugations, have been developed to biofunctionalize titanium implants and improve interfacial osseointegration. However, very few of them took into consideration the clinically challenging osteoporotic condition, as well as dual-functionalization of the implants for improvement of both osteoblast adhesion and osteogenesis. In this work, we combined two mussel-inspired bioactive peptides (i.e., with cell adhesive or osteogenic sequences) for one-step dual-functionalization of Ti screws via a facile self-organized multivalent coordinative interaction. In vitro study indicated that the biomimetic dual-functional coating could efficiently improve the osteogenesis of osteoporosis-derived mesenchymal stem cells despite of their impaired bone metabolism. Moreover, under osteoporotic in vivo condition, the dual-functional peptide coating on Ti screws could also give rise to significant enhancement of interfacial osteogenesis, newly formed bone condition, osseointegration, as well as implant mechanical stability. This is probably due to the integrin-targeted cell adhesive and osteogenic motifs on the modified Ti screws, which recovered the regular bone metabolism equilibrium between osteogenesis and osteoclastogenesis in an osteoporotic condition. We anticipate that the highly biomimetic peptides and one-step dual-functionalized strategy would provide a facile and effective means for improving the clinical outcome of Ti-based implants in patients with a disturbed bone metabolism.