Acute respiratory distress syndrome is an acute respiratory failure caused by cytokine storms; highly pathogenic influenza A virus infection can induce cytokine storms. The innate immune response is vital in this cytokine storm, acting by activating the transcription factor NF-κB. Tissue injury releases a danger-associated molecular pattern that provides positive feedback for NF-κB activation. Exogenous mesenchymal stem cells can also modulate immune responses by producing potent immunosuppressive substances, such as prostaglandin E2. Prostaglandin E2 is a critical mediator that regulates various physiological and pathological processes through autocrine or paracrine mechanisms. Activation of prostaglandin E2 results in the accumulation of unphosphorylated β-catenin in the cytoplasm, which subsequently reaches the nucleus to inhibit the transcription factor NF-κB. The inhibition of NF-κB by β-catenin is a mechanism that reduces inflammation. Acute respiratory distress syndrome (ARDS) is an acute respiratory failure that arises due to acute lung injury which causes a complication ,2. ARDS The mortality of ARDS remains high, although there has been an overall improvement in diagnosis and supportive care. However, no pharmacological therapy is available . AccordiMost studies at present focus on investigations of the regeneration effect of MSCs and its application in treatments that aim to improve tissue injury and regeneration. Knowledge about the crosstalk between regulatory factors in MSCs niches is essential, as it functions in the homeostasis of normal and injured tissue (such as that affected by infection). This pathway is extensively involved in the regulation of adult MSCs’ fate in various tissues. Through the canonical pathway, this Wnt/β-catenin pathway helps the maintenance of MSCs and improves their proliferation ability as a function of regeneration ,30,31. HThe influenza virus is a member of the Orthomyxoviridae family, an enveloped corona virus with a diameter of approximately 80–120 nm. The genome is single-stranded ribonucleic acid (RNA) with a negative sense, consisting of eight (type A and B) or seven (type C) segments. Hundreds of spikes protrude from the surface of the type A virus’s envelope, e.g., hemagglutinin (HA) and neuraminidase (NA). HA is an essential glycoprotein antigen, acting as the site for viral binding and fusion between viral and cell membranes. NA possesses an enzymatic activity, acting to destroy the receptor molecules preventing virus aggregation and releasing the newly assembled virion of the infected cell .The characteristic innate immune response is the primary determinant of the outcome of influenza infection . Three mThe single-stranded RNA (ssRNA) of the influenza A virus is recognized by TLR 7 and 8 via the TLR pathway; it then induces several adaptor molecules and transcription factors, including MyD88, TAK1, TAB1, TAB2, and TRAF6, and activates NF-κB. The interaction between viral protein and TLR activates IKK, which can lead to the phosphorylation and degradation of Iκβ, then release the NF-κB transcription factor subunit p65/p50. Through TKR7/8, TANK binding kinase (TBK) 1 and IKK may also be activated, which then induces the activation of the NF-κB transcription factor and interferon regulatory factor (IRF) 3/7, which expresses the interferon β gene and apoptosis regulation. Influenza A can also be recognized by RIG 1, MDA 5 (melanoma differentiation-associated gene 5), and LGP2. RIG1 and MDA5 act as cytoplasmic viral RNA sensors that play an essential role in innate antiviral immunity ,38.Viral proteins synthesized in the infected cells partially degrade into smaller peptides in the endosomes/lysosomes, and they form a complex with the MHC class I or II peptides at the cell surface. CD4+ helper T cells recognize the complex with MHC class II ,33, wherPro-inflammatory cytokines and chemokines induced by influenza virus infection cause pathological lung damage ,36. HighThe mechanisms of cytokine storm damage proceed through multiple stages. When the highly pathogenic influenza A virus infects alveolar epithelial cells, these cells release various pro-inflammatory cytokines and chemoattractants. HPAI and their infected cells can be recognized by pattern recognition receptors (PRRs) on innate immune cells, such as airway epithelial cells, inflammatory DCs, macrophages, monocytes, and neutrophils. There are several types of cellular PRRs, as follows: membrane-associated toll-like receptors (TLRs), C-type lectin receptors, cytoplasmic nucleotide-binding oligomerization domain-like receptors, and retinoic acid induced gene-like receptors . The virThe TLRs and RIG-I-like receptor on the cellular membrane can interact with their specific extra-cellular and endosomal PAMPs . TLR7 siIn addition to PAMP, DAMP (danger-associated molecular pattern) is a host molecule that functions to maintain PRRs’ activation. High-mobility group box 1 (HMGB1) is a DAMP released from damaged cells due to ARDS ,47. HMGBMSCs are non-hematopoietic stem cells with the ability to differentiate into mesenchymal and non-mesenchymal lineages, and they originate from mesodermal tissues. These cells can be found in all human organs, specifically in the regions of cell populations found in the perivascular area, mainly in bone marrow, the umbilical cord (blood or Whartons jelly), and fatty tissues. MSCs in the lung can differentiate into the epithelial phenotype and structure lung cells . CurrentMSCs, sometimes also referred to as marrow stromal stem cells, were first identified in the guinea pig by Friedenstein in 1968 . The firMSCs are multipotent cells that can differentiate into mesodermal, endodermal, and ectodermal cells . MSCs alAllogeneic MSCs are exogenous stem cells from the same species as MSCs, but they are not genetically identical. The advantages of allogeneic MSCs  in therapeutic applications are manifold compared with autologous MSCs (auto-MSCs). Moreover, auto-MSCs have several associated limitations, including the difficulty of obtaining adequate auto-MSCs from some patients, such as light-weight patients or patients with myelofibrosis. Auto-MSCs isolated from donors of advanced age show decreased biological activity, including activity regarding their differentiation and regenerative potential . Some sySome other limitations of using auto-MSCs include the very time-consuming nature of auto-MSCs extraction, meaning it is hard to use them quickly against acute diseases such as stroke and myocardial infarction. Meanwhile, allo-MSCs are easily obtainable, allowing for immediate administration. As a result, using allo-MSCs represents a promising alternative therapy to auto-MSCs, with advantages in terms of time and cost efficiency, as well as quality assurance . The actMSCs exhibit low levels of MHC class I molecules on their surface and lack expressions of MHC class II as well as the co-stimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40, which may limit immune recognition. However, under infection conditions during which pro-inflammatory factors are released and inflammation occurs, the expression of MHC-II can be up-regulated in antigen-presenting cells (APCs) . KlyushnThere are several techniques for MSCs administration, depending on the underlying clinical indications and pathological abnormalities. The proper selection of the mode of delivering MSCs provides optimal therapeutic efficacy . There aAnother method commonly used to deliver MSCs is via intravascular (IV) infusion, owing to its ease of utilization and low risk . HoweverTheoretically, the intra-arterial (IA) delivery route enables cells to pass through the lungs at least once, thus avoiding the pulmonary “first-pass” effect, and consequently more cells are able to reach the targeted tissues .Several studies have reported the positive therapeutic effects of MSCs; however, there are still a lot of questions and challenges related to the application of MSCs. Cell therapy can induce cell emboli, which may potentially increase mortality. This type of embolism depends on the concentrations and rates of cell administration ,75. OtheThe mechanisms of MSCs as immunomodulatory, improving and regenerating cells, still need to be explored. Some articles have mentioned that paracrine mechanisms are enacted through the secretion of soluble mediators and cell-to-cell contact. Cell-to-cell contact depends on paracrine mechanisms in cells with limited differentiation potential, such as MSCs . The solA study conducted by Ortiz et al.  suggesteKrause et al.  proved tIn addition to the repair and regeneration activities of MSCs in microenvironments where inflammation occurs (inflammation niche), evidence suggests that MSCs have immunomodulatory properties which help in maintaining the immature state of dendritic cells (DCs) through the inhibition of MHC class II, CD1a, CD40, CD80, and CD86 expression and the suppression of pro-inflammatory cytokines’ production . An in vThe immunosuppressive effect produced by MSCs is influenced by several conditions, such as the source of the isolated MSCs, the number of passages in the culture prior to use, the MSC dosages, and the recipient’s specific pathological conditions; however, the immunomodulatory roles of MSCs have attracted great interest on the part of researchers and clinicians .MSCs in situ play an essential role in the cellular homeostasis of host tissue by replacing dead or dysfunctional cells. The capacity of MSCs to modulate immune responses has been shown in an in vitro study . Tissue Tissue damage is commonly accompanied by the activation of immunological or inflammatory cells. The factors produced by cells undergoing apoptosis, necrosis, microvasculature damage, and stroma recruit not only macrophages and neutrophils but also adaptive immune cells, such as CD4 T lymphocytes, CD8 T lymphocytes, and B cells ,92. MeanCytokines, such as TNFα, IL-1β, and IFNγ, toxins from infectious agents, or hypoxia can induce the release of many MSCs growth factors, such as fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), insulin growth factor-1 (IGF-1), angiopoietin-1 (Ang-1), and stromal-cell-derived factor-1 (SDF-1), all of which are induced once the MScs enter the microenvironment ,93. The The inflammatory niche determines the ability of MSCs to inhibit immune cell activity. IFNγ, together with pro-inflammatory cytokines, such as IL-1α, TNFα and IL-1β, the occurrence of a chemokine burst, and the expression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), CCR5 ligand, CXCR3 ligand, and vascular cell adhesion molecule-1 (VCAM-1), can stimulate MSCs to release large amounts of immunosuppressive factors . The co-MSCs have unique immunomodulatory and paracrine properties that are advantageous in treating diseases associated with inflammation and organ injury . SeveralThe study of Ortiz et al.  proved tSeveral studies used MSCs that were not derived from bone marrow, including Shin et al. , with ouA study by Gorman et al.  demonstrDilogo et al.  investigAdas et al.  suggesteMSCs’ administration to ARDS induced by influenza A viral infection has yet to be widely studied; however, investigation on humans is still limited. Several studies focusing on different sub-types of the influenza virus have shown beneficial results. Depending on the stimulation in the microenvironment, MSCs can act in both immune activation and immune suppression. These mechanisms are also known as sensory functions and “switchers” of the body’s immune system . PGE2 plLi et al. suggested that MSCs transfected to overexpress cyclooxygenase (COX)-2 is a potent immunomodulator . Daniel As an essential mediator, PGE2 modulates many biological functions, including immune responses, blood pressure, gastrointestinal integrity, and fertility. PGE2 dysregulation or degradation has been linked to a number of pathological conditions . PGE2 isSome evidence suggests the beneficial effects of PGE in the lung, and its role in several physiological processes, such as the maintenance of barrier integrity ,125. SevThe activation of the β-catenin pathway not only functions in the regeneration process but also is active in immunoregulation mechanisms. According to several studies, the mechanisms of immunoregulation act via the cross-regulation between the Wnt/β-catenin and NF-κB signaling pathways ,133,134.PGE2, produced via the COX pathway, has three identified COX isoforms. One is COX-2, which is undetectable in most tissues but is activated in response to various inflammatory mediators, including pro-inflammatory cytokines. COX-2 is associated with the pathogenesis of several diseases ,136. TheThe β-catenin protein is a subunit of the cadherin protein complex that acts as an intracellular signal transducer, which is also involved in the regeneration and immunoregulation process. β-catenin is a double-function protein that regulates the coordination of cell adhesion and the transcription of genes. β-catenin is homologue with γ-catenin, also known as plakoglobin, and is expressed in various tissues . The WntWnt/β-catenin signaling also acts as the regulation pathway of the stem cells niche in several tissues. However, the role of Wnt signaling in the immunoregulatory function has not been extensively reported, although it is broadly involved in the regulation of adult MSCs’s fate in various tissues. Through the canonical pathway, this Wnt/β-catenin pathway helps the maintenance of MSCs and improves their proliferation ability as a function of regeneration .The available studies suggest that one of the main roles of Wnt signaling is determining the fate of and controlling stem cells . Gehart The elevation of β-catenin under certain tissue damage conditions is indispensable. During viral infection, different transduction signaling pathways control various gene expressions that regulate several molecular and cellular activities required for the eradication of microorganisms and the regulation of inflammation . The infβ-catenin is a CTNNB1 protein encoded by the human CTNNB1 gene that regulates the coordination of adhesion between cells and gene transcription. This protein acts as the main signal transmitter in the Wnt signaling pathway. β-catenin is also detected in adherent junctions, forming a complex with E cadherin and actin filaments from the cytoskeleton .Wnt/β-catenin is a fundamental pathway essential for embryogenesis, cell differentiation, and the maintenance of stem cell self-renewal in humans, lime molds, and worms. Because of the destruction of the complex controlling the level of free β-catenin (which refers to the form unbound to the E-cadherin complex), β-catenin is undetectable in the cytoplasm, nucleus, or unstimulated cells. This complex is made up mainly of the tumor suppressor adenomatous polyposis coli (APC) and Axin, which serves as a protein scaffold for the Ser/Thr protein kinase (casein kinase 1α (CK1α)) and GSK-3β to phosphorylate β-catenin at the N-terminal residues Ser45, Ser33, Ser37, and Thr41. This phosphorylation labels β-catenin, enabling it to be polyubiquitinated by the Skp1-Cdc53-F-Box E3 ubiquitin ligase complex (SCFβ–TRCP) and degraded by the 26S proteasome. GSK-3α is able to regulate β-catenin phosphorylation both in vitro and in vivo, suggesting that both isoforms of GSK-3 contribute to maintaining low levels of β-catenin under baseline conditions .Axin molecules oligomerize with each other in resting cells via the C-terminal DIX domain, which has two interfaces that bind together, forming linear oligomers or even polymers in the cell’s cytoplasm. The only other protein known to have a DIX domain is Dishevelled, which has unique characteristics. A single Dsh protein from Drosophila is related to three paralogous genes in mammals, namely Dvl1, Dvl2, and Dvl3. It corresponds to the cytoplasmic region of the receptor frizzled with its domains, namely PDZ and DEP. When the Wnt molecule binds to the frizzled domain, it starts a cascade that exposes Dishevelled’s DIX domain and creates a site that can completely hook Axin. The oligomer of axin is titrated away, and the β-catenin complex is destroyed by Dsh .Frizzled has essential cytoplasmatic segments containing a GSK-3 pseudo-substrate sequence (Pro-Pro-Pro-Ser-Pro-x-Ser) that is pre-phosphorylated by CKI, namely LRP5 and LRP6 proteins . Once boCanonical Wnt signal activates when the Wnt ligand binds to the Fz receptor and the LRP coreceptor, activating Dvl and inhibiting GSK-3β. Without a Wnt ligand, the activation of Wnt signaling does not occur; GSK-3β, axin, and APC bind β-catenin. This binding stimulates the phosphorylation of β-catenin by GSK-3β and is followed by the ubiquitinylation of β-catenin, which is further degraded by proteasomes. β-catenin accumulates in the nucleus and binds to the T cell factor and lymphoid enhancer factors (TCF/LEF) to regulate gene transcription . Figure PGE2 levels in the lung compared with other organs tend to be higher, as evidenced by the isolation of alveolar epithelial cells that release high amounts of PGE2 . AccordiConsequently, β-catenin is accumulated, and the expression of T cell-4 transcription factor is induced, which forms a transcriptionally active complex with β-catenin. Furthermore, similar to the Wnt/β-catenin pathway, the translocation of β-catenin into the cell nucleus releases transcription factors, which then bind to transcriptional activators and switch to target genes . Figure The cross-regulation between the Wnt/β-catenin and NF-κB signaling pathways is essential to the regulation of diverse genes and active pathways, e.g., many physiological and pathological effects, regulations associated with the areas of growth, immune function, inflammation, tumorigenesis, tumor invasion, and metastasis, as well as cardiovascular and bone disease. However, the activity and signaling consequences are also regulated by direct interactions between the two pathways, resulting in diversity and complexity . β-catenA colonic epithelial cell infection study with Salmonella typhimurium demonstrated the activity of β-catenin, which is able to inhibit NF-κB via two possible mechanisms: by stabilizing IκBα or by binding to NF-κB subunit p50, which prevents its transport from the cytoplasm to the nucleus and also occurs in the nucleus. Both mechanisms can cause a decrease in the transcriptional activity of NF-κB. The inhibition of NF-κB by β-catenin activation reduces inflammation. The study found that the infection of colonic epithelial cells with Salmonella typhimurium caused an elevation in GSK-3β-dependent β-catenin phosphorylation, leading to the up-regulation of Wnt2, IL-6, and IL-8 via TLR5, and thus activating NF-κB . Liu et Based on the available literature, the administration of MSCs could enhance endogenous PGE2, escalate the number of cells expressing β-catenin, and decrease the NF-κB transcription factor and TNFα pro-inflammatory cytokine expression, as well as IL-1β. As previously explained, the characteristics of the innate immune response are the primary determinant of the outcome of influenza infection. PAMPs of the influenza A virus are recognized by three main classes of PRRs, including the cytosolic sensor RIG-I , TLR7 inThe production of active biomolecules of PGE2 increases the accumulation of β-catenin in the cytoplasm. As previously mentioned, PGE2 has been proven to stimulate a double signaling cascade involving the activation of PI3K and protein kinase Akt by the free G-protein βγ subunit and the direct association of the G-protein αs subunit to Axin. The activation of GSK-3β promotes the phosphorylation of β-catenin and subsequent degradation by the 26S proteasome, reducing β-catenin signaling. On the other hand, Akt phosphorylation via PI3K signaling inhibits the activation of GSK-3β and APC in the protein subunits of G-protein-linked PGE2 receptor EP2, as well as the association of the activated α subunit of Gs with the RGS domain of Axin, so that the Axin–GSK–3β complex releases β-catenin ,126,147.In the further course of the disease, other pathways are needed to increase the activation of β-catenin, which serves to maintain homeostatic function in severe lung injury. The increase in β-catenin through the trans-differentiation process of the canonical Wnt/β-catenin pathway, which is usually involved in cell development through frizzled receptors, has a similar effect on PGE2, which functions to decrease the arrangement of the Axin-GSK-3β-APC complex, thereby stabilizing the accumulation of β-catenin in the cytoplasm ,128,147.The β-catenin protein plays a vital role in the process of immunoregulation. The inhibition of NF-κB transcription factor activity is a β-catenin pathway that plays a role in the immunoregulation mechanism. According to previous studies ,131,148,Decreased NF-κB activity also has a significant direct effect on RAGE expression. The activation of the NF-κB pathway through RAGE can provide positive feedback. Furthermore, NF-κB can regulate the activation of RAGE, which causes an elevation of RAGE expression. This positive feedback cycle can further increase inflammation and tissue injury. These conditions occur mainly in diseases with elevated levels of the RAGE ligand ,153. OthThe characteristics of innate immune response are essential to tissue damage caused by highly pathogenic influenza A virus infection. ARDS involves damage in the alveolar–epithelial–endothelial barrier of the lung, which causes the leakage of proteins of intravascular origin into the alveolar lumen. In addition to PAMPs, DAMPs are host molecules that regulate the activation of PRRs during influenza A virus infection. Activation of this molecule, in turn, triggers a signaling cascade that promotes the transcription factor NF-κB, which encodes pro-inflammatory cytokines and chemokines. Allotransplanted MSCs may exert a beneficial effect on tissue injury by inhibiting the transcription factor NF-κB. The effect of allotransplanted MSCs against inhibitory transcription factor NF-κB is known to operate through several related mechanisms, via the release of PGE2 and the accumulation of β-catenin.The β-catenin protein plays a crucial role in the process of immunoregulation. Inhibition of the activity of transcription factor NF-κB is one of the β-catenin pathways that play a role in the mechanism of immunoregulation. The cross-regulatory mechanism of β-catenin in the cytoplasm activates Iκβ, thereby inhibiting NF-κB activity. However, there are still many other pathways that are involved in the mechanism of immunoregulation through inhibiting the innate immune system in addition to inhibiting NF-κB activation. Thus, the utilization of these approaches in acute lung damage caused by the highly pathogenic influenza A virus requires further research both in animal models and humans. Furthermore, the innate immune system is likely to activate its adaptive function to perpetuate the process. Therefore, it is necessary to evaluate the effects of MSCs on the adaptive immune system. Hopefully, a better understanding can be formed of the mechanisms of pro-inflammatory pathways that contribute to lung injury and host defenses, which can inhibit inflammatory damage while maintaining the host defense system. The mechanism of immunoregulation through β-catenin activation requires further research into the development of methods that can modify MSCs in vitro before transplantation, one of which involves genetic modification through the transfection method, such that MSCs can be made to carry the β-catenin gene. Another alternative approach is to provide combination therapy with β-catenin and MSCs.


Embryonic stem cells (ESCs), which are derived from the undifferentiated inner cell mass of the embryo, can differentiate every cell type of the body regarding their pluripotency. Therefore, human or mouse ESCs can be used as an unlimited cell source for numerous researches or therapeutical approaches. However, pluripotency maintenance of ESCs during in vitro culture is challenging because of their endless differentiation capacity. In the current study, the effect of USP7 on pluripotency maintenance of mouse ESCs (mESCs) has been investigated with the help of cell viability assay, morphological analysis, alkaline phosphatase (ALP) staining, qPCR analysis, and Western Blotting. 600 nM P5091 application, which showed no significant toxicity in mESCs, increased the total ubiquitinated protein amount as a proof of the accomplishment of proper USP7 inhibition. Morphological analysis and ALP activity evaluation indicated that dual inhibition of GSK3 and MEK together with leukemia inhibitory factor (LIF) treatment protects the pluripotency in presence of active USP7 enzyme. Yet, inactivation of USP7 reduced the ALP activity and altered the cell morphology in each treatment group. This morphological change and decreased ALP activity refer to differentiated mESCs. These findings were supported by gene expression and protein analysis. Gene expressions and protein amounts of pluripotency related Oct4, Nanog, c-Myc, Sox2 and Klf4 transcription factors decreased significantly after USP7 inhibition. Together with this observation, a remarkable reduction in β-Catenin expression was also noticed. It was also observed that USP7 inactivation shortens the half-live of β-Catenin and GSK3β proteins. This study demonstrates that USP7 activation is crucial for proper pluripotency maintenance, which is provided through β-Catenin stabilization. The number of researches that focused on embryonic stem cells (ESCs) is increasing because of the endless differentiation potential of ESCs. These researches provide a variety of possibilities for developing better techniques to differentiate ESCs into any cell type. For instance, from the perspective of regenerative medicine, ESCs can be used as an unlimited source for various tissue replacements . Keeping+2 pathway. All of the pathways are triggered by the attachment of Wnt ligand to the corresponding receptor (Frizzled family receptor) on the cell membrane , the pluripotency of cells was maintained with feeder-dependent culture technique. Briefly, cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 15% embryonic stem-cell FBS , L-glutamine , 1× MEM nonessential amino acids , 10 ng/mL recombinant mouse leukemia inhibitory factor  and 1% penicillin, streptomycin, and amphotericin B  on mitomycin C  treated mouse embryonic fibroblast (MEF)  feeders. The cells were incubated in humidified incubator at 37 °C and 5% CO3 U/ml LIF ) was used to evaluate whether the 2i application alters the possible effect of P5091 on R1/E cells : Neurobasal Medium  (1:1) supplemented with 0.5% N2 supplement , 1% B-27 supplement , 0.5% of 10% Bovine serum albumin (BSA) in phosphate buffer saline (PBS) , 1% L-glutamine  and 1% Antibiotic-Antimycotic ) and plated on Matrigel –Gelatin  (1:200) coated well plates. The pluripotency of mESCs was maintained by the application of two small-molecule inhibitors (2i)  and 1 μM PD0325901 ) and 10 ng/mL LIF. Furthermore, M15 medium , 15% fetal bovine serum  1% Antibiotic-Antimycotic, 1% non-essential amino acids , 0.1 mM β-mercaptoethanol  and 10/E cells .P5091 , CHIR and PD0325901 were dissolved in dimethyl sulfoxide  to prepare 6 mM, 3 Mm, and 1 mM stock solution, respectively. CHX  was dissolved and diluted in M15 medium to a final working concentration of 50 μg/mL.3 cells/well. After overnight incubation, cells were treated with various P5091 concentrations  for 24 h. The viability of cells was measured by using PBS containing 4.5 g/L glucose and 10% MTS as described previously -5-(3-carboxy-methoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay  was performed. mESCs were seeded on Matrigel-Gelatin coated 96-well plates  at density of 5×10eviously . After 9mESCs were treated with the combinations of 2i, LIF and P5091 in N2B27 (or only P5091 treatment in M15 medium compared with M15 medium control) morphological changes of cells were observed under light microscope. Images were taken via ZEISS microscopy, AxioCam ICc 5 camera and ZEN 2 (blue edition) computer application. Additionally, the percentage of undifferentiated cells were calculated by using ImageJ software.4 cells/well. mESCs were treated with 600 nM P5091 in presence of 2i and/or LIF for 24h. To stain ALP in living cells, mESCs were washed once with DMEM/F12 . After washing step, stock stain (500 X) was diluted to the final concentration of 1 × with DMEM/F12. After 30 min of incubation at 37 °C, mESCs were washed three times with DMEM/F12 before imagining by using fluorescent microscope .To analyze the change in pluripotency of mESCs, alkaline phosphatase staining  was conducted according to manufacturer’s instructions. Briefly, mESCs were seeded on Matrigel-Gelatin coated 12-well plates  at density of 4 × 10Primer-BLAST software  was used to design USP7, SOX2, C-MYC, OCT4, KLF4, Brachyury, SNAIL, CDH1, GAPDH, WNT3a, Vimentin, Axin and β-Catenin primers , which wChanges in pluripotency related Nanog, Oct4, c-Myc and Sox2 proteins were determined by western blot analysis. Additionally, successful decrease in de-ubiquitination activity of USP7 and alterations in Gsk3β and β-catenin proteins were also demonstrated by western blotting. Furthermore, the half-life of proteins was also determined after inhibition of translation by 50 μg/mL CHX treatment via immunoblotting . After eAll experiments were repeated at least three times, and statistical analysis was conducted by using one-way ANOVA and post hoc Tukey test. The statistical significance of P values (less than 0.05) was calculated by using GraphPad Prism 8.0  software.MTS assay was performed to identify the effect of P5091 on viability of mESCs. Various concentrations  of P5091 were applied to mESCs for 24 h. Results showed that application of P5091 at low concentrations  did not affect the cell viability of mESCs . HoweverTo identify the impact of USP7 inhibition on mESCs morphology, cells were treated with 600 nM P5091 in the presence of 2i and/or LIF for 24 h on Matrigel-Gelatin coated plates and morphological alterations were determined by microscopic analysis. Results were determined that morphology of mESCs were affected by P5091 treatment. 2i and LIF application caused no significant change in cell morphologies of mESCs as expected. Yet, the addition of P5091 changed the morphology of mESCs to form spindle-like formation. Additionally, in every group, inactivation of USP7 resulted in impaired cellular morphology. Obtained results also showed that inhibition of ERK and GSK3β by 2i treatment and functional USP7 de-ubiquitination enzyme are required for pluripotency maintenance . FurtherTo examine whether inhibition of USP7 has an effect on pluripotency, SOX2, OCT4, C-MYC, and KLF4 gene expression levels were determined in mESCs by gene expression analysis. Additionally, alterations in gene expression levels of mesoderm marker, Brachyury (T/Bra), ectoderm marker (Vimentin), extra cellular matrix (ECM) related CDH1 and SNAIL were also examined. Lastly, changes in gene expression levels of WNT3a and β-Catenin, which play a critical role in pluripotency homeostasis, were checked by qPCR analysis. All experimental groups were compared to relevant treatment groups including active or inactive USP7 protein to identify any possible change in pluripotency of mESCs. Gene expression profile of USP7 reduced by 10-fold after P5091 treatment in 2i + LIF group. A dramatic decrease was observed in gene expression levels of SOX2 (125-fold), C-MYC (142-fold), OCT4 (13-fold), KLF4 (320-fold), WNT3a (8.5-fold) and β-Catenin (6.25-fold) after inactivation of USP7 in 2i + LIF group. Similarly, in 2i treated group, P5091 application caused a significant decrease in C-MYC, KLF4, AXIN, and β-Catenin gene expression levels by 10-fold, 38-fold, 4.3-fold, and 2.5-fold, respectively. No significant change was observed in expression levels of USP7, SOX2, Brachyury, SNAIL, CHD1, and WNT3a genes. Also, USP7 inactivation reduced SOX2 (37.5-fold), C-MYC (5.5-fold), AXIN (2.12-fold), and WNT3a (3.2-fold) gene expression levels in LIF treatment group. Yet, there was no significant change in USP7, OCT4 and β-Catenin gene expression levels in LIF treatment group. Furthermore, noticeable change was not detected in gene expression levels of Brachyury, Vimentin, CDH1 and SNAIL in all experimental groups . The expTo further examine the effect of USP7 on pluripotency of mESCs, western blot analysis was conducted. Experimental compressions were performed on the same treatment groups with different USP7 activation status. P5091 treatment caused no change in USP7 protein amount significantly in ERK and GSK3β inhibited groups. Interestingly, USP7 protein levels were decreased by 50% after P5091 treatment in LIF treatment group . Nanog pTo evaluate the effect of USP7 inactivation on half-life of USP7, c-Myc, Sox2, Oct4, Nanog, GSK3β and β-Catenin proteins, the protein synthesis was blocked by CHX treatment for different time points . GAPDH protein was used as an internal control. CHX treatment for 4 h did not affect the USP7 levels in both control and P5091 treatment groups. However, the protein amount of Sox2 decreased by approximately 15% after each hour in both control and P5091 groups. The amount of c-Myc protein decreased by 16%, 25%, and 35% after 1 h, 2 h, and 4 h of CHX treatments, respectively. However, no significant decrease was observed after 2 h of CHX treatment, one hour of CHX treatment cause a noteworthy decrease (35%) in c-Myc protein amount in P5091 treatment group. Unsurprisingly, the amount of c-Myc protein dropped significantly after 4 h of CHX treatment (15%) after P5091 treatment. A remarkable decrease in the protein amount of Oct4 was noted after 1h CHX treatment (approximately 64% in both control and P5091 treatment group). CHX treatment for 4 h causes a similar decrease in OCT4 in both experimental groups. Likewise, the protein amount of Nanog protein dropped coherently in both control and P5091 treatment groups. Additionally, the half-life of GSK3β and β-Catenin shortened after P5091 treatment. Although, 1 h of CHX treatment did not change GSK3β protein amount in control group, it caused a significant decrease (37%) in P5091 treated group. Correspondingly, 2 h and 4 h of CHX treatment decreased GSK3β protein amount even more dramatically after USP7 inactivation . Similarly, 1h and 2h of CHX treatment did not change β-Catenin amount in control group. Yet, USP7 inactivation cause a decrease in β-Catenin protein amount even after 1 h and 2 h of CHX treatment . Also, 60% of β-Catenin remained functional in control group while 20% of β-Catenin remained functional in P5091 treatment group after 4 h of CHX treatment . These fPost-translational regulation of proteins is usually controlled by ubiquitination which plays a significant role in almost every molecular pathway such as Wnt/β-catenin pathway  which isIt is known in the literature that pluripotency maintenance can be observed by morphological analysis . MoreoveSeveral studies have focused on transcription factors that are required for keeping embryonic stem cells at pluripotent state . The traThe effect of USP7 inhibition on pluripotency maintenance was further examined by half-life determination of pluripotency related proteins. The literature reveals that cycloheximide (CHX) treatment successfully blocks the protein synthesis in cells ; thus, iPreviously it was clarified that Wnt/β-catenin signaling is vital for pluripotency maintenance of mESCs . Unwantevia its interaction with E3 ligase RNF220. Thus, USP7 also upregulates the β-catenin stability -box 2, OCT4 Octamer-binding transcription factor 4, KLF4 Kruppel Like Factor 4, SNAIL Snail Family Transcriptional Repressor 1, CDH1 Cadherin 1, WNT3a Wnt Family Member 3A, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, mean ± SD; n = 3 independent experiments; one-way ANOVA, *p < 0.05, compared with the M15 medium control group.USP7 inactivation decreases pluripotency of mESCs in M15 medium. A. USP7 inactivation changes the morphology of mESCs in vitro. B. P5091 treatment causes a significant decrease in ALP activity of mESCs in M15 medium. Histogram shows the decrease in ALP activity in each group. C. P5091 treatment decreased the gene expression levels of pluripotency related SOX2, OCT4 and KLF4 genes in M15 medium. However, no significant change was observed in mesoderm (Brachyury) and extra cellular matrix  related gene expression levels. Furthermore, USP7 inactivation caused a decrease in expressions of WNT3a and β-Catenin genes. GAPDH mRNA was used as an external control.


Nanoviridae) that causes one of the most devastating diseases of cultivated bananas and plantains (family Musaceae). It is transmitted by the aphids Pentalonia nigronervosa and P. caladii among host plants of Musaceae and some other families of monocots. Our Illumina sequencing reconstruction of virome components of BBTV-infected banana plants and their neighbor non-banana plants sampled in Vietnam and Laos revealed the monocot Commelina sp. (Commelinaceae) and the dicots Bidens pilosa and Chromolaena odorata (both Asteraceae) as hosts of BBTV and circular ssDNA alphasatellites (family Alphasatellitidae). Counting the proportions and relative abundances of Illumina reads representing BBTV genome components and alphasatellites suggested that Chromolaena and Commelina are poor hosts for BBTV and one to three alphasatellite species, whereas Bidens is a permissive host for BBTV and four alphasatellite species representing two genera of Alphasatellitidae. Our findings provide evidence for the dicot plants of family Asteraceae as alternative hosts of BBTV and its alphasatellites, which warrants further investigation of these and other dicots as a potential refuge and source of BBTV and multiple alphasatellites that become associated with this virus and likely affect its replication, transmission, and host range.Banana bunchy top virus is a multicomponent circular ssDNA virus (family  Babuvirus, family Nanoviridae) causes severe disease in cultivated bananas and plantains (Musa sp.) and represents a serious threat to global food security. Since the beginning of the 19th century, the disease has resulted in devastating epidemics, reducing banana production by up to 95% in several countries in South-East Asia, Oceania, and Africa [Pentalonia nigronervosa and its relative P. caladii [Alphasatellitidae) that are similar in size to BBTV genome components and encode a Rep-like protein that mediates replication of alphasatellite DNA. Alphasatellites depend on their helper viruses for movement, encapsidation, and transmission [Banana bunchy top virus , Zingiberaceae , Araceae (Colocasia esculenta), Cannaceae (Canna indica), and Heliconiaceae (Heliconia aurantiaca) [Pentalonia aphids. The host range of P. nigronervosa and P. caladii is also restricted to the monocot families, including Araceae, Cannaceae, Commelinaceae, Heliconiaceae, Musaceae, Strelitzeaceae, and Zingiberaceae [The host range of BBTV has so far been reported to be restricted to plant species of the monocot families Musaceae (antiaca) ,12,13,14beraceae ,16 and tDuring our surveys in South-East Asia  in 2018 and 2019, leaf samples of wild and cultivated banana plants displaying BBTV symptoms were collected together with leaf samples of non-banana plants grown in close vicinity of the banana plants .Commelina sp. (Commelinaceae) and the dicots Arachis hypogaea (Fabaceae), Bidens pilosa (Asteraceae), Chromolaena odorata (Asteraceae), Ipomoea aquatica (Convolvulaceae), and Phyllanthus sp. (Phyllanthaceae). Notably, B. pilosa plants exhibited leaf chlorosis (The non-banana species included the monocot hlorosis . The tothlorosis . The virA. hypogaea (ALYU-31), I. aquatica (ALYU-28), Phyllanthus sp. (ALYU-30), and 2 of the 26 banana plants (ALYU-46 and ALYU-52), very low numbers of viral  reads were detected; no complete consensus viral component could be reconstructed from these reads. We assumed that these reads represent cross-contamination from other samples multiplexed and sequenced in one flow cell of Illumina HiSeq2500. This assumption was supported by the inspection of read coverage profiles with and without mismatches , B. pilosa , and C. odorata  were considered to be infected with BBTV and coinfected with one or more alphasatellites  sampled in Vietnam shared 100% identical consensus sequences of all the virome components, including six components of BBTV genome and three species of BBTV alphasatellites  , althougP. nigronervosa [Nanovirus, family Nanoviridae) is essential for aphid transmission but not for disease symptom development [C. odorata lacking DNA-N is unlikely to serve as a source for virus transmission, while the self-replicating alphasatellite BBTA5 as the most abundant virome component (The viromes of Chromolaena (ALYU-41) and its two banana neighbors (ALYU-42 and ALYU-43) sampled in Laos all contained BBTV and a single alphasatellite (BBTA5). However, both BBTV and BBTA5 were represented with distinct genetic variants in Chromolaena and each banana neighbor . Most noonervosa . Likewiselopment ,19. Baseomponent B likely Microviridae and Circoviridae  and its banana neighbor (ALYU-37) sampled in Vietnam shared six-component BBTV and one alphasatellite (BBTA2), with all these seven components having 100% identity in their consensus sequences. In addition, the virome of Bidens contained three more alphasatellites  and two Rep-encoding circular ssDNA viruses, both classified as novel species in the non-plant families oviridae ; all theoviridae . Likewisoviridae . Note thoviridae , thus exoviridae . Most nooviridae . These fast Asia .Commelina sp., which represents the monocot family Commelinaceae and falls within the known host range of Pentalonia aphids and BBTV, but also to C. odorata and B. pilosa. Both C. odorata and B. pilosa represent the dicot family Asteraceae, which was unexpected to host BBTV or banana aphids. In the latter case, even a short-term probing and salivation event would be sufficient for the release of viral particles circulating in the aphid body and accumulating in the salivary glands [Pentalonia aphids can feed on BBTV- and alphasatellite-infected Bidens and Chromolaena plants for a prolonged time to acquire BBTV and its alphasatellites and transmit them among these and other Asteraceae plants or back to Musaceae hosts. Previous studies have established a minimal acquisition access period of 4 h for banana aphids on BBTV-infected banana plants to transmit the virus to new plants with a minimal inoculation access period of 15 min [Collectively, our findings suggest that viruliferous banana aphids could have transmitted BBTV and alphasatellites from the respective banana neighbors not only to y glands ,20. It rf 15 min ,22.Muscarsatellite of the subfamily Petromoalphasatellitinae , other alphasatellites represent two new species, BBTA5 and BBTA6, which belong to a tentative genus Banaphisatellite within the subfamily Nanoalphasatellitinae , recently discovered to be associated with BBTV isolates from the banana aphid and plant samples collected in Africa (Democratic Republic of the Congo) [Nicotiana benthamiana (Solanaceae) [Bidens pilosa and Chromolaena odorata.Phylogenetic analysis of the alphasatellites associated with our BBTV isolates from Vietnam, Laos, and China  revealedlitinae  . A hypotanaceae) . This hyB. pilosa (Leaf samples of banana plants displaying characteristic BBTV disease symptoms and non-banana plants grown in close vicinity of the BBTV-infected banana plants see  were col. pilosa . Each ALv/v) (CIAA), followed by centrifugation at 13,000 rpm for 30 min at 4 °C. The supernatant was taken for a second round of extraction with CIAA, followed by centrifugation as described above. The supernatant was mixed with one volume of isopropanol pre-cooled at −20 °C. The mixture was shaken until appearance of a hank and was then spun at 13,000 rpm and 4 °C for 30 min. The pellet was washed twice with 500 µL of 70% ethanol, air-dried, and dissolved in 100 µL of milli-Q water.Dried leaf tissue (100 mg) was ground in liquid nitrogen, and 500 µL extraction buffer  pre-heated at 74 °C and supplemented with 0.4 µL RNase (100 mg/mL) was added to the frozen powder. The mixture was vortexed for 20 s, incubated at 74 °C for 20 min, and then mixed vigorously with one volume of chloroform–isoamyl alcohol  following the manufacturer’s protocol. Briefly, 5 μL sample buffer and 1 μL total DNA extracted from leaf tissues were mixed and heated at 95 °C for 3 min. The samples were cooled in ice, and 5 μL reaction buffer and 0.2 μL enzyme mix were added, followed by incubation at 30 °C for 18 h. The enzyme was inactivated by heating at 65 °C for 10 min. RCA products were purified using NucleoSpin Gel and PCR Clean-up kit  following the manufacturer’s protocol. DNA concentration was measured by Qubit fluorimeter using Qubit dsDNA HS Assay Kit .The RCA products were analyzed by PCR using a pair of primers (5′-GGCGCGATATGTGGTATGCTGG and 5′-CCAAACTCGAAGGGACCTTCG) specific for a conserved region of DNA-R, yielding a 285 bp product. The PCR reaction was performed in a volume of 25 µL containing 1 µL of the RCA-treated total DNA, 2 µL primer mix (10 µM each primer), 1 µL of 2.5 mM dNTPs, 5 µL of 5× GoTaq buffer, and 1U GoTaq DNA polymerase . After denaturation at 94 °C for 5 min, DNA was amplified for 35 cycles of 30 s at 94 °C, 30 s at 56 °C, and 30 s at 72 °C, followed by a final extension at 72 °C for 10 min. The results of the PCR analysis are shown in www.fasteris.com; accessed on 23 October 2023). Libraries were prepared using Nextera XT standard DNA protocol, and all libraries were multiplexed and sequenced in one flow cell of HiSeq2500 with a 2× 125-nt paired-end run. Viral genomes were de novo reconstructed from the sequencing reads of each library by selecting unique inserts sequenced ≥5, ≥10, ≥20, ≥30, ≥40, or ≥50 times and assembling them using Velvet v. 1.2.10 [Fifty ng of the cleaned RCA products were taken for Illumina sequencing at Fasteris AG (. 1.2.10  and visu. 1.2.10 .


Available literature documents that ischemic stroke can disrupt the morphology and function of mitochondria and that the latter in other disease models can be preserved by neuropilin-1 (NRP-1) via oxidative stress suppression. However, whether NRP-1 can repair mitochondrial structure and promote functional recovery after cerebral ischemia is still unknown. This study tackled this very issue and explored the underlying mechanism.Adeno-associated viral (AAV)-NRP-1 was stereotaxically inoculated into the cortex and ipsilateral striatum posterior of adult male Sprague-Dawley (SD) rats before a 90-min transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion. Lentivirus (LV)-NRP-1 was transfected into rat primary cortical neuronal cultures before a 2-h oxygen-glucose deprivation and reoxygenation (OGD/R) injury to neurons. The expression and function of NRP-1 and its specific protective mechanism were investigated by Western Blot, immunofluorescence staining, flow cytometry, magnetic resonance imaging, transmission electron microscopy, etc. The binding was detected by molecular docking and molecular dynamics simulation.Both in vitro and in vivo models of cerebral ischemia/reperfusion (I/R) injury presented a sharp increase in NRP-1 expression. The expression of AAV-NRP-1 markedly ameliorated the cerebral I/R-induced damage to the motor function and restored the mitochondrial morphology. The expression of LV-NRP-1 alleviated mitochondrial oxidative stress and bioenergetic deficits. AAV-NRP-1 and LV-NRP-1 treatments increased the wingless integration (Wnt)-associated signals and β-catenin nuclear localization. The protective effects of NRP-1 were reversed by the administration of XAV-939.NRP-1 can produce neuroprotective effects against I/R injury to the brain by activating the Wnt/β-catenin signaling pathway and promoting mitochondrial structural repair and functional recovery, which may serve as a promising candidate target in treating ischemic stroke. Stroke has been ranked as a primary cause of disability and death in the elderly populace worldwide  and in tMitochondria, a powerhouse of the cell, are rich in a large number of enzymes, which not only participate in cellular oxidative phosphorylation and ATP synthesis, but also regulate intracellular calcium ions and reactive oxygen species to maintain cellular homeostasis. Studies have documented the involvement of mitochondria in the neuronal death following ischemic stroke . In earlAs a multifunctional, transmembrane, non-tyrosine kinase surface glycoprotein, neuropilin-1 (NRP-1) performs important functions in neural and vascular systems , 11. A pNRP-1 has been documented to participate in the maintenance of the stem cell properties of gastric cancer cells by activating the Wnt/β-catenin signaling pathway . Wnt is The current study explored in vivo and in vitro the neuroprotection of NRP-1 against I/R injury to the mitochondrial structure and function in rat models and the related mechanism. The middle cerebral artery occlusion (MCAO) model and OGD/R model were established to investigate NRP-1-conferred neuroprotection against I/R injury to the brain. We found that NRP-1 overexpression restored motor function and structure of mitochondrial, maintained the mitochondrial integrity, and inhibited mitochondrial bioenergetic deficits, neuronal apoptosis, and oxidative stress. These protective effects were reversed by XAV-939, a Wnt/β-catenin signaling pathway inhibitor. These results signify that NRP-1 may serve as a promising candidate target in treating ischemic stroke.Adult Sprague-Dawley (SD) rats  were obtained from Experimental Animal Center of Fujian Medical University and housed in standard filter-top cages on a 12-h light/12-h dark cycle, with free access to water and food. The protocols observed the guidelines of National Institute of Health . The experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Fujian Medical University in Fujian, China.A focal ischemic stroke was established in male SD rats by transiently occluding the middle cerebral artery (MCA) as described . Briefly10gc, respectively) were administered at a rate of 0.2 µl/min. After the injection, the syringe was left in place for at least 15 min to ascertain full virus diffusion and to prevent backflow of the virus. XAV-939   was intraperitoneally injected 2 days before MCAO and on the day of the surgery before MCAO, respectively  before centrifugation of 1500 The peri-infarct cortex tissue or cultured primary neurons was homogenized with RIPA lysis buffer in the presence of 1% protease and phosphatase inhibitor cocktail and 1 mM PMSF. The samples received a centrifugation of 12, 000 r for 20 min at 4 ℃ to collect the supernatant. The concentration of protein was measured with a BCA Protein Assay Kit . An amount of 20–60 µg of protein was then loaded onto an 8–12% SDS-polyacrylamide gel and separated by electrophoresis. The separated protein was transported from the gel to polyvin ylidene difluoride (PVDF) membranes  and blocked using 5% bovine serum albumin  diluted with Tris-buffered saline containing 0.05% Tween 20 (TBST) at RT for 2 h. Subsequently, the membrane was incubated with various primary antibodies at 4 ℃ overnight. The primary antibodies included: rabbit anti-Neuropilin 1 , rabbit anti-Bcl-2 , rabbit anti-Bax , rabbit anti-caspase-3 , rabbit anti-Cyt c , rabbit anti-AIF , rabbit anti-Wnt 3α , rabbit anti-APC , mouse anti-phospho-GSK3β-S9 , rabbit anti-Axin-1 , rabbit anti-Wnt 1 , rabbit anti-Cyclin D1 , rabbit anti-c-Myc , rabbit anti-β-Catenin , rabbit anti-β-actin , rabbit anti-Histone H3 , mouse anti-Tomm20 , rabbit anti-GSK3β , rabbit anti-Phospho-beta catenin (Ser37) . After 3 washes with TBST, HRP-conjugated goat antibodies to rabbit  or mouse  IgG was applied at RT for 60 min. Finally, protein bands were visualized with the FluorChem M system . The expression of protein was quantified with ImageJ software .-100 for 15 min and then blocked with a blocking buffer  at RT for 1 h. Primary antibodies were diluted in PBS at 4℃ overnight, including rabbit anti-Neuropilin 1 , chicken anti-beta III Tubulin , rabbit anti-Phospho-beta catenin (Ser37) . After 3 washes with TBST or PBST (PBS containing 0.2% Triton X-100), the slices or cultured neurons underwent an incubation at RT for 2 h with the secondary antibodies: Donkey anti-Rabbit conjugated with Alexa Fluor 488 , Donkey anti-Mouse conjugated with Alexa Fluor 594 , and Goat anti-Chicken conjugated with Alexa Fluor 594 . After 3 washes with TBST or PBST, brain slices or cultured neurons were stained with the nuclear stain 6-diamin-2-phenylindole-dihydrochloride (DAPI)  at RT for 10 min. After 3 washes with TBST or PBST, the images of slides were visualized under a confocal microscope .Immunofluorescence staining was conducted as described previously . Briefly3) were fixed in a PBS mixture containing 1.5% ferrocyanide and then incubated in 1% osmium tetroxide for 2 h. After washing in 0.1 M PBS, tissues were dehydrated with a graded series of ethanol and acetone, and embedded in Epon and sliced for TEM. Ultrathin slices (90 nm) were prepared and stained with uranyl acetate and lead citrate. The mitochondrial ultrastructure was observed with a Tecnai G2 TEM .The mitochondrial ultrastructure and morphology of cortex tissues in SD rats were observed by TEM. After anaesthetization, rats were perfused with 0.1 M PBS (pH 7.4) and then with precooled PBS containing 1.5% paraformaldehyde and 3% glutaraldehyde. The cortex tissues (1 mm2 flasks (3 × 106 cells/flask) were obtained and then divided into 2 EP tubes (1.5 mL). The pellet in each tube was suspended in 195 µl of 1× annexin V-FITC binding buffer and 5 µl annexin V-FITC and incubated at RT for 10 min. After a centrifugation of 1000 g for 5 min, the pellet was resuspended in 10 µl of propidium iodide (PI) working solution and 190 µl of binding buffer. The samples were analyzed with a BD Accuri C6 Plus flow cytometer  and the data were processed with FlowJo 10.8.1 . The apoptotic percentage of neurons was indicated as the percentage of early apoptotic neurons + the percentage of late apoptotic neurons.Flow cytometry was performed to analyze cell apoptosis with an Annexin V-FITC Apoptosis Detection Kit . Briefly, primary cortical neurons cultured in 25 cmTUNEL staining was performed according to the manufacturer’s instructions . After a fixation with formalin, the slides were embedded in paraffin and sectioned. After deparaffinization, slides were subjected to enzyme reaction with the TUNEL reaction mixture before rinsing. The TUNEL reaction was performed using the freshly-prepared 3, 3-diaminobenzidine (DAB) solution and counter-staining was performed with hematoxylin. Apoptotic cells were observed by fluorescence microscopy  with 20 × objective.2 flasks . The data were processed with FlowJo 10.8.1 , with the results reported as the ratio of the red/green fluorescence.The changes in the mitochondrial membrane potential (ΔΨm) were detected with a JC-1 kit . In brief, primary cortical neurons cultured in 25 cmMalondialdehyde (MDA) was measured with a Lipid Peroxidation MDA Assay Kit . Superoxide Dismutase (SOD) activity was detected with a SOD Assay Kit . Glutathione (GSH)/Glutathione disulfide (GSSG) ratio was measured with a GSH and GSSG Assay Kit . Glutathione peroxidase activity (GSH-PX) was detected with a GSH-PX Assay Kit .The intracellular ATP contents in neurons were detected with an Enhanced ATP Assay kit . Following the manufacturer’s instructions, pre-cooled lysis buffer (200 ml/well) was administered to the 6-well plate; the lysate was scraped with a cell scraper before a centrifugation of 12, 000 g at 4℃ for 5 min; and the supernatants were harvested. The ATP and protein concentrations were measured with a microplate reader  and the ATP content was normalized by protein concentration.4 cells/well). After 7 days, each well of the plate received Seahorse XF Calibrant Solution . The sensor cartridge was soaked in Calibrant Solution at 37 °C overnight in a non-CO2 incubator and received the compounds existing in the Seahorse XF Cell Mito Stress Test Kit  one hour before the measurement according to the protocol. The sensor cartridge was then placed into the Seahorse XFe24 Flux Analyzer for automated calibration and subsequently replaced by the XFe24 culture plates for bioenergetics analysis.The mitochondrial respiration in intact cells was analyzed with a Seahorse XFe24 Extracellular Flux Analyzer . In brief, primary cortical neurons were placed into 24-well XFe24 culture plates (8 × 10+) equilibrium system and a water box. The long-range electrostatic interaction was analyzed by particle mesh Ewald (PME) with elastic simulation calculated respectively by Verlet and cg scheme, at a cut-off value of 1.2 nm. The value of 10Å was used for non-bonded interactions. Energy minimization at a maximum of 50,000 steps was performed by the steepest descent method. Both the van der Waals radius cutoff distance and Coulomb force cutoff distance were set at 1.4 nm. The entire system was equilibrated using canonical (NVT) and the isothermal—isobaric (NPT) ensembles. The MD simulations were carried out for NRP-1, Wnt1, β-catenin, GSK-3β, Axin, APC for 100 ns. During the MD simulation, hydrogen atom-involved bonds were constrained using the LINCS algorithm, with the integration time step set at 2 fs. The temperature was maintained at 300 K with the V-rescale thermostat and the pressure was semi-isotropically kept at 1 bar with a Berendsen barostat. NVT and NPT equilibrations were respectively carried out at 30 ps under 300 K.Briefly, TIP3P water model was selected to add the composite system, consisting of the sodium ion  and visualized with Pymol and analyzed with LigPlot+. The binding free energies for every complex were calculate with the equations below:VDW, ∆Einternal, ∆Eelec refer to, respectively, van der Waals, internal energy, and electrostatic energy. The solvation energy can be divided into the non-polar solvation free energy (GSA) and the electrostatic solvation free energy (GGB). Here, the ∆GGB was calculated by the GB model developed by Nguyen (igb = 8). The ∆GSA was estimated from the SASA using the Gmx_MMPBSA algorithm: ∆GSA = 0.0072 × ∆SASA [The models of NRP-1, Wnt1, β-catenin, GSK-3β, Axin, APC were acquired from the Uniport Protein Data Bank . The str × ∆SASA .p value of less than 0.05 was deemed as statistically different.Graphic presentation and statistical analysis were performed with GraphPad Prism v8.4.3 . Data were presented as mean ± standard error mean (SEM). The Shapiro-Wilk normality test and Brown-Forsythe test were respectively conducted to evaluate the normal distribution of the data and the homogeneity of variance. For data with normality and equal variance, one-way ANOVA with Bonferroni’s post hoc test was employed. Two-way repeated-measures analysis of variance (ANOVA) with Bonferroni’s post hoc test was applied to analyze the treatment effect on the behavior scores . A Western Blot was performed to examine, at different time points, the ischemic responses of NRP-1 in the cultured primary rat cerebral cortical neurons that received OGD/reoxygenation (OGD/R). The analyses indicated that in comparison with that of the Cont group, the protein level of NRP-1 increased after OGD/R, with a significant upregulation at 24 h after OGD/R , swollen and higher electron-dense mitochondria (Class II), and swollen and lower electron-dense mitochondria (Class III) Fig. A. The brThe OGD/R-induced impairment to the mitochondrial function was evaluated by analyzing the oxygen consumption rates (OCRs) of the cortical neurons with a Seahorse XFe24 analyzer Fig. D. At 24 To test the potential effect of NRP-1 on mitochondrial integrity after cerebral I/R injury, we examined Cyt c content in the cytosol and isolated mitochondria. In comparison with the Sham group, the tMCAO and tMCAO + AAV-empty groups reported a marked decrease in mitochondrial Cyt c levels but an increase in nuclear AIF and cytosolic Cyt c levels, indicating that ischemia/reperfusion induces a release of AIF and Cyt c from mitochondria. The overexpression of NRP-1 markedly diminished the AIF levels in the nuclei and Cyt c levels in the cytosol when in comparison with those of the tMCAO + AAV-empty group Fig. A–D. BesiTo explore whether NRP-1 can activate the Wnt/β-catenin signaling pathway, the small molecule inhibitor XAV-939  was employed to suppress the β-catenin-mediated transcription selectively. The expressions of Wnt1, Wnt3α, β-catenin, p-β-catenin Ser 37), C-Myc, Cyclin D1, cytosolic Cyt c, mitochondrial Cyt c, Bcl-2, and nuclear AIF were detected by Western Blot. The analyses showed that compared with the Sham group, the tMCAO group reported a marked decrease in Wnt1, β-catenin, C-Myc, Cyclin D1, mitochondrial Cyt c and Bcl-2, but an increase in cytosolic Cyt c, nuclear AIF and p-β-catenin (Ser 37). Compared with the tMCAO group, the NRP-1 treatment increased the levels of β-catenin, Wnt1, Cyclin D1, C-Myc, mitochondrial Cyt c, and Bcl-2, but decreased the levels of cytosolic Cyt c, nuclear AIF, and p-β-catenin (Ser 37). Interestingly, XAV-939 reversed these changes. Except for the Sham group, all the other groups reported a sharp decrease in Wnt3α  was performed to assess the underlying involvement of NRP-1 in mediating the Wnt/β-catenin signaling pathway. RMSD was adopted to observe the conformational changes of the system during the simulation process and to measure the stability of the system. β-catenin with APC, NRP-1 with Wnt1, and GSK-3β systems were stabilized in the initial simulation phase of 0–10 ns and maintained at 0.6 nm. Rg was employed as a proxy for the compactness of a structure. After initial brief fluctuations, all systems quickly entered a state of equilibrium. SASA was a parameter to measure the fraction of the protein surface interacting with the solvent molecules. The analyses revealed that the SASA of the protein decreased steadily in 0-100 ns, indicating a favorable binding of each system and a gradual protein fluctuation. From the HB change curve, the contact of the β-catenin with GSK-3β system ranged between 10 and 30 times, while that of the β-catenin with Axin system was 5–10 times Fig. A. After The difference in free energies between the two solvated proteins was calculated in the bound and unbound states, respectively, and the free energy binding of different solvated conformations of the same molecule was compared Table . The resThe present study demonstrated that NRP-1 was an important protective factor for the early I/R-induced injury to cerebral cortical neurons. It also showed that after I/R injury, the overexpression of NRP-1 significantly attenuated OGD/R-induced cytotoxicity in neurons and enhanced neurological functional recovery. In addition, the NRP-1 overexpression reduced neuronal apoptosis by downregulating the activation of caspase-3 and Bax and upregulating Bcl-2. Finally, the NRP-1 overexpression ameliorated mitochondrial dysfunction by suppressing mitochondrial bioenergetic deficits and oxidative stress, repairing mitochondrial morphology and ultrastructure, and maintaining mitochondrial integrity in the post-ischemic brain. This NRP-1 overexpression-conferred protection from cerebral I/R insult was accompanied by the up-regulation of Wnt and down-regulation of APC, GSK-3β, Axin, which inactivated the destruction complex, inhibited the phosphorylation of β-catenin at Ser37, and upregulated the β-catenin level in nucleus and cytoplasm. However, this protection was inhibited by β-catenin inhibitor XAV-939, All the findings evidence that after cerebral I/R injury, the NRP-1 overexpression activates the Wnt/β-catenin signaling pathway, signifying the neuronal-specific protective mechanism of the NRP-1/Wnt/β-catenin pathway in ischemic neurons.NRP-1 has been documented in neurons, T-cells, dendritic cells and migrating cancer cells –41. A prNeurological function and brain tissue are very sensitive to ischemia-reperfusion injury . After cApoptosis, a programmed cell death mechanism in neurons characterized by DNA fragmentation, is now recognized as an important factor in accelerating tissue injury and cell death after cerebral ischemia . MitochoOxidative stress is a typical pathological feature of cerebral I/R injury and has been strongly involved in the neuronal death . The strThe disruption of the mitochondrial structure is evident in the neurons following cerebral ischemia . When ceI/R injury results in the loss of mitochondrial integrity and subsequent apoptosis . HypoxiaThe Wnt/β-catenin signaling pathway is crucial for the regulation of various cellular events, including preventing cell apoptosis , 64. RecAvailable studies report that when Wnt is upregulated, the Wnt/β-catenin signaling pathway is activated, inhibiting the phosphorylation of β-catenin . The staNext, we further verified the protective effect of NRP-1 on neuronal cells by activating the Wnt/β-catenin signaling pathway at the cellular level. Firstly, XAV-939 was used to probe into the mechanisms underlying the Wnt/β-catenin signaling pathway-related effects. Our findings indicated that the NRP-1 overexpression promoted the expression of Wnt1, signifying that NRP-1 protects neurons after OGD/R by activating the Wnt/β-catenin signaling pathway. Next, we analyzed the expression of the degradation complex to investigate the molecular mechanism in NRP-1-promoted neuronal viability after OGD injury. The initiation of the canonical Wnt pathway causes dissociation of the β-catenin degradation complex (Axin/APC/GSK-3β). Then, the phosphorylation of β-catenin by GSK-3β at Thr41, Ser33, and Ser37 resulted in the aggregation of β-catenin in the cytoplasm and eventually in the nucleus , 76. A rFurthermore, the results of MD simulation and molecular docking illustrated a tight and stable integration of NRP-1 with Wnt1 and that of β-catenin with GSK-3β, Axin, and APC. Besides, the calculation of free energy revealed that the binding affinity of NRP-1 and β-catenin relies on H-bond and hydrophobic interaction, confirming the reliability of the docking model. Consequently, NRP-1 promotes the depolymerization of APC, Axin, and GSK-3β multimers through activating Wnt1 and increases the cellular deposition of β-catenin.In conclusion, our study demonstrates that the overexpression of NRP-1 ameliorates neurological function in cerebral I/R-injured rats by promoting mitochondrial structural repair and functional recovery, in which NRP-1-conferred neuroprotection is mediated by the Wnt/β-catenin signaling pathway. These findings signify that NRP-1 may act as a promising candidate in treating cerebral I/R injury.


Moreover, HFNP@GOX@PFC also significantly initiates antitumor immune response via re-educating tumor-associated macrophages (TAMs) by activating NF-κB and MAPK signal pathways. In vitro and in vivo results collectively demonstrate that nanosystem not only continuously initiates starvation therapy, but also pronouncedly cascade-amplify CDT and polarize TAMs, consequently efficiently inhibiting tumor growth with good biosafety. The functional nanosystem combined the cascade amplification of starvation and CDT provides a new nanoplatform for tumor therapy.Tumor microenvironment is characterized by the high concentration of reactive oxygen species (ROS), which is an effective key used to open the Pandora’s Box against cancer. Herein, a tumor-targeted nanosystem HFNP@GOX@PFC composed of ROS-cleaved Fe-based metal–organic framework, hyaluronic acid (HA), glucose oxidase (GOX) and perfluorohexane (PFC) has been developed for tumor cascade amplified starvation and chemodynamic therapy (CDT). In response to the high concentration of hydrogen peroxide (HThe online version contains supplementary material available at 10.1186/s12951-023-01878-3. Hence, 2+ with carboxyl groups from ROS-cleaved linker TK using hydrothermal reaction. In order to improve biocompatibility and tumor targeting capacity, HA coat-layer was introduced into the surface of FNP nanoparticles, which was used to prolong the circulation in blood vessels and impart tumor targeting feature to HFNP nanosystem. Transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurement, X-ray diffraction (XRD), Fourier Transform Infrared Spectrometer (FTIR), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (NMR), ultraviolet and visible spectrophotometry (UV–vis) were employed to monitor the functionalization of HFNP nanosystem. Firstly, both FNP and HFNP exhibited the distinctively spherical morphology with good mono-dispersity detected by TEM  were synthesized by conjugation FeTEM Fig. : Fig. S12O2 treatment had no obvious GOX release, proved by the negligible absorption intensity of GOX at 276 nm, indicating the successful GOX loading in the nanosystem. Furthermore, the loading content and encapsulation efficiency of GOX in HFNP were calculated as 2.42% and 82.5% based on the associating standard curve . As shown in Fig. 2O2 and cascade amplification of CDT. Firstly, TEM, DLS and UV–vis spectrophotometry were used to monitor the ROS-responsive Fe-MOF disassembly & drug release traits. As demonstrated in Additional file 2O2  upon 48 h, indicating the good encapsulation, stability and potential biocompatibility of HFNP@GOX@PFC nanosystem (owing to no leakage during the blood circulation). In contrast, the release amount of Fe2+ was dramatically increased in the presence of H2O2, showing the H2O2 concentration-dependence drug release pattern , suggesting that HFNP@GOX@PFC nanosystem actually possesses the ROS-responsive drug release capability. At the same time, GOX also showed H2O2 concentration-dependent release behavior, and the release amount reached 69% and 91%  in 0.1 mM H2O2 and 1 mM H2O2 treatment groups, while the release amount of GOX was negligible in control group as well  and negative HFNP groups, confirming the self-generation of H2O2. Simultaneously, HFNP@GOX@PFC nanosystem could generate gluconic acid via GOX-mediated catalytic reaction, as revealed by the reduction of pH value  for 24 h, HC11 cells showed good cell viability similar to that of the control , and the IC50 was 59.18 × 10–6 M. It was suggested that the HFNP@GOX@PFC nanosystem possesses the superior cytotoxicity against tumor cells, and the friendly cell viability for normal cells. The reasons may be attributed to the HA-endowed actively targeting endocytosis and the cascade activation of the CDT combined starvation therapy in vitro, leading to the effective tumor killing.The cytotoxicity of HFNP@GOX@PFC nanosystem against the HC11 normal breast cells and 4T1 breast cancer cells were both detected by CCK-8 assay. After treatment with a series of concentrations of HFNP@GOX@PFC , free PFC treatment displayed no significant cytotoxicity Fig. g, indicareaction , 22. MorIn order to reveal the active targeting endocytosis of HFNP@GOX@PFC nanosystem with high efficiency above mentioned, GOX conjugated with FITC were first loaded into the nanosystem. Subsequently, 4T1 cells were treated with FNP@GOX@PFC and HFNP@GOX@PFC for 12 h, and the cellular uptake degree of nanosystem was distinctly observed by CLSM. As shown in Fig. 2 supply and CDT amplification were accompanied by ROS generation and hypoxia suppression, which were respectively investigated in the following study. For one thing, 4T1 cells were treated with administrations and the ROS generation level was monitored by FCM using the typical ROS probe of DCFH-DA. As shown in Fig. 2+ diacetic acid were respectively purchased from Sigma-Aldrich  and Ruixi Bio-Technology Co., Ltd . Hyaluronic acid  was offered by Shandong Freda Biochem Co., Ltd . Perfluorohexane (PFC), N-hydroxy succinimide (NHS), 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC) and Glucose oxidase (GOX) were purchased from Macklin Biochem-Technology  and Yuanye Bio-Technology Co., Ltd . Interleukin-6 (IL-6), Interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd . Anti-CD11b, anti-F4/80 and anti-CD86 were purchased from BioLegend Co., Ltd . All the reagents were analytical grade.Ferrous acetate (FeAc2·4H2O (1 mmol) and 2,2'-diacetic acid (1 mmol) were dissolved in 15 mL N, N-Dimethylformamide (DMF) in presence of acetic acid (1 mL), the mixture solution was then placed into a Teflon-liner steel autoclave at 65 °C for 2 h. Next, the obtained sediment was collected by centrifugation at 5000 rpm min−1 for 10 min. The sediment was added to 100 mL ultrapure water overnight, and the blank Fe-MOF (named FNP) was harvested via centrifugation.Firstly, FeAcSubsequently, FNP (20 mg), EDC (115 mg) and NHS (69 mg) were dissolved in 10 mL of PBS (pH 6.0). After stirring for 1.5 h, ethylene diamine (1.67 mL) was added into above mixture at room temperature and continued for 24 h. The functionalized FNP was then collected and mixed with GOX (1.5 mg) and PFC (500 μL) in PBS (10 mL). Above mixture was stirred overnight at room temperature. For one thing, above solution was centrifuged and obtained the drug loaded FNP without HA modification (donated FNP@GOX@PFC). For another thing, 60 mg of HA, 230 mg of EDC and 138 mg of NHS were dissolved in 10 mL of PBS and stirred for 1.5 h. Next, 30 mg of FNP@GOX@PFC was added into above solution and stirred for another 24 h. The final product was obtained by centrifugation and dried by lyophilization, donated as HFNP@GOX@PFC. Notably, the blank nanosystem without drug loading was donated as HFNP.Meanwhile, the drug loading content (DLC) and efficiency (DLE) of GOX in HFNP@GOX@PFC nanosystem were monitored by the ultraviolet and visible spectrophotometry (UV–vis) at 276 nm and the corresponding standard curve, according to previous literature . BesidesThe size and zeta potential of nanoparticles are measured by dynamic light scattering (DLS). The morphology and structure of FNP and HFNP nanosystem were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD), respectively. The conjugation and functionalization processes of HFNP nanosystem were analyzed by Fourier Transform Infrared Spectrometer (FTIR). The drug release behavior of HFNP@GOX@PFC nanosystem was characterized by UV–vis spectrophotometry.−1) was firstly co-cultured with 10% FBS in PBS solution at 37 °C for various time intervals , DLS was used to detect its size and potential change for evaluation the stability of HFNP@GOX@PFC in the short term. As for long-term stability, HFNP@GOX@PFC nanosystem (1 mg mL−1) were cultured in PBS solution at 4 °C for various time intervals , DLS was used to monitor the stability of nanosystem using above same method.Typically, HFNP@GOX@PFC nanosystem  and transferred to dialysis bag (MWCO = 14 kDa). Then, the dialysis bag was immersed in 29 mL PBS solution under gently stirring, 0.8 mL of PBS was taken out at the certain time points  and supplemented with the same amount of fresh PBS containing different doses of H2O2. The release content of GOX and Fe2+ were respectively monitored by the analyzed UV–vis at 276 nm and 506 nm using o-phenanthroline method [HFNP@GOX@PFC nanoparticles (1 mg) were dispersed into 5 mL PBS solution containing different concentrations of He method .2O2 from HFNP@GOX@PFC nanosystem, PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC were respectively added into 8 mL PBS containing glucose (1 mg mL−1). The mixture was then incubated at room temperature under gentle stirring, 0.8 mL of PBS was taken out at 2 h and detected by UV–vis spectrophotometry for characterizing the generation of H2O2 at 240 nm. Besides, the samples harvested from HFNP@GOX@PFC treatment at the certain time points  were also monitored by the UV–vis spectrophotometry for investigating the time-dependent H2O2 generation.In order to detect the self-generation of H−1) were respectively mixed with 0.1 mM H2O2 solution. The above mixture was then stirred under room temperature for 2 h. Next, the samples were detected by UV–vis spectrophotometry at 550 nm using Hydroxyl Free Radical assay kit (Beorebo), according to the manufacturer's protocols.For the self-amplification generation of ·OH detection, PBS (1 mL), HFNP (1 mg), HFNP@GOX (1 mg), HFNP@GOX@PFC (1 mg) and HFNP@GOX@PFC (1 mg) plus glucose (1 mg mL−1)/streptomycin (100 μg mL−1) with 5% CO2 at 37 °C.4T1 breast cancer cells, HC11 mammary gland cells and RAW264.7 macrophages are supplied by Cell Bank of Chinese Academy of Sciences . Cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum  and 1% (w/v) penicillin (100 U mL4 cells well−1) were respectively treated with HFNP@GOX@PFC at different doses  for 24 h. The medium was replaced by the mixture of fresh medium (200 μL) and CCK-8 (20 μL) and incubated for another 2 h. Finally, the supernatant was translated to 96-well plates and the absorbance was recorded at 450 nm by spectrophotometric microplate reader . IC50 was calculated according to the cytotoxicity result.As for the dose-dependent cytotoxicity, 4T1 cells and normal HC11 cells seeded on the 24-well plates (1 × 10−1), HFNP@GOX  and HFNP@GOX@PFC  for 24 h, 48 h and 72 h, respectively. CCK-8 kit was used to determine the corresponding cell viability, according to the above method.As for the time-dependent cytotoxicity, 4T1 cells were treated with PBS, GOX (1.5 μM), PFC (4.85 μM), HFNP (3.3 mg mL5 cells well−1) inoculated in 6-well plates were co-cultured with FNP@GOX@PFC and HFNP@GOX@PFC for 12 h, respectively. Notably, GOX loaded into the above nanosystem was pre-labeled with FITC before drug loading, thus exhibiting green fluorescence upon laser irradiation. Then, the cell was washed, fixed and permeabilized, the nuclei were stained with DAPI and the cytoskeletons were stained with Tetramethyl Rhodamine Isothiocyanate (TRITC). Finally, these cells were imaged by confocal laser scanning microscopy (CLSM) to observe the tumor targeting endocytosis behavior and the cellular distribution of the nanosystem.In order to visualize the tumor targeting endocytosis of 4T1 cells against nanosystem, 4T1 cells (1 × 105 cells well−1) were inoculated in 6-well plates and treated with PBS, HFNP (3.3 mg mL−1), HFNP@GOX (3.4 mg mL−1) and HFNP@GOX@PFC (5.06 mg mL−1) for 24 h. DCFH-DA probes were used to mark the generation of intracellular ROS, which was imaged by CLSM and quantitively analyzed by Image J software. The detailed procedures were referred to previous literature [Briefly, 4T1 cells (1 × 10terature .5 cells well−1) inoculated in 6-well plates were treated with PBS, HFNP (3.3 mg mL−1), HFNP@GOX (3.4 mg mL−1) and HFNP@GOX@PFC (5.06 mg mL−1) for 24 h. The cells were then washed with PBS, collected by centrifugation, followed by staining with CD47 (hypoxia mark) antibody labelled with PE. Finally, the cells were monitored by FCM.For the hypoxia suppression study, 4T1 cells  inoculated in 6-well plates were treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. The cells were then washed with PBS twice, collected by centrifugation, followed by staining with Annexin V-FITC/PI kit, the detailed procedures could refer to our previous literature [As for the apoptosis assay, 4T1 cells , according to the manufacturer's protocols.5 cells well−1) inoculated in 6-well plates were incubated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. Subsequently, cells were lysed, collected and determined the total protein concentration using BCA kit. The expression of p53 protein was measured using Western blot and Image J software. Besides, 4T1 cells treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h were also collected and analyzed the expression of HIF-1α, Glut-1 and CD47 protein using above method.4T1 cells  inoculated in 6-well plates were incubated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC, respectively. After 24 h of incubation, the total RNA was extracted with Trizol reagent, and then reverse transcription using the reverse transcription Kit (TransGen Biotech). The qPCR was carried out for analysis of the mRNA level of BAX, BCL-2, Caspase-3 (Casp3) and Cytochrome C (CytC) according to previous literature [4T1 cells  were inoculated in 6-well plates. After the cell density reached 70–80%, a p200 pipette tip was used to produce a scratching wound. Next, the cell medium was removed and added above collected Solution 1 for 24 h co-incubation. The wound healing levels were finally imaged by a microscope .As for the wound healing assay, 4T1 cells (1 × 105 cells well−1) inoculated in 6-well plates were firstly treated with Solution 1 for 24 h. On the one hand, 4T1 cells (5 × 104 cells) harvested from above system were then seeded in the upper chamber of the Transwell and cultured with serum-free medium, 500 μL of the complete medium was added into the lower chambers. On the other hand, 4T1 cells harvested from above system were seeded in the upper chamber of Transwell pre-coated with Matrigel (BD Biosciences), the medium was chosen as the serum-free medium, and 500 μL of the complete medium was added into the lower chambers. After treatment for 48 h, the cells on the upper station of the chamber were wiped, while cells on the lower surface came from above two systems were stained with crystal violet and observed by microscope to evaluate the migration and invasion level of tumor cells. The detailed experiment procedures were referred to a previous study [As for migration and invasion assays, 4T1 cells (1 × 10us study .6) into the right flank groin of the Balb/c mice. After the tumor size reached about 50 mm3, tumor-bearing mice were randomly divided into 4 groups (n = 6) and intravenously injected with Saline, HFNP, HFNP@GOX and HFNP@GOX@PFC with an equal amount of Fe2+ (60 μmol kg−1), respectively. The above treatments were repeated 3 times per week. The body weight and tumor volume were recorded every 2 days. The calculation formula of tumor volume was as follows: tumor volume = ab2/2 . The mice were euthanized after 18 days, blood was collected for analyzing the biochemical blood index. The survival rate of mice was monitored for another 18 days after the last administration.All animal experiments were strictly conducted in accordance with the Institutional guidelines for animal experimentation and were approved by the Animal Ethics Committee of the Northwestern Polytechnical University. BALB/c mice (5–6 weeks old) were bought from Beijing Institution for Drug Control, China. The model of tumor-bearing mice was constructed by subcutaneous injection of 4T1 cells  and imaged by CLSM. As for the immunofluorescence study, tumor sections were blocked with 5% bovine serum albumin, incubated with CD47, CD86 or Ki67 antibodies, and then stained with Alexa Fluor 488-labeled IgG according to the manufacturer's instructions . Sections were then stained with DAPI and imaged by CLSM.We performed the statistical analysis with the software of Origin (version 9.0) through Student’s t-test and one-way analysis of variance (ANOVA). All data were expressed as means ± standard deviation (SD). P values of * P < 0.05, ** P < 0.01 and *** P < 0.001, respectively. The confidence levels of 95% and 99% were regarded as a significant difference.Additional file 1: Fig. S1. Surface Zeta potential of FNP and HFNP. Fig. S2. XRD of FNP and HFNP. Fig. S3. H-NMR of FNP and HFNP. Fig. S4. FTIR of FNP and HFNP. Fig. S5. Linear relationships between the UV-vis absorbance and the concentration of GOX. Fig. S6. Efflux time of pure PFC and HFNP@GOX@PFC nanoparticles. Fig. S7. Linear relationships between the GC intensity and the concentration of PFC. Fig. S8. TEM images of FNP and HFNP after treatment with H2O2. Fig. S9. DLS curves of FNP and HFNP after treatment with H2O2. Fig. S10. pH values and generated H2O2 concentrations at various time points arisen from GOX and HFNP@GOX@PFC catalyzed disintegration reaction of glucose. Fig. S11. Biostability of HFNP@GOX@PFC in gluconic acid with or without H2O2. Fig. S12. Intracellular ROS fluorescence intensity of 4T1 cells. Fig. S13. Corresponding quantitative MFI analysis of CD47. Fig. S14. Bcl-2, BAX, CytC and Casp3 relative mRNA expressions. Fig. S15. Gate strategy. Fig. S16. Wound healing rate of 4T1 cells. Fig. S17. Migration rate of 4T1 cells. Fig. S18. Invasion rate of 4T1 cells. Fig. S19. Body weight of tumor-bearing mice. Fig. S20. The quantitative IFC intensity analysis of CD47. Fig. S21. The quantitative IFC intensity analysis of CD86. Fig. S22. Biomedical blood index of tumor-bearing BALB/c mice. Fig. S23. H&E staining for major tissues. Table S1. Primer sequences.


Nanocomposite hydrogels offer remarkable potential for applications in bone tissue engineering. They are synthesized through the chemical or physical crosslinking of polymers and nanomaterials, allowing for the enhancement of their behaviour by modifying the properties and compositions of the nanomaterials involved. However, their mechanical properties require further enhancement to meet the demands of bone tissue engineering. Here, we present an approach to improve the mechanical properties of nanocomposite hydrogels by incorporating polymer grafted silica nanoparticles into a double network inspired hydrogel (gSNP Gels). The gSNP Gels were synthesised via a graft polymerization process using a redox initiator. gSNP Gels were formed by grafting 2-acrylamido-2-methylpropanesulfonic acid (AMPS) as the first network gel followed by a sequential second network acrylamide (AAm) onto amine functionalized silica nanoparticles (ASNPs). We utilized glucose oxidase (GOx) to create an oxygen-free atmosphere during polymerization, resulting in higher polymer conversion compared to argon degassing. The gSNP Gels showed excellent compressive strengths of 13.9 ± 5.5 MPa, a strain of 69.6 ± 6.4%, and a water content of 63.4% ± 1.8. The synthesis technique demonstrates a promising approach to enhance the mechanical properties of hydrogels, which can have significant implications for bone tissue engineering and other soft tissue applications. Hydrogels are hydrophilic three-dimensional polymeric networks that hold large amounts of water (up to 99%), giving them their intrinsically soft material properties. They hold numerous properties including flexibility, transparency, permeability, biocompatibility, and low friction ,2,3. HydHydrogel technology has evolved beyond its initial single network systems, driven by the inherent physical and biochemical limitations they possess . This prDNHGs are normally synthesized using a two-step sequential free-radical polymerisation (FRP) where a high relative molecular weight neutral second polymer network is swollen within a lightly cross linked heterogeneous first network polyelectrolyte . DNHGs hN-isopropylacrylamide) (PNIPAAm) with polysiloxane nanoparticles [The introduction of nanocomposite structures such as silica nanoparticles (SNPs) ,15, copparticles . This ex3+ and Ce4+) and reaction by-product H2O2.However, nanocomposite gel structures are challenging to produce due to particle agglomeration, inhomogeneous distribution during synthesis, and a lack of covalent or ionic bonds between particles and the polymer networks ,36,37,38In this work, a method for synthesizing a double network inspired nanocomposite hydrogel is presented. The method involves using a redox initiator, ceric ammonium nitrate, in combination with glucose oxidase (GOx), an oxidoreductase enzyme that can quench oxygen in an open system, eliminating all oxygen inhibition steps that impact FRP and increase the conversion of the polymer . The redASNPs with 100 nm diameter were successfully synthesized by post synthesis functionalization of SNPs using APTES; a schematic representation of this is shown in 1H NMR analysis as described in the Both AMPS and AAm conversion profiles were investigated using samples that were degassed using either argon or GOx. The monomer and polymer peaks were calculated relative to trioxane peaks at 0 h and 24 h samples in order to calculate the final conversion profiles, using 2O2, a GOx by-product, results in free radical formation which impacts the final polymer conversion can be seen by comparing argon and GOx degassed samples of AAm/ASNP. Argon degassed samples resulted in 1% conversion, whereas samples degassed with GOx had 58% conversion. This can occur during the formation of H2O2 and its subsequent in situ degradation back to water where peroxy radicals may form, potentially initiating the polymerization [2O2 plays a part in initiating the polymerisation to a limited extent. This was also evident for AMPS/ASNP in An indication that Hrization ,48. TherIn the experiments, it was observed that samples degassed with argon resulted in inhomogeneous, runny, and wet gel structures. On the other hand, samples degassed with GOx showed textural consistency and more gel-like forms. These results indicate that argon alone would not be sufficient for synthesising the grafted nanocomposite hydrogels, while GOx can be used for this purpose; therefore, only GOx was used in the next part of the study.2O2 by-products [Thermal analysis was performed on dried and ground samples to investigate polymer grafting onto amine silica nanoparticles (ASNP). The mass loss profiles (TGA) in products ,19,50. O2O2 by-products resulting in free radicals on the amine groups of ASNPs. The presence of SDS in the sample suggested successful polymer grafting, but incomplete removal of SDS from the sample even after several washes , tetraethyl orthosilcate , triethoxysilane , deuterium oxide , cerium (IV) ammonium nitrate , sodium dodecyl sulphate . No additional processing and/or purifications were performed. D-glucose (G) and glucose oxidase  were purchased from Sigma-Aldrich  and stored in phosphate buffer saline (PBS) aliquots at −20 °C when received.Reagents were purchased from Sigma-Aldrich  and used as received, unless stated otherwise: acrylamide  and 2-acrylamido-2-methyl-1-propanesulfonic acid ; N, N’-methylenebisacrylamide ; photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone , 1, 3, 5 –trioxane (≥99%); (3-aminopropyl), ammonium hydroxide  synthesis, the procedure for polymer conversion studies of argon degassing and Gox, and the characterisation techniques used in this work can be found in the 2O and sonicating the mixture until it was fully dispersed. Then 4.5 g AMPS was titrated to 5.4 pH using NaOH (0.25 M), to a final concentration of 0.24 M AMPS. ASNPs were then added to the AMPS monomer solution. Then 1 wt. % MBIS and 200 nM GOx/G [The first network was formed by dispersing 150 mg of ASNPs in 5 mL of HnM GOx/G ,47 were nM GOx/G ,67; and nM GOx/G . Once thThe hydrogels were removed from the moulds and soaked in a monomer solution of 2.54 M AAm containing 150 mg ASNP, 0.1 wt. % MBIS, 0.7 g CAN, and 200 nM GOx/G. Once the hydrogels were swollen, they were placed in moulds and put into a 40 °C oven to form the second network. The final hydrogels are referred to as gSNP gels. The gSNP gels were then dried at 60 °C and, at their dry state, placed in water to swell for a week. Each sample is named based on the chemicals used in its polymerization. For instance, if AMPS is polymerized using CAN and SDS on the surface of ASNPs, the sample will be named as AMPS/ASNP/CAN/SDS. If AAm is polymerized on ASNPs without any initiator or stabilizer, the sample will be named as AAm/ASNP.