Cancer immunotherapy currently represents the only promising strategy with the potential to achieve a complete cure for tumors. Enhancing tumor-specific immune responses and overcoming the immunosuppressive TME are key to improving the efficacy of cancer immunotherapy. In this study, we introduce a calcium carbonate-based nanocarrier capable of modulating the tumor microenvironment. This system is coloaded with L-arginine (L-Arg) and the chemotherapeutic agent doxorubicin (DOX), aiming to optimize the conventional chemoimmunotherapy approach by ameliorating the immunosuppressive tumor microenvironment and regulating immune cell activity, thereby promoting synergistic chemoimmunotherapeutic outcomes. Specifically, DOX directly kills tumor cells and induces immunogenic cell death. Moreover, the calcium carbonate nanocarrier not only serves as a drug delivery vehicle but also neutralizes lactic acid in the tumor tissue, thereby alleviating the acidic immunosuppressive microenvironment. Furthermore, it supplies supplemental arginine within the tumor tissue to increase the activation of effector T cells. Ultimately, this strategy achieves highly efficient antitumor efficacy through the combined and synergistic actions of chemotherapy and immunotherapy.

Hypochondroplasia (HCH) is a systemic cartilage disorder caused by gain-of-function mutations in FGFR3, resulting in overactivation of signaling and short stature. Pharmacological therapy remains the primary treatment but provides only partial symptomatic relief, as the avascular and alymphatic nature of growth plate cartilage severely limits drug delivery and accumulation. To address this challenge, we developed CT-CM-NPs, a drug delivery system coated with primary chondrocyte membranes and further modified with the collagen II-binding peptide WYRGRL. This design retains the inherent biological properties of native membranes while incorporating targeting capability, enabling efficient cartilage targeting and penetration. In vivo, CT-CM-NPs effectively delivered the Hedgehog pathway agonist Purmorphamine to the growth plate cartilage in an HCH mouse model, resulting in upregulated Hedgehog signaling, restored impaired ciliogenesis, improved chondrocyte phenotypes, and ultimately increased bone and body length. Collectively, these findings establish CT-CM-NPs as a promising and potentially translatable nanoplatform for the treatment of HCH.

Background and Aims Enhanced inflammatory signalling causally contributes to atrial fibrillation (AF) development. Gasdermin D (GSDMD) is an important downstream effector of several inflammasome pathways. However, the role of GSDMD, particularly the cleaved N-terminal (NT)-GSDMD, in non-immune cells remains elusive. This study aimed to elucidate the function of NT-GSDMD in atrial cardiomyocytes (ACMs) and determine its contribution to atrial arrhythmogenesis.Methods Human atrial appendages were used to assess the protein levels and localization. A modified adeno-associated virus 9 was employed to establish ACM-restricted overexpression of NT-GSDMD in mice.Results The cleavage of GSDMD was enhanced in ACMs of AF patients. Atrial cardiomyocyte-restricted overexpression of NT-GSDMD in mice increased susceptibility to pacing-induced AF. The NT-GSDMD pore formation facilitated interleukin-1 beta secretion from ACMs, promoting macrophage infiltration, while up-regulating 'endosomal sorting complexes required for transport'-mediated membrane-repair mechanisms, which prevented inflammatory cell death (pyroptosis) in ACMs. Up-regulated NT-GSDMD directly targeted mitochondria, increasing mitochondrial reactive oxygen species (ROS) generation, which triggered proarrhythmic calcium-release events. The NT-GSDMD-induced arrhythmogenesis was mitigated by the mitochondrial-specific antioxidant MitoTEMPO. A mutant NT-GSDMD lacking pore-formation capability failed to cause mitochondrial dysfunction or induce atrial arrhythmia. Genetic ablation of Gsdmd prevented spontaneous AF development in a mouse model.Conclusions These findings establish a unique pyroptosis-independent role of NT-GSDMD in ACMs and arrhythmogenesis, which involves ROS-driven mitochondrial dysfunction. Mitochondrial-targeted therapy, either by reducing ROS production or inhibition of GSDMD, prevents AF inducibility, positioning GSDMD as a novel therapeutic target for AF prevention.

INTRODUCTION:Lung cancer progression involves complex interactions between metabolic pathways and the immune microenvironment. The role of serotonin, a tryptophan-derived metabolite, in immune responses to lung tumors remains unclear. METHODS:An orthotopic lung cancer model was established by intravenously injecting KP (KrasG12D/p53-/-) cells into C57BL/6 mice. Metabolomic and flux analyses were conducted on tumor versus normal lung tissues. Serotonin was administered to tumor-bearing mice, followed by immunofluorescence and flow cytometry to assess immune responses. Human lung cancer datasets were analyzed to validate clinical relevance. RESULTS:Tumor tissues exhibited a significant decrease in serotonin levels. Although tryptophan, serotonin, and kynurenine levels were decreased overall, flux analysis revealed a metabolic shift favoring kynurenine synthesis, with a ~10-fold increase in the kynurenine-to-serotonin ratio. Serotonin supplementation significantly prolonged survival and enhanced dendritic cell and CD8⁺ T cell infiltration and activation in tumors. Analysis of public datasets showed that serotonin expression positively correlated with CD8⁺ T cell activation signatures and patient prognosis. DISCUSSION:By revealing serotonin as a potential biomarker and therapeutic target, this study paves new avenues for improving lung cancer treatment strategies through modulation of the immune microenvironment. Moreover, the precise receptor-mediated mechanisms underlying serotonin's immunomodulatory effects remain to be clarified, and translational validation in human tissues is warranted to strengthen clinical relevance. CONCLUSION:Serotonin deficiency in the tumor microenvironment of the lung suppresses antitumor immunity. Its restoration reverses immune dysfunction and limits tumor progression. These findings identify serotonin as a potential metabolic regulator and immunotherapeutic target in lung cancer.

Bone metastases caused by breast cancer pose a major challenge to the successful treatment of breast cancer patients. Many researchers have suggested that herbal medicines are extremely effective at preventing and treating cancer-associated osteolysis. Previous studies have revealed that Morusin (MOR) is cytotoxic to many cancer cells ex vivo. Nevertheless, how MOR contributes to osteolysis induced by breast cancer is still unknown, and the potential mechanism of action against osteolysis is worthy of further study. The protective effect and molecular mechanism of MOR in inhibiting breast cancer cell-induced osteolysis were verified by experiments and network pharmacology. Cell function was assessed by cell proliferation, osteoclast (OC) formation, bone resorption, and phalloidin staining. Tumour growth was examined by micro-CT scanning in vivo. To identify potential MOR treatments, the active ingredient-target pathway of breast cancer was screened using network pharmacology and molecular docking approaches. This study is the first to report that MOR can prevent osteolysis induced by breast cancer cells. Specifically, our results revealed that MOR inhibits RANKL-induced osteoclastogenesis and restrains the proliferation, invasion and migration of MDA-MB-231 breast cells through restraining the PI3K/AKT/MTOR signalling pathway. Notably, MOR prevented bone loss caused by breast cancer cell-induced osteolysis in vivo, indicating that MOR inhibited the development of OCs and the resorption of bone, which are essential for cancer cell-associated bone distraction. This study showed that MOR treatment inhibited osteolysis induced by breast cancer in vivo. MOR inhibited OC differentiation and bone resorption ex vivo and in vivo and might be a potential drug candidate for treating breast cancer-induced osteolysis.

BackgroundRecently, the osteogenic potential of Adiponectin-labeled adipogenic lineage progenitors (Adipoq-lineage progenitors) in bone marrow has been observed to support bone maintenance and repair. However, little is known about the function of Schnurri-3 (SHN3, also known as HIVEP3) in other mesenchymal lineage cells, apart from its negative regulation of bone formation on osteoblasts.MethodIn this study, we used single-cell RNA sequencing (scRNA-seq) profiling to demonstrate that Adipoq-lineage progenitors express higher levels of Shn3 compared to other mesenchymal cell populations in mice and humans. To investigate the role of SHN3 in Adipoq-lineage progenitors, we generated a murine model specifically harboring a Shn3-deficient allele in Adipoq-expressing cells. Information of mice body weight was collected weekly to generate body weight curve. Bone phenotype was analyzed using micro-CT and histomorphometric studies. To eliminate the role of peripheral adipose tissue on bone, we collected adipose wet weight, performed intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests, and conducted a fat-transplantation study. Osteoblast and osteoclast functions were assessed through toluidine blue staining and TRAP staining, respectively. We further investigated the effect of Shn3 depletion on the differentiation of Adipoq-lineage progenitors through immunostaining and in vitro differentiation assays. Finally, we evaluated whether Shn3 deficiency in Adipoq-lineage progenitors affects the fracture healing process by generating bi-cortical femoral fracture models.ResultsDepletion of Shn3 in Adipoq-lineage progenitors resulted in a significant increase in trabecular bone mass and bone formation in vivo, without disrupting whole-body energy metabolism and skeletal development. Consistent with these findings, both cell-lineage tracing and functional assays revealed that Shn3 ablation effectively shifted the cell fate of Adipoq-lineage progenitors towards an osteogenic phenotype in the bone marrow. Furthermore, in vivo studies demonstrated that the lack of Shn3 in Adipoq-lineage progenitors also enhanced bone fracture healing under pathological conditions.ConclusionOverall, our findings provide a novel strategy for targeting the osteoanabolic potential of bone marrow Adipoq-lineage progenitors as a potential treatment for bone loss-related disorders.Translational potential of this articleWe have identified a novel gene target that directs the cell fate of a previously identified non-osteogenic cell population under physiological conditions. This study not only expands the therapeutic value of Shn3 ablation in treating osteoporotic or traumatic bone diseases but also provides new insights into the contribution of bone marrow Adipoq-lineage progenitors to osteogenesis. Thus, this article further supports Shn3 silencing as a valuable approach to treat osteopenia and accelerate fracture healing (see graphical abstract)

The cure rate for patients with osteosarcoma (OS) has stagnated over the past few decades. Penfluridol, a first-generation antipsychotic, has demonstrated to prevent lung and esophageal malignancies from proliferation and metastasis. However, the effect of penfluridol on OS and its underlying molecular mechanism remains unclear. This study revealed that penfluridol effectively inhibited cell proliferation and migration, and induced G2/M phase arrest in OS cells. In addition, penfluridol treatment was found to increased reactive oxygen species (ROS) levels in OS cells. Combined with the RNA-Seq results, the anti-OS effect of penfluridol was hypothesized to be attributed to the induction of ferroptosis. Western blot results showed that penfluridol promoted intracellular Fe2+ concentration, membrane lipid peroxidation, and decreased intracellular GSH level to induce ferroptosis. Further studies showed that p62/Keap1/Nrf2 signaling pathway was implicated in penfluridol-induced ferroptosis in OS cells. Overexpression of p62 effectively reversed penfluridol-induced ferroptosis. In vivo, penfluridol effectively inhibited proliferation and prolonged survival in xenograft tumor model. Therefore, penfluridol is a promising drug targeting OS in the future.

Phosphatidylinositol 3-kinase (PI3K) is frequently hyperactivated in cancer, playing pivotal roles in the pathophysiology of both malignant and immune cells. The impact of PI3K inhibitors on the tumor microenvironment (TME) within lung cancer remains largely unknown. In this study, we explored the regulatory effects of GNE-493, an innovative dual inhibitor of PI3K and mammalian target of rapamycin (mTOR), on the TME of lung cancer. First, through the analysis of The Cancer Genome Atlas-lung squamous cell carcinoma (LUSC) cohort, we found PIK3CA to be related to CD8 T cells, which may affect the overall survival rate of patients by affecting CD8 function. We herein demonstrated that GNE-493 can significantly inhibit tumor cell proliferation and promote cell apoptosis while increasing the expression of the immunogenic death-related molecules CRT and HSP70 using in vitro cell proliferation and apoptosis experiments on the murine KP lung cancer cell line and human A549 lung cancer cell line. Next, through the establishment of an orthotopic tumor model in vivo, it was found that after GNE-493 intervention, the infiltration of CD4+ and CD8+ T cells in mouse lung tumor was significantly increased, and the expression of CRT in tumors could be induced to increase. To explore the mechanisms underlying PI3K inhibition-induced changes in the TME, the gene expression differences of T cells in the control group versus GNE-493-treated KP tumors were analyzed by RNA-seq, and the main effector pathway of anti -tumor immunity was identified. The IFN/TNF family molecules were significantly upregulated after GNE-493 treatment. In summary, our findings indicate that GNE-493 promotes immunogenic cell death in lung cancer cells, and elucidates its regulatory impact on molecules associated with the adaptive immune response. Our study provides novel insights into how PI3K/mTOR inhibitors exert their activity by modulating the tumor-immune interaction.

Single-cell microRNA (miRNA) sequencing has allowed for comprehensively studying the abundance and complex networks of miRNAs, which provides insights beyond single-cell heterogeneity into the dynamic regulation of cellular events. Current benchtop-based technologies for single-cell miRNA sequencing are low throughput, limited reaction efficiency, tedious manual operations, and high reagent costs. Here, a highly multiplexed, efficient, integrated, and automated sample preparation platform is introduced for single-cell miRNA sequencing based on digital microfluidics (DMF), named Hiper-seq. The platform integrates major steps and automates the iterative operations of miRNA sequencing library construction by digital control of addressable droplets on the DMF chip. Based on the design of hydrophilic micro-structures and the capability of handling droplets of DMF, multiple single cells can be selectively isolated and subject to sample processing in a highly parallel way, thus increasing the throughput and efficiency for single-cell miRNA measurement. The nanoliter reaction volume of this platform enables a much higher miRNA detection ability and lower reagent cost compared to benchtop methods. It is further applied Hiper-seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing.

Osteosarcoma is a prevalent kind of primary bone malignancy. Trifluoperazine, as an antipsychotic drug, has anti-tumor activity against a variety of cancers. Nevertheless, the impact of trifluoperazine on osteosarcoma is unclear. Our investigation aimed to explore the mechanism of trifluoperazine's effect on osteosarcoma. We found that trifluoperazine inhibited 143B and U2-OS osteosarcoma cell proliferation in a method based on the dose. Furthermore, it was shown that trifluoperazine induced the accumulation of reactive oxygen species (ROS) to cause mitochondrial damage and induced mitophagy in osteosarcoma cells. Finally, combined with RNA-seq results, we first demonstrated the AMPK/mTOR/ULK1 signaling pathway as a potential mechanism of trifluoperazine-mediated mitophagy in osteosarcoma cells and can be suppressed by AMPK inhibitor Compound C.

Fatty acid binding protein 5 (FABP5) is an intracellular chaperone of fatty acid molecules that regulates lipid metabolism and cell growth. However, its role in intestinal inflammation remains enigmatic. Through examination of human tissue samples and single-cell data, we observed a significant upregulation of FABP5 within the mucosa of patients afflicted with ulcerative colitis (UC) and Crohn’s disease (CD), predominantly localized in intestinal macrophages. Herein, we investigate the regulation of FABP5-IN-1, a FABP5 inhibitor, on various cells of the gut in an inflammatory environment. Our investigations confirmed that FABP5 ameliorates DSS-induced colitis in mice by impeding the differentiation of macrophages into M1 macrophages in vitro and in vivo. Furthermore, following FABP5-IN-1 intervention, we observed a notable restoration of intestinal goblet cells and tuft cells, even under inflammatory conditions. Additionally, FABP5-IN-1 exhibits a protective effect against DSS-induced colitis by promoting the polarization of macrophages towards the M2 phenotype in vivo. In summary, FABP5-IN-1 confers protection against DSS-induced acute colitis through a multifaceted approach, encompassing the reduction of inflammatory macrophage infiltration, macrophage polarization, regulating Th17/Treg cells to play an anti-inflammatory role in IBD. The implications for IBD are underscored by the comprehensive in vivo and in vitro experiments presented in this article, thereby positioning FABP5 as a promising and novel therapeutic target for the treatment of IBD.

Enshi City, in the Hubei Province of China, is known as the world capital of selenium with the most abundant selenium resource. An important selenium hyperaccumulator plant, Cardamine violifolia, was found to naturally grow in this high-selenium ecosystem. However, relatively little is known about the impact of the selenium levels on microbial community and functional shifts in C. violifolia rhizosphere. Here, we tested the hypothesis that underground microbial diversity and function vary along a selenium gradient, including antibiotic resistance genes (ARGs). Comprehensive metagenomic analyses, such as taxonomic investigation, functional detection, and ARG annotation, showed that selenium, mercury, cadmium, lead, arsenic, and available phosphorus and potassium were correlated with microbial diversity and function. Thaumarchaeota was exclusively dominant in the highest selenium concentration of mine outcrop, and Rhodanobacter and Nitrospira were predominant in the high-selenium ecosystem. The plant C. violifolia enriched a high concentration of selenium in the rhizosphere compared to those in the bulk soil, and it recruited Variovorax and Polaromonas in its rhizosphere. Microbial abundance showed a trend of increasing first and then decreasing from low to high selenium concentrations. Annotation of ARGs showed that the multidrug resistance genes adeF, mtrA, and poxtA, the aminoglycoside resistance gene rpsL, and the sulfonamide resistant gene sul2 were enriched in the high-selenium system. It was discovered that putative antibiotic resistant bacteria displayed obvious differences in the farmland and the soils with various selenium concentrations, indicating that a high-selenium ecosystem harbors the specific microbes with a higher capacity to enrich or resist selenium, toxic metals, or antibiotics. Taken together, these results reveal the effects of selenium concentration and the selenium hyperaccumulator plant C. violifolia on shaping the microbial functional community and ARGs. Metalloid selenium-inducible antibiotic resistance is worth paying attention to in future.

BACKGROUND/AIM:As one of the common clinical diseases, fractures have many causes, mechanisms, healing and influencing factors; especially fracture healing is a long-term and complex process. Animal fracture models can simulate the various states of human fractures, and on this basis, the prevention, mechanism, and treatment of fractures can be studied to further guide clinical practice.MATERIALS AND METHODS:Here, we developed a novel and portable device to create a closed fracture model in mice. We then compared this novel closed fracture model with the traditional open model in multiple dimensions to evaluate the modelling process of establishment and healing. The two models were evaluated by imaging, immunostaining, and behavioral tests, which fully demonstrated the stability, universality and operability of the modified fracture model in mice.RESULTS:Surgical quality assessment revealed that the closed fracture model had a shorter operation time and smaller wound than the open model. X-ray and micro-CT results showed no differences between the two models in the evaluation of radiographic and morphological changes during fracture healing. Histological examination revealed the process of the typical intrachondral osteogenic pathway after fracture. Moreover, animal gait analysis indicated reduced postoperative pain in the closed group compared to the open group.CONCLUSION:This study provides a constructive strategy for a closed fracture model in mice and demonstrates the effectiveness and feasibility of the closed fracture model in studying the typical intrachondral osteogenic pathway of fractures from multiple dimensions.

Purpose:To investigate induction of cell death in Osteosarcoma (OS) using the anti-tuberculosis drug, rifampicin, loaded into exosomes.Patients and Methods:BMSC-exosomes were isolated by ultracentrifugation and loaded ultrasonically with rifampicin. Nanoparticle exosome-rifampicin (EXO-RIF) was added to the OS cell-lines, 143B and MG63, in vitro, to observe the growth inhibitory effect. In vivo experiments were conducted by injecting fluorescently labeled EXO-RIF through the tail vein of 143B cell xenograft nude mice and tracking distribution. Therapeutic and toxic side-effects were analyzed systemically.Results:Sonication resulted in encapsulation of rifampicin into exosomes. Exosome treatment accelerated the entry of rifampicin into OS cells and enhanced the actions of rifampicin in inhibiting OS proliferation, migration and invasion. Cell cycle arrest at the G2/M phase was observed. Dynamin-related protein 1 (Drp1) was activated by EXO-RIF and caused mitochondrial lysis and apoptosis. Exosome treatment targeted rifampicin to the site of OS, causing OS apoptosis and improving mouse survival in vivo.Conclusion:The potent Drp1 agonist, rifampicin, induced OS apoptosis and exosome loading, improving OS targeting and mouse survival rates. EXO-RIF is a promising strategy for the treatment of diverse malignancies.

Encouraged by our previous findings and in continuation of our ongoing study project in designing and synthesis of novel Nur77-targeting anti-cancer agents, a series of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carboxamide derivatives were designed, synthesized and biologically evaluated as potent Nur77 modulators. Among synthesized compounds, 8b maintained good potency against different liver cancer cell lines and other types of cancer cell lines while exhibiting lower toxicity than the positive compound celastrol. Moreover, 8b displayed excellent Nur77-binding activity, superior to the lead compound 10g and comparable to the reference compound celastrol. The cytotoxic action of 8b towards cancer cells was associated with its induction of Nur77-mitochondrial targeting and Nur77-dependent apoptosis. Notably, 8b has good in vivo safety and anti-hepatocellular carcinoma (HCC) activity. Altogether, this study reveals that 8b is a novel Nur77 modulator with great promise for further research.

Atrial fibrillation (AF) is the most frequent arrhythmia in adults. The prevalence and incidence of AF is going to increase substantially over the next few decades. Because AF increases the risk of stroke, heart failure, dementia, and others, it severely impacts the quality of life, morbidity, and mortality. Although the pathogenesis of AF is multifaceted and complex, focal ectopic activity and reentry are considered as the fundamental proarrhythmic mechanisms underlying AF development. Over the past 2 decades, large amount of evidence points to the key role of intracellular Ca2+ dysregulation in both initiation and maintenance of AF. More recently, emerging evidence reveal that NLRP3 (NACHT, LRR, PYD domain-containing 3) inflammasome pathway contributes to the substrate of both triggered activity and reentry, ultimately promoting AF. In this article, we review the current state of knowledge on Ca2+ signaling and NLRP3 inflammasome activity in AF. We also discuss the potential crosstalk between these two quintessential contributors to AF promotion.

Aims Obesity, an established risk factor of atrial fibrillation (AF), is frequently associated with enhanced inflammatory response. However, whether inflammatory signaling is causally linked to AF pathogenesis in obesity remains elusive. We recently demonstrated that the constitutive activation of the 'NACHT, LRR, and PYD Domains-containing Protein 3' (NLRP3) inflammasome promotes AF susceptibility. In this study, we hypothesized that the NLRP3 inflammasome is a key driver of obesity-induced AF. Methods and results Western blotting was performed to determine the level of NLRP3 inflammasome activation in atrial tissues of and obese patients, sheep, and diet-induced obese (DIO) mice. The increased body weight in patients, sheep, and mice was associated with enhanced NLRP3-inflammasome activation. To determine whether NLRP3 contributes to the obesity-induced atrial arrhythmogenesis, wild-type (WT) and NLRP3 homozygous knockout (NLRP3(-/-)) mice were subjected to high-fat-diet (HFD) or normal chow (NC) for 10 weeks. Relative to NC-fed WT mice, HFD-fed WT mice were more susceptible to pacing-induced AF with longer AF duration. In contrast, HFD-fed NLRP3(-/-) mice were resistant to pacing-induced AF. Optical mapping in DIO mice revealed an arrhythmogenic substrate characterized by abbreviated refractoriness and action potential duration (APD), two key determinants of reentry-promoting electrical remodeling. Upregulation of ultra-rapid delayed-rectifier K+-channel (Kv1.5) contributed to the shortening of atrial refractoriness. Increased profibrotic signaling and fibrosis along with abnormal Ca2+ release from sarcoplasmic reticulum (SR) accompanied atrial arrhythmogenesis in DIO mice. Conversely, genetic ablation of Nlrp3 (NLRP3(-/-)) in HFD-fed mice prevented the increases in Kv1.5 and the evolution of electrical remodeling, the upregulation of profibrotic genes, and abnormal SR Ca2+ release in DIO mice. Conclusion These results demonstrate that the atrial NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmogenesis and establishes a mechanistic link between obesity-induced AF and NLRP3-inflammasome activation.

Iron is an essential element, closely linked with host immune responses. Nevertheless, the relationship between iron metabolism and virus infection is still unclear in aquatic vertebrates. To address this issue, we employed grass carp (Ctenopharyngodon idella) and its lethal virus, grass carp reovirus (GCRV), a double-strand RNA virus, as models. Our results demonstrate that GCRV infection increases the iron content and alters the expression of iron metabolism-related genes both in vivo and in vitro. Of note, the expression of C. idella transferrin receptor 1 (CiTfR1) rather than transferrin is upregulated upon GCRV infection. To clarify the implications of CiTfR1 upregulation for antiviral immunity, we proved that CiTfR1 was not a helper for GCRV invasion, but instead, it inhibited GCRV infection and promoted cell proliferation by facilitating the accumulation of intracellular labile iron pool (LIP), which increases intracellular oxidative stress. Interestingly, we found that CiTfR1 overexpression inhibited the mRNA expression of C. idella interferon 1 (CiIFN1) and CiIFN3. The present study reveals a novel antiviral defense mechanism in teleost where TfR1 induces the accumulation of LIP, leading to the suppression of virus infection and the proliferation of host cells, indicating that iron can be used as a medicated feed additive for the control of animal viral disease.

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), has been a serious problem in grass carp aquaculture for several decades. Characterization of the primary host factors associated with host-virus interaction is critical for understanding how a virus infects its host cell and these host factors can be antiviral targets. This study aimed to screen host factors that interacted with GCRV in the C. idella kidney (CIK) cells and used them as antiviral targets. Twelve proteins were identified by virus overlay protein binding assay and LC-MS-MS. Among these twelve proteins, Heat Shock Protein 70 (HSP70) was outstanding. Results of flow cytometry and immunofluorescence assay indicated that HSP70 was on the cell membrane. HSP70 was expressed at low levels preceding GCRV infection, but its expression was induced upon GCRV infection. Inhibition of HSP70's function by inhibitors (VER155008 and pifithrin-μ) maintained HSP70 on the cell surface in infected cells, however GCRV quantity was decreased in the CIK cells (compared with the control group, the maximum inhibition rate of the treatment group was close to 85%), suggesting that fully functional HSP70 was required for GCRV infection. Moreover, GCRV showed a dose dependent reduction by inhibiting the entry stage of the viral life cycle following treated with VER155008 and pifithrin-μ. VER + PIF (1:1) were used at 15 μM and the expression of GCRV-VP6 downregulated nearly to 90%, which revealed that HSP70 played an important role in GCRV entering into CIK cells. This work speculated that HSP70 might be a host factor in the process of GCRV infecting CIK cells, therefore, it might be a potential antiviral target for GCRV infection.

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2, while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.