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Schistosomiasis remains an important public health concern. The eggs deposited in livers invoke a Th2-dominant response, which mediates the fibrotic granulomatous response. However, the mechanisms involved in this immunopathological process are still not perfectly clear. Here, we report a single-cell transcriptional landscape of longitudinally collected BALB/c mouse splenocytes at different time points after Schistosoma japonicum infection. We found that exhausted CD4+ T cells were enriched after infection, changing from coproducing multiple cytokines to predominantly producing the Th2 cytokine IL-4. Regulatory B cells had high expression of Fcrl5, Ptpn22, and Lgals1, potentially regulating exhausted CD4+ T cells via direct PD-1–PD-L2 and PD-1–PD-L1 interactions. Within the myeloid compartment, the number of precursor and immature neutrophils sharply increased after infection. Moreover, dendritic cells, macrophages, and basophils showed inhibitory interactions with exhausted CD4+ T cells. Besides, in mouse livers, we found that exhausted CD4+ T cells were distributed around egg granuloma, promoting collagen expression in primary mouse hepatic stellate cells via IL-4 secretion, resulting in liver fibrosis. Our study provides comprehensive characterization of the composition and cellular states of immune cells with disease progression, which will facilitate better understanding of the mechanism underlying liver fibrotic granulomatous response in schistosomiasis.

Fibro-adipogenic progenitors (FAPs) in the muscles have been found to interact closely with muscle progenitor/stem cells (MPCs) and facilitate muscle regeneration at normal conditions. However, it is not clear how FAPs may interact with MPCs in aged muscles. Senolytics have been demonstrated to selectively eliminate senescent cells and generate therapeutic benefits on ageing and multiple age-related disease models.By studying the muscles and primary cells of age matched WT mice and Zmpste24We observed that, compared with muscles of WT mice, muscles of Z24These results indicate that the application of senolytics in the progeria-aged muscles can be an efficient strategy to remove senescent cells, including senescent FAPs, which results in improved function of muscle progenitor/stem cells. The senescent FAPs can be a potential novel target for therapeutic treatment of progeria ageing related muscle diseases.

Rosmarinic acid (RA) and tanshinone IIA (TA) which are effective components in Salvia miltiorrhiza show anti-inflammatory potential against atherosclerosis. Based on polysulfated propylene-polyethylene glycol (PPS-PEG), RA was grafted onto this polymer via amide bonds to form a micelle carrier for TA encapsulation: PPS-PEG-RA@TA. A potent inhibitory effect on lipopolysaccharide (LPS) -induced proliferation of endothelial cells with significant intracellular uptake was observed with this system. This could have been the result of release of TA in a reactive oxygen species (ROS) environment and stronger antioxidant effect of RA. The synergistic effect was optimized when the combination was used in a molar ratio of 1:1. Mechanistic studies showed that, compared with PPS-PEG-RA and TA+RA, PPS-PEG-RA@TA micelles could more effectively regulate the nuclear factor-kappa B (NF-κB) pathway to reduce expression of vascular cell adhesion molecule-1 (VCAM-1), inhibit the inflammatory cascade and reduce endothelial-cell injury. One month after intravenous injection of PPS-PEG-RA@TA micelles, the plaque area in murine aortic vessels was reduced significantly, and serious toxic side-effects were not observed in vivo, along with excellent biocompatibility. In summary, PPS-PEG-RA@TA micelles could achieve synergistic treatment of atherosclerosis.

Traumatic brain injury (TBI)-induced neuroinflammation is closely associated with poor outcomes and high mortality in affected patients, with unmet needs for effective clinical interventions. A series of causal and disseminating factors have been identified to cause TBI-induced neuroinflammation. Among these are cellular microvesicles released from injured cerebral cells, endothelial cells, and platelets. In previous studies, we have put forward that cellular microvesicles can be released from injured brains that induce consumptive coagulopathy. Extracellular mitochondria accounted for 55.2% of these microvesicles and induced a redox-dependent platelet procoagulant activity that contributes to traumatic brain injury-induced coagulopathy and inflammation. These lead to the hypothesis that metabolically active extracellular mitochondria contribute to the neuroinflammation in traumatic brain injury, independent of their procoagulant activity. Here, we found that these extracellular mitochondria induced polarization of microglial M1-type pro-inflammatory phenotype, aggravating neuroinflammation, and mediated cerebral edema in a ROS-dependent manner. In addition, the effect of ROS can be alleviated by ROS inhibitor N-ethylmaleimide (NEM) in vitro experiments. These results revealed a novel pro-inflammatory activity of extracellular mitochondria that may contribute to traumatic brain injury-associated neuroinflammation.

Diabetes-associated liver injury becomes a dominant hepatopathy, leading to hepatic failure worldwide. The current study was designed to evaluate the ameliorative effects of ginsenoside Rh1 (G-Rh1) on liver injury induced by T2DM. A T2DM model was established using C57BL/6 mice through feeding with HFD followed by injection with streptozotocin at 100 mg·kg

Cisplatin is extensively used to treat malignancies. However, its clinical use is always limited due to the serious side effects, especially the nephrotoxicity. Matrine (MAT), a tetracyclic quinolizine alkaloid found in sophora genus, exerts multiple pharmacological roles, including anti-oxidative stress, anti-inflammation and anti-apoptosis, but the role of MAT on acute kidney injury (AKI) has not been evaluated. Here, we found that MAT potently inhibited cell injury induced by cisplatin in HK2 cells in vitro, which was associated with the inhibition of oxidative injury and NF-κB-mediated inflammation. Moreover, MAT treatment could activate the SIRT3/OPA1 axis and subsequently suppress the mitochondrial fragmentation and improve mitochondrial function. More importantly, SIRT3 knockdown suppressed the deacetylation of OPA1, which blocked the protective role of MAT on cisplatin-induced cell injury. In vivo, MAT treatment alleviated renal dysfunction, histological damage and inflammation induced by cisplatin in mice. Furthermore, consistent with the founding in vitro, MAT also activated SIRT3-mediated deacetylation of OPA1 and alleviated mitochondrial dysfunction in AKI mice. Our study proved that MAT protected against cisplatin-induced AKI by synergic anti-oxidative stress and anti-inflammation actions via SIRT3/OPA1-mediated improvement of mitochondrial function, suggesting that MAT may be a novel and effective strategy for AKI.

Allergic rhinitis and allergic asthma are the most common type-2 inflammatory diseases, which are hardly curable and cause heavy burden to general well-being. Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with potential immunomodulatory effects that have been showning to have a therapeutic effect on allergic diseases. Here, we investigated the effects of human induced pluripotent stem cell (iPSC)-derived MSCs on airway hyperresponsiveness and acute type-2-dominated inflammation throughout the upper and lower airways. In this study, human MSCs, MSC cell culture supernatant, and culture medium (control) was injected into the acute airway inflammatory model via the tail vein. Mouse behavioristics were recorded immediately and mouse lung function was measured 24 hours after the last ovalbumin (OVA) challenge. Histological staining, Luminex, Elisa and flow cytometry were employed to evaluate the effects on the production of total/OVA-specific IgG1 and IgE, cytokines expression in lung tissues, and inflammatory cells infiltration in the lung and spleen of the experimental mice. Expressions of eotaxin, IL-4, IL-5, IL-13, IL-33 in nasal and lung lavage were evaluated by Luminex and Elisa. We found that for this acute inflammatory mouse model, human MSC transplantation significantly mitigated the decreased motoring time and the increased lung function Rrs caused by OVA challenge. Serum OVA-IgG1, OVA-IgE, and eosinophil percentages in the splenocytes were significantly decreased. Injection of the MSC supernatant also showed the same trend, but not significantly changed. After treatment, IL-4 and IL-13 were significantly decreased in the lung tissue, and IL-5 and IL-13 were significantly decreased in lung lavage. In conclusion, both human MSC culture supernatant and cell transplantation could alleviate AHR and inflammation in acute inflammatory experimental animals, which demonstrated their potential for clinical therapeutics. Human iPSC-MSCs, MSC cell culture supernatant, or culture medium (control) was injected into the OVA-induced acute airway inflammatory model via the tail vein. Behavioral changes, AHR, serum OVA-specific IgG1 and IgE concentrations, and type-2 inflammations were alleviated.

To explore the mechanisms underlying the specific inhibitor targeting SGLT-2 empagliflozin in alleviating myocardial ischaemia–reperfusion (I/R) injury. A mouse model of I/R injury and H2O2-induced H9C2 cell model were established. The expressions of Bcl-2, Bax, LC3, Beclin1, GRP78, CHOP, PERK, ATF4, ATF6, IREα and P62 were examined by western blot, immunofluorescence or immunohistochemistry staining, respectively. The cardiac function was measured by echocardiography, TCC staining, lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity. Cell apoptosis was analysed by TUNEL, Annexin V/propidium iodide (PI) staining and caspase 3 and 9 activities. CCK-8 assay was used for analysing cell viability. PBA, TUDC and 3-MA were utilised for blocking ER stress and autophagy, respectively. Empagliflozin suppressed myocardial I/R injury in vivo and H2O2-induced cardiomyocyte apoptosis in vitro. Blockade of ER stress and autophagy inhibited H2O2-induced cardiomyocyte apoptosis. ER stress activated autophagy through the PERK signalling in H2O2-treated H9C2 cells. Empagliflozin suppressed ER stress-induced autophagy by inhibiting the PERK/ATF4/Beclin1 signalling. H2O2 and I/R-induced cardiomyocyte apoptosis was restrained by empagliflozin through inhibition of ER stress-induced autophagy. Empagliflozin suppressed ER stress-induced autophagy via suppressing the PERK/ATF4/Beclin1 signalling, thus alleviating myocardial I/R injury and cardiomyocyte apoptosis.

Podocalyxin (PODXL) is a newly identified key negative regulator of human endometrial receptivity, specifically down-regulated in the luminal epithelium at receptivity to permit embryo implantation. Here, we bioinformatically compared the molecular characteristics of PODXL among the human, rhesus macaque, and mouse, determined by immunohistochemistry and in situ hybridization (mouse tissues) whether endometrial PODXL expression is conserved across the three species and examined if PODXL inhibits mouse embryo attachment in vitro. The PODXL gene, mRNA, and protein sequences showed greater similarities between humans and macaques than with mice. In all species, PODXL was expressed in endometrial luminal/glandular epithelia and endothelia. In macaques (n = 9), luminal PODXL was significantly down-regulated when receptivity is developed, consistent with the pattern found in women. At receptivity, PODXL was also reduced in shallow glands, whereas endothelial expression was unchanged across the menstrual cycle. In mice, endometrial PODXL did not vary considerably across the estrous cycle (n = 16); however, around embryo attachment on d4.5 of pregnancy (n = 4), luminal PODXL was greatly reduced especially near the site of embryo attachment. Mouse embryos failed to attach or thrive when co-cultured on a monolayer of Ishikawa cells overexpressing PODXL. Thus, endometrial luminal PODXL expression is down-regulated for embryo implantation in all species examined, and PODXL inhibits mouse embryo implantation. Rhesus macaques share greater conservations with humans than mice in PODXL molecular characteristics and regulation, thus represent a better animal model for functional studies of endometrial PODXL for treatment of human fertility.

DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging. We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity. The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures. The ligand–G4 interaction studied with 1H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures. Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.

Background: β-amyloid peptides (Aβ) induced oxidative damage contributes to the pathogenesis of neurodegenerative diseases, and the cerebrovascular system is more vulnerable to oxidative stress. Our earlier study showed a clue that Genistein (Gen) might activate the Nf-E2 related factor 2 (Nrf2) pathway to protect cerebrovascular cells from oxidative damage induced by Aβ, but the specific mechanisms and regulation targets are unclear. Objective: In this study, the anti-oxidative effects and the possible targets of Gen on regulating the Nrf2 pathway in bEnd.3 cells were investigated. Cells were divided into control, Aβ25-35, Gen, and Gen+Aβ25-35 groups. Methods: Cell viability, levels of malondialdehyde (MDA), Superoxide Dismutase (SOD) activity, and nitrotyrosine were evaluated. Moreover, mRNA and/or protein expressions of Nrf2 and kelchlike ECH-associated protein 1 (Keap1) were measured. Then we transfected Keap1 over-expressed plasmid into bEnd.3 cells and measured the protein expressions of Nrf2 pathway related factors. Results: Data showed that Gen could inhibit the over-production of MDA and nitrotyrosine and activate SOD activity. Furthermore, we discovered that Gen could up-regulate Nrf2 mRNA and protein expression while down-regulating Keap1 protein expression. The Keap1 over-expressed plasmid study revealed that the up-regulation of Nrf2 protein expression induced by Gen pretreatment could be blocked by transfection of Keap1 over-expressed plasmid, and the same results were observed in Nrf2 downstream factors. Conclusion: Gen could alleviate the cerebrovascular cells' oxidative damage induced by Aβ25-35 by regulating the Nrf2 pathway, and Keap1 might be one of the targets of Gen in activating the Nrf2 pathway.

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli—eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

Proper regulation of p53 signaling is critical for the maintenance of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). The hematopoietic cell–specific mechanisms regulating p53 activity remain largely unknown. Here, we demonstrate that conditional deletion of acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) in hematopoietic cells impairs repopulation capacity and postinjury regeneration of HSCs. Mechanistically, ANP32B forms a repressive complex with p53 and thus inhibits the transcriptional activity of p53 in hematopoietic cells, and p53 deletion rescues the functional defect in Anp32b-deficient HSCs. Of great interest, ANP32B is highly expressed in leukemic cells from patients with chronic myelogenous leukemia (CML). Anp32b deletion enhances p53 transcriptional activity to impair LSC function in a murine CML model and exhibits synergistic therapeutic effects with tyrosine kinase inhibitors in inhibiting CML propagation. In summary, our findings provide a novel strategy to enhance p53 activity in LSCs by inhibiting ANP32B and identify ANP32B as a potential therapeutic target in treating CML.

Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Directed differentiation of bone marrow mesenchymal stem cells (BMSCs) toward chondrogenesis plays a predominant role in cartilage repair. However, the uncontrolled inflammatory response to implants is found to impair the stability of scaffolds and the cartilage regeneration outcome. Herein, we fabricated an injectable hydrogel crosslinked by strontium-doped bioglass (SrBG) to modulate both human BMSC (hBMSC) differentiation and the inflammatory response. The results revealed that the introduction of Sr ions could simultaneously enhance the proliferation of hBMSCs, upregulate cartilage-specific gene expression, and improve the secretion of glycosaminoglycan. Moreover, after cultured with SA/SrBG extracts in vitro, a majority of macrophages were polarized toward the M2 phenotype and subsequently facilitated the chondrogenic differentiation of hBMSCs. Furthermore, after the composite hydrogel was injected into a cartilage defect model, neonatal cartilage-like tissues with a smooth surface and tight integration with original tissues could be found. This study suggests that the synergistic strategy based on an enhanced differentiation ability and a regulated inflammatory response is promising and may lead the way to new anti-inflammatory biomaterials.

Background As an efficient tumor immunotherapy, PD-1 antibody has been gradually used in clinical tumor treatment, but the low response rate and excessive immune response limit its extensive application. Results Herein, a therapeutic regime for the reinvigoration and activation of the tumor immune microenvironment is introduced to improve the anti-tumor effect of the PD-1 antibody. To comprehensively improve the effect of the immunotherapy and reduce excessive immune response, a biomimetic cascade targeting nanosystem, siRNA@PLOV, which was fused by photothermal sensitive liposomes (PTSLs) and attenuated Salmonella outer membrane vesicles (OMVs), was administered in the tumor therapy for targeting of tumor tissues and T cells within tumor respectively. The fused PLOVs which not only retained the biological character of the OMVs, but also enhanced the drug loading ability. The results demonstrated that the immunogenicity of OMVs and photothermal effects can obviously increase the infiltration of T cells and the silencing of CD38 can effectively improve the T cell cytotoxicity, especially combining with PD-1 antibody. Conclusions Interesting, this study revealed that anti-PD-1 administration on the 5th day after siRNA@PLOV treatment had the best performance in killing tumors compared with other groups. In addition, this new therapeutic regime also presents a novel strategy for inducing “vaccine effects”, conclusively highlighting its potential in preventing tumor recurrence and improving prognosis. Graphical Abstract

Mutations of the X-linked methyl-CpG-binding protein 2 (MECP2) gene in humans are responsible for most cases of Rett syndrome (RTT), an X-linked progressive neurological disorder. While genome-wide screens in clinical trials have revealed several putative RTT-associated mutations in MECP2, their causal relevance regarding the functional regulation of MeCP2 at the etiologic sites at the protein level requires more evidence. In this study, we demonstrated that MeCP2 was dynamically modified by O-linked-β-N-acetylglucosamine (O-GlcNAc) at threonine 203 (T203), an etiologic site in RTT patients. Disruption of the O-GlcNAcylation of MeCP2 specifically at T203 impaired dendrite development and spine maturation in cultured hippocampal neurons, and disrupted neuronal migration, dendritic spine morphogenesis, and caused dysfunction of synaptic transmission in the developing and juvenile mouse cerebral cortex. Mechanistically, genetic disruption of O-GlcNAcylation at T203 on MeCP2 decreased the neuronal activity-induced induction of Bdnf transcription. Our study highlights the critical role of MeCP2 T203 O-GlcNAcylation in neural development and synaptic transmission potentially via brain-derived neurotrophic factor. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12264-021-00784-8.

High tumor regulatory T (Treg) cell infiltration is associated with poor prognosis of many cancers. CD25 is highly expressed on tumor Treg cells and is a potential target for Treg deletion. Previously characterized anti-CD25 antibodies appear to have limited efficacy in tumor inhibition. Here we identified two human anti-CD25 antibodies, BA9 and BT942, which did not prevent the activation of IL-2R signaling pathway by IL-2. BT942 had weaker binding and cytotoxic activity to human CD25-expressing cell lines than BA9. But both demonstrated significant tumor growth inhibition in early and late-stage animal cancer models. BT942 resulted in a higher expansion of CD8+T cells and CD4+T cells in tumor microenvironment in mouse MC38 model compared to BA9. BT942 also demonstrated significant higher tumor growth inhibition and higher expansion of CD8+T cells and CD4+T cells in combination with an anti-PD1 antibody. Pharmacokinetic study of BT942 in cynomolgus monkeys demonstrated a half-life of 206.97 ± 19.03 h. Structural analysis by cryo-EM revealed that BT942 recognizes an epitope on opposite side of the CD25-IL-2 binding site, consistent with no IL-2 signaling blockade in vitro. BT942 appears to be an excellent candidate for cancer immunotherapy.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.

Amyloid-β (Aβ) accumulating is considered as a causative factor for formation of senile plaque in Alzheimer’s disease (AD), but its mechanism is still elusive. The Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2), a key redox cofactor for energy metabolism, is reduced in AD. Accumulative evidence has shown that the decrease of α-secretase activity, a disintegrin and metalloprotease domain 10 (ADAM10), is responsible for the increase of Aβ productions in AD patient’s brain. Here, we observe that the activity of α-secretase ADAM10 and levels of Nmnat2 are significantly decreased, meanwhile there is a simultaneous elevation of Aβ in Tg2576 mice. Over-expression of Nmnat2 increases the mRNA expression of α-secretase ADAM10 and its activity and inhibits Aβ production in N2a/APPswe cells, which can be abolished by Compound C, an AMPK antagonist, suggesting that AMPK is involved in over-expression of Nmnat2 against Aβ production. The further assays demonstrate that Nmnat2 activates AMPK by up-regulating the ratio of NAD+/NADH, moreover AMPK agonist AICAR can also increase ADAM10 activity and reduces Aβ1-40/1-42. Taken together, Nmnat2 suppresses Aβ production and up-regulates ADAM10 in AMPK activity-dependent manner, suggesting that Nmnat2 may serve as a new potential target in arresting AD.

RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1−/− cells enhances the TCA flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1−/− deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance., RIPK1 is critical for normal development and cell death. Here, the authors identify a metabolic role for RIPK1 in aspartate homeostasis, as increased aspartate levels in RIPK1-deficient cells inhibits starvation-induced autophagy by ULK1.

Still’s disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still’s disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still’s disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still’s disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still’s disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still’s disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still’s disease and macrophage activation syndrome.

Threat and extinction memories are crucial for organisms’ survival in changing environments. These memories are believed to be encoded by separate ensembles of neurons in the brain, but their whereabouts remain elusive. Using an auditory fear-conditioning and extinction paradigm in male mice, here we discovered that two distinct projection neuron subpopulations in physical proximity within the insular cortex (IC), targeting the central amygdala (CeA) and nucleus accumbens (NAc), respectively, to encode fear and extinction memories. Reciprocal intracortical inhibition of these two IC subpopulations gates the emergence of either fear or extinction memory. Using rabies-virus-assisted tracing, we found IC-NAc projection neurons to be preferentially innervated by intercortical inputs from the orbitofrontal cortex (OFC), specifically enhancing extinction to override fear memory. These results demonstrate that IC serves as an operation node harboring distinct projection neurons that decipher fear or extinction memory under the top-down executive control from OFC.

Background: Various potential effect of drugs on alleviating diseases by regulating intestinal microbiome as well as the pharmaceutical excipients on gut microbiota has been revealed. However, the interaction between them is rarely investigated. Methods: Histological analysis, immunohistochemistry analysis, enzyme-linked immunosorbent assay (ELISA) analysis, RT-qPCR, and 16S rRNA analysis were utilized to explore the effect mechanism of the five excipients including hydroxypropyl methylcellulose (HPMC) F4M, Eudragit (EU) S100, chitosan (CT), pectin (PT), and rheum officinale polysaccharide (DHP) on berberine (BBR) to cure UC. Results: The combined BBR with PT and DHP group exhibited better therapeutic efficacy of UC with significantly increased colon length, and decreased hematoxylin-eosin (H&E) scores than other groups. Furthermore, the expression of tight junction ZO-1 and occludin in colon tissue were upregulated, and claudin-2 was downregulated. Ultimately, the serum content of tumor necrosis (TNF)-α, interleukin (IL)-1β, and IL-6 was decreased. Moreover, the combined BBR with PT significantly promoted the restoration of gut microbiota. The relative abundance of Firmicutes and Lactobacillus was significantly increased by the supplement of PT and DHP, and the relative abundance of Proteobacteria was downregulated. Conclusions: Our study may provide a new perspective that the selection of pharmaceutical excipients could be a crucial factor affecting the drugs’ therapeutic efficiency outcome.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1flox/flox mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI.

Promoting residential cells, particularly endogenous neural stem and progenitor cells (NSPCs), for tissue regeneration represents a potential strategy for the treatment of spinal cord injury (SCI). However, adult NSPCs differentiate mainly into glial cells and contribute to glial scar formation at the site of injury. Gsx1 is known to regulate the generation of excitatory and inhibitory interneurons during embryonic development of the spinal cord. In this study, we show that lentivirus-mediated expression of Gsx1 increases the number of NSPCs in a mouse model of lateral hemisection SCI during the acute stage. Subsequently, Gsx1 expression increases the generation of glutamatergic and cholinergic interneurons and decreases the generation of GABAergic interneurons in the chronic stage of SCI. Importantly, Gsx1 reduces reactive astrogliosis and glial scar formation, promotes serotonin (5-HT) neuronal activity, and improves the locomotor function of the injured mice. Moreover, RNA sequencing (RNA-seq) analysis reveals that Gsx1-induced transcriptome regulation correlates with NSPC signaling, NSPC activation, neuronal differentiation, and inhibition of astrogliosis and scar formation. Collectively, our study provides molecular insights for Gsx1-mediated functional recovery and identifies the potential of Gsx1 gene therapy for injuries in the spinal cord and possibly other parts of the central nervous system.

BACKGROUND Oleanolic acid (OA) is a pentacyclic triterpenoid compound that is present at high levels in olive oil and has several promising pharmacological effects, such as liver protection and anti-inflammatory, antioxidant, and anticancer effects. The purpose of the present study was to assess whether OA treatment affects gut health compared to a control condition, including gut microbiota and intestinal epithelial immunity. RESULTS Illumina MiSeq sequencing (16S rRNA gene) was used to investigate the effect of OA on the microbial community of the intestinal tract, while Illumina HiSeq (RNA-seq) technology was used to investigate the regulatory effect of OA on gene expression in intestinal epithelial cells, which allowed for a comprehensive analysis of the effects of OA on intestinal health. The results showed that the consumption of OA initially controlled weight gain in mice and altered the composition of the gut microbiota. At the phylum level, OA significantly increased the relative abundances of cecum Firmicutes but decreased the abundance of Actinobacteria, and at the genus level it increased the relative abundance of potentially beneficial bacteria such as Oscillibacter and Ruminiclostridium 9. Oleanolic acid treatment also altered the expression of 12 genes involved in the Kyoto Encyclopedia of Genes and Genomes（KEGG）pathways of complement and coagulation cascades, hematopoietic cell lineage, and leukocyte transendothelial migration in intestinal epithelial cells to improve gut immunity. CONCLUSION Intake of OA can contribute beneficial effects by optimizing gut microbiota and altering the immune function of intestinal epithelial cells, potentially to improve intestinal health status. © 2021 Society of Chemical Industry.

This study investigated the effects of oleanolic acid (OA) on hepatic lipid metabolism and gut-liver axis homeostasis in an obesity-related non-alcoholic fatty liver disease (NAFLD) nutritional animal model and explored possible molecular mechanisms behind its effects. The results revealed that OA ameliorated the development of metabolic disorders, insulin resistance, and hepatic steatosis in obese rats. Meanwhile, OA restored high-fat-diet (HFD)-induced intestinal barrier dysfunction and endotoxin-mediated induction of toll-like-receptor-4-related pathways, subsequently inhibiting endotoxemia and systemic inflammation and balancing the homeostasis of the gut-liver axis. OA also reshaped the composition of the gut microbiota of HFD-fed rats by reducing the Firmicutes/Bacteroidetes ratio and increasing the abundance of butyrate-producing bacteria. Our results support the applicability of OA as a treatment for obesity-related NAFLD through its anti-inflammatory, antioxidant, and prebiotic integration responses mediated by the gut-liver axis.

LFZ-4-46, that is [2-hydroxy-1-phenyl-1,5,6,10b-tetrahydropyrazolo(5,1-a) isoquinolin-3(2H)-yl](phenyl) methanone, a tetrahydroisoquinoline derivative with a pyrazolidine moiety, was synthetically prepared. The anti-cancer mechanism of the compound has not been clarified yet. In this study, the anticancer effects and potential mechanisms of LFZ-4-46 on human breast and prostate cancer cells were explored. (a) 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide assay was first performed to detect the effects of LFZ-4-46 on the viability of human cancer cells. (b) Comet assay was utilized to evaluate DNA damage. (c) Cell cycle, apoptosis and mitochondrial membrane potential were detected by flow cytometry. (d) The expression of relative proteins was detected by western blotting assay. LFZ-4-46 significantly inhibited the viability of cancer cells in a time- and dose-dependent manner and had no obviously inhibitory effect on the viability of mammary epithelial MCF-10A cells. Mechanistic studies demonstrated that LFZ-4-46-induced cell apoptosis and cycle arrest were mediated by DNA damage. It caused DNA damage through activating γ-H2AX and breaking DNA strands. Further studies showed that mitogen-activated protein kinasess pathway was involved in these activated several key molecular events. Finally, LFZ-4-46 showed a potent antitumor effect in vivo. These results suggest that LFZ-4-46 may be a potential lead compound for the treatment of breast and prostate cancer.

Tumor budding (TB) is considered a histomorphological marker of poor prognosis in patients with breast cancer (BC). Tumor vasculature is disordered and unstable in BC, which causes restricted drug delivery, hypoxia, and tumor metastasis. Traditional anti-angiogenic treatments cause extreme hypoxia, increased invasion, metastasis, and drug resistance due to blood vessel rarefaction or regression. Therefore, the application of anti-angiogenic strategies for vascular normalization in tumors is crucial to improve therapeutic efficacy in BC. In the present study, we found that transgelin (TAGLN) promoted the normalization of tumor vessels by regulating the structure and function of endothelial cells, and knockout of TAGLN in tumor-bearing mice resulted in tumor vessel abnormalization, an increase in epithelial-mesenchymal transition characteristics of tumor cells, and promotion of TB. Moreover, BC cells secrete exosomal miR-22-3p that mediates tumor vessel abnormalization by inhibiting TAGLN. We demonstrated for the first time that TAGLN plays an essential role in tumor vessel normalization, and thus it impairs TB and metastasis. Additionally, the findings of this study indicate that exosomal miR-22-3p is a potential therapeutic target for BC.

Background The echinoderm microtubule-associated protein-like-4 anaplastic lymphoma kinase (EML4-ALK) fusion gene was identified in a subset of non-small cell lung cancer (NSCLC) patients. They responded positively to ALK inhibitors. This study aimed to characterize the mechanisms triggered by EML4-ALK to induce NSCLC transformation. Methods HEK293 and NIH3T3 cells were transfected with EML4-ALK variant 3 or pcDNA3.1-NC. H2228 cells were transfected with siRNA-EML4-ALK or siRNA-NC. Cell viability and proliferation were measured by the CCK-8 and EdU methods, respectively. Flow cytometry revealed apoptosis. Gene expression profiles were generated from a signaling pathway screen in EML4-ALK-regulated lung cancer cells and verified by qPCR and Western blotting. The co-immunoprecipitation and immunohistochemistry/ immunofluorescence determined the interaction and colocalization of JAK2-STAT pathway components with EML4-ALK. Results Microarray identified several genes involved in the JAK2-STAT pathway. JAK2 and STAT6 were constitutively phosphorylated in H2228 cells. EML4-ALK silencing downregulated phosphorylation of STAT6. Expression of EML4-ALK in HEK293 and NIH3T3 cells activated JAK2, STAT1, STAT3, STAT5, and STAT6. In EML4-ALK-transfected HEK293 cells and EML4-ALK-positive H2228 cells, activated STAT6 and JAK2 colocalized with ALK. STAT3 and STAT6 were phosphorylated and translocated to the nucleus of H2228 cells following IL4 or IL6 treatment. Apoptosis increased, while cell proliferation and DNA replication decreased in H2228 cells following EML4-ALK knockdown. In contrast, HEK293 cell viability increased following EML4-ALK overexpression, while H2228 cell viability significantly decreased after treatment with ALK or JAK-STAT pathway inhibitors. Conclusions Our data suggest that the aberrant expression of EML4-ALK leads to JAK2-STAT signaling pathway activation, which is essential for the development of non-small cell lung cancer.

A genetic hallmark of malignant germ cell tumours (GCTs) is isochromosome 12p, but oncogenes located in 12p that are specifically expressed in GCT have not yet been identified. SIN3-HDAC complex-associated factor (SINHCAF) is a subunit of the Sin3/histone deacetylase (HDAC) complex, and it defines a Sin3a-Hdac complex variant that is required for the self-renewal of mouse embryonic stem cells. This study demonstrated that SINHCAF is expressed in a vast majority of malignant GCTs and is rarely expressed in somatic malignancy. Fluorescence in situ hybridisation revealed SINHCAF amplification in malignant GCTs. SINHCAF silencing using shRNA reduced anchorage-dependent cell proliferation and tumoursphere formation and inhibited tumour cell migration and invasion in GCT cell lines. Moreover, in the GCT cell line NTERA2/D1, SINHCAF silencing inhibited the expression of genes associated with embryonic stem cells and induced the expression of genes associated with neuronal and white fat cell differentiation. Compared with somatic cell lines, GCT cell lines were more susceptible to HDAC inhibitor treatment. Thus, we identified SINHCAF to be a potential oncogene located in the amplicon of chromosome 12p and showed that SINHCAF was specifically expressed in malignant GCTs. HDAC inhibitor treatment may counteract the oncogenic activity of SINHCAF and is a promising therapeutic approach for GCTs. © 2022 The Pathological Society of Great Britain and Ireland.

Sonic hedgehog (Shh) signaling plays a critical role in regulating cerebellum development by maintaining the physiological proliferation of granule neuron precursors (GNPs), and its dysregulation leads to the oncogenesis of medulloblastoma. O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuitry. Here, we demonstrate that O-GlcNAc transferase (OGT) in GNPs mediate the cerebellum development, and the progression of the Shh subgroup of medulloblastoma. Specifically, OGT regulates the neurogenesis of GNPs by activating the Shh signaling pathway via O-GlcNAcylation at S355 of GLI family zinc finger 2 (Gli2), which in turn promotes its deacetylation and transcriptional activity via dissociation from p300, a histone acetyltransferases. Inhibition of OGT via genetic ablation or chemical inhibition improves survival in a medulloblastoma mouse model. These data uncover a critical role for O-GlcNAc signaling in cerebellar development, and pinpoint a potential therapeutic target for Shh-associated medulloblastoma.

BackgroundGlioblastoma (GBM) is the most common malignant brain tumor with poor clinical outcomes. Immunotherapy has recently been an attractive and promising treatment of extracranial malignancies, however, most of clinical trials for GBM immunotherapy failed due to predominant accumulation of tumor-associated microglia/macrophages (TAMs).ResultsHigh level of LRIG2/soluble LRIG2 (sLRIG2) expression activates immune-related signaling pathways, which are associated with poor prognosis in GBM patients. LRIG2/sLRIGs promotes CD47 expression and facilitates TAM recruitment. Blockade of CD47–SIRPα interactions and inhibition of sLRIG2 secretion synergistically suppress GBM progression in an orthotropic murine GBM model.ConclusionsGBM cells with high level LRIG2 escape the phagocytosis by TAM via the CD47-SIRPα axis, highlighting a necessity for an early stage of clinical trial targeting LRIG2 and CD47-SIRPα as a novel treatment for patients with GBM.

BackgroundDemyelinating diseases in central nervous system (CNS) are a group of diseases characterized by myelin damage or myelin loss. Transforming growth factor beta1 (TGF-β1) is widely recognized as an anti-inflammatory cytokine, which can be produced by both glial and neuronal cells in CNS. However, the effects of TGF-β1 on demyelinating diseases and its underlying mechanisms have not been well investigated.MethodsA demyelinating mouse model using two-point injection of lysophosphatidylcholine (LPC) to the corpus callosum in vivo was established. Exogenous TGF-β1 was delivered to the lesion via brain stereotactic injection. LFB staining, immunofluorescence, and Western blot were applied to examine the severity of demyelination and pyroptosis process in microglia. Morris water maze test was used to assess the cognitive abilities of experimental mice. Furthermore, lipopolysaccharide (LPS) was applied to induce pyroptosis in primary cultured microglia in vitro, to explore potential molecular mechanism.ResultsThe degree of demyelination in LPC-modeling mice was found improved with supplement of TGF-β1. Besides, TGF-β1 treatment evidently ameliorated the activated proinflammatory pyroptosis of microglia, with downregulated levels of the key pyroptosis effector Gasdermin D (GSDMD), inflammasomes, and cleaved-IL-1β, which effectively attenuated neuroinflammation in vivo. Evaluated by behavioral tests, the cognitive deficit in LPC-modeling mice was found mitigated with application of TGF-β1. Mechanistically, TGF-β1 could reverse pyroptosis-like morphology in LPS-stimulated primary cultured microglia observed by scanning electron microscopy, as well as decrease the protein levels of cleaved-GSDMD, inflammasomes, and cleaved-IL-1β. Activation of ERK1/2 and NF-κB pathways largely abolished the protective effects of TGF-β1, which indicated that TGF-β1 alleviated the pyroptosis possibly via regulating NF-κB/ERK1/2 signal pathways.ConclusionsOur studies demonstrated TGF-β1 notably relieved the demyelinating injury and cognitive disorder in LPC-modeling mice, by attenuating the inflammatory pyroptosis of microglia via ERK1/2 and NF-κB pathways. Targeting TGF-β1 activity might serve as a promising therapeutic strategy in demyelinating diseases.

BackgroundInterleukin-17, the major proinflammatory cytokine secreted by Th17 cells, makes essential contribution to pathogenesis of severe asthma, while the detailed mechanisms, especially the involvement of microRNAs which are also important participants in asthma progression, remains largely unclear.MethodsIn this study, we established a house dust mite (HDM) extract-induced murine asthmatic models and the miRNA expression in the lung tissues of mice were profiled by miRNA microarray assay. The effect of miR-365-3p on IL-17-mediated inflammation was examined by qRT-PCR and immunoblotting analysis. The involvement of ARRB2 as target gene of miR-365-3p was verified by overexpression or RNA interference.ResultsHDM extract-induced asthmatic inflammation was proved to be IL17-mediated and miR-365-3p was screened out to be the only miRNA exclusively responsive to IL-17. miR-365-3p, whose expression was significantly downregulated upon IL-17 stimulation, was demonstrated to exert remarkable anti-inflammatory effect to decrease IL-17-provoked inflammatory cytokines (KC/IL-8 and IL-6) in both airway epithelial cells and macrophages of murine and human origins, verifying its universal antagonizing activity against IL-17-initiated inflammation across the two species. ARRB2 was characterized as the key target of miR-365-3p to negate IL-17-induced inflammatory cytokines.ConclusionTaken together, our data supported the notion that miR-365-3p, which was diminished by IL-17 in murine and human asthmatic pathogenesis, functioned as an essential negative mediator in IL-17-stimuated inflammatory response by targeting ARRB2, which would shed new light to the understanding and therapeutics thereof of asthmatic inflammation.

When the drug induces the organism to produce a type Ⅰ allergic reaction, the combination of IgE and mast cells results in the degranulation of the mast cells. Release of vasoactive substances, increase in vascular permeability, and exudation of intravascular substances outside the blood vessels. Based on this pathophysiological mechanism, a mouse model that can objectively and quantitatively assess the allergic response to the injection has been established. ICR mice were sensitised by intraperitoneal injection of different doses of OVA once every two days for three times. 14 days after the last sensitization, a combination OVA solution of 4 times the sensitizing dose and Evans blue were injected intravenously into mice for the challenge. Compared with the normal group, OVA 0.625/2.5, 1.25/5, 2.5/10, 5/20 mg·kg~(-1) sensitized and challenged can induce allergic reactions mainly manifested by blue staining of the auricle in mice. Direct injection of OVA intravenously did not cause an auricular blue colouration reaction in mice. The passive cutaneous anaphylaxis reaction in mice was conducted with the aforementioned OVA-sensitized mouse serum, and there were obvious blue spots on the mouse's back. In addition, the content of anti-OVA-IgE in 5 mg·kg~(-1) OVA-sensitized mice was significantly increased. Ears and lungs of mice sensitized to OVA showed evident exudation inflammation. Significantly elevated inflammatory factors(VEGF and IL-10) were also detected in the serum of OVA-sensitized mice. The equivalent dose of OVA caused obvious allergic reactions in both guinea pigs and mice. Compared with nude mice, ICR and BALB/c mice are more sensitive to OVA sensitization. Injections of selected TCMI did not induce type Ⅰ allergic reactions in mice and guinea pigs, but there was a risk of inducing pseu-doallergic reactions in mice. The model is problematic and may well reflect the sensitization effect of allergens. It obtains the benefits of simple operation, accuracy, low cost, easy extension, and high repeatability. It is suitable for predicting and researching for IgE-dependent type Ⅰ allergic reactions.

Background Spermine is frequently elevated in tumor tissues and body fluids of cancer patients and is critical for cancer cell proliferation, migration and invasion. However, the immune functions of spermine in hepatocellular carcinoma progression remains unknown. In the present study, we aimed to elucidate immunosuppressive role of spermine in hepatocellular carcinoma and to explore the underlying mechanism. Methods Whole-blood spermine concentration was measured using HPLC. Human primary HCC tissues were collected to examine the expression of CaSR, p-Akt, β-catenin, STT3A, PD-L1, and CD8. Mouse model of tumorigenesis and lung metastasis were established to evaluate the effects of spermine on hepatocellular carcinoma. Western blotting, immunofluorescence, real time PCR, digital Ca2+ imaging, and chromatin immunoprecipitation assay were used to investigate the underlying mechanisms by which spermine regulates PD-L1 expression and glycosylation in hepatocellular carcinoma cells. Results Blood spermine concentration in the HCC patient group was significantly higher than that in the normal population group. Spermine could facilitate tumor progression through inducing PD-L1 expression and decreasing the CD8+ T cell infiltration in HCC. Mechanistically, spermine activates calcium-sensing receptor (CaSR) to trigger Ca2+ entry and thereby promote Akt-dependent β-catenin stabilization and nuclear translocation. Nuclear β-catenin induced by spermine then activates transcriptional expression of PD-L1 and N-glycosyltransferase STT3A, while STT3A in turn increases the stability of PD-L1 through inducing PD-L1 protein N-glycosylation in HCC cells. Conclusions This study reveals the crucial function of spermine in establishing immune privilege by increasing the expression and N-glycosylation of PD-L1, providing a potential strategy for the treatment of hepatocellular carcinoma.

Eye size is a key parameter of visual function, but the precise mechanisms of eye size control remain poorly understood. Here, we discovered that the lipogenic transcription factor sterol regulatory element-binding protein 2 (SREBP2) has an unanticipated function in the retinal pigment epithelium (RPE) to promote eye size in postnatal mice. SREBP2 transcriptionally represses low density lipoprotein receptor-related protein 2 (Lrp2), which has been shown to restrict eye overgrowth. Bone morphogenetic protein 2 (BMP2) is the downstream effector of Srebp2 and Lrp2, and Bmp2 is suppressed by SREBP2 transcriptionally but activated by Lrp2. During postnatal development, SREBP2 protein expression in the RPE decreases whereas that of Lrp2 and Bmp2 increases as the eye growth rate reduces. Bmp2 is the key determinant of eye size such that its level in mouse RPE inversely correlates with eye size. Notably, RPE-specific Bmp2 overexpression by adeno-associated virus effectively prevents the phenotypes caused by Lrp2 knock out. Together, our study shows that rapid postnatal eye size increase is governed by an RPE-derived signaling pathway, which consists of both positive and negative regulators of eye growth.

Background The typical clinical symptoms of allergic rhinitis (AR) are known to be associated with allergen exposure; however, the underlying mechanisms are not fully understood. We wanted to gain a comprehensive view of the molecular mechanisms related to allergen exposure in a well-controlled mouse model of AR. Methods An OVA-induced AR model was developed. Two hours and 4 weeks after the last OVA challenge, AR symptoms and local immune responses were assessed. At the same time, differentially expressed genes (DEG) in nasal mucosa were identified by gene expression microarray and further analyzed by bioinformatics methods. Verification of DEG was done by quantitative RT-PCR and immunohistochemistry. Results The number of nasal rubbings and sneezes, serum OVA-specific IgE concentrations, and the number of neutrophils and eosinophils in the nasal mucosa were significantly increased at 2 h and decreased at 4 weeks after the last allergen challenge compared to controls. A total of 2119 DEG were identified, and their expression dynamics were clustered into 8 profiles. Enriched functions in Profile 5, which had a similar trend to clinical features, were mainly related to inflammatory and immune response to environmental factors, eosinophils and neutrophils chemotaxis, and cell migration. Gene co-expression Network for genes from profile 5 identified BCL3, NFKB2, SOCS3, and CD53 having a higher degree. Profile 6 showed persistence of inflammatory and immune response at 4 weeks after the last allergen challenge. Olfactory and coagulation functions were enriched mainly in profiles with downward trends. Conclusions A wide range of genes with sequential cooperative action were identified to be associated with allergen exposure in AR. BCL3 may be the most vital in symptoms manifestation. Moreover, some inflammatory responses persisted for a period after allergen exposure, supporting a new treatment strategy of targeting inflammation out of season. This study may contribute to a better understanding of AR pathogenesis and provide potential therapeutic targets for AR patients.

Here, we present a detailed protocol for identification and analysis of senescent neuronal stem cells (NSCs) in the developing mouse embryonic neocortex using

Anti-aging is a challenging and necessary research topic.

Oral colon-targeting preparation can achieve targeted drug release in the lesion site and have a great application prospect. In this study, we presented the principle of particle design in the field of materials science into the preparation of colon-targeting preparation. Enzymatic Pulsatilla saponins Colon-targeting composite Microparticles (EPCM) were prepared by mechanical grinding without any organic solvent. Firstly, Response Surface Methodology (RSM) designed by Box-Behnken was used to optimize the preparation process of EPCM, and the structure of EPCM was characterized. All-Atomic and Molecular Dynamics (AAMD) was used to calculate the compatibility between drugs and coating materials before and after release, so as to explain the principle of colon- targeted drug release in this method. Then, colon-targeting characteristics of EPCM in vitro and in vivo were investigated by experiments. Finally, the pharmacodynamics effects of this composite microparticles on Ulcerative Colitis (UC) induced by DSS in C57BL/6 mice were evaluated. The results demonstrated that the colon-targeting composite microparticles could be prepared by mechanical grinding based on particle design principle. The composite microparticles have appropriate colon-targeting drug release performance in vitro and in vivo, and have good anti-ulcerative colitis effect. This study provides a new idea for the preparation of oral colon-targeting preparation of Traditional Chinese medicine, and has evident reference value for the study of coating isolation and powder modification of traditional Chinese medicine for other purposes.