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Objective To investigate the influence of transient receptor potential cation channel 6 (TRPC6) on renal tubulointerstitial inflammation in mice with diabetic kidney disease (DKD) and its mechanism. Methods A total of 36 male C57/BL6J mice, aged 6 weeks, were randomly divided into control group (group A), DKD model group (group B), DKD+normal saline intervention group (group C), DKD+mitophagy activator intervention group (group D), DKD+negative control lentivirus transfection group (group E), and DKD+TRPC6 knockdown lentivirus transfection group (group F), with 6 mice in each group. The mice in groups A and B were respectively given intraperitoneal injection of 0.1 mmol/L citrate buffer and 10 g/L streptozotocin (hereinafter referred to as administration); the mice in groups C and D were respectively given normal saline and 10 mmol/L urolithin A by gavage in addition to the treatment in group B; the mice in groups E and F were respectively injected with negative control lentivirus and TRPC6 knockdown lentivirus via the tail vein in addition to the treatment in group B. The levels of fasting blood glucose (FBG), urinary albumin-to-creatinine ratio (ACR), and blood urea nitrogen (BUN) were measured at week 12 after administration; PAS staining was used for the observation and scoring of renal tubular injury; RT-qPCR was used to measure the mRNA expression levels of inflammatory factors [interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α)] in renal tissue; immunohistochemical staining was used to measure the protein expression level of TRPC6 in renal tissue, and Western blotting was used to measure the relative expression levels of TRPC6, LC3B, P62, PINK1, and Parkin in renal tissue; transmission electron microscopy was used to observe the change in the number of mitophagosomes in renal tubular cells of mice. HK-2 cells were divided into high glucose+TRPC6 siRNA transfection+DMSO intervention group (group G, treated with TRPC6 siRNA+35.0 mmol/L glucose+0.06% DMSO) and high glucose+TRPC6 siRNA transfection+mitophagy inhibitor intervention group (group H, treated with TRPC6 siRNA+35.0 mmol/L glucose+12 μmol/L oroxylin A), and then RT-qPCR was used to measure the mRNA expression levels of inflammatory factors (IL-1β, MCP-1, and TNF-α) in cells. Results At week 12 after administration, compared with group A, group B had significantly higher whole blood FBG, ACR, BUN, renal tubular injury score, mRNA expression levels of inflammatory factors, and protein expression levels of TRPC6 and P62 in renal tissue (t=2.77-13.61,P

Objective To investigate the method for establishing an immortalized mouse corneal epithelial cell line, and to identify the stability of its phenotype. Methods Primary mouse corneal epithelial cells were extracted and transfected with Simian Vacuolating Virus 40 Large T Antigen (SV40-LT) lentivirus to establish an immortalized cell line. HE staining and immunofluorescent staining were used to identify the phenotypes and markers (K12, Pax6, Ki-67) of corneal epithelium. Primary cor-neal epithelial cells were extracted to identify K14 protein in corneal epithelial cell layer, and bright field observation was performed to evaluate cell morphological characteristics. Several markers (K12, Pax6, Ki-67, ABCG2, K14) were identified for P1 and P8 cells after transfection. Crystal violet staining was used to observe the colony-formation ability of P1 and P8 cells after transfection. Results Mouse corneal epithelium was stained positive for K12, Pax6 and Ki-67, while the P1 and P8 generations of mouse cor-neal epithelial cells were stained positive for K12, Pax6, Ki-67, K14, and ABCG2; there were no significant differences in the positive rates of the above markers (P>0.05). There was no significant difference in colony-formation ability between P1 and P8 cells (P>0.05). Conclusion The immortalized mouse corneal epithelial cell line is successfully established by transfection with SV40-LT lentivirus, which has stable phenotype and proliferation.

Objective To investigate the neuroprotective effects and mechanism of CMS121 in a neuron model of oxygen-glucose deprivation/reperfusion (OGD/R) and a mouse model of ischemia/reperfusion (I/R). Methods Cortical neurons were divided into control group (normal culture), OGD/R group (OGD/R model construction), and OGD/R+CMS121 group (OGD/R model construction plus treatment with CMS121). All neurons’ viability and lactate dehydrogenase (LDH) release were assessed after 24 h of reperfusion. Western blotting was performed to measure the expression of protein kinase B (Akt) and phosphorylated Akt (p-Akt) in the neurons after 6 and 24 h of reperfusion. C57BL/6J mice were randomly divided into sham group (sham operation), I/R group (I/R model construction), and I/R+CMS121 group (I/R model construction plus treatment with CMS121). After 24 h of reperfusion, we assessed the neurological damage of the mice with the Neurological Severity Score (NSS), and calculated the brain infarct volume (as a percent of total brain volume) after 2,3,5-triphenyltetrazolium chloride staining. Results There were significant differences in cell viability and LDH release between the three neuron groups (F=71.21,88.93,P0.05). After 6 h reperfusion, a significant difference was observed in neuronal p-Akt expression between the three neuron groups (F=18.78,P0.05). After 24 h reperfusion, p-Akt expression differed significantly between the three neuron groups (F=38.50,P0.05). There were significant differences in the NSS score and infarct volume between the three mouse groups (F=20.24,118.60,P

Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Pure frailty and obese frailty are common types of frailty syndrome. However, the overlapping and distinct characteristics between pure frailty and obese frailty remain unclear. This study aims to reveal the overlapping/distinct physical and biological phenotypes of pure frailty and obese frailty, providing theoretical support for their prevention, diagnosis, and treatment., Method: Mice were fed either a normal or high-fat diet and assessed at 20 months of age. They were assigned to one of the four groups: control, obesity, pure frailty, and obese frailty. Grip strength, walking speed, physical activity, endurance, and body weight were measured to determine pure frailty and obese frailty. Physical and biological phenotypes were assessed., Results: Distinct physical phenotypes were observed between pure frailty and obese frailty in terms of body weight, lean mass, fat mass, fat mass in tissue, grip strength, endurance, and physical activity, while walking speed overlapped. In biological phenotypes, levels of Smad2/3, FoxO3a, P62, LAMP-2, and cathepsin L expression were distinct, while AKT, p-AKT, mTOR, p-mTOR, p-Smad2/3, p-FoxO3a, Beclin-1, ATG7, and LC3 overlapped., Conclusion: Distinct physical phenotypes observed in obese frailty are primarily attributable to the effect of obesity, with further impairment of muscle function resulting from the combined effects of frailty syndromes and obesity. Pure frailty and obese frailty share overlapping biological phenotypes, particularly in the regulation of muscle protein synthesis. Moreover, the interaction between obesity and frailty syndromes gives rise to both overlapping and distinct biological phenotypes, especially in the regulation of specific degradation signaling proteins., (© 2024 The Author(s).)

Altered hepatic glucose fluxes are critical during the pathogenesis of type 2 diabetes. G protein-coupled receptors represent important regulators of hepatic glucose production. Recent studies have shown that hepatocytes express GPCRs that can couple to G 12/13 , a subfamily of heterotrimeric G proteins that has attracted relatively little attention in the past. Here we show, by analyzing several mutant mouse strains, that selective activation of hepatocyte G 12/13 signaling leads to pronounced hyperglycemia and that this effect involves the stimulation of the ROCK1-JNK signaling cascade. Using both mouse and human hepatocytes, we also show that activation of endogenous sphingosine-1-phosphate type 1 receptors strongly promotes glucose release in a G 12/13 -dependent fashion. Studies with human liver samples indicate that hepatic GNA12 (encoding Gα 12 ) expression levels positively correlate with indices of insulin resistance and impaired glucose homeostasis, consistent with a potential pathophysiological role of enhanced hepatic G 12/13 signaling., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Pathological cardiac hypertrophy stands as a pivotal mechanism contributing to diverse cardiovascular diseases, ultimately leading to heart failure. Despite its clinical significance, the intricate molecular mechanisms instigating pathological cardiac hypertrophy remain inadequately understood. In this study, we aim to further reveal its complex pathogenesis by exploring the role of Fas apoptotic inhibitory molecule 2 (FAIM2) in modulating pathological cardiac hypertrophy., Methods and Results: We used phenylephrine-induced hypertrophic cardiomyocytes and also generated cardiac-specific knockout mice and adeno-associated virus serotype 9-Faim2 mice to evaluate the function of FAIM2 in pathological myocardial hypertrophy. Furthermore, unbiased RNA-sequencing analysis was used to identify the direct target and corresponding molecular events contributing to FAIM2 function. Ultimately, our study revealed a downregulation of FAIM2 expression in phenylephrine-induced hypertrophic cardiomyocytes and pressure overload-induced hypertrophic hearts. FAIM2 exhibited a significant attenuation of phenylephrine-induced enlargement of primary neonatal rat cardiomyocytes, whereas FAIM2 knockdown aggravated the hypertrophic response. Furthermore, Faim2 gene knockout significantly exacerbated cardiac hypertrophy and heart fibrosis in vivo. Mechanistic investigations unveiled that FAIM2 exerts its inhibitory effect by suppressing TAK1-JNK1/2-p38 MAPK signaling cascades, thereby mitigating cardiac hypertrophy., Conclusions: Our findings position FAIM2 as a novel negative regulator of pathological cardiac hypertrophy through its inhibitory action on mitogen-activated protein kinase signaling activation. This identification of FAIM2's role provides crucial insights that may pave the way for the development of effective therapeutic strategies aimed at mitigating pathological cardiac hypertrophy, addressing a critical need in cardiovascular disease management.

Background: Drug discovery strongly relies on the thorough evaluation of preclinical experimental studies. In the context of pulmonary fibrosis, micro-computed tomography (µCT) and histology are well-established and complementary tools for assessing, in animal models, disease progression and response to treatment. µCT offers dynamic, real-time insights into disease evolution and the effects of therapies, while histology provides a detailed microscopic examination of lung tissue. Here, we present a semi-automatic pipeline that integrates these readouts by matching individual µCT volume slices with the corresponding histological sections, effectively linking densitometric data with Ashcroft score measurements., Methods: The tool first geometrically aligns the vertical axis of the µCT volume with the cutting plane used to prepare the histological sample. Then, focusing on the left lung, it computes the affine registration that identifies the µCT coronal slice that best matches the histological section. Finally, quantitative µCT imaging parameters are extracted from the selected slice. In a proof-of-concept test, the tool was applied to a bleomycin-induced mouse model of lung fibrosis., Results: The proposed approach demonstrated high accuracy and time effectiveness in matching µCT and histological sections minimizing manual intervention, with an overall success rate of 95%, and reduced time required to align µCT and histological data from 40 to 5 min. Significant correlations were found between quantitative data derived from µCT and histology data., Conclusions: The precise combination of microscopic ex-vivo information with 3D in-vivo data enhances the accuracy and representativeness of tissue analysis and provides a structural context for omic studies, serving as the foundation for a multi-layer platform. By facilitating a detailed and objective view of disease progression and treatment response, this approach has the potential to accelerate the development of effective therapies for lung fibrosis., Competing Interests: Declarations Ethics approval and consent to participate All animal experiments described herein were authorized by the official competent authority and approved by the intramural animal-welfare body (AWB) of Chiesi Farmaceutici and authorized by the Italian Ministry of Health (protocol number: 449/2016-PR). All procedures were conducted in compliance with the European Directive 2010/63 UE, Italian D.Lgs 26/2014, the revised “Guide for the Care and Use of Laboratory Animals” and with the ‘Animal Research: Reporting of In Vivo Experiments’ (ARRIVE) guidelines. Consent for publication Not applicable. Competing interests FFS, GV and EF are employees of Chiesi Farmaceutici S.p.A., which supported the research work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential competing interests., (© 2024. The Author(s).)

Ischemic stroke poses a significant global health risk. Currently, recanalization of blood flow through surgery or medication is the only effective means to control ischemia-reperfusion injury. This study aims to explore the role and molecular mechanism of OGT in regulating neuronal injury and motor deficits following a stroke. The MCAO and OGD/R models were established to validate the therapeutic efficacy of OGT in mitigating neuronal injury and motor dysfunction following stroke. Molecular biological techniques were employed to assess ferroptosis levels, OGT ubiquitination, and SLC7A11 O-GlcNAcylation. OGT has a therapeutic effect on motor deficits and neuronal damage after stroke by regulating SLC7A11 O-GlcNacylation-mediated ferroptosis, while the KDM2B-mediated ubiquitination pathway is responsible for changes in OGT levels. These findings are crucial for target selection and biomarker identification in stroke treatment., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Adoptive T-cell therapy has demonstrated clinical activity in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors experience primary or secondary resistance to this treatment modality. Target antigen loss resulting either from non-uniform antigen expression or defects in antigen processing and presentation machinery is one well-characterized resistance mechanism. Constitutively expressed membrane-anchored interleukin-12 (caIL-12) has demonstrated enhanced antitumor activity and low systemic exposure in multiple preclinical adoptive T-cell treatment models with homogeneous tumor antigen expression. In this study, we assess the therapeutic impact of caIL-12 on target antigen-negative variants in syngeneic mouse models., Methods: Target antigen-positive tumors were generated by transducing B16F10 melanoma cells (B16) or Lewis Lung Carcinoma cells (LLC) with a construct expressing the OVA antigen, SIINFEKL, tagged to ubiquitin (B16-U-OVA, LLC-U-OVA), while B16 or LLC tumors served as antigen-negative variants. C57BL/6J mice were subcutaneously injected with heterogeneous tumors composed of 80% B16-U-OVA and 20% B16. Bilateral tumors were established by injecting the left flank with B16-U-OVA or LLC-U-OVA tumors and the right flank injected with B16 or LLC tumors. The tumor-bearing mice then underwent 5.5 Gy total body irradiation, followed by adoptive transfer of OT-I TCR-T cells engineered with or without caIL-12., Results: TCR-T cells (OT-I) delivered caIL-12 to the B16-U-OVA tumor sites and induced robust tumor control and survival benefits in mice bearing a heterogeneous tumor with OVA-negative variants. caIL-12 exerted its effect on OVA-negative B16 variants primarily by priming and activating endogenous antitumor CD8 T cells via antigen spreading. In addition, antigen spreading induced by OT-I-caIL-12 resulted in controlling OVA-negative tumors implanted at distant sites. This therapeutic effect required antigen-specific TCR-T cells and caIL-12 to colocalize at the tumor site, along with endogenous CD8 T cells capable of recognizing shared tumor antigens., Conclusion: Expression of caIL-12 by tumor-targeting T cells demonstrated therapeutic effect against target-antigen-negative tumor variants, primarily through the induction of antigen spreading. These findings highlight the potential of caIL-12 to address challenges of antigen escape and tumor heterogeneity that may limit the efficacy of T-cell therapy against solid tumors., Competing Interests: Competing interests: LZ is an inventor on NIH patent of inducible IL-12. CSH owns patents and royalties from multiple inventions in the field of immunotherapy and cellular therapy at the NIH and at Rutgers Cancer Institute, serves as a consultant or advisory board for Capstan Therapeutics, Neogene Therapeutics, Vir Biotechnology, sponsored research from Neogene Therapeutics, Iovance Biotherapeutics, equity and company officer for Scarlet TCR. The remaining authors declare no competing financial interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The lymphatic system plays a crucial role in immune function and the removal of cellular waste. Recent studies have highlighted the presence of primo vessels (PVs) inside lymphatic vessels, distinct from conventional lymphatic tissues, yet their structural and functional characteristics remain poorly understood, particularly PVs inside deep cervical lymphatic vessels (dcLVs) connected to the meningeal lymphatics. We used a combination of Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Alcian Blue (AB) and H&E staining techniques to identify and characterize PVs inside the dcLVs of mice. PVs were identified inside the dcLVs, revealing distinct bundle structures composed of 3 to 5 primo subvessels (sub-PV), with diameters of 5.4 ± 2.4 μm. AFM analyses confirmed the presence of nm-sized pores on the sub-PVs, which may facilitate the absorption and retention of nanoparticles. Rod-shaped nuclei were also observed, further distinguishing PVs from other vascular structures. Our study provides new insights into the structural characteristics of PVs inside dcLVs, suggesting their potential role in targeted drug delivery. However, further research is required to explore the functional implications of these structures, especially their therapeutic applications and roles in various lymphatic regions, including the meningeal lymphatics., Competing Interests: Declarations Competing interests The authors declare no competing interests.All authors have no conflicts of interest relevant to this study to disclose. Approval for animal experiments This research was approved by the Wonkwang University Animal Experiment Ethics Committee (WKU23-48)., (© 2024. The Author(s).)

Background: Postoperative cognitive dysfunction (POCD) is a prevalent complication following anesthesia and surgery, particularly in the elderly, leading to increased mortality and reduced quality of life. Despite its prevalence, there are no effective clinical treatments. Exercise has shown cognitive benefits in aging and various diseases, which can be transferred to sedentary animals through plasma. However, it is unclear if exercise-conditioned plasma can replicate these benefits in the context of POCD., Methods: Sixteen-month-old male C57BL/6J mice underwent 30 days of voluntary running wheel training or received systemic administration of exercise-conditioned plasma, followed by tibial fracture surgery under general anesthesia at 17 months of age. Cognitive performance, hippocampal synaptic deficits, neuroinflammation, BDNF/TrkB signaling, and medial septum (MS)-hippocampal cholinergic activity were evaluated through immunohistochemical staining, transmission electron microscopy, Western blotting, and biochemical assays. To investigate the role of hippocampal BDNF signaling and cholinergic activity in the therapeutic effects, the TrkB antagonist ANA-12 and the cholinergic receptor muscarinic 1 (CHRM1) antagonist trihexyphenidyl (THP) were administered via intraperitoneal injection, and adeno-associated virus (AAV) vectors expressing Chrm1 shRNA were delivered via intrahippocampal stereotaxic microinjection., Results: Exercise-conditioned plasma mimicked the benefits of exercise, alleviating cognitive decline induced by anesthesia/surgery, restoring hippocampal synapse formation and levels of regulators for synaptic plasticity, inhibiting neuroinflammatory responses to surgery by microglia and astrocytes, augmenting BDNF production and TrkB phosphorylation in hippocampal neurons, astrocytes, and microglia, upregulating MS expression of choline acetyltransferase (CHAT) and hippocampal expression of CHRM1 in neurons and astrocytes, and enhancing hippocampal cholinergic innervation and acetylcholine release. Conversely, ANA-12 administration blocked TrkB activation and reduced the protective effects on cognition, synaptic deficits, and neuroinflammatory reactivity of glial cells post-surgery. Similarly, THP administration or intrahippocampal delivery of AAV-Chrm1 shRNA inhibited the activation of the hippocampal cholinergic circuit by exercise plasma, negating the cognitive and neuropathological benefits and reducing BDNF/TrkB signaling enhancements., Conclusion: Exercise-conditioned plasma can replicate the protective effects of exercise against anesthesia/surgery-induced neuroinflammation, synaptic, and cognitive impairments, at least partly, through CHRM1-dependent regulation of hippocampal cholinergic activity and BDNF/TrkB signaling., Competing Interests: Declarations Ethic approval and consent to participate All animal handling and use in this study were approved by Institutional Research Board of Harbin Medical University (No. HMUIRB2023016). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological functions through its receptors on the cell membrane. As one of the six LPA receptors, LPA receptor 3 (LPAR3) is highly expressed in mouse kidneys, but its physiological function in the kidney has been poorly explored., Methods: Wild-type (WT) and Lpar3 -/- mice were used to investigate the renal physiological function of LPAR3 under hypoxia. The expression levels of LPA receptors in the kidneys of WT mice with or without exposure to hypoxia (8% O 2 ) were detected by RT‒qPCR. RNA sequencing analysis was performed to identify differences in gene expression profiles between the hypoxic kidneys of WT and Lpar3 -/- mice. The effects of LPAR3 deficiency and treatment with the LPAR1/3 inhibitor Ki16425 or the LPAR3 selective agonist 2S-OMPT on erythropoietin (EPO) production in the kidneys of hypoxic mice were determined by RT‒qPCR and ELISAs. The mechanism of LPAR3-mediated regulation of EPO expression was further studied in vivo with mouse models and in vitro with cultured human cells., Results: LPAR3 is the major LPA receptor in mouse kidneys, and its expression is significantly upregulated under hypoxic conditions. RNA sequencing analysis revealed that, compared with WT mice, Lpar3 -/- mice presented a significant decrease in hypoxia-induced EPO expression in the kidney, together with reduced plasma EPO levels and lower hematocrit and hemoglobin levels. Hypoxic renal EPO expression in WT mice was diminished by the administration of the LPAR1/3 inhibitor Ki16425 and increased by 2S-OMPT, a selective agonist of LPAR3. Hypoxia-induced HIF-2α accumulation in mouse kidneys was impaired by LPAR3 deficiency. Further studies revealed that the PI3K/Akt pathway participated in the regulation of HIF-2α accumulation and EPO expression by LPAR3 under hypoxic conditions., Conclusions: Our study revealed the role of LPAR3 in promoting the HIF-2α‒EPO axis in hypoxic mouse kidneys, suggesting that the LPA receptor may serve as a novel potential pharmaceutical target to regulate renal EPO production in hypoxia-related situations, such as chronic kidney disease and altitude disease., Competing Interests: Declarations Ethical approval All animal experiments in this study were approved by the Ethics and Animal Welfare Committee, College of Life Sciences, Beijing Normal University. The study complied with the relevant ethical regulations pertaining to animal research, and all laboratory animals were cared for and used according to institutional guidelines. Consent for publication All authors have read and agreed with the submission of the manuscript to Lipids in Health and Disease. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Sepsis is a severe condition characterized by multiple organ dysfunction resulting from an imbalanced host immune response to infections. miRNAs play a crucial role in regulating various biological processes. However, the precise role of miR-31 in the immunopathology of sepsis remains poorly understood., Methods: The concentration of hsa-miR-31-5p in patients with sepsis (both survivors and non-survivors) and healthy individuals was assayed. Using an experimental sepsis model of caecal ligation and puncture (CLP), the impact of mmu-miR-31-5p on survival, organ injury, and inflammation was evaluated. Additionally, the effect of mmu-miR-31-5p on macrophage polarization through Chi3l1 was investigated. Lastly, the therapeutic effects of miPEP31 on experimental sepsis were examined., Results: The results of miRNA sequencing (miRNA-seq) and quantitative polymerase chain reaction (q-PCR) analyses identified hsa-miR-31-5p as a potential biomarker for patients with sepsis, with non-survivors showing higher levels of hsa-miR-31-5p in peripheral blood mononuclear cells (PBMCs) compared to survivors. Functional studies conducted on peritoneal elucidated macrophages (PEMs) demonstrated that mmu-miR-31-5p inhibits M2 polarization in macrophages by downregulating Chi3l1. The utilization of miPEP31 as a therapeutic intervention had a substantial impact on reducing mortality rates, mitigating organ damage, inducing macrophage polarization towards the M2 phenotype, and suppressing the inflammatory response in murine models of severe sepsis., Conclusions: The suppression of miR-31 in sepsis plays a protective role in the host defense response by upregulating Chi3l1, highlighting the potential therapeutic efficacy of miPEP31 in sepsis treatment., Competing Interests: Declarations Ethics approval and consent to participate All procedures performed in studies involving human participants were approved by the Ethics Committee of Shanghai General Hospital under ethical approval number 2022SQ098. All animal experiments were conducted under the rules approved by the Experimental Animal Management Ethics Committee of Shanghai Jiao Tong University School of Medicine (ethical approval number: 2022AWS0160). Consent for publication All authors listed have provided their consent for submission, and all data utilized in this study have been obtained with the consent of the individuals responsible for generating the data. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Bipolar disorder is a complex polygenic disorder that is characterized by recurrent episodes of depression and mania, the heterogeneity of which is likely complicated by epigenetic modifications that remain to be elucidated., Methods: We performed transcriptomic analysis of peripheral blood RNA from monozygotic (MZ) twins discordant for bipolar disorder to identify disease-associated differentially expressed long noncoding RNAs (DE-lncRNAs), which were further validated in the PsychENCODE brain RNA-seq dataset. We then performed behavioral tests, electrophysiological assays, chromatin immunoprecipitation, and PCR to investigate the function of DE-lncRNAs in the mouse and cell models. Statistical analyses were performed using GraphPad Prism 9.0 or SPSS., Results: We identified a bipolar disorder-associated upregulated long non-coding RNA (lncRNA), AP1AR-DT. We observed that overexpression of AP1AR-DT in the mouse medial prefrontal cortex (mPFC) resulted in a reduction of both the total spine density and the spontaneous excitatory postsynaptic current (sEPSC) frequency of mPFC neurons as well as depressive and anxiety-like behaviors. A combination of the results of brain transcriptome analysis of AP1AR-DT overexpressing mice brains with the known genes associated with bipolar disorder revealed that NEGR1, which encodes neuronal growth regulator 1, is one of the AP1AR-DT targets and is reduced in vivo upon gain of AP1AR-DT in mice. We further demonstrated that overexpression of recombinant Negr1 in the mPFC neurons of AP1AR-DT OE mice ameliorates depressive and anxiety-like behaviors and normalizes the reduced excitatory synaptic transmission induced by the gain of AP1AR-DT. We finally identified that AP1AR-DT reduces NEGR1 expression by competing for the transcriptional activator NRF1 in the overlapping binding site of the NEGR1 promoter region., Conclusions: The epigenetic and pathophysiological mechanism linking AP1AR-DT to the modulation of depressive and anxiety-like behaviors and excitatory synaptic function provides etiological implications for bipolar disorder., Competing Interests: Declarations Ethics approval and consent to participate All participants in this study provided informed consent prior to the study following presentation of the nature of the procedures. The study was approved by the Medical Ethics Committee of Zhujiang Hospital of Southern Medical University (#2022-KY-086) and the third People’s Hospital of Zhongshan (SSYLL20210301). The study was conducted in accordance with the Declaration of Helsinki. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

TET-family members play a critical role in cell fate commitment. Indeed, TET3 is essential to postnatal development due to yet unknown reasons. To define TET3 function in cell differentiation, we have profiled the intestinal epithelium at single-cell level from wild-type and Tet3 knockout mice. We have found that Tet3 is mostly expressed in differentiated enterocytes. In the absence of TET3, enterocytes exhibit an aberrant differentiation trajectory and do not acquire a physiological cell identity due to an impairment in oxidative phosphorylation, specifically due to an ATP synthase assembly deficiency. Moreover, spatial metabolomics analysis has revealed that Tet3 knockout enterocytes exhibit an unphysiological metabolic profile when compared with their wild-type counterparts. In contrast, no metabolic differences have been observed between both genotypes in the stem cell compartment where Tet3 is mainly not expressed. Collectively, our findings suggest a mechanism by which TET3 regulates mitochondrial function and, thus, terminal cell differentiation at the metabolic level., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Individuals with diabetes frequently face serious challenges, including delayed wound healing and increased risk of infection. Notably, the regeneration of hair follicles plays a crucial role in accelerating diabetic skin damage repair, reducing the risk of infection, and enhancing overall skin health. Research has predominantly emphasized the re-epithelialization of diabetic wounds, with a paucity of in-depth studies on hair follicle regeneration. In the current study, we explored the effects of a bioactive amphibian-derived peptide, Cy RL-QN15 , on promoting hair regeneration in a diabetic skin model. In vivo experiments demonstrated that local treatment with Cy RL-QN15 not only accelerated wound healing of scalded skin on the backs of diabetic Kunming (KM) mice but also improved growth of damaged hair follicles. Additionally, back-shaved diabetic C57BL/6 mice showed a significant increase in the growth of newly formed hair after 28 days of continuous Cy RL-QN15 treatment. Further analysis indicated that the hair-regenerating effects of Cy RL-QN15 were closely associated with the proliferation and migration of hair follicle stem cells (HFSCs). Cy RL-QN15 enhanced intracellular β-catenin expression by binding to the Frizzled-7 receptor on the surface of HFSCs. The up-regulation in β-catenin modulated the levels of downstream proteins, such as c-MYC, Cyclin D1, and Lef1, ultimately inducing hair regeneration. This study not only reveals the robust effects of the bioactive peptide Cy RL-QN15 in hair follicle regeneration but also provides novel avenues for the development of more targeted and effective therapeutics for diabetic wound healing in the future.

Background: Mesenchymal stromal cell (MSC) therapy commonly involves systemic infusion of MSCs, which undergo apoptosis in the lung and induce immunoregulatory macrophages that reduce disease. The relevance of this mode of action, however, is yet to be determined for MSCs administered via other routes. Here, we administered MSCs via subcutaneous (SC) injection into inflamed tissue and investigated the immunomodulatory effects on the local lymph node (LN), which is a major site for the initiation and regulation of immune responses., Methods: A mouse model of localised skin inflammation was established with low-dose lipopolysaccharide (LPS) to in vivo prime adipose-derived MSCs delivered via SC injection. We then analysed the immunomodulatory changes in the LN draining the inflamed tissue, as well as the neutrophil TNF response to LPS re-exposure., Results: When administered directly into the inflamed skin tissue, SC MSC injection induced an expansion of IL-10-producing MerTK + subcapsular sinus macrophages and T cell zone macrophages, as well as activated CD44 + regulatory T cells (Tregs), in the draining LN, which was not observed in the non-draining LN. SC injection of viable, but not apoptotic, MSCs dampened TNF production by inflammatory cells in the draining LN when re-exposed to the inflammatory stimulus. SC injection of MSCs remote to the site of inflammation also did not attenuate the LN response to subsequent inflammatory challenge., Conclusions: MSCs delivered directly into inflamed skin activated immunoregulatory cells in the local LN and inhibited LN responsiveness to subsequent inflammatory challenge. The immunoregulatory effects of SC-injected MSCs in the LN require priming by inflammatory cytokines in the local milieu. Furthermore, SC-injected MSCs exert anti-inflammatory effects in the draining LN prior to their apoptosis, in contrast to intravenously delivered MSCs, where anti-inflammatory effects are linked to their apoptosis., Competing Interests: Declarations Ethics approval and consent to participate All animal experiments received approval from Monash University Animal Ethics Committee (Project title: Mesenchymal stromal cells and the innate immune system; Approval number: 23407; Date of approval: 01/04/2020) and were performed in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Human MSCs were isolated from healthy donor lipoaspirate, performed with informed patient consent in accordance with Monash University Human Research Ethics Committee (Project title: Immunomodulatory properties of different stem cell types; Approval number: 2007/1798; Date of approval: 10/10/2007). Consent for publication Not applicable. Competing interests TSPH had received funding from Regeneus Ltd. MRH had also undertaken other related funded research for Regeneus Ltd. FM, CM and GV were employees of Regeneus Ltd. The funders were not involved in the study design, collection, analysis or interpretation of data, the writing of this article or the decision to submit it for publication. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Messenger RNA (mRNA) vaccines are a key technology in combating existing and emerging infectious diseases. However, improving the immunogenicity and durability of mRNA vaccines remains a challenge. To elicit optimal immune responses, integrating antigen-encoded mRNA and immunostimulatory adjuvants into a single formulation is a promising approach to enhancing the efficacy of mRNA vaccines. Here, we report an adjuvant strategy to enhance the efficacy of mRNA vaccines by co-loading mRNA encoding the antigen (rabies virus glycoprotein, RABV-G) and mRNA encoding IL-7 into lipid nanoparticles, achieving co-delivery to the same antigen-presenting cells. A single immunization with G&IL-7 mRNA vaccine elicited robust humoral immune responses in mice and conferred complete protection against RABV challenge. Notably, the high levels of neutralizing antibody induced by the G&IL-7 mRNA vaccine were maintained for at least 6 months, providing mice with long-term significant and complete protection against RABV. Additionally, IL-7 also enhanced antibody responses against the SARS-CoV-2. These data demonstrate that IL-7 is a potent mRNA vaccine adjuvant that can provide the required immune stimulation in various mRNA vaccine formulations., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Dementia, especially Alzheimer's disease, presents a major clinical challenge, and researchers are still searching for an optimal animal model. To address this gap, we compared male and ovariectomized female C57BL/6 mice treated with 30 mg/kg aftin-4, which induces neurodegeneration, with naturally aged (15-16 months old) mice not treated with aftin-4. We performed a series of behavioral tests; measured postmortem plasma β-amyloid levels (Aβ1-40 and Aβ1-42) and the levels of the oxidative stress indicators glutathione peroxidase (GPx), superoxide dismutase (SOD) and malondialdehyde (MDA); and evaluated astrocytic reactivity in the brain using glial fibrillary acid protein (GFAP) levels. Our results revealed no behavioral changes in the aged or aftin-4-treated mice compared with the control mice. Aftin-4 mice presented lower brain MDA levels and no detectable changes in plasma Aβ levels. In general, female mice had higher GPx and SOD levels and lower Aβ1-42 levels than male mice did. In contrast, aged and aftin-4-treated male mice presented elevated levels of GFAP, indicating astrocyte damage. Our results could not confirm that either aftin-4-treated or aged mice are reliable models for dementia. However, the observed molecular changes suggest that male animals may be more susceptible to oxidative stress and brain damage than females are. This study demonstrates the complexity of modeling dementia in animals and the importance of future studies in this area., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Various signaling pathways are essential for both the innate immune response and the maintenance of cell homeostasis, requiring coordinated interactions among them. In this study, a mutation in the caspase-1 recognition site within MyD88 abolished inflammasome-dependent negative regulation, causing phenotypic changes in mice with some similarities to human NEMO-deficiencies. The MyD88 D162E mutation reduced MyD88 protein levels and colon inflammation in DSS-induced colitis mice but did not affect cytokine expression in bone marrow-derived macrophages (BMDMs). However, compared to MyD88 wt counterparts, MyD88 D162E BMDMs had increased oxidative stress and dysfunctional mitochondria, along with reduced prosurvival Bcl-xL and BTK expression, rendering cells more prone to apoptosis, exacerbated by ibrutinib treatment. NF-κB activation by lipopolysaccharide mitigated this sensitive phenotype. These findings underscore the importance of MyD88 wt signaling for NF-κB activation, protecting against macrophage premature apoptosis at resting state. Targeting MyD88 quantity rather than just its signaling could be a promising strategy for MyD88-driven lymphoma treatment., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Lysosomes play a crucial role in metabolic adaptation to starvation, but detailed in vivo studies are scarce. Therefore, we investigated the changes of the proteome of liver lysosomes in mice starved short-term for 6h or long-term for 24h. We verified starvation-induced catabolism by weight loss, ketone body production, drop in blood glucose and an increase of 3-methylhistidine. Deactivation of mTORC1 in vivo after short-term starvation causes a depletion of mTORC1 and the associated Ragulator complex in hepatic lysosomes, resulting in diminished phosphorylation of mTORC1 target proteins. While mTORC1 lysosomal protein levels and activity in liver were restored after long-term starvation, the lysosomal levels of Ragulator remained constantly reduced. To determine whether this mTORC1 activity pattern may be organ-specific, we further investigated the key metabolic organs muscle and brain. mTORC1 inactivation, but not re-activation, occurred in muscle after a starvation of 12 h or longer. In brain, mTORC1 activity remained unchanged during starvation. As mTORC1 deactivation is known to induce autophagy, we further investigated the more than 150 non-lysosomal proteins enriched in the lysosomal fraction upon starvation. Proteasomal, cytosolic and peroxisomal proteins dominated after short-term starvation, while after long-term starvation, mainly proteasomal and mitochondrial proteins accumulated, indicating ordered autophagic protein degradation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The metabolic reprogramming of macrophages is a potential therapeutic strategy for sepsis treatment, but the mechanism underlying this reprogramming remains unclear. Since glycolysis can drive macrophage phenotype switching, the rate-limiting enzymes in glycolysis may be key to treating sepsis. Here, we found that, compared with other isoenzymes, the expression of 6-phosphofructokinase, muscle type (PFKM) was the most upregulated in monocytes from septic patients. Recombinant thrombomodulin (rTM) treatment downregulated the protein expression of PFKM in macrophages. Both rTM treatment and Pfkm knockout protected mice from sepsis and reduced the production of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and IL-27, whereas PFKM overexpression increased the production of these cytokines. Mechanistically, rTM treatment inhibited glycolysis in macrophages by decreasing PFKM expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. HIF-1α overexpression increased methyltransferase-like 3 (METTL3) expression, elevated the m 6 A level on Pfkm, and upregulated the protein expression of PFKM. METTL3 silence attenuated HIF-1α-mediated PFKM expression. These findings provide insight into the underlying mechanism of macrophage reprogramming for the treatment of sepsis., Competing Interests: Declarations Conflict of interest The authors have no relevant financial or nonfinancial interests to disclose. Ethics approval and consent to participate The study was approved by the Ethics Committee of Xuzhou Medical University (ethical approval number: XYFY2022-KL442-01). All animal studies and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University. Consent for publication All the authors approved the submission of the manuscript to this journal., (© 2024. The Author(s).)

Myocardial ischemia-reperfusion (I/R) injury caused by revascularization treatment is the leading cause of cardiac damage aggravation in ischemic heart disease. Increasing evidence has unraveled the crucial role of pyroptosis in myocardial I/R injury. Of note, lactylation has been validated to be participated in modulating pyroptosis. Hence, this study was aimed to elaborate the potential and mechanism of lactylation in myocardial I/R damage. We established the cell model of I/R through inducing hypoxia/reoxygenation (H/R) of H9c2 cells. It was uncovered that H/R stimulation drove cardiomyocyte pyroptosis and upregulated total lactylation level. Further, we demonstrated that promoting lactylation contributed to H/R-evoked pyroptosis, whereas silencing LDHA led to the opposite results. More than that, LDHA was confirmed to facilitate lactylation of NLRP3 at K245 site and increase its protein stability. Our findings indicated that activation of NLRP3 abolished the function of LDHA deficiency in H/R-treated H9c2 cells. In concert with the aforementioned outcomes, knockout of LDHA attenuated the infarct size and myocardial damage in I/R mice and upregulation of NLRP3 counteracted the effects of LDHA knockout on I/R-evoked injury in vivo. To summarize, the current research provided persuasive evidence that LDHA promoted myocardial I/R damage via enhancing NLRP3 lactylation to induce cardiomyocyte pyroptosis., Competing Interests: Declarations Ethics approval and consent to participate The study was approved by the Ethics Committee of MDKN Biotechnology Co., Lt (MDKN-2023-307, 2023.11.02). All experiments were performed in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Clinical trial number Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Recent studies have highlighted the importance of microglia, the resident macrophages in the brain, in regulating cognitive functions such as learning and memory in both healthy and diseased states. However, there are conflicting results and the underlying mechanisms are not fully understood. In this study, we examined the effect of depleting adult microglia on spatial learning and memory under both physiological conditions and lipopolysaccharide (LPS)-induced neuroinflammation. Our results revealed that microglial depletion by PLX5622 caused mild spatial memory impairment in mice under physiological conditions; however, it prevented memory deficits induced by systemic LPS insult. Inactivating microglia through minocycline administration replicated the protective effect of microglial depletion on LPS-induced memory impairment. Furthermore, our study showed that PLX5622 treatment suppressed LPS-induced neuroinflammation, microglial activation, and synaptic dysfunction. These results strengthen the evidence for the involvement of microglial immunoactivation in LPS-induced synaptic and cognitive malfunctions. They also suggest that targeting microglia may be a potential approach to treating neuroinflammation-associated cognitive dysfunction seen in neurodegenerative diseases., Competing Interests: The authors declare that they have no competing interests., (© 2024 Zong et al.)

Background: SGLT2 inhibitors, a T2DM medication to lower blood glucose, markedly improve cardiovascular outcomes but the underlying mechanism(s) are not fully understood. SGLT2i's produce a unique metabolic pattern by lowering blood glucose without increasing insulin while increasing ketone body and glucagon levels and reducing body weight. We tested if glucagon signaling contributes to SGLT2i induced improvement in CV function., Methods: Cardiac contractility and coronary flow velocity reserve (CFVR) were monitored in ob/ob mice and rhesus monkeys with metabolic syndrome using echocardiography. Metabolic status was characterized by measuring blood ketone levels, glucose tolerance during glucose challenge and Arg and ADMA levels were measured. Baysian models were developed to analyse the data., Results: Dapagliflozin improved CFVR and contractility, co-application of a glucagon receptor inhibitor (GcgRi) blunted the effect on CFVR but not contractility. Dapagliflozin increased the Arg/ADMA ratio and ketone levels and co-treatment with GcgRi blunted only the Dapagliflozin induced increase in Arg/ADMA ratio but not ketone levels., Conclusions: Since GcgRi co-treatment only reduced the Arg/ADMA increase we hypothesize that dapagliflozin via a glucagon-signaling dependent pathway improves vascular function through the NO-signaling pathway leading to improved vascular function. Increase in ketone levels might be a contributing factor in SGLT2i induced contractility increase and does not require glucagon signaling., Competing Interests: Declarations Ethics approval and consent to participate Mouse study: Animal care and experiments comply to the Swedish national regulations L150 for the protection of animals used for scientific purposes. The project protocol was assessed and approved by the Gothenburg Regional Animal Ethics Committee for Animal Experimental permit no 86 2015. NHP study: The present PK and efficacy studies were approved by the Institutional Animal Care and Use Committee of Peking University. The housing and experimental procedures were in accordance with the principles of laboratory animal care of the National Academy of Sciences/National Research Council. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Isolation of adult mouse cardiomyocytes is an essential technique for advancing our understanding of cardiac physiology and pathology, and for developing therapeutic strategies to improve cardiac health. Traditionally, cardiomyocytes are isolated from adult mouse hearts using the Langendorff perfusion method in which the heart is excised, cannulated, and retrogradely perfused through the aorta. While this method is highly effective for isolating cardiomyocytes, it requires specialized equipment and technical expertise. To address the challenges of the Langendorff perfusion method, researchers have developed a Langendorff-free technique for isolating cardiomyocytes. This Langendorff-free technique involves anterograde perfusion through the coronary vasculature by clamping the aorta and intraventricular injection. This method simplifies the experimental setup by decreasing the need for specialized equipment and eliminating the need for cannulation of the heart. Here, we introduce an updated Langendorff-free method for isolating adult mice cardiomyocytes that builds on the Langendorff-free protocols developed previously. In this method, the aorta is clamped in situ, and the heart is perfused using a peristaltic pump, water bath, and an injection needle. This simplicity makes cardiomyocyte isolation more accessible for researchers who are new to cardiomyocyte isolation or are working with limited resources. In this report, we provide a step-by-step description of our optimized protocol. In addition, we present example studies of analyzing mitochondrial structural and functional characteristics in untreated isolated cardiomyocytes and cardiomyocytes treated with the acute inflammatory stimulus lipopolysaccharide (LPS)., Competing Interests: Declarations Ethics approval and consent to participate The animal study protocols were rigorously reviewed and received approval from the Institutional Animal Care and Use Committee (IACUC) at Loyola University Chicago. Our procedures strictly adhered to the guidelines outlined in the National Research Council’s “Guide for the Care and Use of Laboratory Animals.” All methods are reported in accordance with ARRIVE guidelines 2.0 for the reporting of animal experiments [26]. Consent for publication All identifiable individuals have given their informed consent for the publication of data and images in this report. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The mechanisms through which systemic inflammation exerts its effect on the central nervous system (CNS) are still not completely understood. Exosomes are small (30 to 100 nm) membrane-bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell-to-cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs, and cytokines, both locally and systemically to the neighboring as well as distant cells to further modulate the transcriptional and/or translational states and affect the functional phenotype of those cells that have taken up these exosomes. The role of circulating blood exosomes leading to neuroinflammation during systemic inflammatory conditions was hereby characterized. Serum-derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice that were earlier treated with LPS and used to study the effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as a control. In-vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the murine cell lines of microglia (BV-2), astrocytes (C8-D1A), and cerebral microvascular endothelial cells (bEnd.3). To further study their effects in-vivo, SDEL were intravenously injected into normal adult mice. Elevated mRNA expression of pro-inflammatory cytokines was observed in the brains of SDEL recipient mice. Proteomic analysis of the SDEL confirmed the increased expression of inflammatory cytokines in them. Together, these results demonstrate and strengthen the novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication All the authors read and approved the final manuscript for publication. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Preclinical studies indicate that the systemic application of C1-inhibitor, clinically used to treat hereditary angioedema, reduces secondary brain injury after ischemic stroke. This study assessed the effect of C1-inhibitor on secondary brain injury after hemorrhagic stroke., Methods: We used an established striatal whole-blood injection mouse model to mimic intracerebral hemorrhage-related secondary brain injury. Based on the spatiotemporal dynamics in our model, we calculated the necessary sample size (n = 24) and determined the most sensitive time point to detect potential group differences (48 h) prior to the experiments. The experimental setup, tissue processing and image analysis adhered to our published protocol. We randomized mice into three groups: C1-inhibitor treatment, placebo, and sham. Histology was standardized by taking eight anatomically predefined slices across the entire lesion. Lesion size, vascular leakage, and inflammatory responses were assessed using automated thresholding and dextran/ICAM1/CD45 intensity mapping. Investigators were blinded to group allocation during the experiment, tissue processing, and image analysis., Results: Whole blood injection resulted in significantly larger lesion size and more pronounced vascular leakage and cellular inflammation compared to the sham group. However, there was no difference in lesion size or inflammatory markers between the C1-inhibitor and placebo groups. In addition, there was no difference in the inflammatory response of the choroid plexus, which has been identified as a central organ orchestrating inflammation after intracerebral hemorrhage., Conclusion: The protective effect of C1-inhibitor might be isolated to pathophysiological processes with a predominant thromboinflammatory component, as in ischemia-reperfusion, but less so in permanent ischemia or intracerebral hemorrhage., Competing Interests: Declarations Ethics approval and consent to participate All experiments were approved by the Swiss Federal Veterinary Office (ZH89/2019). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: Intestinal flora promotes the pathogenesis of colorectal cancer (CRC) through microorganisms and their metabolites. This study aimed to investigate the composition of intestinal flora in different stages of CRC progression and the effect of fecal microbiota transplantation (FMT) on CRC mice., Methods: The fecal microbiome from healthy volunteers (HC), colorectal adenoma (CRA), inflammatory bowel disease (IBD), and CRC patients were analyzed by 16s rRNA gene sequencing. In an azoxymethane (AOM)/dextran-sulfate-sodium (DSS)-induced CRC mouse, the effect of FMT from HC, CRA, CRC, and IBD patients on CRC mice was assessed by histological analysis. Expression of inflammation- EMT-associated proteins and Wnt/β-catenin pathway were assessed using qRT-PCR and western blot. The ratio of the fecal microorganisms and metabolomics alteration after FMT were also assessed., Result: Prevotella, Faecalibacterium, Phascolarctobacterium, Veillonella, Alistipes, Fusobacterium, Oscillibacter, Blautia, and Ruminococcus abundance was different among HC, IBD, CRC, and CRA patients. HC-FMT alleviated disease progression and inflammatory response in CRC mice, inhibited splenic T help (Th)1 and Th17 cell numbers, and suppressed the EMT and Wnt/β-catenin pathways in tumor tissues of CRC mice. IBD-FMT, CRA-FMT, and CRC-FMT played deleterious roles; the CRC-FMT mice exhibited the most malignant phenotype. Compared with the non-FMT CRC mice, Muribaculaceae abundance was lower after FMT, especially lowest in the IBD-FMT group; while Lactobacillus abundance was higher after FMT and especially high in HC-FMT. Akkermansia and Ileibacterium abundance increased after FMT-HC compared to other groups. Metabolite correlation analysis revealed that Muribaculaceae abundance was significantly correlated with metabolites such as Betaine, LysoPC, and Soyasaponin III. Lactobacillus abundance was positively correlated with Taurocholic acid 3-sulfate, and Ileibacterium abundance was positively correlated with Linoleoyl ethanolamide., Conclusion: The different intestinal microbiota communities of HC, IBD, CRA, and CRC patients may be attributed to the different modulation effects of FMT on CRC mice. CRC-FMT promoted, while HC-FMT inhibited the progress of CRC. Increased linoleoyl ethanolamide levels and abundance of Muribaculaceae, Akkermansia, and Ileibacterium and reduced Fusobacterium might participate in inhibiting CRC initiation and development. This study demonstrated that FMT intervention could restore the intestinal microbiota and metabolomics of CRC mice, suggesting FMT as a potential strategy for CRC therapy., Competing Interests: Declarations Competing interests All the authors declare thare are no competing interests., (© 2024. The Author(s).)

Anesthesia can influence cerebral blood flow by altering vessel diameter. Using in vivo two-photon imaging, we examined the effects of volatile anesthetics, sevoflurane and isoflurane, on vessel diameter in young and adult mice. Our results show that these anesthetics induce robust dilation of cortical arterioles and arteriole-proximate capillaries in adult mice, with milder effects in juveniles and no dilation in infants. This anesthesia-induced vasodilation correlates with decreased cytosolic Ca 2+ levels in NG2 + vascular mural cells. Optogenetic manipulation of these cells bidirectionally regulates vessel diameter, and their ablation abolishes the vasodilatory response to anesthetics. In immature brains, NG2 + mural cells are fewer in number and express lower levels of Kir6.1, a subunit of ATP-sensitive potassium channels. This likely contributes to the age-dependent differences in vasodilation, as Kir6.1 activation promotes, while its inhibition reduces, anesthesia-induced vasodilation. These findings highlight the essential role of NG2 + mural cells in mediating anesthesia-induced cerebral vasodilation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Infiltrated and activated M1 macrophages play a role in kidney injury and fibrosis during chronic kidney disease (CKD) progression. However, the specific ways that M1 macrophage polarization contributes to renal fibrosis are not fully understood. The study seeks to investigate how miR-92a-3p regulates M1 macrophage polarization and its connection to renal fibrosis in the development of CKD. Our results revealed that miR-92a-3p overexpression increased M1-macrophage activation, iNOS, IL-6, and TNF-alpha expression in RAW264.7 upon LPS stimulation. LIN28A overexpression reversed these effects. Moreover, miR-92a-3p overexpression in RAW264.7 exacerbated NRK-52E cell apoptosis induced by LPS, but LIN28A overexpression counteracted this effect. MiR-92a-3p knockout in unilateral ureteral obstruction (UUO) C57BL/6 mice led to reduced renal infiltration and fibrosis, accompanied by decreased iNOS, alpha-SMA, IL-6, TNF-alpha, and increased LIN28A. In summary, our findings suggest that miR-92a-3p may play a role in promoting renal injury and fibrosis both in vitro and in vivo. This effect is potentially achieved by facilitating M1 macrophage polarization through the targeting of LIN28A.

Oncolytic viruses (OV) are designed to selectively infect and kill cancer cells, while simultaneously eliciting antitumour immunity. The mechanism is expected to originate from infected cancer cells. However, recent reports of tumour regression unaccompanied by cancer cell infection suggest a more complex mechanism of action. Here, we engineered vesicular stomatitis virus (VSV) ΔM51 -sensitive and VSV ΔM51 -resistant tumour lines to elucidate the role of OV-infected cancer and non-cancer cells. We found that, while cancer cell infections elicit oncolysis and antitumour immunity as expected, infection of non-cancer cells alone can also contribute to tumour regression. This effect is partly attributed to the systemic production of cytokines that promote dendritic cell (DC) activation, migration and antigen cross-presentation, leading to magnified antitumour CD8 + T cell activation and tumour regression. Such OV-induced antitumour immunity is complementary to PD-1 blockade. Overall, our results reveal mechanistic insights into OV-induced antitumour immunity that can be leveraged to improve OV-based therapeutics., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The post-mortem interval (PMI) is the time elapsed between the death of an individual and its forensic examination. It is a crucial information for judicial authorities, but current techniques still cannot establish a precise time interval. Novel approaches are therefore required. Recently, gingival tissue has emerged as interesting for forensic analysis thanks to the protection offered by lips to this tissue, limiting the influence of environmental factors. It is also easily accessible, and its sampling is minimally invasive even in the presence of rigor mortis. Moreover, the expression of HIF-1α, a master mediator of the hypoxic environment, has been described in gingival samples at different post-mortem (PM) times. We have hypothesized that the time-dependent post-mortem expression of HIF-1α could serve as a biomarker to more accurately predict the PMI. Our analyses were performed in an animal model, the mouse, where environment can be precisely controlled. Therewith, gingival tissue morphology was evaluated through histochemical staining and HIF-1α expression was analyzed by qPCR, western blots and immunofluorescence at different post-mortem times (0h to 100h). Our results showed (a) a global post-mortem stability of gingival tissue (b) a rapid increase in HIF-1α mRNA expression in the short post-mortem times followed by a slow decrease in transcript expression until 100h PM (c) an expression of the HIF- 1α protein and its degradation products, that follows the mRNA pattern (d) the presence of HIF-1α protein in the epithelial and connective layers of the tissue, with signal accumulation in both gingival strata until at least 32h post-mortem. This pilot study thus validated the mouse and the gingival tissue as models for post-mortem analyses, as well as for studying the fate of proteins such as HIF-1α. Transferring these approaches to human subjects may provide a more accurate estimate of PMI., Competing Interests: The authors state that they have no competing interests to declare., (Copyright: © 2024 Mascarell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice., Methods: BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV)., Results: CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic ( P >0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group ( P <0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group ( P <0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group ( P <0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant ( P <0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP ( P <0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group ( P <0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group ( P <0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group ( P <0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased ( P <0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group ( P <0.05) except Tb.Th., Conclusion: Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.

Acute kidney injury (AKI) is a common clinical problem, and patients who survive AKI have a high risk of chronic kidney disease (CKD). The acute protective effects of Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors in AKI have been examined, there is still relatively little known regarding the impact of acute CDK4/6 inhibition on the chronic sequence of AKI. Therefore, we utilized the CDK4/6 inhibitor Palbociclib to examine the long-term effects of CDK4/6 inhibition in a rodent model of ischemic AKI. Palbociclib (Palb) was administered during the acute stage or post the acute stage of AKI and mice were sacrificed 21 days post injury. We found that Palb could cause renal senescence and renal fibrosis. Furthermore, dasatinib (D) plus quercetin (Q) were used to eliminate senescent cells in ischemic AKI murine model and Palb was administered post the treatment of D + Q. We found that Palb could reverse the senolytic and antifibrotic effects induced by D + Q, which indicates that the profibrotic effect of Palb could be ascribed to its pro-senescent effects. Our results demonstrate that CDK4/6 inhibitors treatment might be deleterious on the chronic sequence of AKI., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Post-stroke depression (PSD) affects millions of patients who suffer cerebral stroke. However, the molecular mechanisms and pathophysiology are poorly understood. Previous studies have shown that exosomes have been proven to be involved in neuropsychiatric disorders such as stroke and post-stroke depression in neurotransmitter release, neuronal remodeling, and neuron angiogenesis. Here we extracted and purified hippocampal exosomes from stroke mouse model to investigate mechanisms of hippocampal exocytosis in PSD assessed by using behavioral tests and biochemical methods. Aiming at the effect of hippocampal exosomes from stroke on the development of PSD, behavioral test was compared including sugar water preference experiment, open fields, forced swimming test, to explore it in depth. Further, the expression of depression-related protein (proBDNF and p75NTR) and synapse-associated proteins (Synaptotagmin and PSD95) was evaluated by Western blotting, RT-qPCR or immunofluorescence staining. Density of dendritic protrusions of neurons was assessed by Golgi staining to measure changes in spine density after the treatment of hippocampal exosomes from stroke. Our results revealed that injection of exosomes from stroke models significantly aggravated depressive-like behaviors, increase of depression-related protein (proBDNF and p75NTR) expression and deficiency of synapse-associated proteins (Synaptotagmin and PSD95) expression, and the decreased number of spin density. Our findings together suggest that hippocampal exosomes from stroke cause exacerbation of depressive-like behavior in mice, possibly resulting from the regulation of neurogenesis by its depression-associated proteins (proBDNF and p75NTR). Therefore, hippocampal exosomes from stroke are promising targets for the diagnosis and treatment of PSD., Competing Interests: Declarations Competing interests The authors declare no competing interests. Consent for publication Not applicable. Ethics approval The protocol of this study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University. The study methodology was carried out following ARRIVE guidelines (https://arriveguidelines.org)., (© 2024. The Author(s).)

Metabolic-associated fatty liver disease (MAFLD) is a common chronic condition that poses a significant threat to human health. Mitochondrial dysfunction, particularly involving the mitochondrial Ca 2+ uniporter (MCU), plays a key role in its pathogenesis. This study aimed to investigate the impact of the MCU gene on hepatic lipid metabolism in mice fed a high-fat diet. Using MCU knockout and wild-type mice, subjected to either a high-fat or normal diet for 14 weeks, we observed notable Steatosis and liver weight gain in MCU-deficient mice. Liver function markers, serum triglycerides, very low-density lipoprotein (VLDL) levels, and ApoB protein expression were all significantly elevated. Mechanistic studies revealed that MCU deletion led to mitochondrial dysfunction, increased oxidative stress. These findings highlight the critical role of the MCU gene in maintaining hepatic lipid balance and suggest its potential as a therapeutic target for managing nonalcoholic fatty liver disease., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet the high energy and biosynthetic demands required for their proliferation. High mobility group A1 (HMGA1) is a structural transcription factor and frequently overexpressed in human colorectal cancer (CRC). Here, we show that HMGA1 promotes CRC progression by driving lipid synthesis in a AOM/DSS-induced CRC mouse model. Using conditional knockout (Hmga1 △IEC ) and knock-in (Hmga1 IEC-OE/+ ) mouse models, we demonstrate that HMGA1 enhances CRC cell proliferation and accelerates tumor development by upregulating fatty acid synthase (FASN). Mechanistically, HMGA1 increases the transcriptional activity of sterol regulatory element-binding protein 1 (SREBP1) on the FASN promoter, leading to increased lipid accumulation in intestinal epithelial cells. Moreover, a high-fat diet exacerbates CRC progression in Hmga1 △IEC mice, while pharmacological inhibition of FASN by orlistat reduces tumor growth in Hmga1 IEC-OE/+ mice. Our findings suggest that targeting lipid metabolism could offer a promising therapeutic strategy for CRC., Competing Interests: Competing interests The authors declare no conflict of interest., (© 2024. The Author(s).)

Trimethylamine N-oxide (TMAO), a bioactive metabolite of gut microbes, plays a pivotal role in the pathogenesis of kidney diseases by activating programmed cell death (PCD) pathways. However, whether trimethylamine (TMA) contributes to chronic kidney injury and which kind of PCD is involved in TMA-induced chronic kidney injury has not been previously evaluated. To observe the effect of TMA, male C57BL/6J mice were randomly divided into two groups: the Control group and the TMA group. The mice in the TMA group were intraperitoneally injected with 100 micromol/kg/day TMA for three months, whereas the mice in the Control group were injected with normal saline for the same period. After three months, plasma creatinine and blood urea nitrogen levels, indicators of kidney function, increased significantly in the TMA group as compared with those in the Control group. Furthermore, Masson staining assay showed that TMA treatment led to a larger area of fibrosis than the Control group. TMA treatment did not change the Bax/Bcl-2 ratio, RIP1, RIP3 and MLKL phosphorylation, or iron and malondialdehyde levels in kidney tissues, indicating that apoptosis, ferroptosis and necroptosis were not involved in TMA-induced chronic kidney injury. However, compared with the Control group, TMA treatment significantly upregulated NLRP3, Caspase-1, IL-1beta, cleaved-Caspase 8, Caspase-8, and ZBP1 protein expression in kidney tissues. These results indicated that the ZBP1-NLRP3 inflammasome pathway was involved in TMA-induced chronic kidney injury. In conclusion, our studies revealed that the ZBP1-NLRP3 inflammasome may take part in the progression of TMA induced chronic kidney injury.

Extracellular vesicles (EVs) are involved in the progression of various diseases. Tumor cell-derived EVs (TEVs) are a particular concern, as they can induce fatty liver by promoting liver macrophages to secrete tumor necrosis factor (TNF), thus enhancing the toxicity of chemotherapy. Therefore, reducing pathogenic EV production is a potential strategy for treating EV-related diseases. However, there are currently no effective clinical reagents to obtain this purpose. In addition, EVs are also natural and ideal drug-delivery vehicles. Improving the delivery efficiency of EVs remains a challenge. Proton pump inhibitors (PPIs) have been demonstrated to promote cell uptake of EVs by inducing micropinocytosis. Here, we show that PPIs can accelerate TEV clearance, reduce TEV uptake by liver macrophages and decrease the mRNA expression of TNF in liver macrophages of tumor-bearing mice. Correspondingly, the fatty liver phenotypes are alleviated, and the tolerance to chemotherapy is improved in these mice. Furthermore, our findings indicate that PPIs facilitate the uptake of red blood cell-derived EVs (RBC-EVs) loaded with antisense oligonucleotides of Trim21 (Trim21-ASOs) by the liver macrophages of obesity. Consequently, the inhibition of macrophage inflammatory responses in obese mice mediated by RBC-EVs/Trim21-ASOs was further enhanced by PPIs, resulting in a more profound improvement in obesity and related metabolic disorders. In conclusion, our findings demonstrated that PPIs can effectively clear pathogenic EVs and enhance the delivery efficacy of EV vehicles, making them a highly promising clinical prospect., Competing Interests: Declarations Competing interests The authors declare that they have no competing interests., (© 2024. The Author(s).)

Most non-muscle invasive bladder cancers have been treated by transurethral resection and following intravesical injection of immunotherapeutic agents. However, the delivery efficiency of therapeutic agents into bladder wall is low due to frequent urination, which leads to the failure of treatment with side effects. Here, we report a urease-powered nanomotor containing the agonist of stimulator of interferon genes (STING) for the efficient activation of immune cells in the bladder wall. After characterization, we perform in vitro motion analysis and assess in vivo swarming behaviors of nanomotors. The intravesical instillation results in the effective penetration and retention of nanomotors in the bladder. In addition, we confirm the anti-tumor effect of nanomotor containing the STING agonist (94.2% of inhibition), with recruitment of CD8 + T cells (11.2-fold compared with PBS) and enhanced anti-tumor immune responses in bladder cancer model in female mice. Furthermore, we demonstrate the better anti-tumor effect of nanomotor containing the STING agonist than those of the gold standard Bacille Calmette-Guerin therapy and the anti-PD-1 inhibitor pembrolizumab in bladder cancer model. Taken together, the urease-powered nanomotor would provide a paradigm as a next-generation platform for bladder cancer immunotherapy., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Contrast-induced acute kidney injury (CI-AKI) is one of the main causes of hospital-acquired renal failure, and still lacks of effective treatments. Previously, we demonstrated that αKlotho, which is an anti-aging protein that highly expresses in the kidney, has therapeutic activity in CI-AKI through promoting autophagy. However, the specific mechanism underlying αKlotho-mediated autophagy remains unclear. The RNA sequencing analysis of renal cortex revealed that the differentially expressed genes related to autophagy between αKlotho-treated CI-AKI mice and vehicle-treated CI-AKI mice were found to be associated with mitophagy and apoptosis. In the kidney of CI-AKI mice and HK-2 cells exposed to Iohexol, we revealed that αKlotho promoted mitophagy and decreased cell apoptosis. Mechanistically, αKlotho attenuated mitochondria damage, decreased mitochondrial ROS by upregulating BNIP3-mediated mitophagy. BNIP3 deletion abolished the beneficial effects of αKlotho both in vivo and in vitro. Moreover, we further demonstrated that αKlotho upregulated FoxO3 nuclear expression in Iohexol-treated HK-2 cells. Knockdown of FOXO3 gene inhibited αKlotho-promoted BNIP3-mediated mitophagy and subsequently increased the oxidative injury and cell apoptosis. Taken together, our results indicated a critical role of αKlotho in alleviating CI-AKI via mitophagy promotion involving the FoxO3-BNIP3 pathway., Competing Interests: Declarations Ethical approval All animal experiments were approved by the Animal Care Committee at the Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, and performed according to the Animal Protocol Committee of Shanghai Jiao Tong University. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest., (© 2024. The Author(s).)