Mouse Liver Research Articles

Asprosin aggravates nonalcoholic fatty liver disease via inflammation and lipid metabolic disturbance mediated by reactive oxygen species.

Asprosin (ASP) is a newly-identified adipokine and plays important roles in energy metabolism homeostasis. However, there is no report on whether and how ASP is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Therefore, in the study, we investigated the protective effects of ASP-deficiency on the liver in the NAFLD model mice and the detrimental effects of ASP treatment on the human normal hepatocytes (LO2 cell line). More important, we explored the underlying mechanism from the perspective of lipid metabolism and inflammation. In the in vivo experiments, our data showed that the ASP-deficiency significantly alleviated the high-fat diet-induced inflammation and NAFLD, inhibited the hepatic fat deposition and downregulated the expressions of fat acid synthase (FASN), peroxisome proliferator-activated receptor γ (PPARγ) and forkhead box protein O1 (FOXO1); moreover, the ASP-deficiency attenuated the inflammatory state and inhibited the activation of the IKK/NF-κBp65 inflammation pathway. In the in vitro experiments, our results revealed that ASP treatment caused and even exacerbated the injury of LO2 cells induced by FFA; In contrast, the ASP treatment upregulated the expressions of PPARγ, FOXO1, FASN, ACC and acyl-CoA oxidase 1 (ACOX1) and elevated the reactive oxygen species (ROS) levels. Accordingly, these results demonstrate that ASP causes NAFLD through disrupting lipid metabolism and promoting the inflammation mediated by ROS.

Crosstalk interactions between transcription factors ERRα and PPARα assist PPARα-mediated gene expression

ObjectiveThe peroxisome proliferator-activated receptor α (PPARα) is a transcription factor driving target genes involved in fatty acid β-oxidation. To what extent various PPARα interacting proteins may assist its function as a transcription factor is incompletely understood. An ORFeome-wide unbiased mammalian protein-protein interaction trap (MAPPIT) using PPARα as bait revealed a PPARα-ligand-dependent interaction with the orphan nuclear receptor estrogen-related receptor α (ERRα). The goal of this study was to characterize the nature of the interaction in depth and to explore whether it was of physiological relevance. MethodsWe used orthogonal protein-protein interaction assays and pharmacological inhibitors of ERRα in various systems to confirm a functional interaction and study the impact of crosstalk mechanisms. To characterize the interaction surfaces and contact points we applied a random mutagenesis screen and structural overlays. We pinpointed the extent of reciprocal ligand effects of both nuclear receptors via coregulator peptide recruitment assays. On PPARα targets revealed from a genome-wide transcriptome analysis, we performed an ERRα chromatin immunoprecipitation analysis on both fast and fed mouse livers. ResultsRandom mutagenesis scanning of PPARα’s ligand-binding domain and coregulator profiling experiments supported the involvement of (a) bridging coregulator(s), while recapitulation of the interaction in vitro indicated the possibility of a trimeric interaction with RXRα. The PPARα·ERRα interaction depends on 3 C-terminal residues within helix 12 of ERRα and is strengthened by both PGC1α and serum deprivation. Pharmacological inhibition of ERRα decreased the interaction of ERRα to ligand-activated PPARα and revealed a transcriptome in line with enhanced mRNA expression of prototypical PPARα target genes, suggesting a role for ERRα as a transcriptional repressor. Strikingly, on other PPARα targets, including the isolated PDK4 enhancer, ERRα behaved oppositely. Chromatin immunoprecipitation analyses demonstrate a PPARα ligand-dependent ERRα recruitment onto chromatin at PPARα-binding regions, which is lost following ERRα inhibition in fed mouse livers. ConclusionOur data support the coexistence of multiple layers of transcriptional crosstalk mechanisms between PPARα and ERRα, which may serve to finetune the activity of PPARα as a nutrient-sensing transcription factor.

Dual-Emissive Detection of ATP and Hypochlorite Ions for Monitoring Inflammation-Driven Liver Injury In Vitro and In Vivo.

Reactive oxygen species play a pivotal role in liver disease, contributing to severe liver damage and chronic inflammation. In liver injury driven by inflammation, adenosine-5'-triphosphate (ATP) and hypochlorite ion (ClO-) emerge as novel biomarkers, reflecting mitochondrial dysfunction and amplified oxidative stress, respectively. However, the dynamic fluctuations of ATP and ClO- in hepatocytes and mouse livers remain unclear, and multidetection techniques for these biomarkers are yet to be developed. This study presents RATP-NClO, a dual-channel fluorescent bioprobe capable of synchronously detecting ATP and ClO- ions. RATP-NClO exhibits excellent selectivity and sensitivity for ATP and ClO- ions, demonstrating a dual-channel fluorescence response in a murine hepatocyte cell line. Upon intravenous administration, RATP-NClO reveals synchronized ATP depletion and ClO- amplification in the livers of mice with experimental metabolic dysfunction-associated steatohepatitis (MASH). Through a comprehensive analysis of the principal mechanism of the developed bioprobe and the verification of its reliable detection ability in both in vitro and in vivo settings, we propose it as a unique tool for monitoring changes in intracellular ATP and ClO- level. These findings underscore its potential for practical image-based monitoring and functional phenotyping of MASH pathogenesis.

Discovery and Optimization of N-Arylated Tetracyclic Dicarboximides That Target Primary Glioma Stem-like Cells.

We recently discovered a novel N-aryl tetracyclic dicarboximide MM0299 (1) with robust activity against glioma stem-like cells that potently and selectively inhibits lanosterol synthase leading to the accumulation of the toxic shunt metabolite 24(S),25-epoxycholesterol. Herein, we delineate a systematic and comprehensive SAR study that explores the structural space surrounding the N-aryl tetracyclic dicarboximide scaffold. A series of 100 analogs were synthesized and evaluated for activity against the murine glioma stem-like cell line Mut6 and for metabolic stability in mouse liver S9 fractions. This study led to several analogs with single-digit nanomolar activity in Mut6 glioblastoma cells that were metabolically stable in S9 fractions. In vivo pharmacokinetic analysis of selected analogs identified compound 52a (IC50 = 63 nM; S9 T1/2 > 240 min) which was orally available (39% plasma; 58% brain) and displayed excellent brain exposure. Chronic oral dosing of 52a during a 2-week tolerability study indicated no adverse effect on body weight nor signs of hematologic, liver, or kidney toxicity.

Iron Overload Induces Hepatic Ferroptosis and Insulin Resistance by Inhibiting the Jak2/stat3/slc7a11 Signaling Pathway.

Recent studies showed that patients with iron overload had increased risk of insulin resistance or diabetes. Ferroptosis is a new type of cell death mainly caused by iron-dependent oxidative damage. In the present study, we investigated potential mechanisms of iron overload induced hepatic ferroptosis and insulin resistance through in vivo and in vitro experiments. In vivo, the mice models of iron overload were established by intraperitoneal injection of iron dextran. The changes of body weight, serum ferritin and blood glucose were measured. Hematoxylin-eosin (HE) and Perl's stainings were used to observe the pathological changes and iron deposition in the liver of mice. In vitro, HepG2 cells were treated with ferric ammonium citrate (FAC, 9 mmol/L, 24 h) to establish the cell models of iron overload. The labile iron pool, cell viability, glucose consumption and glycogen contents were measured. The ultrastructure of mitochondria was observed by transmission electron microscope (TEM). The malondialdehyde (MDA) and glutathione (GSH) kits were used to detect lipid peroxidation in liver tissues of mice and HepG2 cells. RT-PCR and Western blot were used to detect the mRNA and protein expression levels of ferroptosis factors and JAK2/STAT3 signaling pathway. In this study, we used the iron chelator deferasirox in mice and HepG2 cells. Iron overload caused weight loss, elevated serum ferritin, fasting blood glucose, fasting insulin, HOMA-IR, impaired glucose tolerance, and decreased insulin sensitivity in mice. HE staining and Perls staining showed clumps of iron deposition in the liver of iron overload mice. Iron overload could reduce the glucose consumption, increase MDA contents of HepG2 cells, while reduce glycogen and GSH contents in liver tissues of mice and HepG2 cells. TEM showed deletion of mitochondrial ridge and rupture of outer membrane in HepG2 cells with iron overload. Iron chelator deferasirox could significantly improve the above indicators, which might be related to the activation of JAK2/STAT3/SLC7A11 signaling pathway and hepatic ferroptosis. Iron overload could induce hepatic ferroptosis and insulin resistance by inhibiting the JAK2/STAT3/SLC7A11 signaling pathway, and the iron chelator deferasirox might improve hepatic insulin resistance induced by iron overload.

Histone demethylase KDM4A mediating macrophage polarization: A potential mechanism of trichloroethylene induced liver injury.

Trichloroethylene (TCE) is a commonly used organic solvent in industry. Our previous studies have found that TCE can cause liver injury accompanied by macrophage polarization, but the specific mechanism is unclear. The epigenetic regulation of macrophage polarization is mainly focused on histone modification. Histone lysine demethylase 4A (KDM4A) is involved in the activation of macrophages. In this study, we used a mouse model we investigated the role of KDM4A in the livers of TCE-drinking mice and found that the expression of KDM4A, M1-type polarization indicators, and related inflammatory factors in the livers of TCE-drinking mice. In the study, BALB/c mice were randomly divided into four groups: 2.5 mg/mL TCE dose group and 5.0 mg/mL TCE dose group, the vehicle control group, and the blank control group. We found that TCE triggered M1 polarization of mouse macrophages, characterized by the expression of CD11c and robust production of inflammatory cytokines. Notably, exposure to TCE resulted in markedly increased expression of KDM4A in macrophages. Functionally, the increased expression of KDM4A significantly impaired the expression of H3K9me3 and H3K9me2 and increased the expression of H3K9me1. In addition, KDM4A potentially represents a novel epigenetic modulator, with its upregulation connected to β-catenin activation, a signal critical for the pro-inflammatory activation of macrophages. Furthermore, KDM4A inhibitor JIB-04 treatment resulted in a decrease in β-catenin expression and prevented TCE-induced M1 polarization and the expression of the pro-inflammatory cytokines TNF-α and IL-1β. These results suggest that the association of KDM4A and Wnt/β-catenin cooperatively establishes the activation and polarization of macrophages and global changes in H3K9me3/me2/me1. Our findings identify KDM4A as an essential regulator of the polarization of macrophages and the expression of inflammatory cytokines, which might serve as a potential target for preventing and treating liver injury caused by TCE.

NHH promotes Sepsis-associated Encephalopathy with the expression of AQP4 in astrocytes through the gut-brain Axis.

Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels.Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE.

Long-term intravital subcellular imaging with confocal scanning light-field microscopy.

Long-term observation of subcellular dynamics in living organisms is limited by background fluorescence originating from tissue scattering or dense labeling. Existing confocal approaches face an inevitable tradeoff among parallelization, resolution and phototoxicity. Here we present confocal scanning light-field microscopy (csLFM), which integrates axially elongated line-confocal illumination with the rolling shutter in scanning light-field microscopy (sLFM). csLFM enables high-fidelity, high-speed, three-dimensional (3D) imaging at near-diffraction-limit resolution with both optical sectioning and low phototoxicity. By simultaneous 3D excitation and detection, the excitation intensity can be reduced below 1 mW mm-2, with 15-fold higher signal-to-background ratio over sLFM. We imaged subcellular dynamics over 25,000 timeframes in optically challenging environments in different species, such as migrasome delivery in mouse spleen, retractosome generation in mouse liver and 3D voltage imaging in Drosophila. Moreover, csLFM facilitates high-fidelity, large-scale neural recording with reduced crosstalk, leading to high orientation selectivity to visual stimuli, similar to two-photon microscopy, which aids understanding of neural coding mechanisms.

Early life interventions metformin and trodusquemine metabolically reprogram the developing mouse liver through transcriptomic alterations.

Recent studies have demonstrated the remarkable potential of early life intervention strategies at influencing the course of postnatal development, thereby offering exciting possibilities for enhancing longevity and improving overall health. Metformin (MF), an FDA-approved medication for type II diabetes mellitus, has recently gained attention for its promising anti-aging properties, acting as a calorie restriction mimetic, and delaying precocious puberty. Additionally, trodusquemine (MSI-1436), an investigational drug, has been shown to combat obesity and metabolic disorders by inhibiting the enzyme protein tyrosine phosphatase 1b (Ptp1b), consequently reducing hepatic lipogenesis and counteracting insulin and leptin resistance. In this study, we aimed to further explore the effects of these compounds on young, developing mice to uncover biomolecular signatures that are central to liver metabolic processes. We found that MSI-1436 more potently alters mRNA and miRNA expression in the liver compared with MF, with bioinformatic analysis suggesting that cohorts of differentially expressed miRNAs inhibit the action of phosphoinositide 3-kinase (Pi3k), protein kinase B (Akt), and mammalian target of rapamycin (Mtor) to regulate the downstream processes of de novo lipogenesis, fatty acid oxidation, very-low-density lipoprotein transport, and cholesterol biosynthesis and efflux. In summary, our study demonstrates that administering these compounds during the postnatal window metabolically reprograms the liver through induction of potent epigenetic changes in the transcriptome, potentially forestalling the onset of age-related diseases and enhancing longevity. Future studies are necessary to determine the impacts on lifespan and overall quality of life.

Euonymus alatus Extract Reduces Insulin Resistance in db/db Mice by Regulating the PI3K–AKT Pathway

In accordance with the usage of Euonymus alatus (EA) as folk medicine in diabetes, the present study employed water and 70% ethanol twig extract to assess its antidiabetic effects in C57BL/KsJ-db/db mice. These effects were then compared with those observed in normal C57BL/6J Jms Slc mice. After 4 weeks of supplementation with 70% ethanolic EA extract or water EA extract by oral gavage at a dose of 500 mg/kg with distilled water (DW) per day, body weight was measured and compared with the diabetic group (Db). HPLC demonstrated that the maximum flavonoids were extracted in the Et.EA extract rather than in the water EA extract. The supplementation of the Et.EA extract significantly increased liver and muscle glycogen content with respect to the Db group. Additionally, the Et.EA extract modulated the expression of glycogen synthase (GS) in the liver and muscles of Db mice, indicating that it plays a promotive role in glycogen synthesis. Mechanistically, Et.EA extract activates insulin receptor substrate (IRS1/IRS2)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) in the liver and muscles of Db mice. In conclusion, Et.EA extract attenuates insulin resistance by regulating the expression of metabolic enzymes and signaling pathways.

Integrated liver and serum proteomics uncover sexual dimorphism and alteration of several immune response proteins in an aging Werner syndrome mouse model.

Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.

Antioxidative Potential and Ameliorative Effects of Rice Bran Fermented with Lactobacillus against High-Fat Diet-Induced Oxidative Stress in Mice

Rice bran is an important byproduct of the rice polishing process, rich in nutrients, but it is underutilized and often used as feed or discarded, resulting in a huge amount of waste. In this study, rice bran was fermented by Lactobacillus fermentum MF423 to obtain a product with high antioxidant activity. First, a reliable and efficient method for assessing the antioxidant capacity of the fermentation products was established using high-performance liquid chromatography (HPLC), which ensured the consistency of the batch fermentation. The fermented rice bran product (FLRB) exhibited significant antioxidant activity in cells, C. elegans, and hyperlipidemic mice. Transcriptome analysis of mouse livers showed that the expression of plin5 was upregulated in diabetic mice administered FLRB, thereby preventing the excessive production of free fatty acids (FFAs) and the subsequent generation of large amounts of reactive oxygen species (ROS). These studies lay the foundation for the application of rice bran fermentation products.

The intervention of curcumin on rodent models of hepatic fibrosis: A systematic review and meta-analysis.

This study aimed to evaluate the intervention effect of curcumin on hepatic fibrosis in rodent models through systematic review and meta-analysis, in order to provide meaningful guidance for clinical practice. A systematic retrieval of relevant studies on curcumin intervention in rats or mice hepatic fibrosis models was conducted, and the data were extracted. The outcome indicators included liver cell structure and function related indicators, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB), ratio of albumin to globulin (A/G), total bilirubin (TBIL), bax protein, bcl-2 protein and index of liver, as well as the relevant indicators for evaluating the degree of hepatic fibrosis, such as hyaluronic acid (HA), laminin (LN), type I collagen (Collagen I), type III collagen (Collagen III), type III procollagen (PCIII), type III procollagen amino terminal peptide (PIIINP), type IV collagen (IV-C), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), α-Smooth muscle actin (α-SMA), hydroxyproline (HYP), platelet derived factor-BB (PDGF-BB), connective tissue growth factor (CTGF) and transforming growth factor-β1 (TGF-β1), and oxidative stress-related indicators, such as superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px). These results were then analyzed by meta-analysis. Studies were evaluated for methodological quality using the syrcle's bias risk tool. A total of 59 studies were included in the meta-analysis, and the results showed that curcumin can reduce the levels of ALT, AST, ALP, TBIL, bax protein, and index of liver in hepatic fibrosis models. It can also reduce HA, LN, Collagen I, Collagen III, PCIII, PIIINP, IV-C, TNF-α, α-SMA, HYP, PDGF-BB, CTGF, TGF-β1 and MDA, and increase the levels of ALB, A/G, SOD, and GSH-Px in the hepatic fibrosis models. However, the effects of curcumin on bcl-2 protein, IL-6 in hepatic fibrosis models and index of liver in mice were not statistically significant. The analysis results indicate that curcumin can reduce liver cell apoptosis by maintaining the stability of liver cell membrane, inhibit the activation and proliferation of hepatic stellate cells by reducing inflammatory response, and alleviate tissue peroxidation damage by clearing oxygen free radicals.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis

ObjectiveAlcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges...

Drug screening on digital microfluidics for cancer precision medicine

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

UBXN1 promotes liver tumorigenesis by regulating mitochondrial homeostasis

BackgroundThe maintenance of mitochondrial homeostasis is critical for tumor initiation and malignant progression because it increases tumor cell survival and growth. The molecular events controlling mitochondrial integrity that facilitate the development of hepatocellular carcinoma (HCC) remain unclear. Here, we report that UBX domain-containing protein 1 (UBXN1) hyperactivation is essential for mitochondrial homeostasis and liver tumorigenesis.MethodsOncogene-induced mouse liver tumor models were generated with the Sleeping Beauty (SB) transposon delivery system. Assessment of HCC cell growth in vivo and in vitro, including tumour formation, colony formation, TUNEL and FACS assays, was conducted to determine the effects of UBXN1 on HCC cells, as well as the involvement of the UBXN1-prohibitin (PHB) interaction in mitochondrial function. Coimmunoprecipitation (Co-IP) was used to assess the interaction between UBXN1 and PHB. Liver hepatocellular carcinoma (LIHC) datasets and HCC patient samples were used to assess the expression of UBXN1.ResultsUBXN1 expression is commonly upregulated in human HCCs and mouse liver tumors and is associated with poor overall survival in HCC patients. UBXN1 facilitates the growth of human HCC cells and promotes mouse liver tumorigenesis driven by the NRas/c-Myc or c-Myc/shp53 combination. UBXN1 interacts with the inner mitochondrial membrane protein PHB and sustains PHB expression. UBXN1 inhibition triggers mitochondrial damage and liver tumor cell apoptosis.ConclusionsUBXN1 interacts with PHB and promotes mitochondrial homeostasis during liver tumorigenesis.

B-lymphocyte-induced maturation protein-1 inhibits inflammation and pyroptosis to alleviate sepsis injury.

Liver and lung tissue damage caused by sepsis is still one of the causes of death. B-lymphocyte-induced maturation protein-1 (Blimp-1) has a protective role in inflammation-related disease. However, whether Blimp-1 can regulate cell pyroptosis and affect disease progression in sepsis is still unclear. Animal and cell models were established by the cecal ligation and puncture method and lipopolysaccharides (LPS)-induced RAW 264.7 cells, respectively, and the role of Blimp-1 in regulation inflammatory response and pyroptosis was verified. The changes of inflammation and pyroptosis in liver and lung tissues of septic mice were determined by the addition of TAK-242 (TLR4 inhibitor). Cell pyroptosis and the level of inflammation was detected after Blimp-1 knockdown and TAK-242 treatment in the cell model. The expression of Blimp-1 was continuously increased in a septic mice model. After treatment with TAK-242, the expression of Blimp-1, pyroptosis and inflammatory levels were reduced in mice. In the LPS-induced cell model, cell injury by knockout Blimp-1 was increased, and cell activity was restored after TAK-242 intervention. Overexpression of Blimp-1 relieved LPS-induced cellular inflammatory damage and pyroptosis. Our study had shown that Blimp-1 could improve septic damage by regulating the level of cellular inflammation and pyroptosis in sepsis.

Diagnosis of Nonalcoholic Fatty Liver Disease via a H2S-Responsive Bioluminescent Probe Combined with Firefly Luciferase mRNA Delivery.

The early detection of nonalcoholic fatty liver disease (NAFLD) through bioluminescent probes is of great significance. However, there remains a challenge to apply them in nontransgenic natural animals due to the lack of exogenous luciferase. To address this issue, we herein report a new strategy for in situ monitoring of endogenous hydrogen sulfide (H2S) in the liver of NAFLD mice by leveraging a H2S-responsive bioluminescent probe (H-Luc) combined with firefly luciferase (fLuc) mRNA delivery. The probe H-Luc was created by installing a H2S recognition moiety, 2,4-dinitrophenol, onto the luciferase substrate (d-luciferin), which is allowed to release cage-free d-luciferin in the presence of H2S via a nucleophilic aromatic substitution reaction. In the meantime, the intracellular luciferase was introduced by lipid nanoparticle (LNP)-mediated fLuc mRNA delivery, rendering it suitable for bioluminescence (BL) imaging in vitro and in vivo. Based on this luciferase-luciferin system, the endogenous H2S could be sensitively and selectively detected in living cells, showing a low limit of detection (LOD) value of 0.72 μM. More importantly, after systematic administration of fLuc mRNA-loaded LNPs in vivo, H-Luc was able to successfully monitor the endogenous H2S levels in the NAFLD mouse model for the first time, displaying a 28-fold higher bioluminescence intensity than that in the liver of normal mice. We believe that this strategy may shed new light on the diagnosis of inflammatory liver disease, further elucidating the roles of H2S.

Efficient high-resolution fluorescence projection imaging over an extended depth of field through optical hardware and deep learning optimizations

Optical microscopy has witnessed notable advancements but has also become more costly and complex. Conventional wide field microscopy (WFM) has low resolution and shallow depth-of-field (DOF), which limits its applications in practical biological experiments. Recently, confocal and light sheet microscopy become major workhorses for biology that incorporate high-precision scanning to perform imaging within an extended DOF but at the sacrifice of expense, complexity, and imaging speed. Here, we propose deep focus microscopy, an efficient framework optimized both in hardware and algorithm to address the tradeoff between resolution and DOF. Our deep focus microscopy achieves large-DOF and high-resolution projection imaging by integrating a deep focus network (DFnet) into light field microscopy (LFM) setups. Based on our constructed dataset, deep focus microscopy features a significantly enhanced spatial resolution of ∼260 nm, an extended DOF of over 30 µm, and broad generalization across diverse sample structures. It also reduces the computational costs by four orders of magnitude compared to conventional LFM technologies. We demonstrate the excellent performance of deep focus microscopy in vivo, including long-term observations of cell division and migrasome formation in zebrafish embryos and mouse livers at high resolution without background contamination.

Adipose stem cells-derived small extracellular vesicles transport Thrombospondin 1 cargo to promote insulin resistance in gestational diabetes mellitus

BackgroundGestational diabetes mellitus (GDM) is a highly prevalent disease and poses a significant risk to the health of pregnant women. Abdominal adipose tissue (AT) contributes to insulin resistance (IR) associated with GDM. However, the underlying mechanisms remain unclear.MethodsIn this study, we developed a mouse model of GDM by subjecting mice to a high-fat diet. We collected adipose-derived stem cells (ADSCs) from the abdominal and inguinal regions and examined their role in inducing IR in normal tissues through the secretion of small extracellular vesicles (sEVs). The sEVs derived from ADSCs isolated from GDM mice (ADSC/GDM) were found to inhibit cell viability and insulin sensitivity in AML12, a normal mouse liver cell line.ResultsThrough proteomic analysis, we identified high levels of the thrombospondin 1 (Thbs1) protein in the sEVs derived from ADSC/GDM. Subsequent overexpression of Thbs1 protein in AML12 cells demonstrated similar IR as observed with ADSC/GDM-derived sEVs. Mechanistically, the Thbs1 protein within the sEVs interacted with CD36 and transforming growth factor (Tgf) β receptors in AML12 cells, leading to the activation of Tgfβ/Smad2 signaling. Furthermore, the administration of LSKL, an antagonistic peptide targeting Thbs1, suppressed Thbs1 expression in ADSC/GDM-derived sEVs, thereby restoring insulin sensitivity in AML12 cells and GDM mice in vivo.ConclusionsThese findings shed light on the intercellular transmission mechanism through which ADSCs influence hepatic insulin sensitivity and underscore the therapeutic potential of targeting the Thbs1 protein within sEVs.