Mouse Liver Research Articles

Partial hepatectomy accelerates colorectal metastasis by priming an inflammatory premetastatic niche in the liver.

Resection of colorectal liver metastasis is the standard of care for patients with Stage IV CRC. Despite undoubtedly improving the overall survival of patients, pHx for colorectal liver metastasis frequently leads to disease recurrence. The contribution of this procedure to metastatic colorectal cancer at a molecular level is poorly understood. We designed a mouse model of orthograde metastatic colorectal cancer (CRC) to investigate the effect of partial hepatectomy (pHx) on tumor progression. CRC organoids were implanted into the cecal walls of wild type mice, and animals were screened for liver metastasis. At the time of metastasis, 1/3 partial hepatectomy was performed and the tumor burden was assessed longitudinally using MRI. After euthanasia, different tissues were analyzed for immunological and transcriptional changes using FACS, qPCR, RNA sequencing, and immunohistochemistry. Mice that underwent pHx presented significant liver hypertrophy and an increased overall metastatic load compared with SHAM operated mice in MRI. Elevation in the metastatic volume was defined by an increase in de novo liver metastasis without any effect on the growth of each metastasis. Concordantly, the livers of pHx mice were characterized by neutrophil and bacterial infiltration, inflammatory response, extracellular remodeling, and an increased abundance of tight junctions, resulting in the formation of a premetastatic niche, thus facilitating metastatic seeding. Regenerative pathways following pHx accelerate colorectal metastasis to the liver by priming a premetastatic niche.

Hepatocyte-specific Selenoi deficiency predisposes mice to hepatic steatosis and obesity.

Selenoprotein I (Selenoi) is highly expressed in liver and plays a key role in lipid metabolism as a phosphatidylethanolamine (PE) synthase. However, the precise function of Selenoi in the liver remains elusive. In the study, we generated hepatocyte-specific Selenoi conditional knockout (cKO) mice on a high-fat diet to identify the physiological function of Selenoi. The cKO group exhibited a significant increase in body weight, with a 15.6% and 13.7% increase in fat accumulation in white adipose tissue (WAT) and the liver, respectively. Downregulation of the lipolysis-related protein (p-Hsl) and upregulation of the adipogenesis-related protein (Fasn) were observed in the liver of cKO mice. The cKO group also showed decreased oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure (p < .05). Moreover, various metabolites of the steroid hormone synthesis pathway were affected in the liver of cKO mice. A potential cascade of Selenoi-phosphatidylethanolamine-steroid hormone synthesis might serve as a core mechanism that links hepatocyte-specific Selenoi cKO to biochemical and molecular reactions. In conclusion, we revealed that Selenoi inhibits body fat accumulation and hepatic steatosis and elevates energy consumption; this protein could also be considered a therapeutic target for such related diseases.

Line-field dynamic optical coherence tomography platform for volumetric assessment of biological tissues.

Dynamic optical coherence tomography (dOCT) utilizes time-dependent signal intensity fluctuations to enhance contrast in OCT images and indirectly probe physiological processes in cells. Majority of the dOCT studies published so far are based on acquisition of 2D images (B-scans or C-scans) by utilizing point-scanning Fourier domain (spectral or swept-source) OCT or full-field OCT respectively, primarily due to limitations in the image acquisition rate. Here we introduce a novel, high-speed spectral domain line-field dOCT (SD-LF-dOCT) system and image acquisition protocols designed for fast, volumetric dOCT imaging of biological tissues. The imaging probe is based on an exchangeable afocal lens pair that enables selection of combinations of transverse resolution (from 1.1 µm to 6.4 µm) and FOV (from 250 × 250 µm2 to 1.4 × 1.4 mm2), suitable for different biomedical applications. The system offers axial resolution of ∼ 1.9 µm in biological tissue, assuming an average refractive index of 1.38. Maximum sensitivity of 90.5 dB is achieved for 3.5 mW optical imaging power at the tissue surface and maximum camera acquisition rate of 2,000 fps. Volumetric dOCT images acquired with the SD-LF-dOCT system from plant tissue (cucumber), animal tissue (mouse liver) and human prostate carcinoma spheroids allow for volumetric visualization of the tissues' cellular and sub-cellular structures and assessment of cellular motility.

Dihydroartemisinin modulated arachidonic acid metabolism and mitigated liver inflammation by inhibiting the activation of 5-LOX and COX-2

BackgroundDihydroartemisinin (DHA), a derivative of Artemisia annua, has been shown to possess anti-inflammatory properties. Besides, Yes-associated protein 1 (YAP1) plays a crucial role in maintaining liver homeostasis. MethodsThis study used Yap1Flox/Flox, Albumin-Cre mice with hepatocyte-specific Yap1 knockout (referred to as Yap1LKO) and their control mice (Yap1Flox/Flox, referred to as Yap1Flox). The effect of Yap1 on lipid metabolism homeostasis was investigated through non-targeted metabolomic analysis of mouse liver. Subsequently, DHA was administered to Yap1LKO mice to assess its potential as a treatment. Liver pathology was evaluated via H&E staining, and the levels of AST, ALT, and TG were quantified using biochemical assays. The contents of arachidonic acid (AA), prostaglandin E1 (PGE1), and leukotrienes (LT) in the liver were measured using ELISA, while the protein expressions of PLIN2, 5-lipoxygenase (5-LOX), and cyclooxygenase-2 (COX-2) were analyzed through IHC staining. ResultsHepatocyte-specific Yap1 knockout activated the AA metabolic pathway, resulting in increased elevated levels of AA, PGE1, and LT levels, along with inflammatory cytokine infiltration. DHA mitigated the elevation of metabolites such as PGE1 and LT caused by the AA metabolic pathway activation by down-regulating the levels of COX-2 and 5-LOX in the liver of Yap1LKO mice. Moreover, it alleviated the accumulation of lipid vacuoles and reduced triglyceride (TG) and perilipin-2 (PLIN2) levels in the liver of Yap1LKO mice. ConclusionsExcessively low YAP1 expression induces liver inflammation and disturbances in lipid metabolism, whereas DHA modulated AA metabolism and mitigated liver inflammation by inhibiting the activation of 5-LOX and COX-2.

Siglec-H-/- Plasmacytoid Dendritic Cells Protect Against Acute Liver Injury by Suppressing IFN-γ/Th1 Response and Promoting IL-21+ CD4 T Cells

Siglec-H is a receptor specifically expressed in mouse plasmacytoid dendritic cells (pDCs), which functions as a negative regulator of interferon-α production and plays a critical role in pDC maturation to become antigen-presenting cells. The function of pDCs in autoimmune and inflammatory diseases has been reported. However, the effect of Siglec-H expression in pDCs in liver inflammation and diseases remains unclear. Using the model of concanavalin A-induced acute liver injury (ALI), we investigated the Siglec-H/pDCs axis during ALI in BDCA2 transgenic mice and Siglec-H-/- mice. Anti-BDCA2 antibody, anti-interleukin (IL)-21R antibody, and Stat3 inhibitor were used to specifically deplete pDCs, block IL21 receptor, and inhibit Stat3 signaling, respectively. Splenocytes and purified naive CD4 T cells and bone marrow FLT3L-derived pDCs were cocultured and stimulated with phorbol myristate acetate/ionomycin and CD3/CD28 beads, respectively. Data showed that specific depletion of pDCs aggravated concanavalin A-induced ALI. Remarkably, alanine aminotransferase, hyaluronic acid, and proinflammatory cytokines IL6 and tumor necrosis factor-α levels were lower in the blood and liver of Siglec-H knockout mice. This was associated with attenuation of both interferon-γ/Th1 response and Stat1 signaling in the liver of Siglec-H knockout mice while intrahepatic IL21 and Stat3 signaling pathways were upregulated. Blocking IL21R or Stat3 signaling in Siglec-H knockout mice restored concanavalin A-induced ALI. Finally, we observed that the Siglec-H-null pDCs exhibited immature and immunosuppressive phenotypes (CCR9LowCD40Low), resulting in reduction of CD4 T-cell activation and promotion of IL21+CD4 T cells in the liver. During T-cell-mediated ALI, Siglec-H-null pDCs enhance immune tolerance and promote IL21+CD4 T cells in the liver. Targeting Siglec-H/pDC axis may provide a novel approach to modulate liver inflammation and disease.

Punicalagin Ameliorates Diabetic Liver Injury by Inhibiting Pyroptosis and Promoting Autophagy via Modulation of the FoxO1/TXNIP Signaling Pathway.

Diabetic liver injury (DLI) is one of the complications of diabetes mellitus, which seriously jeopardizes human health. Punicalagin (PU), a polyphenolic compound mainly found in pomegranate peel, has been shown to ameliorate metabolic diseases such as DLI, and the mechanism needs to be further explored. In this study, a HFD/STZ-induced diabetic mouse model is established to investigate the effect and mechanism of PU on DLI. The results show that PU intervention significantly improves liver histology and serum biochemical abnormalities in diabetic mice, significantly inhibits the expression of pyroptosis-related proteins such as NLRP3, Caspase1, IL-1β, and GSDMD in the liver of diabetic mice, and up-regulated the expression of autophagy-related proteins. Meanwhile, PU treatment significantly increases FoxO1 protein expression and inhibits TXNIP protein expression in the liver of diabetic mice. The above results are further verified in the HepG2 cell injury model induced by high glucose. AS1842856 is a FoxO1 specific inhibitor. The intervention of AS1842856 combined with PU reverses the regulatory effects of PU on pyroptosis and autophagy in HepG2 cells. In conclusion, this study demonstrates that PU may inhibit pyroptosis and upregulate autophagy by regulating FoxO1/TXNIP signaling, thereby alleviating DLI.

Hepatic miR-149-5p upregulation fosters steatosis, inflammation and fibrosis development in mice and in human liver organoids

Background & aimsThe incidence of metabolic dysfunction – associated steatotic liver disease (MASLD) is increasing worldwide. Alterations of hepatic microRNA (miRNA) expression/activity significantly contribute to the development and progression of MASLD. Genetic polymorphisms of miR-149 are associated with an increased susceptibility to develop MASLD in humans. Aberrant expression of miR-149 was also associated with metabolic alterations in several organs, but the impact of hepatic miR-149-5p deregulation in MASLD remains poorly characterized. MethodsMiR-149-5p was downregulated in the liver of mice by in vivo transduction with hepatotropic adeno-associated virus 8 (AAV8) harboring shRNAs specific for miR-149-5p (shmiR149) or scrambled shRNAs (shCTL). MASLD was then induced in mice with a methionine/ choline deficient (MCD, n=7 per group) or a fructose, palmitate and cholesterol-enriched (FPC, n=8-12 per group, per protocol) diet. The impact of miR-149-5p modulation on MASLD development was assessed in vivo and in vitro using multi-lineage 3D human liver organoids (HLOs) and in Huh7 cells. ResultsMiR-149-5p expression was strongly upregulated in mouse liver from different models of MASLD (2-4 fold increase in ob/ob, db/db mice, high-fat and FPC fed mice). In vivo downregulation of miR-149-5p led to an amelioration of diet-induced hepatic steatosis, inflammation/fibrosis, and to increased whole-body fatty acid consumption. In HLOs, miR-149-5p overexpression promoted lipid accumulation, inflammation and fibrosis. In vitro analyses of human Huh7 cells overexpressing miR-149-5p indicated that glycolysis and intracellular lipid accumulation was promoted, while mitochondrial respiration was impaired. Translatomic analyses highlighted deregulation of multiple miR-149-5p potential targets in hepatocytes involved in MASLD development. ConclusionsMiR-149-5p upregulation contributes to MASLD development by affecting multiple metabolic/inflammatory/fibrotic pathways in hepatocytes. Our results further demonstrate that human liver organoids are a relevant 3D in vitro model to investigate hepatic steatosis and inflammation/fibrosis development. Impact and implicationsThis study delves into the escalating global concern of metabolic dysfunction-associated steatotic liver disease (MASLD). Our research shows compelling evidence that miR-149-5p plays a pivotal role in the disease's development and progression. By employing in vivo and innovative in vitro models using multi-lineage human liver organoids (HLOs), we demonstrate that miR-149-5p upregulation significantly impacts hepatocyte energy metabolism, exacerbating hepatic steatosis and inflammation/fibrosis by modulating a wide network of target genes. These findings not only shed light on the intricate miR-149-5p-dependent molecular mechanisms underlying MASLD, but also underscore the importance of HLOs as valuable 3D in vitro models for studying the disease's pathogenesis.

Peritumoral HLJ1-STAT3 pathway investigation in carcinogen-induced hepatocellular carcinoma.

e16251 Background: Hepatocellular carcinoma (HCC) is a major global health issue and its treatment options have been limited. Therefore, it is urgent to develop cancer biomarkers for defining cancer risk, diagnosis, prognosis, and even finding new therapeutic targets. Human Liver DnaJ-Like Protein (HLJ1), which belongs to DnaJ heat shock protein family (HSP40) member B4 (DNAJB4), has been identified as a tumor suppressor in various cancers including lung cancer and colorectal cancer. Though the biological function of HLJ1 in either cancer or the liver has emerged in recent years, the precise role of HLJ1 in HCC carcinogenesis is still unclear. Methods: Samples for gene expression cDNA microarray were from 6-8 weeks wild-type (WT) and HLJ1 knockout (HLJ1-/-) mice liver. For the long-term carcinogenesis study, both mice are injected with 20 mg/kgbw diethylnitrosamine (DEN) at postnatal day 15. After that, 50 mg/kgbw DEN was administrated weekly to mice between the ages of 4 to 12 weeks. Mice were sacrificed at ages 30 and 36 weeks to assess the tumor count and size. For the short-term DEN study, 6-8-week-old mice were injected with 100 mg/kg DEN, and 24 and 48 hours after administration the liver tissues were frozen and fixed for western blotting and IHC analysis. Cancer cell transplantation was performed by intrasplenic injection of HLJ1-wildtype LLC and B16F1 cells into both genotypes. Clinical information of 362 liver cancer patients from the TCGA database was analyzed for the correlation between HLJ1 expression and survival. Results: In cDNA microarray analysis, We demonstrated that HLJ1 deficiency led to HCC gene signatures associated with activated IL-6/STAT3 signaling pathways. We used the DEN as a carcinogen and found STAT3 and H2AX phosphorylation induced by short-term DEN treatment was amplified in HLJ1-deficient mice. In long-term DEN experiments, HLJ1 deletion promoted tumor initiation and proliferation at the early and late stages of HCC progression accompanied by pronounced STAT3 phosphorylation in the normal part instead of the tumor part. Furthermore, transplantation of HLJ1-wildtype B16F1 and LLC cell lines into HLJ1-deficient mice led to more tumorigenic phenotype than into wildtype mice. Clinical information of 362 liver cancer patients from the TCGA database exhibited that higher expression of HLJ1 in the tumor part is correlated to a lower disease-specific survival rate. Conclusions: In summary, HLJ1 protects against DEN-induced HCC hepatocarcinogenesis through the regulation of STAT3 signaling in the peritumoral normal cells. Hence, HLJ1 can be a prognostic biomarker for liver cancer and a promising therapeutic target for HCC in the future.

Skeletal muscle BMAL1 is necessary for transcriptional adaptation of local and peripheral tissues in response to endurance exercise training

ObjectiveIn this investigation, we addressed the contribution of the core circadian clock factor, BMAL1, in skeletal muscle to both acute transcriptional responses to exercise and transcriptional remodeling in response to exercise training. Additionally, we adopted a systems biology approach to investigate how loss of skeletal muscle BMAL1 altered peripheral tissue homeostasis as well as exercise training adaptations in iWAT, liver, heart, and lung of male mice. MethodsCombining inducible skeletal muscle specific BMAL1 knockout mice, physiological testing and standardized exercise protocols, we performed a multi-omic analysis (transcriptomics, chromatin accessibility and metabolomics) to explore loss of muscle BMAL1 on muscle and peripheral tissue responses to exercise. ResultsMuscle-specific BMAL1 knockout mice demonstrated a blunted transcriptional response to acute exercise, characterized by the lack of upregulation of well-established exercise responsive transcription factors including Nr4a3 and Ppargc1a. Six weeks of exercise training in muscle-specific BMAL1 knockout mice induced significantly greater and divergent transcriptomic and metabolomic changes in muscle. Surprisingly, liver, lung, inguinal white adipose and heart showed divergent exercise training transcriptomes with less than 5% of ‘exercise-training’ responsive genes shared for each tissue between genotypes. ConclusionsOur investigation has uncovered the critical role that BMAL1 plays in skeletal muscle as a key regulator of gene expression programs for both acute exercise and training adaptations. In addition, our work has uncovered the significant impact that altered exercise response in muscle and its likely impact on the system plays in the peripheral tissue adaptations to exercise training. Our work also demonstrates that if the muscle adaptations diverge to a more maladaptive state this is linked to increased gene expression signatures of inflammation across many tissues. Understanding the molecular targets and pathways contributing to health vs. maladaptive exercise adaptations will be critical for the next stage of therapeutic design for exercise mimetics.

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.

Lipid nanoparticle-encapsulated DOCK11-siRNA efficiently reduces hepatitis B virus cccDNA level in infected mice

The hepatitis B virus (HBV) infects many people worldwide. As HBV infection frequently leads to liver fibrosis and carcinogenesis, developing anti-HBV therapeutic drugs is urgent. Therapeutic drugs for preventing covalently closed circular DNA (cccDNA) production, which can eliminate HBV infection, are unavailable. The host factor dedicator of cytokinesis 11 (DOCK11) is involved in the synthesis and maintenance of HBV cccDNA in vitro. However, the effectiveness of DOCK11 as a target for the in vivo elimination of HBV cccDNA remains unclear. In this study, we assessed whether DOCK11 inhibitors suppressed HBV cccDNA production in mouse models of HBV infection. The tocopherol-conjugate hetero gapmer, a DNA/RNA duplex of gapmer/complementary RNA targeting the DOCK11 sequence, partially reduced the expression of DOCK11 but not that of HBV cccDNA, in the livers of HBV-infected human hepatocyte chimeric mice, along with weight loss and decreased serum human albumin levels. Lipid nanoparticle-encapsulated chemically modified siRNAs specific for DOCK11 suppressed DOCK11 expression and decreased HBV cccDNA levels without adverse effects in the mice. Therefore, nucleic acid-based drugs targeting DOCK11 in hepatocytes are potentially effective anti-HBV therapeutics, which can reduce HBV cccDNA levels in vivo.

Histopathological changes of mice tissues in course of the envenomation by the Macrovipera lebetina obtusa venom and the neutralizing effect of the ovine-derived experimental antivenom

Viper bites pose a significant public health issue in Armenia, even within urban areas, often resulting in clotting disorders, hypofibrinogenemia, and tissue necrosis in humans. This study investigates histopathological changes in various tissues during mice envenomation by West-Asian blunt-nosed viper (Macrovipera lebetina obtusa) venom, as well as the recovery process aided by experimental antivenom derived from sheep. The high venom dose caused substantial damage to the heart, lungs, liver, and kidneys in mice, indicating systemic harm. While antivenom administration can prevent mortality in mice envenomation, it may not fully mitigate histological damage in affected organs. Additionally, the study highlights the importance of timing antivenom administration, as the severity of tissue alterations can vary depending on the duration of envenomation. These findings shed light on antivenom's effects on viper envenomation and stress the need for further research to optimize its timing and dosage for minimizing histological damage and enhancing clinical outcomes.

Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver: A Focus on Genomic Imprinting by Tissue and Sex.

Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. Female mice were exposed to human relevant doses of either Pb () via drinking water or DEHP () via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes ( and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types ( and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.

Light-controlled crosslinked multifunctional "Band-Aids" as dual-stage wound dressing for dynamic wound closure

The objective of regenerative wound healing dressings is to accelerate skin tissue regeneration and restore normal physiological function at wound sites. Achieving this goal requires biomaterials capable of repairing distinct phases of wound healing in a way that balances material function, degradation, safety, and tissue growth. In this study, we introduced a novel dual-stage wound dressing system comprising methacrylic anhydride-modified recombinant humanized type III collagen (rhCol III-MA) and methacrylic anhydride-modified dopamine (DMA) (RMDM), which was synthesized through free radical polymerization and π-π stacking. Within this system, RMDM was formulated into two forms with identical compositions: hydrogel and sponge, tailored for application across various stages of wound repair. These materials displayed favorable hemocompatibility, biocompatibility, antioxidant properties, and angiogenic potential in vitro. Moreover, the in vivo experiments also demonstrated that sponges could rapidly stop the bleeding of wounds in mouse tail amputation and liver incision models. Notably, the sponge/gel (S/G) system accelerated wound healing compared to individual sponge and gel treatments in a rat full-thickness skin wound model, underscoring the synergistic benefits of combining sponge and gel materials for wound repair at different stages. Therefore, this research provides valuable insights into designing advanced biomaterials that can be tailored to specific stages of wound healing, which may have significant potential for biomedical applications.Graphical

Collagen/Curdlan composite sponge for rapid hemostasis and skin wound healing

Collagen's unique properties promise hemostatic potential, but its sponge form's stability and mechanics need improvement. In this study, we developed a series of homeostatic sponges by co-assembling collagen and curdlan at different ratios into hydrogels, followed by freeze-drying treatment. The incorporation of curdlan into collagen sponges has been found to significantly enhance the sponge's properties, including increased porosity, elevated water uptake, improved elasticity, and enhanced resistance to degradation. In vitro cytotoxicity and hemolysis assays have demonstrated the biocompatibility and nontoxicity of composite sponges. In mouse liver perforation and incision models, the composite sponges achieved rapid coagulation within 67 s and 75 s, respectively, outperforming gauze and gelatin sponge in reducing blood loss. Furthermore, composite sponges demonstrated superior wound healing potential in mice full-thickness skin defects model, with accelerated healing rates observed at days 3, 7, and 14 compared to the control group. Overall, collagen/curdlan composite sponge show promise for hemostasis and wound healing applications.

BTLA deficiency promotes HSC activation and protects against hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is unavoidable even despite the development of more effective surgical approaches. During hepatic IRI, activated HSC (aHSC) are involved in liver injury and recovery. A proportion of aHSC increased significantly both in the mouse liver tissues with IRI and in the primary mouse HSCs and LX-2 cells during hypoxia-reoxygenation. "Loss-of-function" experiments revealed that depleting aHSC with gliotoxin exacerbated liver damage in IRI mice. Subsequently, we found that the transcription of mRNA and the expression of B and T lymphocyte attenuator (BTLA) protein were lower in aHSC compared with quiescent HSCs. Interestingly, overexpression or knockdown of BTLA resulted in opposite changes in the activation of specific markers for HSCs such as collagen type I alpha 1, α-smooth muscle actin, and Vimentin. Moreover, the upregulation of these markers was also observed in the liver tissues of global BLTA-deficient (BTLA-/-) mice and was higher after hepatic IRI. Compared with wild-type mice, aHSC were higher, and liver injury was lower in BTLA-/- mice following IRI. However, the depletion of aHSC reversed these effects. In addition, the depletion of aHSC significantly exacerbated liver damage in BTLA-/- mice with hepatic IRI. Furthermore, the TGF-β1 signaling pathway was identified as a potential mechanism for BTLA to negatively regulate the activation of HSCs in vivo and in vitro. These novel findings revealed a critical role of BTLA. Particularly, the receptor inhibits HSC-activated signaling in acute IRI, implying that it is a potential immunotherapeutic target for decreasing the IRI risk.

MLKL deficiency alleviates acute alcoholic liver injury via inhibition of NLRP3 inflammasome

Mixed lineage kinase domain-like protein (MLKL) is identified as the terminal executor of necroptosis. However, its role in acute alcoholic liver injury remains unclear. This study elucidates that MLKL can contribute to acute alcoholic liver injury independently of necroptosis. Although the expression of MLKL was upregulated, no significant increase in its phosphorylation or membrane translocation was observed in the liver tissues of mice treated with ethanol. This finding confirms that alcohol intake does not induce necroptosis in mouse liver tissue. Additionally, the deletion of Mlkl resulted in the downregulation of NLRP3 expression, which subsequently inhibited the activation of the NLRP3 inflammasome and the ensuing inflammatory response, thereby effectively mitigating liver injury induced by acute alcohol consumption. The knockout of Nlrp3 did not affect the expression of MLKL, further confirming that MLKL acts upstream of NLRP3. Mechanistically, inhibiting the nuclear translocation of MLKL reduced the nuclear entry of p65, the principal transcriptional regulator of NLRP3, thereby limiting the transcription of Nlrp3 mRNA and subsequent NLRP3 expression. Overall, this study unveils a novel mechanism of MLKL regulates the activation of NLRP3 inflammasomes in a necroptosis independent way in acute alcoholic liver injury

Single-cell transcriptomics reveal the microenvironment landscape of perfluorooctane sulfonate-induced liver injury in female mice

Epidemic and animal studies have reported that perfluoroalkyl and polyfluoroalkyl substances (PFASs) are strongly associated with liver injury; however, to date, the effects of PFASs on the hepatic microenvironment remain largely unknown. In this study, we established perfluorooctane sulfonic acid (PFOS)–induced liver injury models by providing male and female C57BL/6 mice with water containing PFOS at varying doses for 4 weeks. Hematoxylin and eosin staining revealed that PFOS induced liver injury in both sexes. Elevated levels of serum aminotransferases including those of alanine aminotransferase and aspartate transaminase were detected in the serum of mice treated with PFOS. Female mice exhibited more severe liver injury than male mice. We collected the livers from female mice and performed single-cell RNA sequencing. In total, 36,529 cells were included and grouped into 10 major cell types: B cells, granulocytes, T cells, NK cells, monocytes, dendritic cells, macrophages, endothelial cells, fibroblasts, and hepatocytes. Osteoclast differentiation was upregulated and the T cell receptor signaling pathway was significantly downregulated in PFOS-treated livers. Further analyses revealed that among immune cell clusters in PFOS-treated livers, Tcf7+CD4+T cells were predominantly downregulated, whereas conventional dendritic cells and macrophages were upregulated. Among the fibroblast subpopulations, hepatic stellate cells were significantly enriched in PFOS-treated female mice. CellphoneDB analysis suggested that fibroblasts interact closely with endothelial cells. The major ligand–receptor pairs between fibroblasts and endothelial cells in PFOS-treated livers were Dpp4_Cxcl12, Ackr3_Cxcl12, and Flt1_complex_Vegfa. These genes are associated with directing cell migration and angiogenesis. Our study provides a general framework for understanding the microenvironment in the livers of female mice exposed to PFOS at the single-cell level.

Covalent Binding of Reactive Anhydride of Cantharidin to Biological Amines.

Cantharidin is a terpenoid from coleoptera beetles. Cantharidin has been used to treat molluscum contagiosum and some types of tumors. Cantharidin is highly toxic, and cantharidin poisoning and fatal cases have been reported worldwide. The mechanisms underlying cantharidin-induced toxicity remain unclear. Cantharidin contains anhydride, which may react with biologic amines. This study aimed to examine the chemical reactivity of cantharidin toward nucleophiles and characterize adducts of cantharidin with biologic amines in vitro and in mice. Here two types of conjugates were formed in the incubation of cantharidin under physiologic conditions with free amino acids, a mimic peptide, or amine-containing compounds, respectively. Amide-type conjugates were produced by the binding of cantharidin anhydride with the primary amino group of biologic amines. Imide-type conjugates were generated from the dehydration and cyclization of amide-type conjugates. The structure of the conjugates was characterized by using high-resolution mass spectrometry. We introduced the 14N/15N and 79Br/81Br isotope signatures to confirm the formation of conjugates using L-(ε)15N-lysine, L-lysine-15N2, and bromine-tagged hydrazine, respectively. The structure of imide conjugate was also confirmed by nuclear magnetic resonance experiments. Furthermore, the amide and imide conjugates of cantharidin with amino acids or N-acetyl-lysine were detected in mouse liver and urine. Cantharidin was found to modify lysine residue proteins in mouse liver. Pan-cytochrome P450 inhibitor 1-aminobenzotriazole significantly increased the urine cantharidin-N-acetyl-lysine conjugates, whereas it decreased cantharidin metabolites. In summary, cantharidin anhydride can covalently bind to biologic amines nonenzymatically, which facilitates a better understanding of the role of nonenzymatic reactivity in cantharidin poisoning. SIGNIFICANCE STATEMENT: Anhydride moiety of cantharidin can covalently bind to the primary amino group of biological amines nonenzymatically. Amide and imide conjugates were generated after the covalent binding of cantharidin anhydride with the primary amino groups of amino acids, a mimic peptide, and protein lysine residues. The structure of conjugates was confirmed by 14N/15N and 79Br/81Br isotope signatures using isotope-tagged reagents and nuclear magnetic resonance experiments. This study will facilitate the understanding of the role of nonenzymatic reactivity in cantharidin poisoning.

From genes to phenotype: investigating the link between smad2rs29725537:c&amp;gt;a and high take off in mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación, Gobierno de España. Introduction and Purpose High take off (HTO) is a congenital anomaly with a prevalence of 0.36% that can cause severe ischemia and sudden cardiac death. The C57Bl/6 (B6) mouse strain shows around 60% incidence of HTO, whereas it is absent in the Balb/c (BC) strain. The aim of this study was to identify the genes responsible for the development of HTO. Methods Following an inheritance study, a genetic linkage study was performed in B6C mice using 134 microsatellites (MIT). After selection of Smad2 as a candidate gene, a genotype-phenotype study was performed by PCR-RFLP genotyping of 293 B6C mice. The process of coronariogenesis was examined in E13.5 and E14.5 mouse embryos of B6 (n = 27) and BC (n = 23) strains with specific markers for the vascular plexuses involved in coronariogenesis (PROX1 and ERG 1/2/3) and for SMAD2. Finally, the SMAD2 and pSMAD2 protein expression levels were analyzed by western blot in ventricle, liver, and kidney of B6C mice. Results The incidence of HTO in B6, BC and B6C mice were concordant with an inheritance pattern mediated by a dominant causal allele and dominant modifier in the BC strain that reduces the incidence of HTO. The genetic linkage study revealed the association of a region between markers MIT-49 and 106 on chromosome 18 with the presence of HTO. This region contained a total of 10 genes, of which Smad2 was selected as a candidate gene because of its functions in aortic root formation. Comparative sequence analysis of Smad2 between the B6 and BC mouse strains uncovered an intronic SNP (Smad2rs29725537:C&amp;gt;A or Smad2C&amp;gt;A) that predicts and error in the splicing of intron 10-11. A statistically significant association (p&amp;lt;0.05) between the Smad2C&amp;gt;A allele and the presence of HTO was found. pSMAD2 was detected in embryonic coronary primordia, the subepicardial aortic plexus (SAP) and the primary coronary plexus. Around 60% of B6 examined embryos showed an exacerbated SAP compared to the BC embryos. Additionally, we detected a differential expression of total SMAD2, but not of pSMAD2, in different organs according to the Smad2C&amp;gt;A allele genotype (C/C &amp;gt; A/C &amp;gt; A/A). Conclusions We conclude that: 1) Smad2C&amp;gt;A allelic variant is associated with HTO in mice, suggesting it as a major causal factor. 2) Smad2 altered expression may affect the establishment of the embryonic coronary primordia leading to a mispositioned ostium. 3) the Smad2C&amp;gt;A allele has a systemic effect on total SMAD2 expression without affecting its phosphorylation level, which suggest a compensatory mechanism of expression that maintain the normal activity of pSMAD2 in most tissues.