Healthy Mouse Liver Research Articles

Molecular exploration of the diurnal alteration of glycogen structural fragility and stability in time-restricted-feeding mouse liver

The structure of glycogen α particles in healthy mouse liver has two states: stability and fragility. In contrast, glycogen α particles in diabetic liver present consistent fragility, which may exacerbate hyperglycemia. Currently, the molecular mechanism behind glycogen structural alteration is still unclear. In this study, we characterized the fine molecular structure of liver glycogen α particles in healthy mice under time-restricted feeding (TRF) mode during a 24-h cycle. Then, differentially expressed genes (DEGs) in the liver during daytime and nighttime were revealed via transcriptomics, which identified that the key downregulated DEGs were mainly related to insulin secretion in daytime. Furthermore, GO annotation and KEGG pathway enrichment found that negative regulation of the glycogen catabolic process and insulin secretion process were significantly downregulated in the daytime. Therefore, transcriptomic analyses indicated that the structural stability of glycogen α particles might be correlated with the glycogen degradation process via insulin secretion downregulation. Further molecular experiments confirmed the significant upregulation of glycogen phosphorylase (PYGL), phosphorylated PYGL (p-PYGL), and glycogen debranching enzyme (AGL) at the protein level during the daytime. Overall, we concluded that the downregulation of insulin secretion in the daytime under TRF mode facilitated glycogenolysis, contributing to the structural stability of glycogen α-particles.

Protective Potential of Ashwagandha, Against Pyrifluquinazon Induced Hepato-And Nephrotoxicity in Albino Mice

Globally, due to rise in dependence on the agricultural sector for food supply, the use of insecticides i.e., “Chemicals used to kill insects”, has been increasing day by day. One of these is Pyrifluquinazon. The emerging concern is that after field spray, this insecticide has the tendency to be incorporated into surface water and become a part of the food chain and so becomes toxic for both aquatic and terrestrial organisms. Objective: To investigate the protective potential of Ashwagandha against damaging effect of Pyrifluquinazon (PQZ) on liver and kidney of mature and healthy male albino mice. Methods: Albino mice were divided into four groups, each group contained five mice. First group was control, supplied with water while, three were treated groups (Dose, Dose + Antidote and Antidote group respectively) to check protective potential of Ashwagandha against PQZ induced hepato and nephrotoxicity in mice. The dose group was supplied with PQZ while Dose + Antidote group with PQZ and Ashwagandha as an antidote while the Antidote group was supplied with Ashwagandha. This was accomplished for 20 days trial. Blood sampling and dissection was done at 20th day. Blood sample were taken using intra cardiac sampling method for biochemistry while liver and kidney were separated from each group for histopathological studies. Results: The liver function test and kidney function were normal and indicated restoration of their normal function. Histopathological findings also revealed regeneration in damaged areas of liver and kidney tissues. Conclusions: The Findings of this study clearly revealed the protective potential of Ashwagandha against PQZ induced hepato and nephrotoxicity.

In vivo evaluation of liver function by multimodal imaging in an alcohol-induced liver injury model.

Visually evaluating liver function is a hot topic in hepatology research. There are few reliable and practical visualization methods for evaluating the liver function in vivo in experimental studies. In this study, we established a multimodal imaging approach for in vivo liver function evaluation and compared healthy mice with chronic alcoholic liver injury (cALI) model mice to explore its potential applicability in experimental research. In vivo fluorescence imaging (IVFI) technology was utilized to visually represent the clearance of indocyanine green from the liver of both healthy mice and mice with cALI. The reserve liver function was evaluated via IVFI using the Cy5.5-galactosylated polylysine probe, which targets the asialoglycoprotein receptor of hepatocytes. Hepatic microcirculation was assessed through laser speckle perfusion imaging of hepatic blood perfusion. The liver microstructure was then investigated by in vivo confocal laser endomicroscopy imaging. Finally, hepatic asialoglycoprotein receptor expression, histology, and the levels of serum alanine aminotransferase and aspartate aminotransferase were measured. In vivo multimodal imaging results intuitively and dynamically showed that indocyanine green clearance [mean ± standard deviation (SD): 30.83±14.71, 95% confidence interval (CI): 20.3 to 41.35], the fluorescence signal intensity (mean ± SD: 1,217.92±117.63; 95% CI: 1,148.38 to 1,290.84) and fluorescence aggregation area (mean ± SD: 5,855.80±1,271.81; 95% CI: 5,051.57 to 6,653.88) of Cy5.5-galactosylated polylysine targeting the asialoglycoprotein receptor, and hepatic blood perfusion (mean ± SD: 1,494.86±299.33; 95% CI: 1,316.98 to 1,690.16) in model mice were significantly lower than those in healthy mice (all P<0.001). Compared to healthy mice, the model mice exhibited a significant decline in liver asialoglycoprotein receptor expression (mean ± SD: 219.03±16.34; 95% CI: 208.97 to 230.69; P<0.001), increased serum alanine aminotransferase (mean ± SD: 149.70±47.89 U/L; 95% CI: 81.75 to 128.89; P=0.01) and aspartate aminotransferase levels (mean ± SD: 106.30±36.13 U/L; 95% CI: 122.01 to 180.17; P=0.021), hepatocyte swelling and deformation, disappearance of the hepatic cord structure, partial necrosis, and disintegration of hepatocytes. The imaging features of fluorescence signals in liver regions, hepatic blood perfusion and microstructure were biologically related to hepatic asialoglycoprotein receptor expression, serum indices of liver function, and histopathology in model mice. Utilizing in vivo multimodal imaging technology to assess liver function is a viable approach for experimental research, providing dynamic and intuitive visual evaluations in a rapid manner.

Superparamagnetic Nanocrystals Clustered Using Poly(ethylene glycol)-Crosslinked Amphiphilic Copolymers for the Diagnosis of Liver Cancer.

Superparamagnetic iron oxide (SPIO) nanocrystals have been extensively studied as theranostic nanoparticles to increase transverse (T2) relaxivity and enhance contrast in magnetic resonance imaging (MRI). To improve the blood circulation time and enhance the diagnostic sensitivity of MRI contrast agents, we developed an amphiphilic copolymer, PCPZL, to effectively encapsulate SPIO nanocrystals. PCPZL was synthesized by crosslinking a polyethylene glycol (PEG)-based homobifunctional linker with a hydrophobic star-like poly(ε-benzyloxycarbonyl-L-lysine) segment. Consequently, it could self-assemble into shell-crosslinked micelles with enhanced colloidal stability in bloodstream circulation. Notably, PCPZL could effectively load SPIO nanocrystals with a high loading capacity of 66.0 ± 0.9%, forming SPIO nanoclusters with a diameter of approximately 100 nm, a high cluster density, and an impressive T2 relaxivity value 5.5 times higher than that of Resovist®. In vivo MRI measurements highlighted the rapid accumulation and contrast effects of SPIO-loaded PCPZL micelles in the livers of both healthy mice and nude mice with an orthotopic hepatocellular carcinoma tumor model. Moreover, the magnetic micelles remarkably enhanced the relative MRI signal difference between the tumor and normal liver tissues. Overall, our findings demonstrate that PCPZL significantly improves the stability and magnetic properties of SPIO nanocrystals, making SPIO-loaded PCPZL micelles promising MRI contrast agents for diagnosing liver diseases and cancers.

Elaborately engineering of lipid nanoparticle for targeting delivery of siRNA and suppressing acute liver injury

Small interfering RNA (siRNA)-based gene silencing has been considered as a potential therapy modality against inflammatory diseases. Nevertheless, the effective delivery of siRNA to desired destination still remains challenging due to poor stability, high molecular weight and negative charge. Currently, ionizable lipid nanoparticle (LNP) has been extensively used as vector for effective delivery of siRNA. Herein, we report a mannose-modified LNP (M-MC3 LNP@TNFα) loading tumor necrosis factor α (TNFα) siRNA for targeting liver macrophages, achieving effectively inhibit acute liver injury. The M-MC3 LNP@TNFα not only increases the internalization of LNP by macrophages, but also enhances the gene silencing efficiency of TNFα in vitro. Additionally, the M-MC3 LNP@TNFα exhibits higher accumulation in liver of healthy mice than that of MC3 LNP@TNFα (un-modified LNP) owing to the targeting effect of mannose. As expected, the M-MC3 LNP@TNFα significantly suppresses the expression of TNFα and ameliorates liver damage in acute liver injury model. Such a LNP targeting siRNA delivery holds great potential for the treatment of diseases associated with liver in the future.

Hepatic Symbiotic Bacterium L. reuteri FLRE5K1 Inhibits the Development and Progression of Hepatocellular Carcinoma via Activating the IFN-γ/CXCL10/CXCR3 Pathway.

Symbiotic bacteria participate in the formation of the structure and function of the tissues and organs in which they live, and play an essential role in maintaining the balance between health and disease. Lactobacillus reuteri FLRE5K1 was isolated from the liver of healthy mice and proved to be a probiotic with anti-melanoma activity in previous studies. The relationship between hepatic symbiotic probiotics and hepatocellular carcinoma (HCC) has not been reported yet. In the present study, L. reuteri FLRE5K1 was initially confirmed to successfully enter the liver after being administered by gavage, and the efficacy of probiotic feeding on HCC and its potential mechanism of inhibiting tumor progression were investigated by an orthotopic liver cancer model established. The results showed that L. reuteri FLRE5K1 significantly reduced the tumor formation rate and inhibited tumor growth in mice. From the perspective of mechanism, activation of the IFN-γ/CXCL10/CXCR3 pathway, as well as its positive feedback on the secretion of IFN-γ, induced the polarization of Th0 cell to Th1 cells and inhibited the differentiation of Tregs, which played a key role in the inhibitory effect of L. reuteri FLRE5K1 on the development and progression of HCC.

Attenuation of phenobarbital-induced cytochrome P450 expression in carbon tetrachloride-induced hepatitis in mice models.

Certain pathological conditions, such as inflammation, are known to affect basal cytochrome P450 (CYP) expression by modulating transcriptional regulation, and the pharmacokinetics of drugs can vary among patients. However, changes in drug-induced CYP expression under pathological conditions have not been elucidated in detail. Here, we investigated the effects of hepatic inflammation and injury on phenobarbital-induced expression of CYP isoforms in mice. Phenobarbital was administered once as a CYP inducer in the carbon tetrachloride-induced hepatitis model mice. The mRNA expression levels of Cyp3a11 and Cyp2b10 in the liver and small intestine were measured using reverse transcription polymerase chain reaction. The enzymatic activity of CYP3A in liver S9 was evaluated using midazolam as the substrate. Phenobarbital increased the mRNA expression of Cyp3a11 and Cyp2b10 in the liver of healthy mice, but not in the small intestine. Increased mRNA expression of hepatic Cyp3a11 and Cyp2b10 by phenobarbital was significantly suppressed in the hepatitis model mice. Hepatitis also suppressed the increased CYP3A enzymatic activity induced by phenobarbital in liver S9, consistent with the results of Cyp3a11 mRNA expression. These results suggest that the inducibility of CYP by phenobarbital may vary in patients with hepatitis, indicating that pharmacokinetic drug-drug interactions can be altered under certain pathological conditions.

Comparison of hepatic responses to glucose perturbation between healthy and obese mice based on the edge type of network structures

Interactions between various molecular species in biological phenomena give rise to numerous networks. The investigation of these networks, including their statistical and biochemical interactions, supports a deeper understanding of biological phenomena. The clustering of nodes associated with molecular species and enrichment analysis is frequently applied to examine the biological significance of such network structures. However, these methods focus on delineating the function of a node. As such, in-depth investigations of the edges, which are the connections between the nodes, are rarely explored. In the current study, we aimed to investigate the functions of the edges rather than the nodes. To accomplish this, for each network, we categorized the edges and defined the edge type based on their biological annotations. Subsequently, we used the edge type to compare the network structures of the metabolome and transcriptome in the livers of healthy (wild-type) and obese (ob/ob) mice following oral glucose administration (OGTT). The findings demonstrate that the edge type can facilitate the characterization of the state of a network structure, thereby reducing the information available through datasets containing the OGTT response in the metabolome and transcriptome.

Oral supplementation of a cell preparation of Enterococcus faecalis strain EC-12 stimulates superoxide dismutase production in the livers of healthy and arthritis-induced mice.

Hepatitis, a major human chronic inflammation disease, has been linked to oxidative stress, which can be initiated by radicals produced during the oxidative metabolism. Oxidative damage has been also observed in arthritis-induced mice. Here we evaluated whether supplementation of a cell preparation of Enterococcus faecalis EC-12 could induce superoxide dismutase activity and/or damage in the livers of healthy mice or mice with arthritis. In Experiment 1, both healthy and arthritis-induced mice were orally given a saline solution, or a solution with a low (0.2 mg/mouse/day) or a high (2.0 mg/mouse/day) concentration of E. faecalis EC-12 for 49 consecutive days. Manganese superoxide dismutase activity increased in E. faecalis EC-12-supplemented mice but with no arthritis. In Experiment 2, mice received orally either a saline or an E. faecalis EC-12 suspension (10 mg/kg of body weight/day) for 28 consecutive days. No changes in tissues and levels of function markers and 8-hydroxy-2'-deoxyguanosine were observed in mouse livers, inferring that E. faecalis EC-12 supplementation caused no damage. While mRNA expression of copper/zinc superoxide dismutase remained unaltered, that of manganese superoxide dismutase increased in E. faecalis EC-12 administration mice. In conclusion, at least in healthy mice, E. faecalis EC-12 supplementation stimulated manganese superoxide dismutase activity in liver tissues with no side effects.

In vivo micro-computed tomography imaging in liver tumor study of mice using Fenestra VC and Fenestra HDVC

Contrast agents are used to enhance the visibility of rodent organs during in vivo micro-computed tomography imaging. Specifically, this non-invasive technique can study liver tumor growth and progression in small animals. Fenestra VC and the novel Fenestra HDVC were compared for enhancement in the liver of healthy and tumor-bearing mice, and the images were compared for their ability to define the tumor border, volume and quantity of tumors. Fenestra VC and Fenestra HDVC were injected into healthy eight-week-old female mice (C57BL/6) via the tail vein then imaged at seven different time points. The experimental results showed that 0.005 mL/g of Fenestra HDVC resulted in the same enhancement for all eight organs as 0.01 mL/g of Fenestra VC across all time points. For the tumor study, B16F10 tumors were surgically introduced into ten eight-week-old female mice (C57BL/6) then imaged in vivo over a 3 day period. Ex vivo micro-CT images of the excised livers were also obtained. The tumor volume and quantity were measured in each image, and the tumour progression observed over 3 days. We showed Fenestra HDVC is effective for in vivo imaging in rodents because the optimal enhancement level in organs is maintained at a reduced injection volume.

Poloxamers-based nanomicelles as delivery vehicles of hypericin for hepatic photodynamic therapy

Polymeric micelles in oxyalkylic blocks have been investigated as carriers of hydrophobic photosensitizers for intravenous administration in photodynamic therapy. This study evaluated the uptake of hypericin carried by polymeric micelles formed with Pluronics® P123, F127 and P84 and their photoinduced actions on gluconeogenesis in perfused livers of mice. Uptake and photoinduced action of F127-carried hypericin were also evaluated against cultured human hepatoblastoma HepG2 cells. Respiratory activity was assessed in isolated hepatic mitochondria. Hypericin is also investigated as a promising photosensitizer and it was herein photoinduced with red light. This procedure should activate this agent to a lower extension and prevent damage against healthy tissues. Hypericin carried by P123, P84 and F127 micelles was substantially taken up by perfused livers of healthy mice. However, P123 and P84 inhibited hepatic gluconeogenesis by impairing the aerobic energy supply of the cell, effects probably associated with their more hydrophobic character. Hypericin carried by F127, a more hydrophilic micelle, did not inhibit gluconeogenesis and did not modify the mitochondrial respiration at concentrations lower than 2 μM in the dark or when photoinduced with red light. What is more, F127-carried hypericin was substantially taken up by HepG2 cells and, when photoinduced with red light, considerably decreased the viability of these cells. The results show that F127-carried hypericin could be a suitable photosensitizer system to be used in photodynamic therapy against liver tumors, especially if it is photoinduced with red light, which should activate the molecule to a lower extension and prevent damage to the healthy tissue.

Synthesis of [11C]carbonyl-labeled cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate ([11C-carbonyl]PK68) as a potential PET tracer for receptor-interacting protein 1 kinase

BackgroundReceptor-interacting protein 1 kinase (RIPK1) is a key enzyme in the regulation of cellular necroptosis. Recently, cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate (PK68, 5) has been developed as a potent inhibitor of RIPK1. Herein, we synthesized [11C]carbonyl-labeled PK68 ([11C-carbonyl]PK68, [11C]PK68) as a potential PET tracer for imaging RIPK1 and evaluated its brain uptake in vivo.ResultsWe synthesized [11C]PK68 by reacting amine precursor 14 with [11C]acetyl chloride. At the end of synthesis, we obtained [11C]PK68 of 1200–1790 MBq with a radiochemical yield of 9.1 ± 5.9% (n = 10, decay-corrected to the end of irradiation) and radiochemical purity of > 99%, and a molar activity of 37–99 GBq/μmol starting from 18–33 GBq of [11C]CO2. The fully automated synthesis took 30 min from the end of irradiation. In a small-animal PET study, [11C]PK68 was rapidly distributed in the liver and kidneys of healthy mice after injection, and subsequently cleared from their bodies via hepatobiliary excretion and the intestinal reuptake pathway. Although there was no obvious specific binding of RIPK1 in the PET study, [11C]PK68 demonstrated relatively high stability in vivo and provided useful structural information further candidate development.ConclusionsIn the present study, we successfully radiosynthesized [11C]PK68 as a potential PET tracer and evaluated its brain uptake. We are planning to optimize the chemical structure of [11C]PK68 and conduct further PET studies on it using pathological models.

Survivin expression is essential for early activation of hepatic stellate cells and fibrosis progression in chronic liver injury

AimHepatic fibrosis in injured liver is characterized by the activation of hepatic stellate cells (HSCs) from their quiescent state. Survivin (BIRC5) is one of the key genes that are upregulated during activation of HSCs but their role in HSC activation and fibrosis progression is unknown. Here, we have investigated the role of survivin protein in early fibrogenic activation of HSCs and fibrosis progression in chronic liver injury. Materials & methodsPrimary quiescent HSCs were isolated from healthy mice liver through perfusion and cultured for fibrogenic activation. Survivin expression was suppressed by its pharmacological suppressant, YM155. We developed chronic liver injury induced fibrotic mice model through administrating repeated dose of CCl4 for 2 weeks and 4 weeks. Mice were pre-treated with YM155 a week before CCl4 administration till 2nd week of dosing and then discontinued. Hepatic parameters were characterized and underlying mechanisms were investigated. Key findingsSurvivin expression gradually increased along with the expression of αSMA, collagen I activation maker in HSCs during their activation from quiescent state. Survivin suppression through YM155 downregulated αSMA, collagen I. Pre-treatment of YM155 in mice ceased the early activation of HSCs and onset of fibrosis in injured liver. However, discontinuation of YM155 initiated the activation of HSCs and fibrosis progression that shows survivin expression in HSCs is essential for their early activation and onset of liver fibrosis. SignificanceSurvivin expression induces with activation of HSCs and drives onset of liver fibrosis in injured liver. Targeting survivin protein in activated HSCs could be a potential anti-fibrotic therapeutic approach in chronic liver injury.

Single-cell RNA transcriptome landscape of hepatocytes and non-parenchymal cells in healthy and NAFLD mouse liver

SummaryNonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers.

Photothermal treatment by PLGA–gold nanorod–isatin nanocomplexes under near-infrared irradiation for alleviating psoriasiform hyperproliferation

Psoriasis is a chronic autoimmune skin disorder that involves keratinocyte hyperproliferation and inflammatory cell recruitment. A strategy to mitigate psoriatic lesions is to induce keratinocyte apoptosis for proliferation suppression. Herein we designed a nanoformulation capable of treating psoriasis via hyperthermia-induced apoptosis in response to near-infrared (NIR) irradiation. To this end, gold nanorods (GNRs) and isatin, which is an anti-inflammatory agent for synergizing antipsoriatic activity, were loaded into a poly (lactic-co-glycolic acid) (PLGA) matrix to form the nanocomplexes. The physicochemical and photothermal properties of the nanocomplexes were determined in terms of size, surface charge, NIR-absorbing feature, isatin release, keratinocyte uptake, and cytotoxicity. The nanocomplexes showed a spherical shape with an average size of about 180 nm. The GNR-loaded nanoparticles can efficiently convert NIR light at 0.42 W/cm2 into heat with an increased temperature of 10 °C. When combined with NIR exposure, the nanocomplexes were internalized into keratinocyte cytoplasm with an inhibition of keratinocyte viability to about 60%. Live/dead cell assay and flow cytometry confirmed that the nanocomplexes could serve as NIR-absorbers to specifically elicit keratinocyte apoptosis through caspase and poly ADP-ribose polymerase (PARP) pathways. The in vivo psoriasiform murine model indicated that the combined nanocomplexes and NIR inhibited epidermal hyperplasia and neutrophil infiltration. The overexpressed cytokines in the lesion could be recovered to normal baseline level after the photothermal management. The subcutaneous nanocomplexes remained in the skin for at least 5 days. The nanocomposites produced a negligible toxicity in the skin or liver of healthy mice. The photothermal nanosystems, as designed in this study, shed new light on the therapeutic approach against psoriasis.

Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein.

Citrullinemia type I (CTLN-I) results from the absence or deficiency of argininosuccinate synthetase (AS), a 46 kDa enzyme that acts in the cytosol of hepatocytes to convert aspartic acid and citrulline into argininosuccinic acid. AS is an essential component of the urea cycle, and its absence or deficiency results in the harmful accumulation of ammonia in blood and cerebrospinal fluid. No disease-modifying treatment of CTLN-I exists. Here we report that the cell-permeant miniature protein (CPMP) ZF5.3 (ZF) can deliver AS to the cytosol of cells in culture and the livers of healthy mice. The fusion protein ZF-AS is catalytically active in vitro, stabilized in plasma, and traffics successfully to the cytosol of cultured Saos-2 and SK-HEP-1 cells, achieving cytosolic concentrations greater than 100 nM. This value is 3–10-fold higher than the concentration of endogenous AS (11 ± 1 to 44 ± 5 nM). When injected into healthy C57BL/6 mice, ZF-AS reaches the mouse liver to establish concentrations almost 200 nM above baseline. These studies demonstrate that ZF5.3 can deliver a complex enzyme to the cytosol at therapeutically relevant concentrations and support its application as an improved delivery vehicle for therapeutic proteins that function in the cytosol, including enzyme replacement therapies.

Tissue-specific murine neutrophil activation states in health and inflammation.

Neutrophils are quickly recruited to tissues in response to proinflammatory cues; however, little is known about tissue neutrophil phenotypes in health. We employ a multicolor flow cytometric approach to assess surface markers of activation on neutrophils from the bone marrow, blood, peritoneum, spleen, liver, fat, colon, and oral cavity of healthy mice. Cell preparations were promptly fixed to preserve native surface marker expression levels. Peritoneal, colonic, and oral neutrophils were also assessed in the setting of pHrodo-induced peritonitis, dextran sodium sulfate-induced colitis, and ligature-induced periodontal disease, respectively. Our results demonstrate consistent detectable neutrophil populations in various sterile and nonsterile tissues of healthy mice, and these cells had tissue-specific neutrophil immunophenotypes. Neutrophils derived from biofilm-associated mucosal tissues had 2- to 3-fold higher expression of surface markers of activation, including CD66a, CD11b, and CD62L, compared to neutrophils derived from both sterile healthy tissues as well as tissues in animals treated with broad-spectrum antibiotics. Furthermore, the unique cluster of differentiation (CD) marker activation signatures of tissue-specific neutrophils from the peritoneum, colon, and oral cavity were altered to a proinflammatory immunophenotype with the presence of an inflammatory stimulus. Based on our results, we propose a model whereby a hierarchy of tissue neutrophil immunophenotypes, based on the differential expression of CD markers of activation, correlates with sterile, healthy commensal biofilm-associated and inflamed tissue states.

Transcriptomic landscape profiling of metformin-treated healthy mice: Implication for potential hypertension risk when prophylactically used.

Recently, the first‐line anti‐diabetic drug metformin shows versatile protective effects against several diseases and is potentially prescribed to healthy individual for prophylactic use against ageing or other pathophysiological processes. However, for healthy individuals, it remains unclear what effects metformin treatment will induce on their bodies. A systematic profiling of the molecular landscape of metformin treatment is expected to provide crucial implications for this issue. Here, we delineated the first transcriptomic landscape induced by metformin in 10 tissues (aorta, brown adipose, brain, eye, heart, liver, kidney, skeletal muscle, stomach and testis) of healthy mice by using RNA‐sequencing technique. A comprehensive computational analysis was performed. The overrepresentation of cardiovascular disease‐related gene sets, positive correlation with hypertension‐related transcriptomic signatures and the associations of drugs with hypertensive side effect together indicate that although metformin does exert various beneficial effects, it would also increase the risk of hypertension in healthy mice. This prediction was experimentally validated by an independent animal experiments. Together, this study provided important resource necessary for investigating metformin's beneficial/deleterious effects on various healthy tissues, when it is potentially prescribed to healthy individual for prophylactic use.

Long-term mildronate treatment increased Proteobacteria level in gut microbiome, and caused behavioral deviations and transcriptome change in liver, heart and brain of healthy mice

Mildronate is a cardiac and neuroprotective drug that is widely used in some countries. By inhibiting carnitine biosynthesis, mildronate impairs the fatty acids transport into mitochondria, thereby decreasing the β-oxidation intensity. Since 2016, it has been prohibited by the World Anti-Doping Agency (WADA). However, the information on its safety and its influence on the athletes' health is scarce. There are no published studies on whether mildronate-induced long-term metabolism “rearrangement” may cause negative effects on high-metabolic-rate organs and on the whole organism. Here, we demonstrate that long-term mildronate treatment of healthy mice induced global metabolism change at the transcriptome level in liver, heart, and brain. Mildronate treatment also induced some behavioral changes such as anxiety-related behavior and diminished explorative behavior. We also found that mildronate induced a dysbiosis, as manifested by an increase in Proteobacteria level in gut microbiome. At the same time, the absence of a statistically significant increase in mouse strength and endurance procedures suggests that mildronate effect on productivity is negligible. The sum of our data suggests that long-term treatment of healthy mice with mildronate induces dysbiosis and behavioral deviations despite the effectiveness of mildronate for cardiac and neurological diseases. Thus, we suggest that long-term mildronate treatment is undesirable or at the very least should be accompanied by prebiotics treatments, but this issue should be studied further.

Reevaluation of Lung Injury in TNF-Induced Shock: The Role of the Acid Sphingomyelinase.

Tumor necrosis factor (TNF) is a well-known mediator of sepsis. In many cases, sepsis results in multiple organ injury including the lung with acute respiratory distress syndrome (ARDS). More than 20-year-old studies have suggested that TNF may be directly responsible for organ injury during sepsis. However, these old studies are inconclusive, because they relied on human rather than conspecific TNF, which was contaminated with endotoxin in most studies. In this study, we characterized the direct effects of intravenous murine endotoxin-free TNF on cardiovascular functions and organ injury in mice with a particular focus on the lungs. Because of the relevance of the acid sphingomyelinase in sepsis, ARDS, and caspase-independent cell death, we also included acid sphingomyelinase-deficient (ASM−/−) mice. ASM−/− and wild-type (WT) mice received 50 μg endotoxin-free murine TNF intravenously alone or in combination with the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD) and were ventilated at low tidal volume while lung mechanics were followed. Blood pressure was stabilized by intra-arterial fluid support, and body temperature was kept at 37°C to delay lethal shock and to allow investigation of blood gases, lung histopathology, proinflammatory mediators, and microvascular permeability 6 hours after TNF application. Besides the lungs, also the kidneys and liver were examined. TNF elicited the release of inflammatory mediators and a high mortality rate, but failed to injure the lungs, kidneys, or liver of healthy mice significantly within 6 hours. Mortality in WT mice was most likely due to sepsis-like shock, as indicated by metabolic acidosis, high procalcitonin levels, and cardiovascular failure. ASM−/− mice were protected from TNF-induced hypotension and reflex tachycardia and also from mortality. In WT mice, intravenous exogenous TNF does not cause organ injury but induces a systemic inflammatory response with cardiovascular failure, in which the ASM plays a role.