C57 Mice Research Articles

Protective effects of nicotinamide mononucleotide on radiation-related cardiac injury

Abstract Background Radiation therapy for cancer treatment can cause damage to the heart. There are numerous possible mechanisms related to radiation-related cardiac dysfunction, among which increased oxidative stress is an important factor. However, the detailed mechanisms remain unclear and effective treatments on radiation-related cardiac dysfunction are still lacking. Nicotinamide mononucleotide (NMN), the most direct precursor of nicotinamide adenine dinucleotide (NAD+), can effectively reduce reactive oxygen species (ROS) production and thus could be a candidate to alleviate radiation-induced damage. However, the protective effects and the underlying mechanisms of NMN on radiation-related cardiac injury remain to be elucidated. Therefore, this study was designed to identify the mechanisms of radiation-related cardiac injury and investigate the protective actions of NMN. Methods Wild-type c57 mice were divided into radiation exposure (7.2Gy radiation) and non-exposure groups. Wild-type C57BL6/J mice supplied with NMN for 12 months before radiation. Echocardiography to detect mice cardiac function. Epicardial electrical mapping technique to detect electrical conduction in the heart. Non-targeted metabolomics was used to detect cardiac different metabolites. In parallel, we observed the effect of radiation on survival and mitochondrial function (ROS production and ATP content) of H9C2 cells with or without NMN. Results In the radiation exposure group, echocardiography showed that the left ventricular ejection fraction (LVEF) and fractional shortening (FS) were reduced, whilst epicardial mapping demonstrated slowed conduction velocity and change in the direction of propagation. Non-targeted metabolomics showed the different metabolites between the radiation group and the control group were mainly in nucleotide and amino acid metabolism. Metabolic pathway enrichment analysis showed that different metabolites were implicated in the metabolism of pyrimidine, folate, arginine and proline, and tryptophan. Mice with NMN before radiation showed reduced mechanical and electrical dysfunction, with non-targeted metabolomic results showed NMN rescued the radiation-induced down-regulation of 1-(4-aminobutyl)urea, 4-aminobenzoic acid, 3-hydroxyanthranilic acid, which ultimately exerted cardioprotective effects. We also found that radiation can reduce the survival, increase ROS production, and reduce ATP content of H9C2 cells in vitro, and these harmful effects can be partially reversed by NMN. Conclusion The mechanical and electrical dysfunction caused by radiation were associated with altered nucleotide and amino acid metabolism in the myocardium, and these harmful effects could be partially mitigated by NMN supplement.

The role of the transsulfuration pathway in spermatogenesis of vitamin D deficient mice

Vitamin D deficiency is a global health problem and has been linked to defective spermatogenesis and male infertility. In this study, we aimed to investigate the main enzymes involved in the transsulfuration pathway of 1-carbon metabolism, and spermatogenesis function. Therefore, sixteen male C57 mice were addressed to a control (standard diet) or vitamin D deficient (VDD) diet for 14 weeks. The results show that compared to the standard diet, VDD increased final body weight and reduced sperm quality, caused damage to the testicular structure, and decreased the serum levels of testosterone. In addition, serum concentrations of homocysteine, vitamin B12, and sperm oxidative stress markers increased. In testicular tissues, the CBS and CSE protein levels were down-regulated whereas HO-1 was up-regulated at both mRNA and protein expression levels. Within a mice deprivation model, VDD deeply suppressed testosterone and impaired spermatogenesis with oxidative stress-mediated mechanisms. The effects of the deprivation appeared to be at least in part independent of genomic and receptor-mediated vitamin D actions and suggest a specific impairment of the alternative transsulfuration pathway.

A Comparative Study of Antitumor Immunity Induced by Radiofrequency Microwave and Cryoablation in Hepatocellular Carcinoma.

This study aimed to compare the immune responses induced by microwave ablation (MWA), radiofrequency ablation (RFA), and cryoablation (CRYO) in hepatocellular carcinoma (HCC) and identify differences in immune responses and the timing of immune changes. A bilateral subcutaneous model was established in C57 mice, and the successfully modeled mice were divided into the microwave (n = 15), radiofrequency (n = 15), CRYO (n = 15), control (n = 9), and blank groups (n = 3). Mice in the control group were dissected before ablation, whereas mice in the three ablation groups underwent ultrasound-guided ablation of one axillary tumor. Three mice were sacrificed and dissected at 1-4 weeks after ablation. After tissue processing, flow cytometry was used to detect the levels of CD8 + T and regulatory T (Treg) cells in the tissue, and western blotting was used to assess the level of programmed cell death ligand 1 (PD-L1) protein in the tumor tissue. The pattern of immune changes after the three types of ablation was consistent, with immune changes occurring at 3-4 weeks. CRYO induced the most significant increase in the percentage of CD8 + T cells. There were no significant differences in the levels of Treg cells and the level of PD-L1 protein among the three types of ablation (p > 0.05), but the decline in Treg cells and PD-L1 protein level caused by CRYO was the most pronounced. In the HCC mouse model, the immune changes following the three types of ablation were consistent, with immune changes occurring at 3-4 weeks. Among them, CRYO elicited the strongest adaptive immune response, and RFA outperformed MWA.

Sting mutation attenuates acetaminophen-induced acute liver injury by limiting NLRP3 activation

Acetaminophen (N-acetyl-p-aminophenol; APAP), a widely used effective nonsteroidal anti-inflammatory drug, leads to acute liver injury at overdose worldwide. Evidence showed that the severity of liver injury associated with the subsequent involvement of inflammatory mediators and immune cells. The innate immune stimulator of interferon genes protein (STING) pathway was critical in modulating inflammation. Here, we show that STING was activated and inflammation was enhanced in the liver in APAP-overdosed C57BL/6J mice, and Sting mutation (Stinggt/gt) mice exhibited less liver damage. Multiplexing flow cytometry displayed that Sting mutation changed hepatic recruitment and replacement of macrophages/monocytes in APAP-overdosed mice, which was inclined to anti-inflammation. In addition, Sting mutation limited NLRP3 activation in the liver in APAP-overdosed mice, and inhibited the expression of inflammatory cytokines. Finally, MCC950, a potent and selective NLRP3 inhibitor, significantly ameliorated APAP-induced liver injury and inflammation. Besides, pretreatment of MCC950 in C57 mice resulted in changes of immune cells infiltration in the liver similar to Stinggt/gt mice. Our study revealed that STING played a crucial role in APAP-induced acute liver injury, possibly by maintaining liver immune cells homeostasis and inhibiting NLRP3 inflammasome activation, suggesting that inhibiting STING-NLRP3 pathway might be a potential therapeutic strategy for acute liver injury.

Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease characterised by severe muscle wasting. The mechanisms underlying the DMD pathology likely involve the interaction between inflammation, oxidative stress and impaired Ca2+ signalling. Hypochlorous acid (HOCl) is a highly reactive oxidant produced endogenously via myeloperoxidase; an enzyme secreted by neutrophils that is significantly elevated in dystrophic muscle. Oxidation of Ca2+ -handling proteins by HOCl may impair Ca2+ signalling. This study aimed to determine the effects of HOCl on skeletal muscle function and its potential contribution to the dystrophic pathology. Extensor digitorum longus (EDL), soleus and interosseous muscles were surgically isolated from anaesthetised C57 (wild-type) and mdx (dystrophic) mice for measurement of ex vivo force production and intracellular Ca2+ concentration. In whole EDL muscle, HOCl (200μM) significantly decreased maximal force and increased resting muscle tension which was only partially reversible by dithiothreitol. The effects of HOCl (200μM) on maximal force in slow-twitch soleus were lower than found in the fast-twitch EDL muscle. In single interosseous myofibres, HOCl (10μM) significantly increased resting intracellular Ca2+ concentration and decreased Ca2+ transient amplitude. These effects of HOCl were reduced by the application of tetracaine, Gd3+ or streptomycin, implicating involvement of ryanodine receptors and transient receptor potential channels. These results demonstrate the potent effects of HOCl on skeletal muscle function potentially mediated by HOCl-induced oxidation to Ca2+ signalling proteins. Hence, HOCl may provide a link between chronic inflammation, oxidative stress and impaired Ca2+ handling that is characteristic of DMD and presents a potential therapeutic target for DMD. KEY POINTS: Duchenne muscular dystrophy is a fatal genetic disease with pathological mechanisms which involve the complex interaction of chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations. Hypochlorous acid can be endogenously produced by neutrophils via the enzyme myeloperoxidase. Both neutrophil and myeloperoxidase activity are increased in dystrophic mice. This study found that hypochlorous acid decreased muscle force production and increased cytosolic Ca2+ concentrations in isolated muscles from wild-type and dystrophic mice at relatively low concentrations of hypochlorous acid. These results indicate that hypochlorous acid may be key in the Duchenne muscular dystrophy disease pathology and may provide a unifying link between the chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations observed in Duchenne muscular dystrophy. Hypochlorous acid production may be a potential target for therapeutic treatments of Duchenne muscular dystrophy.

In Silico Analyses, Experimental Verification and Application in DNA Vaccines of Ebolavirus GP-Derived pan-MHC-II-Restricted Epitopes.

(1) Background and Purpose: Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), which causes extremely high mortality and widespread epidemics. The only glycoprotein (GP) on the surface of EBOV particles is the key to mediating viral invasion into host cells. DNA vaccines for EBOV are in development, but their effectiveness is unclear. The lack of immune characteristics resides in antigenic MHC class II reactivity. (2) Methods: We selected MHC-II molecules from four human leukocyte antigen II (HLA-II) superfamilies with 98% population coverage and eight mouse H2-I alleles. IEDB, NetMHCIIpan, SYFPEITHI, and Rankpep were used to screen MHC-II-restricted epitopes with high affinity for EBOV GP. Further immunogenicity and conservation analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis and binding affinity analysis of EBOV GP epitopes and selected MHC-II molecules were performed using data from NetMHCIIpan. The selective GP epitopes were verified by the enzyme-linked immunospot (ELISpot) assay using splenocytes of BALB/c (H2d), C3H, and C57 mice after DNA vaccine pVAX-GPEBO immunization. Subsequently, BALB/c mice were immunized with Protein-GPEBO, plasmid pVAX-GPEBO, and pVAX-LAMP/GPEBO, which encoded EBOV GP. The dominant epitopes of BALB/c (H-2-I-AdEd genotype) mice were verified by the enzyme-linked immunospot (ELISpot) assay. It is also used to evaluate and explore the advantages of pVAX-LAMP/GPEBO and the reasons behind them. (3) Results: Thirty-one HLA-II-restricted and 68 H2-I-restricted selective epitopes were confirmed to have high affinity, immunogenicity, and conservation. Nineteen selective epitopes have cross-species reactivity with good performance in MHC-II molecular docking. The ELISpot results showed that pVAX-GPEBO could induce a cellular immune response to the synthesized selective peptides. The better immunoprotection of the DNA vaccines pVAX-LAMP/GPEBO coincides with the enhancement of the MHC class II response. (4) Conclusions: Promising MHC-II-restricted candidate epitopes of EBOV GP were identified in humans and mice, which is of great significance for the development and evaluation of Ebola vaccines.

Effects of Fmr1 Gene Mutations on Sex Differences in Autism-Like Behavior and Dendritic Spine Development in Mice and Transcriptomic Studies

Fragile X syndrome (FXS) is the most common single gene disorder contributing to autism spectrum disorder (ASD). Although significant sex differences are observed in FXS, few studies have focused on the phenotypic characteristics as well as the differences in brain pathological changes and gene expression in FXS by sex. Therefore, we analyzed sex differences in autism-like behavior and dendritic spine development in two-month-old male and female Fmr1 KO and C57 mice and evaluated the mechanisms at transcriptome level. Results suggest that Fmr1 KO mice display sex differences in autism-like behavior and dendritic spine density. Compared to females, male had more severe effects on anxiety, repetitive stereotype-like behaviors, and socializing, with higher dendritic spine density. Furthermore, two male-biased and five female-biased expressed genes were screened based on KEGG pathway enrichment and protein–protein interaction (PPI) analyses. In conclusion, our findings show mutations in the Fmr1 gene lead to aberrant expression of related genes and affect the sex-differentiated behavioral phenotypes of Fmr1 KO mice by affecting brain development and functional architecture, and suggest future studies should focus on including female subjects to comprehensively reflect the differentiation of FXS in both sexes and develop more precise and effective therapeutic strategies.

A fat- and sucrose-enriched diet causes metabolic alterations in mdx mice.

Duchenne muscular dystrophy (DMD), a progressive muscle disease caused by the absence of functional dystrophin protein, is associated with multiple cellular, physiological, and metabolic dysfunctions. As an added complication to the primary insult, obesity/insulin resistance (O/IR) is frequently reported in patients with DMD; however, how IR impacts disease severity is unknown. We hypothesized a high-fat, high-sucrose diet (HFHSD) would induce O/IR, exacerbate disease severity, and cause metabolic alterations in dystrophic mice. To test this hypothesis, we treated 7-wk-old mdx (disease model) and C57 mice with a control diet (CD) or an HFHSD for 15 wk. The HFHSD induced insulin resistance, glucose intolerance, and hyperglycemia in C57 and mdx mice. Of note, mdx mice on CD were also insulin resistant. In addition, visceral adipose tissue weights were increased with HFHSD in C57 and mdx mice though differed by genotype. Serum creatine kinase activity and histopathological analyses using Masson's trichrome staining in the diaphragm indicated muscle damage was driven by dystrophin deficiency but was not augmented by diet. In addition, markers of inflammatory signaling, mitochondrial abundance, and autophagy were impacted by disease but not diet. Despite this, in addition to disease signatures in CD-fed mice, metabolomic and lipidomic analyses demonstrated a HFHSD caused some common changes in C57 and mdx mice and some unique signatures of O/IR within the context of dystrophin deficiency. In total, these data revealed that in mdx mice, 15 wk of HFHSD did not overtly exacerbate muscle injury but further impaired the metabolic status of dystrophic muscle.

A novel approach to the assessment of higher-order rule learning in male mice.

Historically, the development of valid and reliable methods for assessing higher-order cognitive abilities (e.g., rule learning and transfer) has been difficult in rodent models. To date, limited evidence supports the existence of higher cognitive abilities such as rule generation and complex decision-making in mice, rats, and rabbits. To this end, we sought to develop a task that would require mice to learn and transfer a rule. We trained mice to visually discriminate a series of images (image set, six total) of increasing complexity following three stages: (1) learn a visual target, (2) learn a rule (ignore any new images around the target), and finally (3) apply this rule in abstract form to a comparable but new image set. To evaluate learning for each stage, we measured (1) days (and performance by day) to discriminate the original target at criterion, (2) days (and performance by day) to get back to criterion when images in the set were altered by the introduction of distractors (rule learning), and (3) overall days (and performance by day) to criterion when experienced versus naïve cohorts of mice were tested on the same image set (rule transfer). Twenty-seven wild-type male C57 mice were tested using Bussey-Saksida touchscreen operant conditioning boxes (Lafayette Instruments). Two comparable black-white image sets were delivered sequentially (counterbalanced for order) to two identical cohorts of mice. Results showed that all mice were able to effectively learn their initial target image and could recall it >80 d later. We also found that mice were able to quickly learn and apply a "rule" : Ignore new distractors and continue to identify their visual target embedded in more complex images. The presence of rule learning was supported because performance criterion thresholds were regained much faster than initial learning when distractors were introduced. On the other hand, mice appeared unable to transfer this rule to a new set of stimuli. This is supported because visual discrimination curves for a new image set were no better than an initial (naïve) learning by a matched cohort of mice. Overall results have important implications for phenotyping research and particularly for the modeling of complex disorders in mice.

Radiotherapy Combined with PD-L1 Antibody Exerts a Synergistic Anti-tumor Effect in a Mouse Model of Esophageal Squamous Cell Carcinoma

The combination of radiotherapy and immune checkpoint inhibitors (ICIs) has been shown to exert synergistic anti-tumor effects in various tumors. In esophageal squamous cell carcinoma (ESCC), several clinical trials of radiotherapy combined with ICIs are undergoing or have been finished. However, the efficacy and action mechanisms of radiotherapy combined with ICIs is still not clear in ESCC. Our study aimed to investigate whether radiotherapy improved the anti-cancer effect of PD-L1 antibody and the effect of this combination therapy on tumor-immune microenvironment. The mouse esophageal cancer cell line mEC25 was implanted into the armpits of female C57 mice. When the tumor volume grew to 150-250 mm3, the mice were randomly divided into different groups including control group (administration of IgG antibody), radiotherapy group (12 Gy at 4 Gy per fraction), PD-L1 antibody group (200 mg/kg i.p. for three times) and combination group (fractionated radiation combined with PD-L1 antibody was delivered on day 1, day 4 and day 7, the day when fractionated radiation began was recorded as day 1).The mice were sacrificed on the 1st, 3rd and 7th day after the treatment was ended. The tumor, spleen and draining lymph nodes of the mice were collected and analyzed using flow cytometry (FCM), and the tumor volume and survival time of mice were calculated. Compared with radiotherapy or PD-L1 antibody alone, the combination therapy significantly prolonged the survival time of mice and decreased the growth rate of xenograft tumors FCM analysis showed that the combination therapy significantly increased the infiltration and cytotoxicity of effector T cells and reduced the proportion of M2 macrophages in tumor, spleen and lymph nodes. In addition, in tumor and spleen, the proportion of regulatory T cells (Tregs) decreased in the group of combination therapy. In tumor, spleen, and lymph nodes, the proportion of central memory T cell (TCM) was increased in the group of combination therapy. The anti-tumor effect of radiotherapy combined with PD-L1 antibody is superior to single treatment in the mouse model of esophageal cancer. Radiotherapy can shape tumor-immune microenvironment, allowing the infiltration of effector T cells and exclusion of immune-suppressive cells such as M2 macrophages and Tregs.

Astragaloside IV inhibits AOM/DSS-induced colitis-associated tumorigenesis via activation of PPARγ signaling in mice

BackgroundColitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD), resulting from long-term inflammation in the intestines. The primary cause of CAC is the imbalance of oxidative metabolism in intestinal cells, triggered by excessive reactive oxygen (ROS) and nitrogen (NO) species production due to prolonged intestinal inflammation. This imbalance leads to genomic instability caused by DNA damage, eventually resulting in the development of intestinal cancer. Previous studies have demonstrated that astragaloside IV is effective in treating dextran sulfate sodium salt (DSS)-induced colitis, but there is currently no relevant research on its efficacy in treating CAC. MethodsTo investigate the effect of astragaloside IV against CAC and the underlying mechanism, C57 mice were treated with (20, 40, 80 mg/kg) astragaloside IV while CAC was induced by intraperitoneal injection of 10 mg/kg azoxymethane (AOM) and ad libitum consumption of 2% dextran sulfate sodium salt (DSS). We re-verified the activating effects of astragaloside IV on PPARγ signaling in IEC-6 cells, which were reversed by GW9662 (the PPARγ inhibitor). ResultsOur results showed that astragaloside IV significantly improved AOM/DSS-induced CAC mice by inhibiting colonic shortening, preventing intestinal mucosal damage, reducing the number of tumors and, the expression of Ki67 protein. In addition, astragaloside IV could activate PPARγ signaling, which not only promoted the expression of Nrf2 and HO-1, restored the level of SOD, CAT and GSH, but also inhibited the expression of iNOS and reduced the production of NO in the intestine and IEC-6 cells. And this effect could be reversed by GW9662 in vitro. Astragaloside IV thus decreased the level of ROS and NO in the intestinal tract of mice, as well as reduced the damage of DNA, and therefore inhibited the occurrence of CAC. ConclusionAstragaloside IV can activate PPARγ signaling in intestinal epithelial cells and reduces DNA damage caused by intestinal inflammation, thereby inhibiting colon tumourigenesis. The novelty of this study is to use PPARγ as the target to inhibit DNA damage to prevent the occurrence of CAC.

Aflatoxin B1 exposure triggers inflammation and premature skin aging via ERMCS/Ca2+/ROS signaling cascade

Aflatoxin B1 (AFB1) is a recognized hazard environmental contaminant mainly found in cereal and fruits. The toxicity of AFB1 exposure to various organs has been revealed in some literature. In current study, we explored the effect of AFB1 exposure on premature aging/senescence of skin. In vivo, 8-week-old C57 mice were used as models to evaluate the effect of dietary AFB1 exposure on premature skin aging. The results showed that AFB1 exposure caused premature skin aging by testing aging markers. Additionally, AFB1 led to oxidative stress and inflammatory response. In vitro, AFB1 exposure triggered premature cellular senescence in mouse skin fibroblasts cells (L929 cells) by assessing a range of cellular senescence-related markers. Further, the potential molecular mechanism by which AFB1 induce the premature skin aging was studied. ROS and Ca2+ is proven to be the key molecules in AFB1-induced cellular senescence. Further, through eliminating Ca2+, AFB1-caused oxidative stress and cellular senescence were both attenuated, suggesting that Ca2+ overload in the mitochondria results in cellular senescence by increasing ROS production. Next, we analyzed the causes of Ca2+ overload, and results showed that AFB1 exposure induces Ca2+ overload through increasing the formation of mitoguardin (Miga) and vesicle-associated membrane protein (VAMP)-associated protein (Vap33)-mediated endoplasmic reticulum (ER)–mitochondria contact sites (ERMCS). AFB1 exposure also inhibited mitophagy, leading to accelerate L929 cell senescence. In short, combining in vivo and in vitro results, we demonstrate that exposure to AFB1 causes premature skin aging, which is dependent on ERMCS/Ca2+/ROS/ signaling axis. The current study suggests that prolonged exposure to AFB1 makes skin more vulnerable to damage.

Galectin-3 contributes to acute cardiac dysfunction and toxicity by increasing oxidative stress and fibrosis in doxorubicin-treated mice

BackgroundDoxorubicin (DOX) leads to cardiovascular toxicity through direct cardiomyocyte injury and inflammation. We aimed to study the role of Galectin-3 (Gal-3), a β-galactosidase binding lectin associated with inflammation and fibrosis in DOX-induced acute cardiotoxicity in mice. MethodsMale C57 and Gal-3 knockout (KO) mice were given a single dose of DOX (15 mg/kg, i.p) or placebo. Serum creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and cardiac thiobarbituric acid-reactive substance (TBARS) were measured at 3 days to assess cardiac injury and oxidative stress. Cardiac remodeling and function were studied by echocardiography and catheterization at 7 days. Myocardial fibrosis was quantified in picrosirius red stained slices. ResultsAbsence of Gal-3 tended to reduce the mortality after DOX. DOX significantly increased CPK, LDH, AST and TBARS while treated Gal-3 KO mice showed reduced injury and oxidative stress. After 7 days, adverse remodeling, fibrosis and dysfunction in treated-C57 mice were severely affected while those effects were prevented by absence of Gal-3. ConclusionIn summary, genetic deletion of Gal-3 prevented cardiac damage, adverse remodeling and dysfunction, associated with reduced cardiac oxidative stress and fibrosis. Understanding the contribution of GAL-3 to doxorubicin-induced cardiac toxicity reinforces its potential use as a therapeutic target in patients with several cancer types.

Agarwood extract mitigates alcoholic fatty liver in C57 mice via anti‑oxidation and anti‑inflammation.

Alcoholic fatty liver disease (AFLD) is a disease with a high incidence rate among individuals who drink alcohol. Our previous study found that agarwood alcohol extracts (AAEs) have a protective effect against drug‑induced liver damage via anti‑inflammatory and antioxidant mechanisms. Therefore, we hypothesized that agarwood may have a protective effect against AFLD. The present study assessed the potential protective effects and the underlying mechanism of action of AAEs for the treatment of an AFL invivo model. The AFLD mouse model was established by continuous high fat diet and alcohol gavage in C57 mice. After treatment with AAEs, blood was collected, liver and adipose tissues were removed and liver and adipose indexes were analyzed. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and cholesterol (CHO) in serum were detected. The liver tissue was assessed using pathological sections. Biochemical methods were used to detect the levels of oxidative stress in the supernatant of liver tissue homogenate. The levels of pro‑inflammatory cytokines in the serum were detected by ELISA. The protein expression levels of nuclear erythroid 2‑related factor 2 (Nrf2) and nuclear factor kappa‑B (NF‑κB) in liver tissues were detected using western blotting. AAE treatment decreased the liver and adipose indexes, reduced the levels of AST, ALT, TG and CHO, improved the liver pathological characteristics and enhanced antioxidant and anti‑inflammatory activities. In addition, AAEs increased the protein expression level of Nrf2 and decreased the protein expression level of NF‑κB compared with AFL mice. AAE‑treated animals exhibited reduced metabolic enzyme and blood lipid levels, demonstrated improved liver function and relieved the pathological damage of AFLD induced by consuming a high fat and alcohol diet. AAEs have potential protective effects in AFLD via antioxidant and anti‑inflammatory mechanisms.

Deferoxamine promotes recovery of bone marrow hematopoietic function in mice exposed to a sublethal dose of X-ray irradiation

To evaluate the effect of deferoxamine (DFO) on bone marrow hematopoietic function in C57 mice exposed to a sublethal dose of X-ray irradiation. C57 mice exposed to a sublethal dose (5.4 Gy, 1.0 Gy/min) of total body X-ray irradiation (TBI) were treated with subcutaneous injection of 100 mg/kg DFO, with normal saline as the control, on a daily basis for 10 and 20 consecutive days. Body weight changes of the mice were monitored every 3 days. Five mice were selected from each group at 10 and 20 days for examination of blood cell counts, bone marrow nucleated cell counts, percentage of bone marrow CD34+ cells, bone marrow pathology, and expressions of cleaved PARP-1, cleaved caspase-3, VEGF, GPX4, and SLC7A11 in the nucleated cells. The body weight of the mice decreased significantly on day 3 in TBI and DFO groups (P<0.05), and to the lowest on day 6 in TBI group (P<0.01). Blood cell counts and bone marrow nucleated cell counts of the mice were significantly decreased at 10 and 20 days following TBI (P<0.01). On day 10 following TBI, the mice showed significantly decreased nucleated cells and the presence of adipocytes in the bone marrow, where increased expressions of cleaved PARP-1 and cleaved caspase-3 and lowered expressions of GPX4 and SLC7A11 were detected in the nucleated cells (P<0.05). In the mice exposed to TBI, treatment with DFO significantly increased CD34+ cell percentage (P<0.001), decreased the expressions of cleaved PARP-1 and cleaved caspase-3, and increased the expressions of GPX4, SLC7A11 and VEGF in the bone marrow nucleated cells (P<0.05). DFO treatment significantly increased blood cell counts and bone marrow nucleated cells in mice at 20 days following TBI (P<0.05). DFO improves bone marrow hematopoiesis in mice with sublethal-dose TBI by inhibiting apoptosis and ferroptosis of bone marrow nucleated cells and promoting VEGF expression and CD34+ cell proliferation.

Quercetin Attenuates Atherosclerosis via Modulating Apelin Signaling Pathway Based on Plasma Metabolomics.

To interpret the pharmacology of quercetin in treatment of atherosclerosis (AS). Fourteen apolipoprotein E-deficient (ApoE-/-) mice were divided into 2 groups by a random number table: an AS model (ApoE-/-) group and a quercetin treatment group (7 in each). Seven age-matched C57 mice were used as controls (n=7). Quercetin [20 mg/(kg·d)] was administered to the quercetin group intragastrically for 8 weeks for pharmacodynamic evaluation. Besides morphological observation, the distribution of CD11b, F4/80, sirtuin 1 (Sirt1) and P21 was assayed by immunohistochemistry and immunofluorescence to evaluate macrophage infiltration and tissue senescence. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MSC/MS) was performed to study the pharmacology of quercetin against AS. Then, simultaneous administration of an apelin receptor antagonist (ML221) with quercetin was conducted to verify the possible targets of quercetin. Key proteins in apelin signaling pathway, such as angiotensin domain type 1 receptor-associated proteins (APJ), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), tissue plasminogen activator (TPA), uncoupling protein 1 (UCP1) and angiotensin II receptor 1 (AT1R), were assayed by Western blot. Quercetin administration decreased lipid deposition in arterial lumen and improved the morphology of ApoE-/- aortas in vivo. Quercetin decreased the densities of CD11b, F4/80 and P21 in the aorta and increased the level of serum apelin and the densities of APJ and Sirt1 in the aorta in ApoE-/- mice (all P<0.05). Plasma metabolite profiling identified 118 differential metabolites and showed that quercetin affected mainly glycerophospholipids and fatty acyls. Bioinformatics analysis suggested that the apelin signaling pathway was one of the main pathways. Quercetin treatment increased the protein expressions of APJ, AMPK, PGC-1α, TPA and UCP1, while decreased the AT1R level (all P<0.05). After the apelin pathway was blocked by ML221, the effect of quercetin was abated significantly, confirming that quercetin attenuated AS by modulating the apelin signaling pathway (all P<0.05). Quercetin alleviated AS lesions by up-regulation the apelin signaling pathway.

Edible exosome-like nanoparticles from portulaca oleracea L mitigate DSS-induced colitis via facilitating double-positive CD4+CD8+T cells expansion

Plant-derived exosome-like nanoparticles (PDENs) have been paid great attention in the treatment of ulcerative colitis (UC). As a proof of concept, we isolated and identified Portulaca oleracea L-derived exosome-like nanoparticles (PELNs) from edible Portulaca oleracea L, which exhibited desirable nano-size (~ 160 nm) and a negative zeta potential value (-31.4 mV). Oral administration of PELNs effectively suppressed the expressions of pro-inflammatory cytokines (TNF-α, IL-6, IL-12, and IL-1β) and myeloperoxidase (MPO), increased levels of the anti-inflammatory cytokine (IL-10), and alleviated acute colitis in dextran sulfate sodium (DSS)-induced C57 mice and IL-10−/− mice. Notably, PELNs exhibited excellent stability and safety within the gastrointestinal tract and displayed specific targeting to inflamed sites in the colons of mice. Mechanistically, oral administration of PELNs played a crucial role in maintaining the diversity and balance of gut microbiota. Furthermore, PELNs treatment enhanced Lactobacillus reuteri growth and elevated indole derivative levels, which might activate the aryl-hydrocarbon receptor (AhR) in conventional CD4+ T cells. This activation downregulated Zbtb7b expression, leading to the reprogramming of conventional CD4+ T cells into double-positive CD4+CD8+T cells (DP CD4+CD8+ T cells). In conclusion, our findings highlighted the potential of orally administered PELNs as a novel, natural, and colon-targeted agent, offering a promising therapeutic approach for managing UC.Graphic abstractSchematic illustration of therapeutic effects of oral Portulaca oleracea L -derived natural exosome-like nanoparticles (PELNs) on UC. PELNs treatment enhanced Lactobacillus reuteri growth and elevated indole derivative levels, which activate the aryl-hydrocarbon receptor (AhR) in conventional CD4+ T cells leading to downregulate the expression of Zbtb7b, reprogram of conventional CD4+ T cells into double-positive CD4+CD8+T cells (DP CD4+CD8+ T cells), and decrease the levels of pro-inflammatory cytokines.

Long-term exposure to polystyrene microplastics triggers premature testicular aging

BackgroundPlastic pollution is greatly serious in the ocean and soil. Microplastics (MPs) degraded from plastic has threatened animals and humans health. The accumulation of MPs in the tissues and blood in animals and humans has been found. There is therefore a need to assess the toxicological effects of MPs on the reproductive system.ResultsIn this study, we explored the effect of polystyrene microplastics (PS-MPs) on premature testicular aging in vitro and in vivo. In vitro, we found that testicular sertoli cells (TM4 cells) was prematurely senescent following PS-MPs treatment by the evaluation of a range of aging marker molecules (such as Sa-β-gal, p16 and 21). TM4 cells were then employed for in vitro model to study the potential molecular mechanism by which PS-MPs induce the premature senescence of TM4 cells. NF-κB is identified as a key molecule for PS-MPs-induced TM4 cellular senescence. Furthermore, through eliminating reactive oxygen species (ROS), the activation of nuclear factor kappa B (NF-κB) was blocked in PS-MPs-induced senescent TM4 cells, indicating that ROS triggers NF-κB activation. Next, we analyzed the causes of mitochondrial ROS (mtROS) accumulation induced by PS-MPs, and results showed that Ca2+ overload induced the accumulation of mtROS. Further, PS-MPs exposure inhibits mitophagy, leading to the continuous accumulation of senescent cells. In vivo, 8-week-old C57 mice were used as models to assess the effect of PS-MPs on premature testicular aging. The results illustrated that PS-MPs exposure causes premature aging of testicular tissue by testing aging markers. Additionally, PS-MPs led to oxidative stress and inflammatory response in the testicular tissue.ConclusionIn short, our experimental results revealed that PS-MPs-caused testicular premature aging is dependent on Ca2+/ROS/NF-κB signaling axis. The current study lays the foundation for further exploration of the effects of microplastics on testicular toxicology.

Dermatophagoides Pteronyssinus 1 (DerP1) May Trigger NLRP3-Mediated Corneal Epithelial Cell Pyroptosis by Elevating Interleukin-33 Expression Levels

Purpose To characterize the in vivo effects of Dermatophagoides pteronyssinus 1 (DerP1) in mice and determine the underlying NLRP3 inflammasome-mediated pyroptosis signaling mechanisms in the human corneal epithelial cells (HCECs). Methods DerP1 was used to induce allergic conjunctivitis in C57 mice. HCECs were sensitized with DerP1 in vitro to mimic their condition observed in allergic conjunctivitis in vivo. Transmission electron microscopy was used to evaluate pyroptosis in the HCECs, enzyme-linked immunosorbent assays to assess interleukin (IL)-33, IL-1β and IL-4 levels, flow cytometry to detect the proportion of Th2 cells, MTT assays to assess cell metabolic activity, immunofluorescence to evaluate the effects of DerP1 on functional HCEC phenotypes, and Western blot assays to detect the expression of NOD-like receptor family pyrin domain-containing 3 (NLRP3), gasdermin D (GSDMD), N-terminal fragment of GSDMD (GSDMD-N), pro-caspase-1, cleaved caspase-1, IL-1β, and IL-33. IL-33 expression in the HCECs was knocked down via lentivirus transfection. Results In vivo, DerP1 promotes pyroptosis, production of Th2 inflammatory cytokines and IL-33, and NLRP3 activation in mouse corneas. In vitro, pyroptotic bodies were found in the HCECs after sensitization with DerP1. Various concentrations of DerP1 increased the expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved caspase-1, and IL-1β in the HCECs, with the largest increase observed after exposure to 20 µM DerP1. In vitro, recombinant human IL-33 mediated the expression of pyroptotic biomarkers in the HCECs, whereas IL-33 silencing diminished 20 µM DerP1-induced increase in their expression levels. Conclusions DerP1 induces pyroptosis and allergic conjunctivitis, the expression of Th2 inflammatory cytokines, NLRP3 activation, and IL-33 in mouse corneas in our model. These effects would attribute to its activating NLRP3–GSDMD signaling pathway axis via enhancing IL-33 expression in HCECs.

Inulin alleviates neuroinflammation and oxidative stress induced by perinatal 2-ethylhexyl diphenyl phosphate (EHDPHP) exposure in female mice and offspring

Organophosphorus flame retardants (OPFRs), including 2-ethylhexyl diphenyl phosphate (EHDPHP), are prevalent in everyday life due to their broad usage in fields such as healthcare, electronics, industry, and sports. These compounds, added to polymers through physical mixing, can leach into the environment, posing a risk to humans through direct contact or the food chain. Despite known associations with health issues like endocrine disruption, neurotoxicity, and reproductive toxicity, the implications of perinatal EHDPHP exposure on both mothers and offspring are still unclear. This study aimed to investigate the neuroinflammatory effects of EHDPHP and the potential mitigating role of inulin. Pregnant C57 mice were administered either a corn oil control or an EHDPHP solution (300 μg/kg bw/d) from gestation day 7 (GD7) to postnatal day 21 (PND21). Concurrently, mice were provided either regular drinking water or water supplemented with 1% inulin. We found that EHDPHP significantly increased the serum levels of IL-1β, IL-6, and MDA, but decreased SOD levels in both mothers and pups. These effects were reversed by inulin supplementation. RNA-sequencing revealed that EHDPHP induced inflammation and oxidative stress through the TLR4/NF-κB pathway, which was mitigated by inulin. In conclusion, inulin ameliorated EHDPHP-induced neuroinflammation and oxidative stress in both mothers and offspring, highlighting its potential therapeutic role.