Blood Of Mice Research Articles

Development and Validation of a New High-Performance Liquid Chromatography-Ultraviolet Assay for Quantification of Mitoxantrone in Plasma of BALB/c-nu Mice.

The concentration of mitoxantrone in the blood of mice was determined by a high-performance liquid chromatography-ultraviolet method with aloe-emodin as the internal standard. The separation was performed on a Hypersil BDS2 column (4.6 × 250mm, 5μm) as the analytical column, the mobile Phase A was acetonitrile, and B was 20-mM potassium dihydrogen phosphate (adding 1% triethylamine and adjusting the pH to 2.8 with phosphoric acid) and 4.6-mM sodium octyl sulfonate. The flow rate was 1.0mL·min-1, the detection wavelength was 243nm, the column temperature is 25 ± 5°C and the injection amount was 20μL. Finally, the linear range of mitoxantrone was 5-200μg·mL-1, and the correlation coefficient was r = 0.9999. The recovery rate of the method was 91.93-105.5%, and the extraction recovery rate was 91.45-105.5%. The intraday precision and interday precision were <3.29% (limit of detection = 0.3μg·mL-1). The HPLC method established in this paper was simple, rapid, sensitive and accurate, and can be used to determine the content of mitoxantrone in mouse plasma after tail vein injection.

Antidepressant-like effects of hyperoside on chronic stress-induced depressive-like behaviors in mice: Gut microbiota and short-chain fatty acids

BackgroundThe antidepressant effect of hyperoside (HYP), which is the main component of Hypericum perforatum, is not established. This study aimed to determine the effects of HYP on depression. MethodsThe antidepressant-like effect of HYP was studied in mice induced by chronic restraint stress (CRS). The effects of HYP on behavior, inflammation, neurotransmitters, gut microbiota, and short-chain fatty acids (SCFAs) were studied in CRS mice. ResultsHYP improved depressive-like behavior in mice induced by CRS. Nissl staining analysis showed that HYP improved neuronal damage in CRS mice. Western blot (WB) analysis showed that HYP increased the expression levels of BDNF and PSD95 in the hippocampus of CRS mice. The results of ELISA showed that HYP down-regulated the expression levels of IL-6, IL-1β, TNF-α, and CORT in the hippocampus, blood, and intestinal tissues of mice and up-regulated the expression levels of 5-HT and BDNF. Hematoxylin and eosin (HE) staining results indicate that HYP can improve the intestinal histopathological injury of CRS mice. The results of 16S rRNA demonstrated that HYP attenuated the dysbiosis of the gut microbiota of depressed mice, along with altering the concentration of SCFAs. LimitationsIn the present study, direct evidence that HYP improves depressive behaviors via gut microbiota and SCFAs is lacking, and only female mice were evaluated, which limits the understanding of the effects of HYP on both sexes. ConclusionsHYP can improve CRS-induced depressive-like behaviors in mice, which is associated with regulating the gut microbiota and SCFAs concentration.

A newly developed UPLC-MS/MS method for simultaneous quantitative analysis of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-acetylharpagide derived from Ajuga plants in mice blood and the in vivo pharmacokinetics

Objective To develop a sensitive and fast detection method via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to assess the concentration of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-Acetylharpagide primarily derived from Ajuga plants in mice blood and their pharmacokinetics. Methods Single protein precipitation with high-proportioned acetonitrile is chosen for sample clean-up. The UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column with a mobile phase in gradient elution mode at the flow rate of 0.4 mL/min was used for sample separation. Acetonitrile was selected as the organic phase solution and water containing 0.1% formic acid was chosen as the aqueous solution. A tandem mass spectrometer containing an electrospray ionization (ESI) source in the positive ionization mode was used to detect four compounds via multiple reaction monitoring (MRM). Results The calibration curves (5–1000 ng/mL) of four compounds were linear with correlation coefficients > 0.997. The matrix effects, accuracy, precision, and recovery were all within permissible scope. Conclusions In this approach, the corresponding pharmacokinetic parameters were successfully clarified in mouse for the first time, which provided a theoretical basis for the improvement of the standard of Ajuga plants and the safety of clinical medication. Furthermore, this method may provide the UPLC-MS/MS evidence for the differentiation of the main close relative varieties of genus Ajuga according to these plants contain different mixtures of the four marker compounds.

Unveiling the mechanisms of trichloroethylene hypersensitivity syndrome: Exploring the role of connexin 43 gap junctions in severe skin damage

Trichloroethylene (TCE), extensively used as an organic solvent in various industrial applications, has been identified as a causative factor in inducing hypersensitivity syndrome (THS). Currently, there is no specific treatment for THS, and most patients experience serious adverse outcomes due to extensive skin damage leading to severe infection. However, the pathogenesis of THS-associated skin damage remains unclear. This study aims to elucidate the mechanism underlying skin damage from the perspective of intercellular communication and gap junctions in THS. Our results verified that hyperactivation of connexin43 gap junctions, caused by the aberrantly elevated expression of connexin43, triggers a bystander effect that promotes apoptosis and inflammation in THS via the TNF-TNFRSF1B and mitochondria-associated pathways. Additionally, we identified the gap junction inhibitor Carbenoxolone disodium (CBX) as a promising agent for the treatment of skin damage in THS. CBX protects against inflammatory cell infiltration in the skin and decreases immune cell imbalance in the peripheral blood of THS mice. Furthermore, CBX reduces connexin43 expression, apoptosis and inflammation in THS mice. The study reveals new insights into the mechanisms underlying TCE-induced skin damage, offering a potential treatment strategy for the development of effective therapies targeting severe dermatitis induced by chemical exposure.

Quantitative proteomics reveals tissue-specific, infection-induced and species-specific neutrophil protein signatures

Neutrophils are one of the first responders to infection and are a key component of the innate immune system through their ability to phagocytose and kill invading pathogens, secrete antimicrobial molecules and produce extracellular traps. Neutrophils are produced in the bone marrow, circulate within the blood and upon immune challenge migrate to the site of infection. We wanted to understand whether this transition shapes the mouse neutrophil protein landscape, how the mouse neutrophil proteome is impacted by systemic infection and perform a comparative analysis of human and mouse neutrophils. Using quantitative mass spectrometry we reveal tissue-specific, infection-induced and species-specific neutrophil protein signatures. We show a high degree of proteomic conservation between mouse bone marrow, blood and peritoneal neutrophils, but also identify key differences in the molecules that these cells express for sensing and responding to their environment. Systemic infection triggers a change in the bone marrow neutrophil population with considerable impact on the core machinery for protein synthesis and DNA replication along with environmental sensors. We also reveal profound differences in mouse and human blood neutrophils, particularly their granule contents. Our proteomics data provides a valuable resource for understanding neutrophil function and phenotypes across species and model systems.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

Targeting metastasis-initiating cancer stem cells in gastric cancer with leukaemia inhibitory factor

Gastric cancer’s (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC’s high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.

Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2.

The overexpression of sialic acids on glycans, called hypersialylation, is a common alteration found in cancer cells. Sialylated glycans can enhance immune evasion by interacting with sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on tumor-infiltrating immune cells. Here, we investigated the effect of sialylated glycans and their interaction with Siglec receptors on myeloid-derived suppressor cells (MDSCs). We found that MDSCs derived from the blood of lung cancer patients and tumor-bearing mice strongly express inhibitory Siglec receptorsand are highly sialylated. In murine cancer models of emergency myelopoiesis, Siglec-E knockout in myeloid cells resulted in prolonged survival and increased tumorinfiltration of activated T cells. Targeting suppressive myeloid cells by blocking Siglec receptors or desialylation strongly reduced their suppressive potential. We further identified CCL2 as a mediator involved in T-cell suppression upon interaction between sialoglycans and Siglec receptors on MDSCs. Our results demonstrated that sialylated glycans inhibit anticancer immunity by modulating CCL2 expression.

Photoprotective effect of topical treatment with Lopezia racemosa extract against deleterious UVB irradiation effects in the skin of hairless mice.

Lopezia racemosa is known as a "mosquito flower or perlilla." It is commonly found in corn crops. In traditional Mexican medicine, this plant is used to treat stomach cancer and urinary tract infections. Likewise, compounds and extracts isolated from plants have shown cytotoxic and anti-inflammatory effects. Theobjective of this study was to evaluate the photochemoprotective effect of topical treatment with the methanolic extract of L. racemosa (MELR) as a photochemoprotective agent against the harmful effects of UV irradiation (UVR) on a bacterial model and hairless mice. The MELR components were separated and analyzed via HPLC-UV-ESI-MS. Antioxidant activity was evaluated by the ability of MERL to scavenge DPPH and ABTS free radicals and by its FRAP capacity. The toxicity of MELR was evaluated in keratinocyte cultures. The photoprotective capacity of MELR was assessed through challenge experiments using models with bacteria and hairless CD1 et/et mice; cytokines related to the damage caused by UVR were also measured. In the methanolic extract of L. racemosa, five metabolites were detected and identified: two isomers of quercetin 6-C glycoside, orientin, quercetin 3-(6″-acetylglycoside) and quercetin 3-(6″-galloylglycoside) 7-(2,3-dihydroxytetrahydro-2H-pyran-4-yl acetate). MELR exhibited DPPH and ABTS radical scavenging properties, in addition to Fe ion reducing activity. MELR showed a photoprotective effect against UVB radiation-induced death in Escherichia coli bacteria. At the histological level, topical treatment of CD-1 et/et mice with MERL reduced the damage caused by UVR. Quantification of interleukins in the blood of mice revealed that the expression of IL-12 was greater in the control group treated with ultraviolet radiation than in the group protected with MELR. The methanolic extract of L. racemosa has photochemoprotective properties.

Effects of Dityrosine on Lactic Acid Metabolism in Mice Gastrocnemius Muscle During Endurance Exercise via the Oxidative Stress-Induced Mitochondria Damage.

Dityrosine (Dityr) has been detected in commercial food as a product of protein oxidation and has been shown to pose a threat to human health. This study aims to investigate whether Dityr causes a decrease in lactic acid metabolism in the gastrocnemius muscle during endurance exercise. C57BL/6 mice were administered Dityr or saline by gavage for 13 weeks and underwent an endurance exercise test on a treadmill. Dityr caused a severe reduction in motion displacement and endurance time, along with a significant increase in lactic acid accumulation in the blood and gastrocnemius muscle in mice after exercise. Dityr induced significant mitochondrial defects in the gastrocnemius muscle of mice. Additionally, Dityr induced serious oxidative stress in the gastrocnemius muscle, accompanied by inflammation, which might be one of the causes of mitochondrial dysfunction. Moreover, significant apoptosis in the gastrocnemius muscle increased after exposure to Dityr. This study confirmed that Dityr induced oxidative stress in the gastrocnemius muscle, which further caused significant mitochondrial damage in the gastrocnemius muscle cell, resulting in decreased capacity of lactic acid metabolism and finally affected performance in endurance exercise. This may be one of the possible mechanisms by which highly oxidized foods cause a decreased muscle energy metabolism.

Assessment of wound healing activity in diabetic mice treated with a novel therapeutic combination of selenium nanoparticles and platelets rich plasma

Diabetic wound healing is sluggish, often ending in amputations. This study tested a novel, two-punch therapy in mice—Selenium nanoparticles (Se NPs) and platelet-rich plasma (PRP)—to boost healing. First, a mouse model of diabetes was created. Then, Se NPs were crafted for their impressive antioxidant and antimicrobial powers. PRP, packed with growth factors, was extracted from the mice's blood. Wound healing was tracked for 28 days through photos, scoring tools, and tissue analysis. Se NPs alone spurred healing, and PRP added extra fuel. Furthermore, when used in combination with PRP, the healing process was accelerated due to the higher concentration of growth factors in PRP. Notably, the combination of Se NPs and PRP exhibited a synergistic effect, significantly enhancing wound healing in diabetic mice. These findings hold promise for the treatment of diabetic wounds and have the potential to reduce the need for lower limb amputations associated with diabetic foot ulcers. The innovative combination therapy using Se NPs and PRP shows great potential in expediting the healing process and addressing the challenges of impaired wound healing in individuals with diabetes. This exciting finding suggests this therapy could change diabetic wound management, potentially saving limbs and improving lives.

Innovative freeze-drying technique in the fabrication of dissolving microneedle patch: Enhancing transdermal drug delivery efficiency.

Microneedle patch (MNP) has become a hot research topic in the field of transdermal drug delivery due to its ability to overcome the stratum corneum barrier. Among the various types of microneedles, dissolving microneedles represent one of the most promising transdermal delivery methods. However, the most used method for preparing dissolving microneedles, namely microfabrication, suffers from issues such as long drying time, susceptibility to humidity, and large batch-to-batch variability, which limit the development of dissolving microneedles. In this study, we report for the first time a method for preparing dissolving microneedles using freeze-drying technology. We screened substrates suitable for freeze-dried microneedle patch (FD-MNP) and used coating technology to enhance the mechanical strength of FD-MNP, allowing them to meet the requirements for skin penetration. We successfully prepared FD-MNP using hyaluronic acid as the substrate and insulin as the model drug. Scanning electron microscopy revealed that the microneedles had a porous structure. After coating, the mechanical strength of the microneedles was 0.61N/Needle, and skin penetration rate was 97%, with a penetration depth of 215μm. The tips of the FD-MNP dissolved completely within approximately 60s after skin penetration, which is much faster than conventional MNP (180s). In vitro transdermal experiments showed that the FD-MNP shortened the lag time for transdermal delivery of rhodamine 123 and insulin compared to conventional MNP, indicating a faster transdermal delivery rate. Pharmacological experiments showed that the FD-MNP lowered mouse blood glucose levels more effectively than conventional MNP, with a relative pharmacological availability of 96.59 ± 2.84%, higher than that of conventional MNP (84.34 ± 3.87%), P = 0.0095. After storage under 40℃ for two months, the insulin content within the FD-MNP remained high at 95.27 ± 4.46%, which was much higher than that of conventional MNP (58.73 ± 3.71%), P < 0.0001. In conclusion, freeze-drying technology is a highly valuable method for preparing dissolving microneedles with potential applications in transdermal drug delivery.

A novel GARP humanized mouse model for efficacy assessment of GARP-targeting therapies

Although breakthroughs have been achieved with immune checkpoint inhibitors (ICI) therapy, some tumors do not respond to those therapies due to primary or acquired resistance. GARP, a type I transmembrane cell surface docking receptor mediating latent transforming growth factor-β (TGF-β) and abundantly expressed on regulatory T lymphocytes and platelets, is a potential target to render these tumors responsive to ICI therapy, and enhancing anti-tumor response especially combined with ICI. To facilitate these research efforts, we developed humanized mouse models expressing humanized GARP (hGARP) instead of their mouse counterparts, enabling in vivo assessment of GARP-targeting agents. We created GARP-humanized mice by replacing the mouse Garp gene with its human homolog. Then, comprehensive experiments, including expression analysis, immunophenotyping, functional assessments, and pharmacologic assays, were performed to characterize the mouse model accurately. The Tregs and platelets in the B-hGARP mice (The letter B is the first letter of the company's English name, Biocytogen.) expressed human GARP, without expression of mouse GARP. Similar T, B, NK, DCs, monocytes and macrophages frequencies were identified in the spleen and blood of B-hGARP and WT mice, indicating that the humanization of GARP did not change the distribution of immune cell in these compartments. When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP/TGF-β1 complexes demonstrated enhanced in vivo anti-tumor activity compared to monotherapy with either agent. The novel hGARP model serves as a valuable tool for evaluating human GARP-targeting antibodies in immuno-oncology, which may enable preclinical studies to assess and validate new therapeutics targeting GARP. Furthermore, intercrosses of this model with ICI humanized models could facilitate the evaluation of combination therapies.

MiR-335-3p improves type II diabetes mellitus by IGF-1 regulating macrophage polarization.

Previous studies have found that miR-335 is highly expressed in type II diabetes mellitus (T2DM) models and is related to insulin secretion, but there are few studies on the regulatory effects of miR-335-3p on insulin resistance and macrophage polarization in T2DM patients. This study aims to explore the effects of miR-335-3p on insulin resistance and macrophage polarization in T2DM patients. Blood glucose (insulin tolerance tests, glucose tolerance tests) and body weight of the T2DM model were measured; macrophages from adipose tissue were isolated and cultured, and the number of macrophages was detected by F4/80 immunofluorescence assay; the Real-time quantitative polymerase chain reaction (qPCR) assay and Western blot assay were used to detect the miR-335-3p expression levels, insulin-like growth factor 1 (IGF-1), M1-polarizing genes (inducible nitric oxide synthase [iNOS] and TNF-α) as well as M2-polarizing genes (IL-10 and ARG-1). The targeting link between miR-335-3p and IGF-1 was confirmed using bioinformatics and dual luciferase assay. The results showed that miR-335-3p expression level in adipose tissue of the T2DM model was significantly decreased, and the mice's body weight and blood glucose levels dropped considerably, miR-335-3p inhibited the number of macrophages, inhibiting the iNOS and TNF-α relative mRNA expression levels, and up-regulated the IL-10 and ARG-1 relative mRNA expression levels, miR-335-3p negatively regulated target gene IGF-1, IGF-1 significantly increased the iNOS and TNF-α mRNA and protein expression levels, decreasing the IL-10 and ARG-1 mRNA and protein expression levels, indicating that miR-335-3p could affect the T2DM process by regulating macrophage polarization via IGF-1.

Podocyte Ercc1 is indispensable for glomerular integrity

As life expectancy continues to increase, age-related kidney diseases are becoming more prevalent. Chronic kidney disease (CKD) is not only a consequence of aging but also a potential accelerator of aging process. Here we report the pivotal role of podocyte ERCC1, a DNA repair factor, in maintaining glomerular integrity and a potential effect on multiple organs. Podocyte-specific ERCC1-knockout mice developed severe proteinuria, glomerulosclerosis, and renal failure, accompanied by a significant increase in glomerular DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). ERCC1 gene transfer experiment in the knockout mice attenuated proteinuria and glomerulosclerosis with reduced DNA damage. Notably, CD44+CD8+ memory T cells, indicative of T-cell senescence, were already elevated in the peripheral blood of knockout mice at 10 weeks old. Additionally, levels of senescence-associated secretory phenotype (SASP) factors were significantly increased in both the circulation and multiple organs of the knockout mice. In older mice and human patients, we observed an accumulation of DSBs and an even greater buildup of SSBs in glomeruli, despite no significant reduction in ERCC1 expression with age in mice. Collectively, our findings highlight the crucial role of ERCC1 in repairing podocyte DNA damage, with potential implications for inflammation in various organs.

Label-free analysis of the β-hydroxybutyricacid drug on mitochondrial redox states repairment in type 2 diabetic mice by resonance raman scattering

BackgroundMitochondrial redox imbalance underlies the pathophysiology of type2 diabetes mellitus (T2DM), and is closely related to tissue damage and dysfunction. Studies have shown the beneficial effects of dietary strategies that elevate β-hydroxybutyrate (BHB) levels in alleviating T2DM. Nevertheless, the role of BHB has not been clearly elucidated. MethodsWe performed a spectral study to visualize the preventive effects of BHB on blood and multiorgan mitochondrial redox imbalance in T2DM mice via using label-free resonance Raman spectroscopy (RRS), and further explored the impact of BHB therapy on the pathology of T2DM mice by histological and biochemical analyses. FindingsOur data revealed that RRS-based mitochondrial redox states assay enabled clear and reliable identification of the improvement of mitochondrial redox imbalance by BHB, evidenced by the reduction of Raman peak intensity at 750 cm−1, 1128 cm−1 and 1585 cm−1 in blood, tissue as well as purified mitochondria of db/db mice and the increase of tissue mitochondrial succinic dehydrogenase (SDH) staining after BHB treatment. Exogenous supplementation of BHB was also found to attenuate T2DM pathology related to mitochondrial redox states, involving organ injury, blood glucose control, insulin resistance and systemic inflammation. InterpretationOur findings provide strong evidence for BHB as a potential therapeutic strategy targeting mitochondria for T2DM.

Study based on the acetaldehyde dehydrogenase 2 gene polymorphism and acetaminophen-induced liver injury

Objective: To explore the association between aldehyde dehydrogenase 2 (ALDH2) gene polymorphisms and abnormal liver function-induced by acetaminophen (APAP) drugs. Methods: An ALDH2 gene knockout mouse model was constructed using CRISPR/Cas9 gene editing technology. The obtained heterozygous mice were mated with opposite sex of heterozygotes. Genomic DNA was extracted from the tail of the offspring mouse. The polymerase chain reaction (PCR) method was used to determine the ALDH2 genotype. APAP was further used to induce acute drug-induced liver injury models in wild-type and ALDH2 knockout mice. Blood and liver tissues of mice were collected for liver function index, HE staining, F4/80 immunohistochemistry, and other detections. The intergroup mean was compared using a one-way ANOVA. The LSD- t test was used for pairwise comparison. Results: ALDH2 knockout mice were bred successfully. The genotyping of the offspring was segregated into the wild-type (ALDH2(+/+)), heterozygous mutant (ALDH2(+/-)), and homozygous mutant (ALDH2(-/-)), respectively. Biochemical and histological results after APAP modeling showed that the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) was not significantly increased in the blank control group (P < 0.05), while the ALT, AST,ALP, and TBil were all elevated in the APAP experimental group. The levels of ALT (P = 0.004), AST (P = 0.002), and TBil (P = 0.012) were significantly elevated among the mutant group compared to those in the wild-type group, and the expression levels of these indicators were also significantly elevated among the homozygous mutant group compared to those in the heterozygous mutant group (P = 0.003, 0 and 0.006). In addition, the ALP levels were higher in the heterozygous mutation group than those in the homozygous mutant group (P = 0.085) and wild-type group mice, but the difference was only statistically significant compared to wild-type mice (P = 0.002). HE staining results showed that mice in the APAP experimental group had hepatocyte degeneration, necrosis, and increased inflammatory cell infiltration, which was mostly evident in mutant mice. Simultaneously, the F4/80 immunohistochemical staining results showed that brown granules were visible in the liver tissue of APAP experimental group mice, and its expression levels were significantly enhanced compared to the blank control group. Conclusion: APAP-induced liver function abnormalities were associated with the ALDH2 gene polymorphism. The liver injury symptoms were increased in ALDH2 mutant mice following APAP modeling, and the ALDH2 gene defect may alleviate, to some extent, APAP-induced liver function abnormalities.

A multilocus-dendritic boronic acid functionalized magnetic nanoparticle for capturing circulating tumor cells in the peripheral blood of mice with metastatic breast cancer

BackgroundDynamic fluctuation of circulating tumor cells (CTCs) can serve as an indicator of tumor progression. However, the sensitive isolation of CTCs remains extremely challenging due to their rarity and heterogeneity. Against this dilemma, dendritic boronic acid-modified magnetic nanoparticles (MNPs) were prepared in this study, and polyethyleneimine (PEI) was utilized as a scaffold to significantly increase the number of boronic acid moieties. Then the novel developed material was applied to monitor the number of CTCs in mice with metastatic breast cancer to evaluate the therapeutic effects of matrine (Mat), doxorubicin (Dox), and Mat in combination with Dox. ResultsCompared to the low binding capacity of a single boronic acid ligand, dendritic boronic acid shows enhanced sensitivity in binding to sialic acid (SA), which is overexpressed in CTCs. The results showed that the capture efficiency of this modified material could achieve 94.7% and successfully captured CTCs in blood samples from mice with metastatic breast cancer. The CTC counts were consistent with the results of the pathologic examination, demonstrating the reliability and utility of the method. SignificanceThe dendritic boronic acid nanomaterials prepared in this study showed high specificity, sensitivity, and accuracy for cancer cell capture. The approach is expected to provide new insights into cancer diagnosis, personalized therapy, and optimization of treatment regimens.

Preclinical evaluation of new GRPR-antagonists with improved metabolic stability for radiotheranostic use in oncology

BackgroundThe gastrin-releasing peptide receptor (GRPR) has been extensively studied as a biomolecular target for peptide-based radiotheranostics. However, the lack of metabolic stability and the rapid clearance of peptide radioligands, including radiolabeled GRPR-antagonists, often impede clinical application. Aiming at circumventing these drawbacks, we have designed three new GRPR-antagonist radioligands using [99mTc]Tc-DB15 ([99mTc]Tc-N4-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt; AMA: p-aminomethylaniline; DIG: diglycolate) as a motif, due to its high GRPR-affinity and stability to neprilysin (NEP). The new analogues carry the DOTAGA-chelator (1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid) through different linkers at the N-terminus to allow for labeling with the theranostic radionuclide pair In-111/Lu-177. After labeling with In-111 the following radioligands were evaluated: (i) [111In]In-AU-SAR-M1 ([111In]In-DOTAGA-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt), (ii) [111In]In-AU-SAR-M2 ([111In]In-[DOTAGA-Arg]AU-SAR-M1) and (iii) [111In]In-AU-SAR-M3 ([111In]In-[DOTAGA-DArg]AU-SAR-M1).ResultsThese radioligands were compared in a series of in vitro assays using prostate adenocarcinoma PC-3 cells and in murine models. They all displayed high and GRPR-specific uptake in PC-3 cells. Analysis of mice blood collected 5 min post-injection (pi) revealed similar or even higher metabolic stability of the new radioligands compared with [99mTc]Tc-DB15. The stability could be further increased when the mice were treated with Entresto® to in situ induce NEP-inhibition. In PC-3 xenograft-bearing mice, [111In]In-AU-SAR-M1 displayed the most favourable biodistribution profile, combining a good tumor retention with the highest tumor-to-organ ratios, with the kidneys as the dose-limiting organ.ConclusionsThese findings strongly point at AU-SAR-M1 as a promising radiotherapeutic candidate when labeled with Lu-177, or other medically appealing therapeutic radiometals, especially when combined with in situ NEP-inhibition. To this goal further investigations are currently pursued.

Qijiao Shengbai Capsule alleviated leukopenia by interfering leukotriene pathway: Integrated network study of multi-omics

BackgroundLeukopenia could be induced by chemotherapy, which leads to bone marrow suppression and even affects the therapeutic progression of cancer. Qijiao Shengbai Capsule (QSC) has been used for the treatment of leukopenia in clinic, but its bioactive components and mechanisms have not yet been elucidated clearly. PurposeThis study aimed to elucidate the molecular mechanisms of QSC in treating leukopenia. Study designSerum pharmacochemistry, multi-omics, network pharmacology, and validation experiment were combined to study the effect of QSC in murine leukopenia model. MethodsFirst, UPLC-QTOF-MS was used to clarify the absorbed components of QSC. Then, cyclophosphamide (CTX) was used to induce mice model with leukopenia, and the therapeutic efficacy of QSC was assessed by an integrative approach of multi-omics and network pharmacology strategy. Finally, molecular mechanisms and potential therapeutic targets were identified by validated experiments. Results121 compounds absorbed in vivo were identified. QSC significantly increase the count of white blood cells (WBCs) in peripheral blood of leukopenia mice with 15 days treatment. Multi-omics and network pharmacology revealed that leukotriene pathway and MAPK signaling pathway played crucial roles during the treatment of leukopenia with QSC. Six targets (ALOX5, LTB4R, CYSLTR1, FOS, JUN, IL-1β) and 13 prototype compounds were supposed to be the key targets and potential active components, respectively. The validation experiment further confirmed that QSC could effectively inhibit the inflammatory response induced by leukopenia. The inhibitors of ALOX5 activity can significantly increase the number of WBCs in leukopenia mice. Molecular docking of ALOX5 suggested that calycosin, daidzein, and medicarpin were the potentially active compounds of QSC. ConclusionLeukotriene pathway was found for the first time to be a key role in the development of leukopenia, and ALOX5 was conformed as the potential target. QSC may inhibit the inflammatory response and interfere the leukotriene pathway, it is able to improve hematopoiesis and achieve therapeutic effects in the mice with leukopenia.