Canine mammary tumours (CMTs) and human breast cancer (HBC) share highly similar pathological characteristics. Cancer stem cells (CSCs) are critical to breast cancer invasion, metastasis, drug resistance, and recurrence. The experiment utilised CMT cell line CMT-U27 and HBC cell line MDA-MB-231. Cancer stem cell spheres were isolated from the two cell lines by serum-free culture respectively, named as CMT-U27 microspheres (CMT-U27S) and MDA-MB-231 microspheres (MDA-MB-231S), and the proportion of CSCs with the CD44+/CD24- phenotype was identified by flow cytometry. Monensin (MON), one of Polyether ionophore antibiotics, has been demonstrated to effectively suppress various types of CSCs, while erlotinib (ERL), as a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR), effectively inhibits cancer cell growth. This study investigates the synergistic inhibitory effects of MON-ERL combination on CSCs derived from CMTs. Cell viability was analysed by CCK-8 assay, while cell invasion and mammosphere formation assays were conducted to evaluate changes. An orthotopic tumour model in nude mice using CMT-U27S was established for in vivo validation. The results suggested that MON and ERL combination synergistically inhibited CSCs viability and significantly suppress their invasion and mammosphere formation abilities. The expression of key proteins of the EGFR pathway-p-EGFR, PI3K, and p-AKT was significantly reduced. In animal experiments, tumour volumes in the combination treatment group were markedly reduced compared to those in all other groups (p < 0.05), and no lung metastases were observed only in the combination group. Immunofluorescence and immunohistochemistry results indicated a marked reduction of CD44+/CD24- cells in the combination group, along with suppressed expression of stem cell markers and proliferation/apoptosis proteins. In conclusion, MON combined with ERL effectively inhibits the proliferation, self-renewal, and metastatic capabilities of CSCs from CMTs by synergistically downregulating the EGFR/PI3K pathway, demonstrating promising antitumor potential and offering a new strategy for CMTs treatment.

Ferroptosis is a promising programmed cell death modality for cancer therapy, driven by iron overload and the accumulation of phospholipid peroxides that culminate in lethal membrane damage. Over the past decade, emerging evidence supports the concept that ferroptosis can be harnessed as an effective strategy to suppress tumor growth, particularly in therapy-resistant cancer cells undergoing epithelial–mesenchymal transition and in cancer stem cells. Given that ferroptosis is mechanistically and morphologically different from other known programmed cell death forms, increasing critical findings have shed light on mechanisms by which ferroptosis is regulated, and context-dependent cancer phenotype which is clinical relevant to ferroptosis. In this review, we summarize the basic biology of ferroptosis, including iron regulation and lipid metabolism, as well as key molecular mechanisms such as the system Xc⁻-GSH-GPX4, NADPH-FSP1-CoQ10 and GCH1-BH4 axis in fighting cancer. We also discuss crosstalk between ferroptosis and cuproptosis, disulfidptosis and autophagy, and outline how ferroptosis shapes the tumor immune microenvironment and responses to immunotherapy. More importantly, we highlight the clinical potential of ferroptosis induction via chemotherapy, radiotherapy, immunotherapy and nanomedicine-based delivery strategies, while summarizing common resistance mechanisms and safety considerations. Finally, we outline major challenges and pressing questions for clinical translation, including what are the molecular bases of ferroptosis, how can ferroptosis be leveraged for cancer therapy, how can ferroptosis be integrated with conventional therapies, and how to balance benefits and risks of ferroptosis-based therapy. Collectively, this review connects mechanistic insights with actionable intervention points for developing ferroptosis-based cancer therapies.

Poloxamers are thermosensitive triblock copolymers valued for their safety and sustained-release properties. Using China's prevalent CPV-2c strain (JSLYG-18) of canine parvovirus (CPV), we produced virus-like particles (VLPs) via prokaryotic expression and formulated a thermosensitive poloxamer gel vaccine. The results showed that the optimal formulation of the poloxamer thermosensitive gel consisted of 17% poloxamer 407 (P407), 3.2% poloxamer 188 (P188), and 0.3% hydroxypropyl methylcellulose (HPMC), with a gelation temperature (Tm) of 35.8 °C, demonstrating excellent stability and ease of preparation. In vitro drug release studies revealed that BSA was released from the poloxamer gel over 48 h, with a cumulative release rate of 98.2%, following the Weibull kinetic model, indicating a synergistic mechanism of drug diffusion and gel erosion. The poloxamer-based VLPs vaccine exhibited minimal tissue irritation and enhanced immune responses in mice, including activation of splenic lymphocytes. Post-immunization, serum IgG levels reached 1:3 × 105, and neutralizing antibody titers reached 1:300. Both were significantly higher than those in the naked VLPs protein group, with prolonged antibody persistence. These findings demonstrate that the poloxamer thermosensitive gel-based CPV VLPs vaccine not only possesses excellent immunogenicity but also serves as a promising vaccine delivery platform for further application.

Methuosis represents a novel cell death modality characterized by catastrophic cytoplasmic vacuolization in normal and malignant cells. However, the critical role and the underlying mechanism of cytoskeleton and plasma membrane damage in methuotic cells are largely unknown. We found that cytoskeleton protein F-actin, α-tubulin, β-tubulin and filamin A/B were disrupted in a reversible-dependent manner. In addition, RhoA-ROCK1 signaling pathway mediated cytoskeleton disruption in methuotic cells. Excessive cytoplasmic vacuolization triggered cellular plasma membrane damage and the release of damage associated molecular patterns (DAMPs), including lactate dehydrogenase (LDH), adenosine triphosphate (ATP) and calreticulin (CRT). Furthermore, at the end phase of methuotic cells, plasma membrane was damaged independent of pore-forming protein phosphorylation mixed lineage kinase domain-like (p-MLKL) and gasdermin D (GSDMD). Endosomal sorting complex required for transport (ESCRT)-III especially its subunit charged multivesicular body protein 3 (CHMP3) and charged multivesicular body protein 5 (CHMP5) negatively regulated excessive vacuolization-induced plasma membrane damage in cells undergoing methuosis. The critical role and potential mechanism of cytoskeleton and plasma membrane damage in methuotic cells are known, which would facilitate the employment of methuosis in life science and pharmacology.

Cancer cells possess a negatively charged surface that significantly influences key biological processes, including motility and invasiveness. Understanding the cellular membrane at the nanoscale level, especially in deciphering tumor migration mechanisms, is crucial yet complex. In this study, we explored the impact of maduramicin (MAD), a polyether ionophore antibiotic, on the surface charge distribution in tumor cell membranes. Our findings demonstrated that modulating calcium channels and disrupting charge distributions, imaged by a current difference-based scanning ion conductance microscopy (Delta I-SICM) method, resulted in significant reductions in both cell invasiveness and viability. These results emphasize the cell membrane's considerable role at the nano-bio interface, highlighting the intricate relationship between biophysical properties and cellular behaviors. Our research suggests that targeting the charge distribution of cell membrane may offer a novel therapeutic strategy to mitigate cell migration and enhance treatment outcomes in cancer therapy.

Nanoparticle-based transdermal drug delivery systems (TDDS) have emerged as a revolutionary approach for antiparasitic therapy, addressing key challenges such as poor bioavailability, systemic toxicity, and drug resistance. This review highlights the advancements in nanotechnology-driven TDDS for combating zoonotic parasitic diseases, including leishmaniasis, malaria, and infections treated by broad-spectrum drugs like ivermectin and albendazole. By leveraging nanocarriers such as liposomes, nanoemulsions, and microneedles, which enhance skin permeation, enable controlled drug release, and improve targeting specificity. For instance, deformable transfersomes and ethosomes achieve high transdermal efficiency without chemical adjuvants, while microneedle arrays physically bypass the stratum corneum for precise delivery. Furthermore, sustained-release hydrogels and stimuli-responsive nanoparticles optimize therapeutic efficacy and reduce adverse effects. Despite promising results, clinical translation faces challenges in manufacturing scalability, long-term safety, and accessibility in resource-limited settings. Future directions include bioinspired nanocarriers, artificial intelligence (AI)-driven design, and integration with global health initiatives like “One Health”, all aimed at ensuring equitable implementation. This review highlights the transformative potential of nanotechnology in achieving sustainable antiparasitic solutions for zoonotic diseases.

Ionophore polyether antibiotics (IPAs) exhibit remarkable therapeutic potential in combating parasitic diseases and cancer, yet their clinical utility is significantly hampered by severe hepatotoxicity. Despite widespread documentation of IPAs-induced hepatotoxicity, the precise molecular mechanisms underlying this phenomenon remain elusive. This study elucidates the role of ferroptosis in IPAs-induced liver injury and delineates the associated regulatory pathways. Through comprehensive in vitro (HepG2 cells) and in vivo (mice) investigations, we demonstrate that IPAs, particularly the highly toxic maduramicin (Mad), induce hepatocyte ferroptosis. Mechanistic studies employing lipid reactive oxygen species (ROS) quantification, intracellular Fe2+ assays, and Western blot analysis revealed that IPAs-induced ferroptosis occurs through an autophagy-dependent pathway. Surface plasmon resonance (SPR) and molecular docking analyses confirmed direct binding and regulation of transcription factor EB (TFEB) by maduramicin. This interaction activates TFEB, subsequently mediating nuclear receptor coactivator 4 (NCOA4)-regulated lysosomal degradation processes that culminate in ferroptosis-mediated hepatotoxicity. Importantly, our findings extend beyond maduramicin, as other IPAs including monensin and salinomycin similarly targeted TFEB, triggering hepatocyte ferroptosis. Crucially, adeno-associated virus serotype 8 (AAV8)-mediated TFEB knockdown in mice conferred protection against IPAs-induced liver injury and attenuated hepatocyte ferroptosis. These findings establish TFEB-mediated NCOA4-dependent ferritinophagy and ferroptosis as central mechanisms in IPAs-induced hepatotoxicity, thereby identifying TFEB as a promising therapeutic target for mitigating IPAs-induced liver damage. This study provides critical insights into the molecular mechanisms of IPAs-induced liver injury and offers a novel strategy for therapeutic intervention.

Postoperative distant metastasis and high recurrence rate causes a dilemma in treating triple-negative breast cancer (TNBC) owing to its unforeseeable invasion into various organs or tissues. The wealth of nutrition provided by vascular may facilitate the proliferation and angiogenesis of cancer cells, which further enhance the rates of postoperative metastasis and recurrence. Chemotherapy, as a systemic postoperative adjuvant therapy, is generally applied to diminish recurrence and metastasis of TNBC. Herein, an halofuginone-silver nano thermosensitive hydrogel (HTPM&AgNPs-gel) was prepared via a physical swelling method. The in vitro anticancer efficacy of HTPM&AgNPs-gel was analyzed by investigating cell proliferation, migration, invasion, and angiogenesis capacity. Furthermore, the in vivo anti-cancer activity of HTPM&AgNPs-gel was further appraised through the tumor suppression, anti-metastatic, anti-angiogenic, and anti-inflammatory ability. The optimized HTPM&AgNPs-gel, a thermosensitive hydrogel, showed excellent properties, including syringeability, swelling behavior, and a sustained release effect without hemolysis. In addition, HTPM&AgNPs-gel was confirmed to effectively inhibit the proliferation, migration, invasion, and angiogenesis of MDA-MB-231 cells. An evaluation of the in vivo anti-tumor efficacy demonstrated that HTPM&AgNPs-gel showed a stronger tumor inhibition rate (68.17%) than did HTPM-gel or AgNPs-gel used alone and exhibited outstanding biocompatibility. Notably, HTPM&AgNPs-gel also inhibited lung metastasis induced by residual tumor tissue after surgery and further blocked angiogenesis-related inflammatory responses. Taken together, the suppression of inflammation by interdicting the blood vessels adjoining the tumor and inhibiting angiogenesis is a potential strategy to attenuate the recurrence and metastasis of TNBC. HTPM&AgNPs-gel is a promising anticancer agent for TNBC as a local postoperative treatment.

Canine mammary tumor (CMT) is one of the relevant models of human breast cancer (HBC) with histopathological, epidemiological, and clinical characteristics similar to those of humans. This study aimed to establish and characterize a new canine cell line CMT-N7. CMT-N7 tumor is a complex canine mammary carcinoma that stained negative for human epidermal growth receptor-2 (HER2) and progesterone receptors (PR), and positive to estrogen receptor (ER). Cell growth, ultrastructure, doubling time, metastasis capacity, and biomarker characteristics of CMT-N7 were assessed. Xenograft transplantation was conducted to evaluate tumorigenicity. The cell morphology of CMT-N7 was generally epithelioid, with large and irregular nuclei and obvious multinucleation. The established CMT-N7 cell line underwent over 120 generations of subculture, exhibiting a rapid proliferation rate with a doubling time of 20.34h and a chromosome number ranging from 70 to 90. Transwell and wound healing assays demonstrated the CMT-N7 cells had invasive ability. Immunofluorescence analysis revealed positive expression of ER, alpha-SMA, CK-14, SOX-2, Vimentin, Ki-67, E-cadherin, and COX-2 in CMT-N7 cells. Following inoculation with CMT-N7 cells for two weeks, all mice developed tumors. Immunohistochemical analysis showed negative expression of HER-2 and PR, and positive expression of ER, Ki-67, E-cadherin, Vimentin, and COX-2. Consequently, the establishment of the canine mammary cancer cell line CMT-N7 provides a good model for investigating the mechanism of epithelial-mesenchymal transition (EMT) in both dogs and humans.

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.

Contamination by pathogens, such as bacteria, can irritate a wound and prevent its healing, which may affect the physical fitness of the infected person. As such, the development of more novel nano-biomaterials able to cope with the inflammatory reaction to bacterial infection during the wound healing process to accelerate wound healing is required. Herein, a halofuginone‑silver nano thermosensitive hydrogel (HTPM&AgNPs-gel) was prepared via a physical swelling method. HTPM&AgNPs-gel was characterized based on thermogravimetric analysis, differential scanning calorimetry, morphology, injectability, and rheological mechanics that reflected its exemplary nature. Moreover, HTPM&AgNPs-gel was further tested for its ability to facilitate healing of skin fibroblasts and exert antibacterial activity. Finally, HTPM&AgNPs-gel was tested for its capacity to accelerate general wound healing and treat bacterially induced wound damage. HTPM&AgNPs-gel appeared spherical under a transmission electron microscope and showed a grid structure under a scanning electron microscope. Additionally, HTPM&AgNPs-gel demonstrated excellent properties, including injectability, temperature-dependent swelling behavior, low loss at high temperatures, and appropriate rheological properties. Further, HTPM&AgNPs-gel was found to effectively promote healing of skin fibroblasts and inhibit the proliferation of Escherichia coli and Staphylococcus aureus. An evaluation of the wound healing efficacy demonstrated that HTPM&AgNPs-gel had a more pronounced ability to facilitate wound repair and antibacterial effects than HTPM-gel or AgNPs-gel alone, and exhibited ideal biocompatibility. Notably, HTPM&AgNPs-gel also inhibited inflammatory responses in the healing process. HTPM&AgNPs-gel exhibited antibacterial, anti-inflammatory, and scar repair features, which remarkably promoted wound healing. These findings indicated that HTPM&AgNPs-gel holds great clinical potential as a promising and valuable wound healing treatment.

The small-molecule alkaloid halofuginone (HF) is obtained from febrifugine. Recent studies on HF have aroused widespread attention owing to its universal range of noteworthy biological activities and therapeutic functions, which range from parasite infections and fibrosis to autoimmune diseases. In particular, HF is believed to play an excellent anticancer role by suppressing the proliferation, adhesion, metastasis, and invasion of cancers. This review supports the goal of demonstrating various anticancer effects and molecular mechanisms of HF. In the studies covered in this review, the anticancer molecular mechanisms of HF mainly included transforming growth factor-β (TGF-β)/Smad-3/nuclear factor erythroid 2-related factor 2 (Nrf2), serine/threonine kinase proteins (Akt)/mechanistic target of rapamycin complex 1(mTORC1)/wingless/integrated (Wnt)/β-catenin, the exosomal microRNA-31 (miR-31)/histone deacetylase 2 (HDAC2) signaling pathway, and the interaction of the extracellular matrix (ECM) and immune cells. Notably, HF, as a novel type of adenosine triphosphate (ATP)-dependent inhibitor that is often combined with prolyl transfer RNA synthetase (ProRS) and amino acid starvation therapy (AAS) to suppress the formation of ribosome, further exerts a significant effect on the tumor microenvironment (TME). Additionally, the combination of HF with other drugs or therapies obtained universal attention. Our results showed that HF has significant potential for clinical cancer treatment.

BACKGROUND:Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are recognized as a highly challenging subset of cells, renowned for their heightened propensity for relapse and unfavorable prognosis. Monensin, an ionophoric antibiotic, has been reported to exhibit significant therapeutic efficacy against various cancers, especially CSCs. Erlotinib is classified as one of the EGFR-TKIs and has been previously identified as a promising therapeutic target for TNBC. Our research aims to assess the effectiveness of combination of monensin and erlotinib as a potential treatment strategy for TNBC. METHODS:The combination of monensin and erlotinib was assessed for its potential anticancer activity through various in vitro assays, including cytotoxicity assay, colony formation assay, wound healing assay, transwell assay, mammosphere formation assay, and proportion of CSCs assay. Additionally, an in vivo study using tumor-bearing nude mice was conducted to evaluate the inhibitory effect of the monensin and erlotinib combination on tumor growth. RESULTS:The results indicated that combination of monensin with erlotinib synergistically inhibited cell proliferation, the migration rate, the invasion ability and decreased the CSCs proportion, and CSC markers SOX2 and CD133 in vivo and in vitro. Furthermore, the primary proteins involved in the signaling pathways of the EGFR/ERK and PI3K/AKT are simultaneously inhibited by the combination treatment of monensin and erlotinib in vivo and in vitro. CONCLUSIONS:The simultaneous inhibition of the EGFR/ERK and PI3K/AKT/mTOR signaling pathways by the combination of monensin and erlotinib exhibited a synergistic effect on suppressing tumor proliferation and cancer cell stemness in TNBC.

In female dogs, the highest morbidity and mortality rates cancer are the result of mammary adenocarcinoma, which presents with metastases in the lung. Other than early surgical removal, however, no special methods are available to treat mammary adenocarcinoma. Because human breast cancer and canine mammary carcinoma share clinical characteristics and heterogeneity, the canine model is a suitable spontaneous tumor model for breast cancer in humans. In this study, the physical swelling method was used to prepare halofuginone-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles nano-thermosensitive hydrogels (HTPM-gel). Furthermore, HTPM-gel was investigated via characterization, morphology, properties such as swelling experiment and in vitro release with reflecting its splendid nature. Moreover, HTPM-gel was further examined its capability to anti-proliferation, anti-migration, and anti-invasion. Ultimately, HTPM-gel was investigated for its in vivo anticancer activity in the post-operative metastatic and angiogenic canine mammary carcinoma. HTPM-gel presented spherical under transmission electron microscope (TEM) and represented grid structure under scanning electron microscope (SEM), with hydrodynamic diameter (HD) of 20.25 ± 2.5 nm and zeta potential (ZP) of 15.10 ± 1.82 mV. Additionally, HTPM-gel own excellent properties comprised of pH-dependent swelling behavior, sustained release behavior. To impede the migration, invasion, and proliferation of CMT-U27 cells, we tested the efficacy of HTPM-gel. Evaluation of in vivo anti-tumor efficacy demonstrates HTPM-gel exhibit a splendid anti-metastasis and anti-angiogenic ability, with exhibiting ideal biocompatibility. Notably, HTPM-gel also inhibited the scar formation in the healing process after surgery. In summary, HTPM-gel exhibited anti-metastasis and anti-angiogenic and scar repair features. According to the results of this study, HTPM-gel has encouraging clinical potential to treat tumors with multifunctional hydrogel.

Background: Serum phosphate levels remain insufficiently controlled in chronic kidney disease (CKD) patients, and novel therapeutic strategies are needed.Blocking intestinal phosphate absorption mediated by sodiumdependent phosphate cotransporter type 2b (NPT2b) holds promise as one such strategy.Methods: The in vitro cellular potency of DZ1462 was evaluated using a radioactive Pi uptake assay on stable Chinese hamster ovary (CHO) cell clones transfected with human NPT2b (hNPT2b) or rat NPT2b (rNPT2b).The ability of DZ1462 to inhibit phosphate absorption was studied in vivo in an acute model after oral bolus challenge with 33 PO 4 and in an adenineinduced chronic hyperphosphatemia rat model.PK and minitox was also evaluated.Results: The cellular assays with the hNPT2b-CHO and rNPT2b-CHO clones showed that DZ1462 significantly and potently inhibited phosphate uptake.In vivo, in a chronic Pi-fed rat model, DZ1462 effectively inhibited intestinal Pi uptake.In a hyperphosphatemia rat model, DZ1462 significantly inhibited Pi uptake, and DZ1462 in combination with sevelamer had a synergistic effect.The pharmacokinetics (PK) study confirmed that DZ1462 is a gastrointestinal (GI)-restricted compound that can remain in the intestine for a sufficient duration.In addition, DZ1462 also reduced cardiovascular events and ameliorated osteoporosis in a CKD animal model.Conclusions: This study revealed that a GI-restricted NPT2b inhibitor DZ1462 potently inhibits NPT2b in vitro and blocks intestinal phosphate uptake in multiple animal models with potential to reduce various cardiovascular events in CKD models.Therefore, DZ1462 may be useful to treat renal disease patients who have shown an unsatisfactory response to phosphate binders.

Methuosis, a novel cell death phenotype, is characterized by accumulation of cytoplasmic vacuolization upon external stimulus. Methuosis plays a critical role in maduramicin-induced cardiotoxicity despite the underlying mechanism is largely unknown. Herein, we aimed to investigate the origin and intracellular trafficking of cytoplasmic vacuoles, as well as the molecular mechanism of methuosis caused by maduramicin (1 & mu;g/mL) in myocardial cells. H9c2 cells and broiler chicken were used and were exposed to maduramicin at doses of 1 & mu;g/mL in vitro and 5 ppm-30 ppm in vivo. Morphological observation and dextran-Alexa Fluor 488 tracer experiment showed that endosomal compartments swelling and excessive macropinocytosis contributed to madurdamcininduced methuosis. Cell counting kit-8 assay and morphology indicated pharmacological inhibition of macropinocytosis largely prevent H9c2 cells from maduramicin-triggered methuosis. In addition, late endosomal marker Rab7 and lysosomal associated membrane protein 1 (LAMP1) increased in a time-dependent manner after maduramicin treatment, and the recycling endosome marker Rab11 and ADP-ribosylation factor 6 (Arf6) were decreased by maduramicin. Vacuolar-H+-ATPase (V-ATPase) was activated by maduramicin, and pharmacological inhibition and genetic knockdown V0 subunit of V-ATPase restore endosomal-lysosomal trafficking and prevent H9c2 cells methuosis. Animal experiment showed that severe cardiac injury included the increase of creatine kinase (CK) and creatine kinase-MB (CK-MB), and vacuolar degeneration resembled methuosis in vivo after maduramicin treatment. Taken together, these findings demonstrate that targeting the inhibition of VATPase V0 subunit will prevent myocardial cells methuosis by restoring endosomal-lysosomal trafficking.

Coccidiosis is a worldwide epidemic intestinal disease with high incidence, which causes huge economic losses. Halofuginone hydrobromide (HF) is widely applied as an effective anticoccidial drug in the poultry industry. However, its therapeutic efficacy is severely restrained due to toxic effects, poor aqueous solubility and low permeability. Nanotechnology can improve the biological effect of drugs, and thus, reduce administered doses and toxic effects. The objective of this study was to investigate the therapeutic and preventive potential of novel HF-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles (HTPM) for preventing coccidiosis in chickens. The HTPM were approximately spherical with a hydrodynamic diameter of 12.65 ± 0.089 nm, a zeta potential of 8.03 ± 0.242 mV, a drug loading of 14.04 ± 0.12%, and an encapsulation efficiency of 71.1 ± 4.15%. HF was encapsulated in the polymer micelles through interactions with TPGS, as characterized by X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Cellular take up assays showed that TPGS polymer micelles could enhance drug internalization to alleviate intestinal apoptosis induced by coccidiosis and promote the necrosis of second-generation merozoites of E. tenella. Notably, clinical trials proved that 1.5 mg L-1 HTPM had a stronger anticoccidial effect on E. tenella than that of 3 mg kg-1 HF premix. Amplicon sequencing identified that HTPM could alleviate coccidiosis by restoring the structure of the gut microbiome. These findings indicated that the anticoccidial efficacy of HF was significantly enhanced after being encapsulated in polymer micelles, and further demonstrated the potential protective application of nano-encapsulating anticoccidial drugs as a promising approach to control coccidiosis in poultry. In summary, HTPM hold huge potential as an effective therapeutic agent for coccidiosis.

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24 h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.

[目的]建立一种绵羊组织中地昔尼尔及其代谢产物2,4,6-三氨基-5-氯基嘧啶质量浓度的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法.[方法]对采集的绵羊肌肉、脂肪、肝脏、肾脏组织进行前处理,以乙腈和七氟丁酸水溶液为流动相,利用Acquity UPLC Beh C18色谱柱进行梯度洗脱;流速为0.15 mL/min,柱温为30 ℃,进样量为5 μL.在正离子扫描模式下通过多离子反应监测采集数据,采用外标法测定地昔尼尔及其代谢产物浓度,并考察该检测方法的线性范围、检测限、定量限、回收率、批内与批间精密度、准确度和稳定性.[结果]建立的检测方法灵敏度高,检测限为10,μg/kg,定量限为20 μg/kg,地昔尼尔和2,4,6-三氨基-5-氰基嘧啶的添加浓度为20～1 000 μg/kg,标准曲线的线性关系良好,相关系数均≥0.99.将地昔尼尔和2,4,6-三氨基-5-氰基嘧啶以高、中、低、定量限4个水平分别添加到各空白组织中,测得地昔尼尔和2,4,6-三氨基-5-氰基嘧啶的回收率为70％～110％,批内和批间变异系数均＜15％.在考察的条件下,地昔尼尔和2,4,6-三氨基-5-氰基嘧在各组织中的稳定性良好.[结论]建立的UPLC-MS/MS检测方法专属性强、重复性好、灵敏度高,可适用于地昔尼尔乳剂在绵羊组织内的药物代谢动力学和残留研究.

Objective To study the efficacy of DZ1462, a novel sodium-phosphate transporter inhibitor, on rat hyperphosphatemia models established by 5/6 nephrectomy.Methods Totally 156 rats were randomly selected into four groups. Rats fed a normal diet were control group, named as group Ⅰ (n=6); rats fed a normal diet after 5/6 nephrectomy were named as group Ⅱ (n=60); rats fed a high phosphate diet after 5/6 nephrectomy were named as group Ⅲ (n=60); rats fed a high phosphate diet after sham surgery were named as group Ⅳ (n=30). The molding cycle was 10 weeks. Serum Pi was detected and the number of animal deaths was recorded every two weeks. Hematoxylin-eosin (HE) staining was performed to observe the change in kidney pathology, and to screen animal models with high phosphorus blood syndrome. Totally 18 model rats that met the inclusion criteria (all of group Ⅲ) were selected and randomly assigned to three groups: the model control group recorded as the G2 group; the DZ1462 administration group (30 mg/kg, tid, 21 d) recorded as the G3 group; the Sevelamer administration group (250 mg/kg, tid, 21 d) recorded as the G4 group. In addition, the normal control group was set as the G1 group. Serum phosphate levels were measured using a kit.Results In the 8th and 10th weeks, compared to group Ⅰ, serum phosphorus in group Ⅲ showed a significant difference (P < 0.01). The kidneys in group Ⅲ had obvious glomerular sclerosis, renal tubular atrophy, degeneration, interstitial inflammation, fibrosis, and calcification. Similarly to chronic kidney disease accompanied by hyperphosphatemia, the animal model was established successfully. At each time point, the serum phosphorus inhibition rate of the G3 group was significantly higher than that of the G4 group (P < 0.05).Conclusion DZ1462, as a novel small-molecule inhibitor of intestinal sodium and phosphorus transporter, can effectively inhibit intestinal phosphorus ion absorption in rat hyperphosphatemia model, and is expected to become a potential drug for the clinical treatment of hyperphosphatemia.