Objective To investigate the temporal changes in pathological damage and macrophage polarization in liver and spleen tissues of mice infected with Angiostrongylus cantonensis, and to preliminarily unravel the peripheral immune responses during the early stage of A. cantonensis infection. Methods Forty female BALB/c mice at ages of 6 to 8 weeks were randomly divided into four groups, including the control group and 7-, 14-, and 21-day infection groups, with 10 mice in each group. Each mouse in the infection groups was inoculated with 30 third-stage (L3) larvae of A. cantonensis by oral gavage, and five mice were randomly selected from each infection group on days 7, 14, and 21 post-infection, while mice in the control group were given the same volume of physiological saline and five mice were randomly selected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled. The histopathological changes of mouse liver and spleen tissues were observed using hematoxylin and eosin (HE) staining, and the percentage of positive staining area and the co-localization positive rates of the macrophage surface antigens F4/80, CD86, and CD206 were quantified in mouse liver and spleen tissues using immunohistochemical and immunofluorescence staining. In addition, five mice were collected from each infection group on days 7, 14, and 21 post-infection, and five mice were collected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled for detection of macrophage markers CD86 and CD206 and macrophage phenotyping using flow cytometry, and the expression of M1 macrophage markers, including inducible nitric oxide synthase (Nos2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and M2 markers, including arginase 1 (Arg1), mannose receptor C-type 1 (Mrc1) and chitinase-like protein 3 (Chil3) was quantified in mouse liver and spleen tissues using real-time quantitative PCR (RT-qPCR) assay. Results Proliferative lesions of the hepatocyte were observed in mouse liver tissues and the follicular structures of the mouse spleen white pulp were disrupted 21 days post-infection with A. cantonensis. Immunohistochemical staining showed that there were significant differences in the percentages of F4/80, CD86 and CD206 positive staining areas in the liver and spleen tissues among the four groups of mice (F = 242.40, 197.14, 183.19, 157.65, 242.35 and 146.24; all P values < 0.001), and the percentages of positive staining in the liver and spleen tissues of mice in the 14-day infection group [(4.45 ± 0.51)%, (3.74 ± 0.67)%, (8.32 ± 0.72)%, (16.56 ± 1.14)%, (11.62 ± 0.52)%, and (8.29 ± 0.72)%, respectively] and the 21-day infection group [(3.70 ± 0.11)%, (3.22 ± 0.43)%, (11.53 ± 1.03)%, (12.59 ± 1.05)%, (9.02 ± 0.83)%, and (11.67 ± 1.10)%, respectively] were higher than in the control group [(0.35 ± 0.16)%, (0.40 ± 0.02)%, (0.93 ± 0.05)%, (2.78 ± 0.26)%, (2.33 ± 0.20)%, and (1.85 ± 0.20)%, respectively] (all P values < 0.05). Immunofluorescence staining showed significant differences in the positive rates of F4/80 co-localization with CD86 and CD206 in mouse liver and spleen tissues among the four groups (F = 24.42, 25.28, 54.51 and 130.55; all P values < 0.001). Flow cytometry detected significant differences in the proportions of CD86⁺ and CD206⁺ macrophages in mouse liver and spleen tissues among the four groups (F = 67.98, 18.41, 29.77, 172.80; all P values < 0.001), and the proportions of CD206⁺ macrophages in the liver and spleen of the 21-day infection group were significantly higher than those in the control group [(9.25 ± 2.55)% vs (3.83 ± 0.72)%, and (4.22 ± 0.56)% vs (0.47 ± 0.18)%, respectively] (both P values < 0.05). In addition, RT-qPCR assay quantified significant differences in the relative mRNA expression of M1 macrophage markers (IL-1β, TNF-α and Nos2) and M2 macrophage markers (Arg1, Chil3 and Mrc1) in mouse liver and spleen tissues among the four groups (F = 41.30, 31.82, 199.33, 19.96, 62.01, 119.76, 23.67, 95.90, 72.27, 82.59, 123.41 and 29.75; all P values < 0.05). Conclusions A. cantonensis infection may cause progressive pathological damage in mouse liver and spleen tissues, accompanied by dynamic temporal changes in macrophage polarization. M1 macrophage polarization predominates at the early stage of A. cantonensis infection and shifts towards M2 polarization at the later stages, suggesting that M2 polarization may participate in immune regulation at late stages of A. cantonensis infection by suppressing excessive inflammatory responses and promoting tissue repair.

Objective:To evaluate the toxicity of foscarnet sodium injection into the anterior chamber and intravitreal cavity on the cornea and retina.Methods:Thirty-six adult New Zealand White rabbits were randomly divided into control group, intravitreal injection group, and intracameral injection group, with 12 rabbits in each group.In the control group, 0.1 ml of balanced salt solution (BSS) was injected into the vitreous cavity of one eye, and an equal volume of BSS was injected into the anterior chamber of the other eye.In the intracameral injection group and intravitreal injection group, 0.1 ml of sodium foscarnet 1.2 mg was injected into the anterior chamber and vitreous cavity of one eye, respectively.Slit-lamp microscopy, ophthalmoscope, optical coherence tomography (OCT), and in vivo confocal laser scanning microscopy were performed on 3 experimental rabbits from each group on days 1, 7, 14, and 28 after injection.After sacrifice, both eyeballs were removed, and the corneas and retinas were examined using optical microscopy, scanning electron microscopy and transmission electron microscopy to evaluate the toxicity to the cornea and retina comprehensively.The use and care of the animals complied with the ARVO Statement.The study protocol was approved by an Ethics Committee of Peking University Third Hospital (No.IRB00006761-2015197). Results:Slit-lamp microscopy and OCT showed no corneal edema, intraocular inflammation, or other abnormalities in the intravitreal injection and control groups.Mild corneal edema was observed in intracameral injection group 1 day after injection, which resolved 7 days after injection. In vivo confocal laser scanning microscopy revealed normal hexagonal corneal endothelial cell morphology in the intravitreal injection and control groups.There was no significant difference in endothelial cell density at baseline and 1, 7, and 14 days after injection among the three groups ( Fgroup=1.21, P=0.32; Ftime=1.21, P=0.32).Light microscopy revealed no obvious corneal abnormalities.On days 1 and 7 after injection, retinal nerve fiber layer vacuolization and inflammatory cell infiltration were observed in the intravitreal injection and control groups.In the intravitreal injection of BSS group, inflammatory cell infiltration occurred in the retina without vacuolization 1 day after injection.There were no structural changes in the photoreceptor layer, and the nuclear layer was well-organized.Scanning electron microscopy showed no significant abnormalities in the corneal endothelium in the intravitreal injection group 1 day after injection.In the intracameral injection group, a large number of inflammatory cells were deposited and adhered to the corneal endothelium 1 day after injection and disappeared 7 days after injection.Transmission electron microscopy revealed that in the intravitreal injection group, 1 day after injection swelling of corneal endothelial cells, dilatation of the endoplasmic reticulum, and partial mitochondrial swelling were observed, which normalized 14 days after injection and vacuolization was present in the retina and interstitial fluid accumulation persisted until the 28 days after injection.In the intracameral injection group, swollen mitochondrial and endoplasmic reticulum of corneal endothelial cells was observed and resolved by 14 days after injection.However, structural abnormalities in the membranous discs of the photoreceptor outer segments and interstitial fluid accumulation in the optic nerve fiber layer persisted 1 day after injection and did not fully recover 28 days after injection. Conclusions:Intracameral intravitreal and injection of foscarnet sodium have transient toxic effects on the retina, which gradually weaken over time.Intracameral injection of foscarnet sodium was more toxic to corneal endothelial cells than intravitreal injection.

G protein-coupled receptors (GPCRs) are significant drug targets, but their potential in cancer therapy remains underexplored. Conventional GPCR agonists or antagonists have shown limited effectiveness in cancer treatment, necessitating new GPCR-targeting strategies for more effective therapies. This study discovers that Yersinia pestis LcrV, a crucial linker protein for plague infection, acts as a biased agonist of a GPCR, the formyl peptide receptor 1 (FPR1). The LcrV protein induces unique conformational changes in FPR1, resulting in G proteins being activated in a distinctive state without subunit dissociation. This leads to a biased signaling profile characterized by cyclic adenosine monophosphate (cAMP) responses and β-arrestin2 recruitment, but not calcium mobilization. In FPR1-expressing triple-negative breast cancer (TNBC) cells, LcrV bi-directionally modulates intracellular signaling pathways, downregulating extracellular signal-regulated kinases (ERK1/2) and Akt pathways while upregulating Jun N-terminal kinase (JNK) and p38 pathways. This dual modulation results in cell cycle arrest and the inhibition of TNBC cell proliferation. In TNBC xenograft mouse models, long-term LcrV treatment inhibits tumor growth more effectively than a conventional FPR1 antagonist. Additionally, LcrV treatment reprograms tumor cells by reducing stemness-associated proteins OCT4 and c-MYC. Our findings highlight the potential of biased GPCR agonists as a novel GPCR-targeting strategy for cancer treatment.

OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity. METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants. RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes. CONCLUSION The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.
Legionella pneumophila/pathogenicity* ; CRISPR-Cas Systems ; Biofilms/growth & development* ; Phenotype ; Bacterial Proteins/metabolism* ; Gene Deletion

Aim To investigate the expression of or-ganic anion transporting polypeptide 4C1(Oatp4c1)-P-glycoprotein(P-gp)in the kidneys of obese mice in-duced by high-fat diet(HFD),and its impact on the pharmacokinetic changes of digoxin.Methods C57BL/6 mice were randomly divided into the Chow group and the HFD group.Body weight and blood glu-cose were recorded weekly.After successful model es-tablishment,digoxin was intraperitoneally injected,and blood was collected at different time points.Part of the blood samples was used for LC-MS/MS detection,and the other part was used for the detection of other bio-chemical indicators.After 16 weeks,the organs were removed and weighed.HE and immunohistochemical staining was used to observe the renal pathology and the expression of Villin,a marker of proximal tubules.Western blot and qPCR were combined to detect the expression of Villin,Oatp4c1 and P-gp.Results In the HFD group,body weight and blood glucose in-creased significantly.The blood concentration of digox-in rose,the area under the curve increased,and the half-life was prolonged.The proximal tubular epithelial cells shed,and the protein expression of Villin,Oatp4c1 and P-gp decreased significantly.Conclu-sions The down-regulation of Oatp4c1-P-gp expres-sion in the kidneys of HFD mice leads to an increase in the blood concentration of digoxin and a decrease in re-nal clearance.

Objective:To evaluate the toxicity of foscarnet sodium injection into the anterior chamber and intravitreal cavity on the cornea and retina.Methods:Thirty-six adult New Zealand White rabbits were randomly divided into control group, intravitreal injection group, and intracameral injection group, with 12 rabbits in each group.In the control group, 0.1 ml of balanced salt solution (BSS) was injected into the vitreous cavity of one eye, and an equal volume of BSS was injected into the anterior chamber of the other eye.In the intracameral injection group and intravitreal injection group, 0.1 ml of sodium foscarnet 1.2 mg was injected into the anterior chamber and vitreous cavity of one eye, respectively.Slit-lamp microscopy, ophthalmoscope, optical coherence tomography (OCT), and in vivo confocal laser scanning microscopy were performed on 3 experimental rabbits from each group on days 1, 7, 14, and 28 after injection.After sacrifice, both eyeballs were removed, and the corneas and retinas were examined using optical microscopy, scanning electron microscopy and transmission electron microscopy to evaluate the toxicity to the cornea and retina comprehensively.The use and care of the animals complied with the ARVO Statement.The study protocol was approved by an Ethics Committee of Peking University Third Hospital (No.IRB00006761-2015197). Results:Slit-lamp microscopy and OCT showed no corneal edema, intraocular inflammation, or other abnormalities in the intravitreal injection and control groups.Mild corneal edema was observed in intracameral injection group 1 day after injection, which resolved 7 days after injection. In vivo confocal laser scanning microscopy revealed normal hexagonal corneal endothelial cell morphology in the intravitreal injection and control groups.There was no significant difference in endothelial cell density at baseline and 1, 7, and 14 days after injection among the three groups ( Fgroup=1.21, P=0.32; Ftime=1.21, P=0.32).Light microscopy revealed no obvious corneal abnormalities.On days 1 and 7 after injection, retinal nerve fiber layer vacuolization and inflammatory cell infiltration were observed in the intravitreal injection and control groups.In the intravitreal injection of BSS group, inflammatory cell infiltration occurred in the retina without vacuolization 1 day after injection.There were no structural changes in the photoreceptor layer, and the nuclear layer was well-organized.Scanning electron microscopy showed no significant abnormalities in the corneal endothelium in the intravitreal injection group 1 day after injection.In the intracameral injection group, a large number of inflammatory cells were deposited and adhered to the corneal endothelium 1 day after injection and disappeared 7 days after injection.Transmission electron microscopy revealed that in the intravitreal injection group, 1 day after injection swelling of corneal endothelial cells, dilatation of the endoplasmic reticulum, and partial mitochondrial swelling were observed, which normalized 14 days after injection and vacuolization was present in the retina and interstitial fluid accumulation persisted until the 28 days after injection.In the intracameral injection group, swollen mitochondrial and endoplasmic reticulum of corneal endothelial cells was observed and resolved by 14 days after injection.However, structural abnormalities in the membranous discs of the photoreceptor outer segments and interstitial fluid accumulation in the optic nerve fiber layer persisted 1 day after injection and did not fully recover 28 days after injection. Conclusions:Intracameral intravitreal and injection of foscarnet sodium have transient toxic effects on the retina, which gradually weaken over time.Intracameral injection of foscarnet sodium was more toxic to corneal endothelial cells than intravitreal injection.

Nuclear receptor co-activator 4 (NCOA4) acts as a selective cargo receptor that binds to ferritin, a cytoplasmic iron storage complex. By mediating ferritinophagy, NCOA4 regulates iron metabolism and releases free iron in the body, thus playing a crucial role in a variety of biological processes, including growth, development, and metabolism. Recent studies have shown that NCOA4-mediated ferritinophagy is closely associated with the occurrence and development of iron metabolism-related diseases, such as liver fibrosis, renal cell carcinoma, and neurodegenerative diseases. In addition, a number of clinical drugs have been identified to modulate NCOA4-mediated ferritinophagy, significantly affecting disease progression and treatment efficacy. This paper aims to review the current research progress on the role of NCOA4-mediated ferritinophagy in related diseases, in order to provide new ideas for targeted clinical therapy.
Humans ; Nuclear Receptor Coactivators/physiology* ; Ferritins/metabolism* ; Animals ; Neurodegenerative Diseases/metabolism* ; Iron/metabolism* ; Autophagy/physiology* ; Liver Cirrhosis/metabolism* ; Carcinoma, Renal Cell/metabolism* ; Kidney Neoplasms/physiopathology*

The study aimed to construct the second and third generation chimeric antigen receptor T cells (CAR-T) targeting the c-mesenchymal-epithelial transition factor (c-Met) protein, and observe their killing effect on human serous ovarian cancer cell SKOV-3. The expression of MET gene in ovarian serous cystadenocarcinoma, the correlation between MET gene expression and the abundance of immune cell infiltration, and the effect of MET gene expression on the tissue function of ovarian serous cystadenocarcinoma were analyzed by bioinformatics. The expression of c-Met in ovarian cancer tissues and adjacent tissues was detected by immunohistochemical staining. The second and third generation c-Met CAR-T cells, namely c-Met CAR-T(2G/3G), were prepared by lentivirus infection, and the cell subsets and infection efficiency were detected by flow cytometry. Using CD19 CAR-T and activated T cells as control groups and A2780 cells with c-Met negative expression as Non target groups, the kill efficiency on SKOV-3 cells with c-Met positive expression, cytokine release and cell proliferation of c-Met CAR-T(2G/3G) were explored by lactate dehydrogenase (LDH) release, ELISA and CCK-8 respectively. The results showed that MET gene expression was significantly up-regulated in ovarian cancer tissues compared with normal tissues, which was consistent with the immunohistochemistry results. However, in all pathological stages, there was no obvious difference in MET expression and no correlation between MET gene expression and the race and age of ovarian cancer patients. The second generation and third generation c-Met CAR-T cells were successfully constructed. After lentivirus infection, the proportion of CD8⁺ T cells in c-Met CAR-T(2G) was upregulated, while there was no significant change in the cell subsets of c-Met CAR-T(3G). The LDH release experiment showed that the kill efficiency of c-Met CAR-T(2G/3G) on SKOV-3 increased with the increase of effect-target ratio. When the effect-target ratio was 20:1, the kill efficiency of c-Met CAR-T(2G) reached (42.02 ± 5.17)% (P < 0.05), and the kill efficiency of c-Met CAR-T(3G) reached (51.40 ± 2.71)% (P < 0.05). ELISA results showed that c-Met CAR-T released more cytokine compared to CD19 CAR-T and activated T cells (P < 0.05). Moreover, the cytokine release of c-Met CAR-T(3G) was higher than c-Met CAR-T(2G) (P < 0.01). The CCK-8 results showed that after 48 h, the cell number of c-Met CAR-T(2G) was higher than that of c-Met CAR-T(3G) (P < 0.01). In conclusion, both the second and third generation c-Met CAR-T can target and kill c-Met-positive SKOV-3 cells, with no significant difference. c-Met CAR-T(2G) has stronger proliferative ability, and c-Met CAR-T(3G) releases more cytokines.
Humans ; Female ; Ovarian Neoplasms/immunology* ; Proto-Oncogene Proteins c-met/metabolism* ; Receptors, Chimeric Antigen/immunology* ; Cell Line, Tumor ; Cystadenocarcinoma, Serous/immunology* ; T-Lymphocytes/immunology*

This study aims to explore whether Naoxintong Capsules improve multiple cerebral infarctions and myocardial injury via promoting angiogenesis, thereby exerting a simultaneous treatment effect on both the brain and heart. Male SD rats were randomly divided into six groups: sham-operated group, model group, high-dose, medium-dose, and low-dose groups of Naoxintong Capsules(440, 220, and 110 mg·kg~(-1)), and nimodipine group(10.8 mg·kg~(-1)). Rat models of multiple cerebral infarctions were established by injecting autologous thrombus, and samples were collected and tested seven days after modeling. Evaluations included multiple cerebral infarction model assessments, neurological function scores, grip strength tests, and rotarod tests, so as to evaluate neuromotor functions. Morphological structures of brain and heart tissue were observed using hematoxylin-eosin(HE) staining, Nissl staining, and Masson staining. Network pharmacology was employed to screen the mechanisms of Naoxintong Capsules in improving multiple cerebral infarctions and myocardial injury. Neuronal and myocardial cell ultrastructures were observed using transmission electron microscopy. Apoptosis rate in brain neuronal cells was detected by TdT-mediated dUTP nick end labeling(TUNEL) staining, and reactive oxygen species(ROS) levels in myocardial cells were measured. Immunofluorescence was used to detect the expression of platelet endothelial cell adhesion molecule-1(CD31), antigen identified by monoclonal antibody Ki67(Ki67), hematopoietic progenitor cell antigen CD34(CD34), and hypoxia inducible factor-1α(HIF-1α) in brain and myocardial tissue. Western blot, and real-time quantitative polymerase chain reaction(RT-qPCR) were used to detect the expression of HIF-1α, vascular endothelial growth factor(VEGF), vascular endothelial growth factor receptor 2(VEGFR2), sarcoma(Src), basic fibroblast growth factor(bFGF), angiopoietin-1(Ang-1), and TEK receptor tyrosine kinase(Tie-2). Compared with the model group, the medium-dose group of Naoxintong Capsules showed significantly lower neurological function scores, increased grip strength, and prolonged time on the rotarod. Pathological damage in brain and heart tissue was reduced, with increased and more orderly arranged mitochondria in neurons and cardiomyocytes. Apoptosis in brain neuronal cells was decreased, and ROS levels in cardiomyocytes were reduced. The microvascular density and endothelial cells of new blood vessels in brain and heart tissue increased, with increased overlapping regions of CD31 and Ki67 expression. The relative protein and mRNA expression levels of HIF-1α, VEGF, VEGFR2, Src, Ang-1, Tie-2, and bFGF were elevated in brain tissue and myocardial tissue. Naoxintong Capsules may improve multiple cerebral infarctions and myocardial injury by mediating HIF-1α/VEGF expression to promote angiogenesis.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Cerebral Infarction/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Capsules ; Signal Transduction/drug effects* ; Humans ; Brain/metabolism* ; Myocardium/metabolism* ; Apoptosis/drug effects*

PURPOSE: This study aims to identify the prevalence and risk factors of military training-related abdominal injuries and help plan and conduct training properly. METHODS: This questionnaire survey study was conducted from October 2021 to May 2022 among military personnel from 6 military units and 8 military medical centers and participants' medical records were consulted to identify the training-related abdominal injuries. All the military personnel who ever participated in military training were included. Those who refused to participate in this study or provided an incomplete questionnaire were excluded. The questionnaire collected demographic information, type of abdominal injury, frequency, training subjects, triggers, treatment, and training disturbance. Chi-square test and t-test were used to compare baseline information. Univariate and multivariate regression analyses were used to explore the risk factors associated with military training-related abdominal injuries. RESULTS: A total of 3058 participants were involved in this study, among which 1797 (58.8%) had suffered training-related abdominal injuries (the mean age was 24.3 years and the service time was 5.6 years), while 1261 (41.2%) had no training-related abdominal injuries (the mean age was 23.1 years and the service time was 4.3 years). There were 546 injured patients (30.4%) suspended the training and 84 (4.6%) needed to be referred to higher-level hospitals. The most common triggers included inadequate warm-up, fatigue, and intense training. The training subjects with the most abdominal injuries were long-distance running (589, 32.8%). Civil servants had the highest rate of abdominal trauma (17.1%). Age ≥ 25 years, military service ≥ 3 years, poor sleep status, and previous abdominal history were independent risk factors for training-related abdominal injury. CONCLUSION More than half of the military personnel have suffered military training-related abdominal injuries. Inadequate warm-up, fatigue, and high training intensity are the most common inducing factors. Scientific and proper training should be conducted according to the factors causing abdominal injuries.
Humans ; Military Personnel ; Risk Factors ; Prevalence ; Male ; Abdominal Injuries/etiology* ; Female ; Adult ; Surveys and Questionnaires ; Young Adult