Objective To investigate the in vivo anti-inflammatory effects of the triterpenoid saponin-enriched fraction Fr.B4 from the She medicine Clematis florida (C. florida) in a rat model of phenol-induced cervicitis and to preliminarily explore its potential mechanisms of action. Methods Thirty 6–8-week-old female SPF-grade SD rats were randomly assigned to three groups: control, cervicitis model, and C. florida treatment group, with 10 rats in each group. The model and C. florida treatment groups were induced with cervicitis by vaginal injection of 25% phenol gel (once every 2 d, for a total of three injections), and the vulva and cervix were examined to assess the effectiveness of model establishment. After successful modeling, the C. florida treatment group was administered Fr.B4 at 290 mg/kg daily by gavage (dissolved in normal saline), whereas the model group received an equal volume of normal saline; both treatments continued for 2 weeks. The control group received no modeling procedure and was administered normal saline for 2 weeks. After the last intervention, fresh feces and urine samples were collected from all rats. The rats were euthanized after anesthesia with 40 mg/kg Zoletil 50, and the serum and cervical tissue samples were harvested. ELISA was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in rat serum and cervical tissue. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in cervical tissue. Liquid chromatography-mass spectrometry was employed to analyze differential metabolites in feces and urine, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and core metabolite analysis. 16S rDNA sequencing was used to assess the gut microbiota structure and diversity in each group. Statistical analysis was conducted using GraphPad Prism 9.0. Results Compared with the control group, rats in the model group exhibited cervical redness, swelling, and erosion, with significant increases in TNF-α, IL-1β, and IL-6 levels in both serum and cervical tissue (P<0.05 or P<0.01). Pathological examination of cervical tissue showed thickening of the squamous epithelium, infiltration of inflammatory cells, and edema. Compared with the model group, rats with C. florida treatment showed marked alleviation of cervicitis symptoms, with significantly reduced levels of pro-inflammatory cytokines in serum and cervical tissue (P<0.05 or P<0.01). Cervical tissue edema was alleviated, inflammatory cells were reduced, and squamous epithelium thickening was ameliorated. Metabolomics analysis revealed 346 and 564 differential metabolites in the feces and urine between the model and control groups, respectively. After treatment with Fr.B4, 236 and 451 metabolites in the feces and urine showed significant shifts toward control levels, involving pathways including cyclic adenosine monophosphate (cAMP) signaling, AMP-activated protein kinase (AMPK) signaling, and glutathione metabolism. Anti-inflammatory metabolites like boswellic acid and oleanolic acid were increased (P<0.01 or P<0.001). Gut microbiota analysis showed dysbiosis in the model group, with an imbalance in the Firmicutes/Bacteroidetes ratio. The Fr.B4 treatment remodeled the gut microbiota. The α-diversity of microbiota shifted toward that of the control group. Specific bacterial genera were found to correlate significantly with metabolites (P<0.05 or P<0.01). Conclusion The triterpenoid saponin-enriched fraction Fr.B4 from She medicine C. florida may exert anti-cervicitis effects by reducing pro-inflammatory cytokine levels, modulating the gut microbiota balance, and remodeling metabolism.
Objective To explore the effect and mechanism of Gegen Qinlian decoction (GQD) on myocardial injury induced by high-fat diet combined with streptozotocin in type 2 diabetic mice through network pharmacology and animal experiments. Methods Network pharmacology was applied to screen the potential signaling pathways of GQD in the intervention of diabetic heart disease (DHD). A total of 36 specific pathogen-free (SPF) male C57BL/6J mice aged 8 weeks were acclimatized for 1 week and randomly divided into a normal control group (n=6) and a model group (n=30). Mice in the model group were fed a 60% high-fat diet and 5% glucose water for 4 weeks, and were intraperitoneally injected with 100 mg/kg streptozotocin after 12 h of fasting to establish the type 2 diabetes model. Mice in the normal control group were given a conventional diet and drinking water, and were intraperitoneally injected with an equal volume of citrate buffer after 12 h of fasting at the same time point. After successful establishment of the diabetic mouse model, the model mice were randomly divided into five groups according to body weight: model control group, GQD low-dose group, GQD medium-dose group, GQD high-dose group, and metformin positive control group, with 6 mice in each group. After 6 weeks of intragastric administration of corresponding doses of GQD, metformin, and ddH2O respectively, all mice in the six groups (one normal control group and five model groups) were anesthetized with inhaled 2% isoflurane, blood samples were collected via eyeball enucleation, and heart tissues were harvested after euthanasia by cardiac exsanguination. Hematoxylin-eosin (HE) staining and wheat germ agglutinin (WGA) staining were performed to observe the pathological damage of myocardial tissues and measure the cross-sectional area of cardiomyocytes. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of pro-inflammatory cytokines in serum. Immunohistochemical staining was adopted to determine the protein expression levels of receptor for advanced glycation end products (RAGE), nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3), and phosphorylated nuclear factor-κB p65 (p-p65) in myocardial tissues. Results Network pharmacology analysis revealed that the targets of GQD against DHD were significantly enriched in inflammatory pathways, including the advanced glycation end products (AGEs)-RAGE axis, tumor necrosis factor (TNF) and interleukin-17 (IL-17) signaling pathways. HE and WGA staining results showed that, compared with the normal control group, the model control group exhibited myocardial fiber rupture, cardiomyocyte swelling, and significantly increased cardiomyocyte cross-sectional area (P<0.05). Compared with the model control group, GQD intervention significantly ameliorated the pathological changes, including myocardial fiber rupture, cell swelling, and cardiomyocyte hypertrophy. The results of immunohistochemical staining and ELISA indicated that compared with the model control group, GQD intervention markedly downregulated the protein expression of RAGE, NLRP3, and p-p65 in myocardial tissues, as well as the levels of serum pro-inflammatory cytokines (P<0.05). Conclusion GQD can effectively alleviate myocardial pathological injury in diet-induced type 2 diabetic mice, and its mechanism may be related to inhibiting the activation of the RAGE/p-p65/NLRP3 signaling pathway and reducing myocardial inflammatory response.
Objective To investigate the protective effect of artemitin against ozone (O3)-induced pulmonary inflammation in mice and to explore its underlying mechanism. Methods Thirty-two specific pathogen-free (SPF) male C57BL/6 mice were randomly divided into a blank control group, an artemitin-only control group, an O3-induced model group, and an artemitin-treated group, with 8 mice in each group. In the artemitin-treated group, animals were injected intraperitoneally with artemitin (20 mg/kg) daily, followed by a 1 h exposure to 0.000 25% O3. The artemitin-only control group received the same artemitin injection but was exposed to clean air. The O3-induced model group was injected intraperitoneally with an equal volume of normal saline daily, followed by a 1-h exposure to 0.000 25% O3. The blank control group received the saline injection and was exposed to clean air. All treatments were administered for 7 consecutive days. Pulmonary function indices of the mice in each group were measured using a whole-body plethysmography system. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the histopathological changes in the lung tissue, and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for untargeted metabolomic analysis to examine the changes in overall metabolic pathways in plasma. Results Compared with the O3-induced model group, artemitin treatment significantly improved O3-induced lung function impairments (P<0.05), lowered the levels of TNF-α and IL-1β in BALF (P<0.05) and alleviated inflammatory cell infiltration and structural damage in lung tissues. Metabolomics results indicated that artemitin treatment compensatorily enhanced energy supply and oxidized lipid signaling, while inhibiting purine metabolism and tryptophan-derived pathways. Conclusion Artemitin exhibits a significant protective effect against O3-induced pulmonary inflammation in mice. It corrects upstream metabolic disorders by synergistically modulating tryptophan metabolism, energy metabolism, and lipid inflammatory mediators, thereby inhibiting downstream inflammatory responses and airway hyperresponsiveness. This study not only reveals the pleiotropic mechanism of artemitin in treating airway inflammation but also provides potential metabolic biomarkers and a theoretical basis for its clinical translation.
Objective To investigate the time-dependent pattern of blue light-induced retinal damage in mice, establish a suitable animal model, and evaluate the protective effects of a compound formula containing lutein esters, DHA algal oil, Lycium ruthenicum, and other ingredients used as both food and medicine. Methods Thirty-two of the 56 SPF-grade male C57BL/6J mice were randomly divided into a control group and model groups (1 h, 3 h, and 6 h irradiation) (n=8 per group) to determine the optimal blue light irradiation time. First, mice were exposed to blue light from a 467 nm light-emitting diode at (5 000±200) lx for 1 h, 3 h, and 6 h, respectively. Retinal histopathology and thickness were examined using hematoxylin‑eosin (HE) staining. Serum malondialdehyde (MDA) and glutathione (GSH) levels were measured using assay kits. Retinal inflammatory cytokine mRNA expression was assessed by real-time fluorescent quantitative reverse transcription PCR. After 3 h of blue light exposure was confirmed as the appropriate modeling condition, the remaining 24 mice were used in an intervention experiment to evaluate the protective effect of the eye-protective compound formula on blue light-induced retinal damage. The mice were administered the compound formula (1 000 mg·kg-1·d-1) by gavage for 14 days, followed by 3 h of blue light irradiation, and the same indicators were evaluated as described above. Results With prolonged irradiation time, the thickness of each retinal layer gradually decreased (P<0.05 to P<0.01) and cell arrangement became disordered. Serum MDA levels progressively increased (P<0.01), while GSH levels gradually decreased (P<0.01). The mRNA expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in retinal tissue was significantly upregulated with increasing irradiation time. The 1 h irradiation group showed only minor changes (P>0.05), whereas the 6 h irradiation group exhibited the most pronounced changes (P<0.000 1). Therefore, 3 h of blue light irradiation (P<0.001), which showed significant yet moderate differences in all indicators, was selected as the subsequent modeling condition. After intervention with the eye-protective compound formula, compared with the model group, serum MDA level was significantly reduced (P<0.01), and serum GSH level was significantly increased (P<0.05). The expression levels of retinal inflammatory cytokines IL-1β (P<0.000 1), IL-6 (P<0.001), and TNF-α (P<0.01) were significantly downregulated. Total retinal thickness (P<0.01), as well as the thicknesses of the inner plexiform layer (P<0.05), inner nuclear layer (P<0.05), outer plexiform layer (P<0.05), and outer nuclear layer (P<0.01), were significantly increased compared with the model group. Histopathological examination also showed that the regularity and density of retinal cell arrangement were markedly better than those in the model group. Conclusion Blue light irradiation at a specific illuminance for 3 h can establish a stable mouse model of retinal damage. The compound formula alleviates retinal damage through antioxidant and anti-inflammatory pathways, providing an experimental basis for the development of related functional foods.
Objective This study aims to conduct a 28-day oral toxicity test to observe the subacute toxic effects of Ganoderma lucidum spore oil in rats, elucidate its impacts on physiological indicators in rats, and provide a scientific basis for the safety evaluation of this substance. Methods Eighty 4-week-old specific pathogen-free (SPF) SD rats were randomly divided into a control group and three dose groups: low-dose (0.83 g/kg), medium-dose (1.67 g/kg), and high-dose (3.33 g/kg), with 10 males and 10 females in each group. The experimental groups were given Ganoderma lucidum spore oil by gavage at the corresponding doses, while the control group was given the same volume of edible corn oil. Administration lasted for 28 consecutive days, during which the rats had free access to food and water. The general condition of the rats was observed, and body weight, food intake, and food utilization rate were measured. After the treatment period, urine was collected from rats in each group for routine urinalysis. Blood samples were collected from the abdominal aorta under anesthesia induced by intraperitoneal administration of Zoletil 50 (5 mg/kg) and xylazine hydrochloride (10 mg/kg) for complete blood count, coagulation testing, and serum biochemical analyses. Gross necropsy was performed in all rats. Organs were excised and weighed, organ coefficients of major organs were calculated, and histopathological examinations of major organs were conducted in the control and high-dose groups. Results During the entire experimental period, all rats in the control and the three dose groups thrived without any mortality. No abnormal behaviors or clinical signs were observed in any of the groups. In the serum biochemistry, a statistically significant increase (P<0.05) in total protein was found in the high-dose female group compared with the control group. However, there was no dose-response relationship, and the values remained within the normal range of historical data in our laboratory; therefore, the increase was considered not biologically significant. Rats in the remaining groups showed no statistically significant differences (all P>0.05) in complete blood count, serum biochemical parameters, coagulation parameters, urinalysis parameters, or organ coefficients compared with the control group. Histopathological examination revealed no significant abnormalities. Conclusion The results indicate that Ganoderma lucidum spore oil does not exhibit significant subacute toxicity in SD rats at the tested dose levels.
Objective To establish and evaluate a rat model of chronic obstructive pulmonary disease (COPD) associated with the traditional Chinese medicine (TCM) phlegm-dampness syndrome. Methods Eighteen SPF-grade 8-week-old male SD rats were randomly assigned to a control group (n=8) and a model group (n=10). The modeling procedure lasted for 19 weeks. Rats in the control group received no intervention. Lipopolysaccharide (LPS; 40 μg per rat per instillation) was instilled intratracheally into rats in the model group at 09:00 on the first day of weeks 1, 5, 9, 13, and 17, for a total of five instillations. Except for the instillation weeks, rats were exposed daily to cigarette smoke in a self-made high-humidity smoke exposure chamber (15 cigarettes/session before week 9 and 20 cigarettes/session from week 9 onward; 30 min/session/day). Body weight was recorded weekly, and general signs and manifestations of TCM phlegm-dampness syndrome were observed. On the day after modeling, rats were anesthetized for pulmonary ventilatory function testing. Tumor necrosis factor-α (TNF-α) and mucin 5AC (MUC5AC) levels in serum and bronchoalveolar lavage fluid (BALF) were measured using enzyme-linked immunosorbent assay (ELISA). Lung tissues were subjected to hematoxylin and eosin (HE) and alcian blue and periodic acid-Schiff staining (AB-PAS) to assess lung tissue morphology and goblet cell hyperplasia. Western blotting was performed to determine TNF-α and MUC5AC protein expression in lung tissue. Results General signs and overall condition: Compared with the control group, body weight in the model group was significantly decreased from week 3 onward (P<0.01). Rats showed yellowish fur, sluggish activity, reduced movement, rapid/labored breathing, and increased oral/nasal secretions. Pulmonary ventilatory function: Compared with the control group, minute ventilation and tidal volume (both P<0.01) and dynamic compliance (P<0.05) were decreased, whereas elastic resistance was markedly increased (P<0.01) in the model group. Inflammatory factors and mucus-related proteins: Compared with the control group, TNF-α and MUC5AC levels in serum and BALF, as well as their protein levels in lung tissue, were significantly elevated in the model group (all P<0.01). Histopathology: HE staining showed obvious alveolar dilatation and rupture with partial coalescence into bullae, and inflammatory cell infiltration in bronchiolar lumens. AB-PAS staining revealed increased goblet cells in bronchiolar mucosal epithelium and mucus plug formation in the lumen. Conclusion This protocol can reproduce a COPD rat model with manifestations consistent with TCM phlegm-dampness syndrome, reduced pulmonary ventilation function, and pathological features of emphysema, inflammation, and chronic bronchitis, thereby providing reliable support for subsequent studies on TCM treatment of COPD with phlegm-dampness syndrome.
Objective To evaluate the effect of sex on phenotypic stability in mouse models of chemotherapy-induced alopecia (CIA) induced by cyclophosphamide (CYP) or paclitaxel (PTX), and to explore potential mechanisms underlying phenotypic heterogeneity in female mice. Methods Twenty-eight wild-type specific pathogen-free (SPF) C57BL/6J mice (half male and half female) aged 6-8 weeks were selected for synchronous back hair removal. On the 9th day after hair removal (T0), the mice were randomly divided into a wild-type female CYP group, a wild-type male CYP group, a female negative control group and a male negative control group. Mice in the CYP groups were intraperitoneally injected with a single dose of 120 mg/kg CYP, and mice in the negative control groups were intraperitoneally injected with the same volume of normal saline. Subsequently, the phenotypic changes of the CIA model induced by CYP were continuously observed and photographed. Eleven 6-week-old SPF female C57BL/6J mice underwent bilateral ovariectomy (OVX group). Following successful surgery, these mice and five XY (Sry⁻) mice bred from Four Core Genotypes breeding stock were used to establish the chemotherapy-induced alopecia (CIA) model using the same protocol. Serum was collected from mice in the OVX group, wild-type female CYP group, and wild-type male CYP group on T7 after CYP administration for steroid hormone analysis by mass spectrometry to evaluate the effects of gonadal estrogen and sex chromosome background changes on the stability of the CYP-induced female alopecic phenotype. Finally, twenty SPF C57BL/6J mice aged 6-8 weeks (half male and half female) were randomly divided into a PTX low-dose group and a PTX high-dose group. On the 9th day after simultaneous hair removal, PTX was injected intraperitoneally at a single dose of 80 mg/kg or 120 mg/kg, respectively. Mice from the PTX low-dose group and the PTX high-dose group were continuously observed and photographed to compare the intra-group consistency of the phenotypes of female and male mice. Six SPF C57BL/6J mice (half male and half female) aged 6-8 weeks were used to repeat the construction method of the PTX low-dose group, and the hair follicle histological examination was performed at T0, T4, T7, T14 and T21, respectively, to observe the damage caused by PTX to hair follicle regeneration. Results The phenotypes of CYP-induced male CIA mice are stable and reproducible. There are obvious individual differences in the alopecia phenotype in the CYP-induced female CIA model mice, which may be mainly caused by the differences in the relevant estrogens in the estrous cycle. The difference between OVX+CYP group and XY (Sry⁻) +CYP group disappeared, suggesting that ovarian-derived hormones and XY (Sry⁻) sex chromosome background may be involved in the formation of phenotypic heterogeneity of CYP-induced alopecia in female mice. The mice in the high-dose PTX group and the low-dose PTX group showed a relatively consistent alopecia and regrowth process, and there was no significant sex difference. Conclusion CYP-induced CIA is strongly influenced by sex, and ovariectomy or alteration of the sex chromosome background can improve the consistency of CYP-induced alopecic phenotypes in female mice. PTX-induced CIA shows good phenotypic consistency in both female and male mice, indicating broad applicability across sexes. Taken together, the choice of animal sex in CIA model construction, mechanistic studies, and evaluation of anti-alopecia interventions should take into account the type of chemotherapeutic agent, the study objective, and the required degree of phenotypic stability. Sex should therefore be considered an important variable in model standardization and interpretation of experimental findings.
Objective To compare the effects of the Rho-associated protein kinase (ROCK) inhibitors Fasudil and Y27632 on promoting angiogenesis in vivo and in vitro and on flap survival in mice. Methods Thirty-six SPF-grade adult male ICR mice were randomly divided into the Y27632 group, Fasudil group, and control group, with 12 mice in each group. From 3 d before surgery, the mice were intraperitoneally injected daily with Y27632 solution, Fasudil solution, and an equal volume of normal saline, respectively. Then, a peninsular flap model pedicled on the iliolumbar vessels on the back of the mice was established surgically. Laser speckle contrast imaging was used to evaluate flap blood perfusion immediately after surgery (day 0) and on day 5, and the pedicle artery diameter was measured and proximal and distal blood flow were calculated. On day 5 after surgery, the flap necrosis rate was calculated, and CD31-positive microvessel density in the distal part of the flap was detected by immunofluorescence. In addition, EdU cell proliferation, scratch, Transwell, and Matrigel tube formation assays were used to compare the effects of Fasudil and Y27632 on the proliferation, migration, invasion, and tube formation ability of human umbilical vein endothelial cells (HUVECs) in vitro, with DMSO treatment as the control. Results On day 5 after surgery, the flap necrosis rate in the Fasudil group was (2.0±1.5)%, which was significantly lower than that in the control group and the Y27632 group (P<0.001), and the necrosis rate in the Y27632 group was also significantly lower than that in the control group (P<0.001). The pedicle iliolumbar artery diameter in the Fasudil group on day 0 and day 5 after surgery was (143.8±4.2) μm and (177.6±7.6) μm, respectively, both significantly larger than those in the control group and the Y27632 group (P<0.001). On day 5 after surgery, the proximal and distal blood flow in the Fasudil group were (453.0±12.5) PU and (79.2±4.8) PU, respectively, both significantly higher than those in the control group(P<0.001) and the Y27632 group (P<0.05). The microvessel density in the distal surviving tissue of the flap was (71.6±3.5) vessels/region, significantly higher than that in the control group (P<0.001) and also significantly higher than that in the Y27632 group (P＜0.05). On day 0 after surgery, the pedicle iliolumbar artery diameter in the Y27632 group was smaller than that in the control group (P＜0.05), while on day 5 after surgery, there was no statistically significant difference between the two groups (P>0.05). The proximal and distal blood flow on day 5 after surgery were also significantly higher than that in the control group (P<0.001), and the microvessel density was also significantly higher than that in the control group (P<0.05), but the distal blood flow showed no significant difference compared with the control group (P>0.05). In vitro experiments showed that the proliferation rate, migration rate, and number of invasive cells of HUVECs in the Fasudil and Y27632 groups were significantly increased compared with the control group (P<0.001), but there was no statistically significant difference between the two drug groups (P>0.05), whereas the tube formation ability of the Fasudil group was significantly better than that of the Y27632 group (P<0.05). Conclusion Both Fasudil and Y27632 can effectively promote angiogenesis and improve flap survival rate, but the effect of Fasudil is more significant, which may be related to its sustained regulation of hemodynamics and vascular stability.
Objective To observe the dynamic changes in heparin-binding protein (HBP), coagulation function indicators, and vascular endothelial injury markers in SD rats with sepsis, analyze the associations among these indicators, and elucidate the regulatory role of HBP in sepsis-associated coagulation disorders. Methods Eighteen specific pathogen-free (SPF) male SD rats aged 8–12 weeks were selected to establish a sepsis model using cecal ligation and puncture (CLP). Blood samples were collected from the heart at 0 hours before modeling and at 3, 6, and 9 hours post-modeling. Thromboelastography (TEG) with standard and heparinase cups was used to determine the difference in reaction time (R value) between the two cups (ΔR), thereby evaluating coagulation function and the endogenous heparin effect. Plasma levels of HBP, heparan sulfate (HS), syndecan-1 (SDC-1), and thrombomodulin (TM) were measured by enzyme-linked immunosorbent assay (ELISA). Samples with ΔR≥2 min were grouped as the ΔR≥2 min group, while those with ΔR<2 min were grouped as the ΔR<2 min group. Correlation and receiver operating characteristic (ROC) curve analyses were performed to evaluate the relationship between HBP and various indicators, as well as its predictive efficacy for ΔR values. Results ΔR, HS, SDC-1 and HBP significantly increased after modeling compared with 0 hours (P<0.01). The HS, SDC-1, and HBP levels in the ΔR≥2 min group were significantly higher than those in the ΔR<2 min group (P<0.05 or P<0.01). HBP showed a significant positive correlation with ΔR and HS (P<0.05), while ΔR was significantly positively correlated with HS (r=0.799, P<0.01) and weakly positively correlated with SDC-1 (r=0.289, P<0.05). HS, HBP, and SDC-1 demonstrated significant predictive value for ΔR≥2 min (P<0.01). Conclusion In the early stages of sepsis, the release of HS-like anticoagulant substances from the glycocalyx following its degradation can lead to coagulation disorders, while HBP can reflect the pathological process and play an important regulatory role in the occurrence and development of sepsis-related coagulation disorders.
Staphylococcus aureus is a highly prevalent Gram-positive pathogenic bacterium in clinical settings, capable of causing skin and soft tissue infections, pneumonia, sepsis, infective endocarditis and other illnesses. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) pose formidable challenges to clinical treatment. Animal models serve as vital tools for dissecting its pathogenic and drug resistance mechanisms, as well as evaluating novel anti-infective strategies. Four well-established models for S. aureus infection research include skin and soft tissue infection models, osteomyelitis and implant-associated infection models, pneumonia models, and infective endocarditis models. This review systematically elaborates on laboratory animal selection criteria, mainstream modeling protocols, application scenarios, and the respective advantages and limitations of these four model types. Common laboratory animals encompass mice, rats, rabbits, pigs and so forth. Skin and soft tissue infection models feature rapid construction and low costs yet struggle to recapitulate complex chronic lesions. Osteomyelitis and implant-associated infection models are primarily utilized to mimic bacterial biofilm formation and the process of chronic infection. Pneumonia models are applied to investigate acute lung injury and assess drug efficacy. Infective endocarditis models are primarily used to investigate the mechanisms of vegetation formation and evaluate the efficacy of drugs against deep-seated infection. Due to the inherent differences in immune systems between laboratory animals and humans, these existing models generally have limitations. For instance, most animal models employ relatively high bacterial inoculation doses with short experimental cycles, making it difficult to fully recapitulate the clinical process of chronic infection. Future improvements can be realized by developing humanized animal models and organ-on-a-chip in vitro systems to overcome interspecies differences, integrating comorbid factors to enhance clinical relevance, adopting multimodal dynamic assessment systems, and establishing non-animal alternatives. This review aims to provide systematic theoretical references for the rational selection, optimization, and application of animal models of S. aureus infection, thereby contributing to advances in the field of anti-infective research.
Objective To evaluate the feasibility of using near-infrared-Ⅱ (NIR-Ⅱ) rare-earth down-conversion nanoparticles (DCNPs) as a novel contrast agent for labeling adipose-derived mesenchymal stem cells (ADSCs), and to explore their potential application for in vivo tracking in a mouse model of subacute liver injury. Methods Core-shell DCNPs (NaYF₄:Yb³⁺,Er³⁺@NaYF₄) were synthesized via a high-temperature thermal decomposition method. Water-soluble DCNPs were prepared through surface modification with DSPE-PEG2000, and further conjugated with fluorescein isothiocyanate (FITC) to observe cellular uptake. The CCK-8 assay was used to detect cell viability after co-incubation of ADSCs with DCNPs at 24 h and 48 h. Confocal laser scanning microscopy was employed to evaluate the labeling efficiency of DCNPs in ADSCs across various probe concentrations and incubation periods. Flow cytometry and differentiation assays were employed to evaluate the effects of DCNPs labeling on ADSCs surface antigen phenotype and multidirectional differentiation potential. NIR-Ⅱ fluorescence imaging was adopted to evaluate the dynamic changes of NIR‑Ⅱ fluorescence signals of labeled ADSCs during continuous in vitro culture. The in vivo toxicity of DCNP-labeled ADSCs transplanted via a single tail vein injection into SPF-grade male Sprague-Dawley (SD) rats was evaluated one month after transplantation through complete blood count, serum biochemical analysis, and multi-organ histopathological examination. A subacute liver injury model was induced in SPF-grade male BALB/c mice using carbon tetrachloride (CCl₄) administration. DCNP-labeled ADSCs were administered via tail vein injection, and the homing of ADSCs in vivo was monitored using NIR-Ⅱ fluorescence imaging. The effects of tracking on ADSC therapy were assessed using biochemical analysis of liver function indicators and hematoxylin and eosin (HE) staining. Results The NaYF₄:Yb³⁺/Er³⁺ nanoparticles synthesized in this study exhibited a spherical crystal structure with particle sizes ranging from approximately 20 to 30 nm, and demonstrated good dispersion stability in water. CCK-8 results showed that DCNPs labeling exerted no cytotoxic effect on ADSCs viability after 24 and 48 h of labeling within the concentration range of 0 to 500 μg/mL used in this study. The cell labeling efficiency of DCNPs increased with dosage, while the cellular uptake remained essentially stable within 16 h. The NIR-Ⅱ fluorescence signal of DCNPs remained stable during the early stage of in vitro ADSCs culture. Although the fluorescence intensity decreased on day 3 of culture (P=0.003), detectable fluorescence signals were still observed in the cells on day 8. Flow cytometry and differentiation assays confirmed that DCNPs labeling had no significant adverse effects on the phenotype and differentiation capacity of ADSCs. One month after the transplantation of DCNP-labeled ADSCs into normal SD rats, blood routine tests, serum biochemical analysis, and multi-organ histopathological examination all revealed no obvious toxic reactions in the body. After the DCNP-labeled ADSCs were injected via the tail vein into mice with subacute liver injury, the serum levels of the liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly reduced (both P<0.05), and the infiltration of inflammatory cells in the liver tissue was markedly decreased. Moreover, in vivo tracing results showed that the labeled ADSCs could home and migrate to the liver within 1 h, with the fluorescence signal gradually weakening over time. By 24 h, the fluorescence signal enriched in the liver had significantly decreased. Conclusion DCNPs possess good biocompatibility for ADSC labeling without affecting stem cell therapy. They may enable real-time, high signal-to-noise ratio in vivo tracking of stem cell homing and migration in ADSC therapy for subacute liver injury.
Objective To develop a carbon nanoparticle-manganese dioxide (CN-MnO2) injection integrating visually identifiable staining and magnetic resonance (MR) T1/T2 dual-modal imaging, and to systematically evaluate its physicochemical properties, critical quality attributes, biocompatibility, and efficacy for gastrointestinal marking and localization. Methods CN-MnO2 was prepared using a mild liquid-phase in situ redox deposition strategy and characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). The pH of CN-MnO2 was directly measured using a pH meter. The osmolality pressure of CN-MnO2 was determined by the freezing point depression method. The bacterial endotoxin was detected using the gel-clot Limulus amebocyte lysate test, which did not rely on absorbance readings. The appearance of the CN-MnO2 injection solution stored at 4 ℃ under light shielding and sealed for 0, 5, and 10 days was observed, and its hydrated particle size, polydispersity index, zeta potential, and pH were also measured. With gadopentetate dimeglumine (Gd-DTPA) as the positive control, different concentration gradients were established, and T1 and T2 relaxation times were measured to construct concentration-dependent plots of the longitudinal and transverse relaxation rates (R1 and R2) and calculate the corresponding relaxivities (r1 and r2), compare relaxivity performance, and determine the working concentration for subsequent in vivo experiments. Cytocompatibility was assessed using the methyl thiazolyl tetrazolium (MTT) assay and live/dead staining. Sixteen male 6-week-old SD rats were randomly assigned to an experimental group and a control group (n=8 per group) and subcutaneously injected with CN-MnO2 injection at a total Mn concentration of 0.80 mmol/L or an equal volume of normal saline, respectively. Short-term in vivo safety was evaluated by regular body weight monitoring, hematoxylin-eosin (HE) staining of major organs, hematological and serum biochemical analyses, inflammatory cytokine detection, and inductively coupled plasma optical emission spectrometry (ICP-OES) analysis. Eight male 5–6-month-old Bama minipigs were randomly assigned to a CN-MnO2 group (n=6) and a carbon nanoparticle suspension injection (CNSI) group (n=2). CN-MnO2 injection or CNSI was injected subserosally into the stomach, small intestine, and colon. Within 5 min after each injection, the maximum staining diameter at each injection site was measured and recorded. MR T1WI/T2WI scans were performed on days 3 and 7 after injection to evaluate MR visualization at the injection sites. At the end of observation, the injection sites and major organs were collected for histopathological evaluation by hematoxylin-eosin staining. Results CN-MnO2 appeared as a black suspension, with no obvious aggregation or sedimentation after standing at room temperature (20-25 ℃) for 15 days. Its average hydrodynamic diameter was approximately (228.7±15.3) nm, the polydispersity index was 0.184±0.021, and the zeta potential was (-24.6±2.8) mV. XPS showed that CN-MnO2 was mainly composed of C, O, and Mn, with oxygen accounting for approximately 68.82%. The Mn 2p spectrum exhibited two characteristic peaks at 642.4 and 653.1 eV. XRD indicated that low-crystallinity δ-MnO2 was dispersed within the carbon matrix. The pH was 6.82±0.06, the osmolality was (298.4±6.7) mOsm/kg, and the bacterial endotoxin level was below the detection limit. In vitro MR imaging showed a clear concentration-dependent response, with r1 and r2 values of 9.6 L·mmol-1·s-1 and 17.2 L·mmol-1·s-1, respectively, indicating favorable T1/T2 contrast performance superior to that of Gd-DTPA. At 0.80 mmol/L, CN-MnO2 showed no obvious cytotoxicity in vitro. Short-term safety evaluation in SD rats revealed no obvious inflammatory response, impairment of liver or renal function, or abnormal manganese accumulation in major organs. In Bama minipigs, the mean staining diameters of the CN-MnO2 group in the stomach, small intestine, and colon were (0.65±0.12) cm, (0.61±0.11) cm, and (0.58±0.10) cm, respectively, with no significant difference among the three injection sites (P>0.05). The overall mean staining diameter was smaller in the CN-MnO2 group than in the CNSI group (P<0.05). MR examinations showed that the injection sites were identifiable on both day 3 and day 7, with stronger signals observed on day 3. At the end of observation, no obvious pathological abnormalities were observed at the injection sites or in major organs. Conclusion A CN-MnO2 injection with uniform particle size and stable physicochemical properties can be successfully prepared. This formulation integrates visible gastrointestinal staining with MR T1/T2 dual-modal imaging, providing a new strategy for preoperative gastrointestinal marking and localization as well as precise intraoperative resection.
Objective This study attempts to establish a cysteine bacterial endotoxin test as an in vitro alternative method for cysteine pyrogen detection, so as to reduce the use of rabbits in pyrogen detection and ensure the welfare of experimental animals. Methods According to the Chinese Pharmacopoeia (2025 edition) Volume Ⅳ, General Chapters 1142 and 1143, and Guidelines 9251 and 9301, methodological studies on pyrogen and bacterial endotoxin testing were conducted on three batches of cysteine raw material. We used the pyrogen test (rabbit method, RPT) and the in vitro pyrogen test (monocyte activation test, MAT) to detect pyrogens in cysteine and assessed cell viability during the MAT for one batch. Bacterial endotoxin testing was performed using the gel-clot method (limit test) and the bacterial endotoxin photometric assay (microkinetic chromogenic assay, kinetic chromogenic assay, kinetic turbidimetric assay). Results Pyrogen testing of cysteine using the two pyrogen test methods yielded false-negative results. In the RPT, all the test articles spiked with endotoxin did not show a positive pyrogen response. The MAT results showed that cysteine inhibited interleukin-6 (IL-6) secretion by human peripheral blood mononuclear cells (hPBMCs) after incubation, with recovery rates below 50%. Interference was observed in bacterial endotoxin testing using the gel-clot method: all three batches showed interference at dilutions not exceeding the maximum valid dilution (MVD). In the interference test using the microkinetic chromogenic method, when cysteine was prepared at a concentration of 1.05 mg/mL, the recovery rates for all three batches were between 50% and 200%, indicating that the interference was eliminated and the method was feasible. The recovery rates measured by the microkinetic chromogenic assay, kinetic chromogenic assay and kinetic turbidimetric assay fell within the acceptable range of 50%–200% when the cysteine concentration was 1.05 mg/mL. The bacterial endotoxin levels in the three batches met the specified limits. Conclusion This study establishes a bacterial endotoxin assay for cysteine and preliminarily demonstrates that the photometric techniques can replace RPT for the bacterial endotoxin testing of cysteine.
Objective To optimize the technical parameters of mouse embryo transfer (including oviduct opening method, pseudopregnant recipient body weight, number of embryos transferred per recipient, and unilateral vs. bilateral oviduct transfer), improve the efficiency of embryo transfer (the pregnancy rate of recipient female mice after embryo transfer is used as the evaluation index), and improve the utilization rate of high-quality embryo germplasm resources (the birth rate of mice after embryo transfer is used as the evaluation index). Methods 560 SPF-grade ICR mice and 440 C57BL/6J mice were used as experimental animals. 2-cell embryos were obtained through in vitro fertilization. A single-factor controlled variable method was used to systematically investigate the effects of four factors, including oviduct opening method, pseudopregnant recipient body weight, number of embryos transferred per recipient, and unilateral vs. bilateral oviduct transfer, on the pregnancy rate and birth rate of mouse embryo transfer. Results The pregnancy rate of recipients with an opening made in the oviduct using a 26G syringe needle reached 90%, which was significantly higher than the 43% in the group with oviduct openings cut by Vannas scissors (P<0.05). Pseudopregnant recipients weighing 29-35 g exhibited optimal embryo transfer outcomes, with a pregnancy rate of 90% and a birth rate of 44%, and no pups were crushed by the recipient mothers. The highest birth rate of 42% was observed in the group receiving 16-25 embryos per recipient. Transferring 26-35 embryos per recipient achieved a 90% pregnancy rate and maximized embryo utilization efficiency. However, transfer efficiency decreased significantly when more than 35 embryos were transferred. Transfer of 24-26 embryos per recipient balanced relatively high pregnancy and birth rates. Bilateral oviduct transfer of mouse embryos yielded slightly higher pregnancy and birth rates than unilateral transfer, but the difference was not statistically significant (P>0.05). Conclusion Selecting pseudopregnant recipient female mice weighing 29-35 g, using a 26G syringe needle to make an opening in the oviduct, and controlling the number of transferred embryos at 24-26 per recipient can effectively improve the success rate of embryo transfer and achieve efficient utilization of high-quality embryo germplasm resources.
Genetically engineered mice are important tools for elucidating gene function and disease mechanisms and for advancing drug development. With the growing demand in biomedical research for complex genetic modifications, rapid model generation, and high-throughput functional analysis, technologies for generating genetically engineered mice continue to evolve. This review systematically summarizes the evolution and applications of these technologies, with a focus on conventional approaches, including early transgenic technologies and embryonic stem cell (ESC)-based homologous recombination for gene targeting, as well as the use of random mutagenesis approaches, such as N-ethyl-N-nitrosourea (ENU) mutagenesis and transposon insertion, in forward genetic screening. It further discusses the principles and characteristics of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas systems, as well as their applications in generating genetically engineered mice. With advances in genome editing, Cas9-derived systems, base editing, prime editing, and RNA editing further improve editing precision, expand the repertoire of genetic modifications and precise regulatory approaches, and provide additional options for generating genetically engineered mice. This review also compares the advantages, limitations, and suitability of different technologies for specific applications and summarizes quality-control strategies addressing practical issues that may affect model reliability, including off-target effects, mosaicism, genetic background, and genetic stability. Finally, future directions in technologies for generating genetically engineered mice are discussed to inform the generation and application of related models.
Objective To summarize the application experience of the "Three-Separation" model in the pre-service training of laboratory animal practitioners and scientifically evaluate its training effectiveness. Methods A retrospective analysis was conducted on 8 009 practitioners who participated in pre-service training at the Fujian Provincial Laboratory Animal Training Base from January 2010 to December 2025. Among them, 1 250 trainees from January 2010 to December 2017 were included in the control group (traditional training model), and 6 759 trainees from January 2018 to December 2025 were included in the evaluation group ("Three-Separation" model, based on separating job requirements, differentiating exam content, and categorizing exam methods). Meanwhile, a questionnaire survey was conducted to evaluate training effectiveness, yielding 1 624 valid questionnaires (585 from the control group and 1 039 from the evaluation group). The "Three-Separation" model simultaneously optimized fee structures to improve affordability and inclusiveness. The comprehensive competencies and training satisfaction of the two groups after training were compared and analyzed using the independent sample t-test, χ² test, and Wilcoxon rank-sum test. Results The overall satisfaction rate of the evaluation group was 92.49% (961/1 039), significantly higher than that of the control group (77.78%, 455/585), with a highly significant difference (χ²=72.50, P<0.001). The evaluation group showed a more favorable distribution of positive, neutral, and negative ratings in dimensions such as course pertinence, time flexibility, assessment rationality, and reasonableness of fees, and the positive rating rates were highly significantly higher than those in the control group (P<0.001). The comprehensive pass rate of the evaluation group (95.53%) was significantly higher than that of the control group (86.24%), with a highly significant difference (P<0.001). Conclusion The scientific application of the "Three-Separation" model can accurately match the competency requirements of different positions, optimize the training and assessment system, reduce participation barriers by making training fees more affordable and accessible, effectively improve the comprehensive quality and training satisfaction of laboratory animal practitioners, achieve better teaching outcomes, and provide replicable and promotable practical references for the standardized upgrading of the training system in the laboratory animal industry and other industries.
