Traumatic brain injury (TBI) is a common disorder of the nervous system and has become a leading cause of death and disability worldwide, imposing a substantial burden on patients and their social circles. Its main symptoms include dyskinesia, language acquisition difficulties, and cognitive decline. Because of its complexity and diversity, the diagnosis and treatment of TBI have consistently been key areas of focus in medical research. Traditional imaging methods, including X-ray computed tomography (CT) scans and magnetic resonance imaging (MRI), are proficient at identifying overt structural abnormalities; however, their sensitivity in detecting subtle or hidden brain injuries is somewhat restricted. CT is insensitive to nonhemorrhagic lesions and cannot accurately evaluate the degree of injury. The detection rate of microhemorrhagic foci and specific types of TBI by MRI is low; other technologies have complicated operation and high equipment requirements. Therefore, the utilization of biological indicators, or biomarkers, in assessing and predicting the course and outcome of TBI holds immense importance in both diagnostic and prognostic evaluations. This overview reviews the research progress of biomarkers in TBI. Recognizing the significance of TBI-related biomarkers, understanding the pertinent key molecular pathways, and staying informed about the latest advancements in treatment methods are of utmost importance. However, research on TBI biomarkers continues to face challenges. To advance the field, future efforts should focus on delving deeper into the mechanisms, refining more sensitive and tailored detection methodologies, conducting extensive clinical validations, and exploring the potential of personalized treatment plans.

Background: Liujing Toutong tablet (LTT) is a traditional Chinese patent medicine. Previous studies have demonstrated that LTT exerts a protective effect in permanent cerebral ischemia and cerebral ischemia–reperfusion injury (CIRI). However, the active compounds and underlying mechanisms remain unclear. This investigation aimed to integrate the analyses of serum pharmacochemistry, network pharmacology, and metabolomics to elucidate the therapeutic effects of LTT on CIRI.

Methods: The therapeutic effects of LTT were evaluated in the CIRI rat model, which was induced using the thread embolism method. The prototype active compounds of LTT absorbed into the serum (LPCs) were identified using ultra-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive-Orbitrap/MS). Potential targets of LPCs were determined using network pharmacological analysis. A combination of metabolomics and network pharmacology was employed to identify upstream key targets and downstream endogenous metabolites. The predicted mechanisms were then verified by molecular docking. Finally, key targets and signaling pathways were examined using enzyme-linked immunosorbent assay (ELISA) and Western blot.

Results: A total of 27 LPCs were identified as the active ingredients. Furthermore, glycerophospholipid and arachidonic acid (AA) metabolism were also identified, with PTGS2, ALOX5, and AchE as the upstream key targets, and phosphorylcholine, AA, phosphatidate (PA), hosphatidylcholines (PCs), and lysophosphatidylcholines (LysoPCs) as the core endogenous metabolites. Additionally, LPCs binding with these key targets might reduce the inflammatory response via nuclear factor Kappa-B (NF-κB) signaling pathway.

Conclusion: LTT may effectively alleviate symptoms of CIRI by acting on PTGS2, ALOX5, and AchE, improving the glycerophospholipid and AA metabolism and reducing the inflammatory response by inhibiting NF-κB signaling pathway-related proteins.

Background: The underlying mechanisms for exacerbated brain injury and poor recovery observed in patients with diabetes and ischemic stroke (IS) remain undetermined. We explored the role of microRNA-34a (miR-34a) in diabetic IS (DMIS) and ischemic postconditioning (IPOC)'s neuroprotective effects in tree shrews.

Methods: We established a tree shrew DMIS model and exposed it to interventions, including miR-34a inhibition (antagomir), IPOC, and miR-34a overexpression (agomir). Infarct size and pathology were assessed via staining. Cellular/molecular changes (astrocytes, neurons, brain-derived neurotrophic factor [BDNF], Sine oculis homeobox 3 [SIX3], proliferation, apoptosis, axon formation) were analyzed using immunofluorescence, polymerase chain reaction (PCR), and Western blotting. In vitro, miR-34a's targeting of BDNF/SIX3 was validated, with rescue experiments testing regulation via these factors.

Results: Infarct size and neuronal damage were greater in the DMIS group than in the nondiabetic IS group. miR-34a inhibition or IPOC reduced infarcts, alleviated injury, improved cell survival, upregulated BDNF/SIX3, enhanced proliferation/axon formation, and reduced apoptosis. miR-34a overexpression reversed IPOC's benefits. In vitro, miR-34a directly targeted BDNF/SIX3, suppressing their expression; exogenous BDNF/SIX3 rescued neurotoxicity and restored function.

Conclusion: IPOC exerts partial neuroprotection through miR-34a downregulation, highlighting miR-34a as a potential therapeutic target.

The escalating global incidence of end-stage renal disease has exacerbated the critical shortage of kidneys from human donors. Porcine kidney xenotransplantation has emerged as the most promising alternative solution to providing an unlimited organ supply. In this review, we examine the historical evolution, current breakthroughs and future directions of kidney xenotransplantation. We probe the milestones from early attempts and non-human primate (NHP) experiments to recent clinical trials involving both brain-dead and living human recipients. The core of this review provides an in-depth discussion of the significant barriers in kidney xenotransplantation, including immune rejection, physiological incompatibilities and the risk of cross-species infection. Next, we systematically outline the multifaceted strategies developed to overcome these barriers. The rapid development of gene editing technology has enabled the establishment of multigene-edited pigs. These donors feature knockout of key carbohydrate antigen genes and expression of various human proteins, including complement regulators, anticoagulants, and immunomodulators. These genetic modifications have extended xenograft survival in NHP models to over 750 days. This is synergized with novel immunosuppressive regimens, tolerance-induction protocols, cellular therapies, and emerging adjuncts like bioengineering materials and organoid-on-a-chip technologies. Finally, we discuss future directions, raising concerns about potential complications arising from the biomechanical incompatibility between pigs and human in xenotransplantation, highlighting the need to deploy advanced multi-omics to identify unknown xenoantigens, optimize bioengineering materials for local immunomodulation, and validate extracellular vesicles as non-invasive biomarkers. While challenges for long-term xenograft survival remain, kidney xenotransplantation is rapidly advancing from preclinical research to clinical reality, holding huge potential to resolve the organ shortage crisis.

Rapidly emerging infectious and genetic diseases demand robust vertebrate models to investigate pathogenesis and accelerate therapeutic discovery. Zebrafish (Danio rerio) offer substantial translational value owing to their conserved physiology, optical transparency, rapid reproduction, and the presence of orthologs for approximately 70% of human genes and approximately 82% of disease-associated genes. The integration of CRISPR/Cas9 technology has transformed zebrafish research, enabling efficient generation of targeted knockouts, knockins, and high-throughput mutagenesis screens. This synergy supports mechanistic dissection and modeling of cardiovascular, oncologic, viral, and other genetic disorders. Despite these advantages, rigorous allele validation, consideration of paralog redundancy, maternal contribution, and off-target analysis remain essential to ensure translational accuracy. This review summarizes current applications, methodological advances, limitations, and best-practice recommendations for combining zebrafish models with genome editing to improve understanding and treatment of human diseases.

Hepatitis A virus (HAV) is an important pathogen that has continuously posed a threat to global public health for over 5000 years. The development of accessible and reliable small animal models, especially murine models, is essential for elucidating HAV pathogenesis and advancing preventive and therapeutic strategies. The first mouse model for HAV infection was established using human-liver chimeric mice, which supported robust viral replication and high viral loads. Later, an Ifnar1^−/− mice model was established with a specific mouse-adapted strain, recapitulating key clinical features of human hepatitis A. Recently, to overcome limitations associated with the difficulty in obtaining and amplifying stocks of HAV for animal models, our laboratory established a novel hepatitis A mouse model using lipid nanoparticle-encapsulated viral genomic RNA (LNP-vRNA). This approach provides a new strategy for modeling infections of hard-to-culture RNA viruses. In this review, we systematically summarize and compare these mouse models, respectively highlighting their advantages and limitations, and offer guidance for their application in future HAV research.

Experimental animal models have historically been pivotal in molecular biology, providing insights into gene function, disease causes, and therapeutic advancements. However, their translational relevance is increasingly scrutinized due to interspecies variations, ethical dilemmas, and restricted reproducibility. The objective of this study is to critically analyze the obstacles and ethical considerations related to animal models in molecular biology, while highlighting the promise of human-relevant and combinatorial techniques to enhance translational outcomes. A narrative synthesis of literature published from 2013 to 2025 was performed via PubMed, Scopus, Web of Science, and Google Scholar. Research focusing on model development, translational obstacles, ethical implications, and disease-specific applications—encompassing neurodegenerative, metabolic, oncological, hepatic, renal, and pain models—was prioritized. Animal models are indispensable but insufficient for precisely mirroring human disease. Principal challenges include species variability, infrastructural limitations, and insufficient reproducibility. Innovative approaches such as humanized models, organoids, and CRISPR/Cas9 technologies, when combined with rigorous methodologies, controlled heterogeneity, and ethical safeguards, offer a means to improve the predictability and reliability of preclinical research. Progress in molecular biology needs to include strict planning, long-lasting infrastructure, and new ideas from interdisciplinary fields. Integrating human-relevant and combinatorial approaches can improve the scientific integrity and ethical underpinning of biomedical research.

Background: The contribution of gut microbiota to the early stage of type 2 diabetes (T2D) remains incompletely understood. This study established a germ-free mouse model colonized with gut microbiota from donors with T2D to determine whether diabetes-associated microbiota could induce early diabetes-like phenotypes.

Methods: Human fecal samples were collected from 18 healthy donors and 14 donors with T2D for microbiota profiling. Based on these features, samples from 12 healthy donors and 3 donors with T2D were selected, pooled within each group, and transplanted into germ-free mice, generating a healthy microbiota recipient group (HM, n = 14) and a diabetes-associated microbiota recipient group (DM, n = 13). Glucose homeostasis was assessed over 10 weeks using fasting blood glucose and intraperitoneal glucose tolerance tests. Gut microbial succession was analyzed by 16S rRNA sequencing. Fecal and plasma metabolomics were performed to identify metabolic and their associations with microbial changes.

Results: DM mice developed an early diabetes-like phenotype characterized by progressive impairment of glucose tolerance, reduced insulin levels, and mild renal alterations, without sustained overt fasting hyperglycemia. Microbial divergence emerged before stable metabolic dysfunction and was accompanied by persistent dysbiosis in DM mice. Integrated metabolomic analysis identified coordinated alterations in fecal and plasma metabolites, with cholic acid and L-Dopa decreased in both compartments. L-Dopa showed consistent associations with altered genera, particularly Ruminococcus and Sellimonas.

Conclusions: Transplanting diabetes-associated human gut microbiota effectively induced early glucose dysregulation in germ-free recipient mice. This model provides a framework for studying microbiota-associated events during early T2D-related metabolic deterioration.

Background: This study revealed the function and mechanism of the intestinal microbiota in knee osteoarthritis (KOA) rats treated using transcutaneous electrical nerve stimulation (TENS).

Methods: KOA model rats were randomly divided into low−/medium−/high-intensity TENS groups, sodium hyaluronate (SH)-positive control group (SH), and model control group (KOA rat). After 1, 2, and 3 weeks of treatment, the improvement in KOA severity was assessed, and the expression of interleukin-1β (IL-1β)/IL-6/IL-8/bone morphogenetic protein 2 (BMP-2)/transforming growth factor β (TGF-β) was analyzed. The diversity of the intestinal microbiota in KOA rats was analyzed via 16S ribosomal DNA (rDNA) sequencing. After fecal microbiota transplantation (FMT), which was induced by TENS, the improvement in the intestinal microbiota in KOA rats was analyzed.

Results: After 3 weeks of treatment using TENS, compared to those in the model control group, the biomechanical parameters increased in the SH and TENS groups (p < 0.05); the gait parameters improved in the SH and TENS groups; the bone mineral density (BMD) increased in the TENS group (p < 0.05); the Mankin scores of the distal femur and proximal tibiofibular muscles decreased in the SH and TENS groups (p < 0.05). IL-1β/6/8 expression levels decreased in the SH and TENS groups (p < 0.05). BMP-2/TGF-β expression in the distal femur increased in the TENS group (p < 0.05). 16S rDNA sequencing revealed that the intestinal microbiota of KOA rats was changed after TENS treatment, including increases in Escherichia-Shigella, Lachnospira, Eubacterium, Gastranaerophilales, and Rikenellaceae RC9 and decreases in Fusicatenibacter and Mycoplasma. After FMT, which is induced by TENS, similar improvements in KOA rats were obtained.

Conclusions: TENS promoted anti-inflammatory and osteogenic effects by downregulating the Il-1β/6/8 expression levels and upregulating the BMP-2/TGF-β signaling pathway. 16S rDNA sequencing revealed that the intestinal microbiota of KOA rats was changed after TENS treatment via the gut–knee joint axis, and that these dominant genera of FMT elicited improvements in KOA rats. TENS caused improvements in KOA rats by regulating the intestinal microbiota; thus, TENS and induced FMT altered intestinal microbiota suggest a potential novel therapeutic avenue for KOA in clinical settings.

Background: The ErZhi formula (EZF) exhibits sound therapeutic effects on osteosarcopenia (OS). However, EZF's therapeutic effects on skeletal muscles are rarely reported. This study explored the mechanism of EZF in skeletal muscle during OS by integrating energy metabolism and metabolomics.

Methods: After an ovariectomized rat model was established for 4 weeks, the rats were subjected to a 12-week intervention of EZF and alendronate. The rats' body weight, gastrocnemius muscle mass, degree of myofiber fibrosis, and myofiber cross-sectional area (CSA) were measured to evaluate the pathological state of the gastrocnemius muscle. The mitochondrial membrane potential and reactive oxygen species (ROS) levels were detected to assess mitochondrial function. Then, energy metabolite analysis and metabolomics were performed on the gastrocnemius muscle.

Results: Compared to the model group, EZH increased CSA by 5.58% and decreased myofiber fibrosis by 15.11%. Notably, compared to the model group, EZH exhibited a 52.66% increase in mitochondrial membrane potential and a 44.32% reduction in ROS levels. Starch and sucrose metabolism, insulin secretion, insulin resistance, and galactose metabolism were the most significantly affected pathways in energy metabolism. Thirty-five differential metabolites were found in the metabolomics of the gastrocnemius muscle, and EZF could effectively inhibit the sphingolipid metabolism pathway. Correlation analysis identified 13 differential metabolites that were significantly associated with skeletal muscle mass, ROS production, muscle fibrosis, and mitochondrial function, suggesting that these metabolites may play important roles in the progression of skeletal muscle lesions.

Conclusions: EZF alleviates OS by regulating skeletal muscle physiological indicators, mitochondrial function, and energy metabolism.

The simultaneous characterization of histological alterations and distinct cell death pathways associated with diabetic cardiomyopathy (DCM) has not been comprehensively explored and may differ depending on the experimental model. We evaluated cardiac histological changes and markers of apoptosis, cell death associated with mitochondrial permeability transition pore (mPTP) opening (necrosis), and autophagy in early and advanced stages of streptozotocin (STZ)-induced diabetes in rats receiving insulin daily to prevent mortality while maintaining hyperglycemia. Adult male Sprague–Dawley rats (n = 4–5) were randomly assigned to receive STZ (diabetic group) or saline (mock) and followed up for 4 or 12 weeks. Hearts were processed for histological examination and immunohistochemical detection of active caspase-3, beclin-1, and cyclophilin D. STZ significantly increased glycemia at both time points, whereas body weight reduction was observed only at 12 weeks. No evidence of cardiac hypertrophy, fibrosis, or structural injury was detected in diabetic rats at either stage. Expression of caspase-3, beclin-1, and cyclophilin D decreased at 12 weeks compared with 4 weeks, regardless of treatment; however, cyclophilin D was elevated in diabetic hearts at 4 weeks. These results suggest an early susceptibility to mPTP opening under persistent hyperglycemia, despite the absence of overt histological damage. They also underscore the critical importance of insulin dosing and study duration when interpreting data from STZ-based models. Further studies are warranted to determine how even minimal insulin administration may shape the temporal dynamics of cell death during the progression of DCM.

Brachycephalic obstructive airway syndrome (BOAS) in dogs shares pathophysiological features with human obstructive sleep apnea, presenting different degrees of airway obstruction. Thus, the severity of the disorder may result in negative pregnancy consequences differently. This study aimed to compare the effects of BOAS severity throughout gestation on maternal clinical status, uteroplacental hemodynamics, and fetal development. Bitches were grouped according to BOAS symptomatic manifestation as mild (n = 5), severe BOAS (n = 5), and non-BOAS groups (control; n = 5). Along gestation (early, mid, and late stage), females were examined for clinical evaluations, arterial blood acid–base balance, and obstetric B-mode and uterine and umbilical artery Doppler velocimetry. Mild and severe BOAS had lower blood pressure, considered hypotension, and remained tachycardiac. All bitches had tachypnea, but mild BOAS group had higher (p < 0.05) respiratory rate. Peripheral oximetry decreased (p < 0.05) with time in severe BOAS, in a manner that females presented severe hypoxia (87.8% ± 4.6%) and hypocapnia (30.7 ± 2.37 mmHg) in late pregnancy. Blood glucose concentration was higher (p < 0.05) in mild BOAS (123.8 ± 5.79 mg/dL), in addition to persistent hyperglycemia until mid-pregnancy (121.8 ± 7.37 mg/dL). Severe BOAS bitches had higher (p < 0.05) lactatemia and hyperlactatemia throughout gestation. Fetal abdominal diameter, femur length, and humerus length were lower (p < 0.05) in severe BOAS compared to control. Uterine artery hemodynamic indexes, indicative of vascular resistance, decreased (p < 0.05) over time only in control, whereas BOAS degree groups remained unchanged. Severe BOAS had higher (p < 0.05) umbilical artery resistance in mid and late pregnancy (0.84 ± 0.02 and 0.77 ± 0.01) compared to mild BOAS (0.76 ± 0.01 and 0.64 ± 0.04, respectively). In conclusion, the degree of respiratory impairment in BOAS dogs has an adverse impact on maternal health and the fetoplacental unit, despite tentative attempts of physiological adaptations during pregnancy to maintain gestational homeostasis.

Knee osteoarthritis (KOA) is a prevalent and disabling disease with limited nonsurgical options for pain management. Magnetic resonance–guided focused ultrasound (MRgFUS) is a noninvasive technique for targeted thermal ablation and has emerged as a potential therapy for pain control. Large-animal models that better replicate human joint anatomy are needed for translational evaluation. Although monosodium iodoacetate (MIA) is widely used to induce KOA in small animals, its application in sheep remains limited. KOA was induced in the right knees of nine sheep using intra-articular MIA injections on days 8 and 29, with contralateral knees serving as controls. Animals were monitored for 12 weeks with serial behavioral and radiographic assessments. MRgFUS treatment was performed between weeks 6 and 8 after the first MIA injection. At the study endpoint, knee joints were obtained for macroscopic and histological evaluation. MIA-treated knees demonstrated radiographic features of KOA, including joint space narrowing and osteophyte formation. Gross and histological analyses confirmed cartilage degeneration, with surface erosion, reduced proteoglycan staining, and disrupted cartilage architecture. Despite consistent structural changes, only two animals developed persistent pain-related behaviors. In these animals, MRgFUS treatment was associated with improved pain scores and activity levels. No treatment-related increases in pain, functional impairment, or tissue damage were observed. Intra-articular MIA injection produced a reproducible structural model of KOA in sheep. MRgFUS was well tolerated and exhibited preliminary potential for pain relief, supporting further evaluation in larger controlled studies.