Background: Advances in intensive care have markedly improved survival after severe brain injury, leading to a growing population of patients with prolonged disorders of consciousness (pDOC). Current management of pDOC remains largely supportive, and evidence-based neuromodulatory interventions are limited; moreover, existing guidelines provide insufficiently explicit recommendations regarding mechanisms of action and objective biomarkers of treatment response. Transcutaneous auricular vagus nerve stimulation (taVNS) has emerged as a potential noninvasive intervention; however, its modulatory effects on brain function in pDOC are not yet well characterized, and the paucity of integrative mechanistic evidence has constrained its translation into routine clinical practice. Objectives: Within a multimodal assessment framework, this study aims to systematically elucidate the neurobiological mechanisms by which taVNS modulates brain function and autonomic activity in patients with pDOC, and to evaluate its clinical potential to enhance levels of consciousness. Methods: In this randomized controlled trial, 60 patients with vegetative state/minimally conscious state will be enrolled and randomly allocated to a taVNS group, a transcutaneous nonauricular vagus nerve stimulation group (sham), or a control group (n = 20 per group) for a 4-week intervention. The primary outcome will be changes in the Coma Recovery Scale-Revised scores from baseline to weeks 1, 2, and 4 of treatment. Secondary outcomes will include functional brain activity assessed by electroencephalography and functional near-infrared spectroscopy, as well as autonomic modulation indexed by heart rate variability. Functional prognosis will be evaluated using the Glasgow Outcome Scale-Extended at the end of treatment and at a 6-month follow-up. Safety will be assessed by continuous monitoring and documentation of adverse events throughout the study period. Results and discussion: By integrating electroencephalography–functional near-infrared spectroscopy with heart rate variability, this study will characterize the effects of taVNS on functional brain networks and consciousness recovery in pDOC across complementary behavioral, electrophysiological, hemodynamic, and autonomic domains, while interrogating potential sources of clinical and neurobiological heterogeneity. The findings are expected to provide a mechanistic and evidence-based foundation for the mechanism-driven clinical implementation of taVNS and the optimization of stimulation protocols in pDOC. Clinical trial registration: International Traditional Medicine Clinical Trial Registry, ITMCTR20250021041, https://itmctr.ccebtcm.org.cn.

OBJECTIVES: Recent evidence suggests that the gut may be a primary site of metformin action. However, studies on the effects of metformin on gut microbiota remain limited, and its impact on gut microbial metabolites such as short-/medium-chain fatty acids is unclear. This study aims to investigate the effects of metformin on gut microbiota, short-/medium-chain fatty acids, and associated metabolic benefits in high-fat diet rats. METHODS: Twenty-four Sprague-Dawley rats were randomly divided into 3 groups: 1) Normal diet group (ND group), fed standard chow; 2) high-fat diet group (HFD group), fed a high-fat diet; 3) high-fat diet + metformin treatment group (HFD+Met group), fed a high-fat diet for 8 weeks, followed by daily intragastric administration of metformin solution (150 mg/kg body weight) starting in week 9. At the end of the experiment, all rats were sacrificed, and serum, liver, and colonic contents were collected for assessment of glucose and lipid metabolism, liver pathology, gut microbiota composition, and the concentrations of short-/medium-chain fatty acids. RESULTS: Metformin significantly improved HFD-induced glucose and lipid metabolic disorders and liver injury. Compared with the HFD group, the HFD+Met group showed reduced abundance of Blautia, Romboutsia, Bilophila, and Bacteroides, while Lactobacillus abundance significantly increased (all P<0.05). Colonic contents of butyric acid, 2-methyl butyric acid, valeric acid, octanoic acid, and lauric acid were significantly elevated (all P<0.05), whereas acetic acid, isoheptanoic acid, and nonanoic acid levels were significantly decreased (all P<0.05). Spearman correlation analysis revealed that Lactobacillus abundance was negatively correlated with body weight gain and insulin resistance, while butyrate and valerate levels were negatively correlated with insulin resistance and liver injury (all P<0.05). CONCLUSIONS Metformin significantly increases the abundance of beneficial bacteria such as Lactobacillus and promotes the production of short-/medium-chain fatty acids including butyric, valeric, and lauric acid in the colonic contents of HFD rats, suggesting that metformin may regulate host metabolism through modulation of the gut microbiota.
Animals ; Metformin/pharmacology* ; Rats, Sprague-Dawley ; Diet, High-Fat/adverse effects* ; Rats ; Gastrointestinal Microbiome/drug effects* ; Male ; Fatty Acids, Volatile/metabolism* ; Fatty Acids/metabolism*

OBJECTIVE: To observe the neuroprotective effect of 6-shogaol (6-SH) in global cerebral ischemia/reperfusion injury (CIRI) following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in rats. METHODS: Computer-aided molecular docking was used to determine whether 6-SH could spontaneously bind to death-associated protein kinase 1 (DAPK1). SPF-grade male SD rats were randomly divided into a sham group (n = 5), a CPR group (n = 7), and a CPR+6-SH group (n = 7). The CPR group and CPR+6-SH group were further divided into 12-, 24-, and 48-hour subgroups based on observation time points. A rat model of global CIRI after CA-CPR was established by asphyxiation. In the sham group, only tracheal and vascular intubation was performed without asphyxia and CPR induction. The CPR group was intraperitoneally injected with 1 mL of normal saline immediately after successful modeling. The CPR+6-SH group received an intraperitoneal injection of 20 mg/kg 6-SH (1 mL) immediately after successful modeling, followed by administration every 12 hours until the endpoint. Neurological Deficit Score (NDS) was recorded at each time point after modeling. After completion of observation at each time point, rats were anesthetized and sacrificed, and brain tissue specimens were collected. Histopathological changes of neurons were observed under light microscopy after hematoxylin-eosin (HE) staining. Ultrastructural changes of hippocampal neurons and autophagy were observed by transmission electron microscopy (TEM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression levels of DAPK1, vacuolar protein sorting 34 (VPS34), Beclin1, and microtubule-associated protein 1 light chain 3 (LC3) in brain tissues. Western blotting was used to detect protein expression levels of DAPK1, phosphorylated DAPK1 at serine 308 (p-DAPK1 ser308), VPS34, Beclin1, and LC3. Immunofluorescence was used to observe Beclin1 and LC3 expression in brain tissues under a fluorescence microscope. RESULTS: Molecular docking results indicated that 6-SH could spontaneously bind to DAPK1. Compared with the sham group, the NDS scores of the CPR group rats were significantly increased at all modeling time points; under light microscopy, disordered cell arrangement, widened intercellular spaces, and edema were observed in brain tissues, with pyknotic and necrotic nuclei in some areas; under TEM, mitochondria were markedly swollen with intact membranes, dissolved matrix, reduced or disappeared cristae, vacuolization, and increased autophagosomes. Compared with the CPR group, the NDS scores of the CPR+6-SH group rats were significantly decreased at all modeling time points; under light microscopy, local neuronal edema and widened perinuclear space were observed; under TEM, mitochondria were mostly mildly swollen with intact membranes, fewer autophagosomes, and alleviated injury. RT-qPCR results showed that compared with the sham group, mRNA expression levels of DAPK1, VPS34, Beclin1, and LC3 in brain tissues were significantly upregulated in all CPR subgroups, with the most pronounced changes at 24 hours. Compared with the CPR group, the CPR+6-SH group showed significantly lower mRNA expression of the above indicators at each time point [24 hours post-modeling (relative expression): DAPK1 mRNA: 3.41±0.68 vs. 4.48±0.62; VPS34 mRNA: 3.63±0.49 vs. 4.66±1.18; Beclin1 mRNA: 3.08±0.49 vs. 4.04±0.22; LC3 mRNA: 2.60±0.36 vs. 3.67±0.62; all P < 0.05]. Western blotting results showed that compared with the sham group, the protein expression levels of DAPK1, VPS34, Beclin1, and LC3 in all CPR subgroups were significantly increased, while the expression of p-DAPK1 ser308 was significantly decreased, with the most pronounced changes observed in the CPR 24-hour subgroup. Compared with the CPR group, the CPR+6-SH subgroups exhibited significantly reduced protein expression of DAPK1, VPS34, Beclin1, and LC3 [24-hour post-modeling: DAPK1/β-actin: 1.88±0.22 vs. 2.47±0.22; VPS34/β-actin: 2.55±0.06 vs. 3.46±0.05; Beclin1/β-actin: 2.12±0.03 vs. 2.87±0.03; LC3/β-actin: 2.03±0.24 vs. 3.17±0.23; all P < 0.05]. Conversely, the expression of p-DAPK1 ser308 was significantly upregulated in the CPR+6-SH group compared to the CPR group [24-hour post-modeling: p-DAPK1 ser308/β-actin: 0.40±0.02 vs. 0.20±0.07, P < 0.05]. Under the fluorescence microscope, fluorescence intensities of Beclin1 and LC3 in the CPR 24-hour group were significantly higher than those in the sham 24-hour group; compared with the CPR 24-hour group, the CPR+6-SH 24-hour group showed significantly reduced fluorescence intensities of Beclin1 and LC3. CONCLUSION 6-SH inhibited the expression of DAPK1, alleviated excessive autophagy after global CIRI following CA-CPR in rats, and exerted neuroprotective effects. The mechanism may be related to phosphorylation at the DAPK1 ser308 site.
Animals ; Rats, Sprague-Dawley ; Male ; Rats ; Cardiopulmonary Resuscitation ; Autophagy/drug effects* ; Heart Arrest/therapy* ; Death-Associated Protein Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Disease Models, Animal ; Neuroprotective Agents/pharmacology* ; Brain Ischemia/metabolism*

This study aims to explore the mechanism of Buyang Huanwu Decoction(BHD) in promoting angiogenesis after oxygen-glucose deprivation/reoxygenation(OGD/R) of mouse brain microvascular endothelial cell line(brain-derived Endothelial cells.3, bEnd.3) based on the caveolin-1(Cav1)/Yes-associated protein 1(YAP1)/hypoxia-inducible factor-1α(HIF-1α) signaling pathway. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to analyze the blood components of BHD. The cell counting kit-8(CCK-8) method was used to detect the optimal intervention concentration of drug-containing serum of BHD after OGD/R injury of bEnd.3. The lentiviral transfection method was used to construct a Cav1 silent stable strain, and Western blot and polymerase chain reaction(PCR) methods were used to verify the silencing efficiency. The control bEnd.3 cells were divided into a normal group(sh-NC control group), an OGD/R model + blank serum group(sh-NC OGD/R group), and an OGD/R model + drug-containing serum group(sh-NC BHD group). Cav1 silent cells were divided into an OGD/R model + blank serum group(sh-Cav1 OGD/R group) and an OGD/R model + drug-containing serum group(sh-Cav1 BHD group). The cell survival rate was detected by the CCK-8 method. The cell migration ability was detected by a cell migration assay. The lumen formation ability was detected by an angiogenesis assay. The apoptosis rate was detected by flow cytometry, and the expression of YAP1/HIF-1α signaling pathway-related proteins in each group was detected by Western blot. Finally, co-immunoprecipitation was used to verify the interaction between YAP1 and HIF-1α. The results showed astragaloside Ⅳ, formononetin, ferulic acid, and albiflorin in BHD can all enter the blood. The drug-containing serum of BHD at a mass fraction of 10% may be the optimal intervention concentration for OGD/R-induced injury of bEnd.3 cells. Compared with the sh-NC control group, the sh-NC OGD/R group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, significantly increased cell apoptotic rate, significantly lowered phosphorylation level of YAP1 at S127 site, and significantly elevated nuclear displacement level of YAP1 and protein expression of HIF-1α, vascular endothelial growth factor(VEGF), and vascular endothelial growth factor receptor 2(VEGFR2). Compared with the same type of OGD/R group, the sh-NC BHD group and sh-Cav1 BHD group had significantly increased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly decreased cell apoptotic rate, a further decreased phosphorylation level of YAP1 at S127 site, and significantly increased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC OGD/R group, the sh-Cav1 OGD/R group exhibited significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC BHD group, the sh-Cav1 BHD group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at the S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. YAP1 protein was present in the protein complex precipitated by the HIF-1α antibody, and HIF-1α protein was also present in the protein complex precipitated by the YAP1 antibody. The results confirmed that the drug-containing serum of BHD can increase the activity of YAP1/HIF-1α pathway in bEnd.3 cells damaged by OGD/R through Cav1 and promote angiogenesis in vitro.
Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Signal Transduction/drug effects* ; Glucose/metabolism* ; Caveolin 1/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; YAP-Signaling Proteins ; Oxygen/metabolism* ; Endothelial Cells/metabolism* ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics* ; Neovascularization, Physiologic/drug effects* ; Cell Hypoxia/drug effects* ; Angiogenesis

Objective:To explore the genetic and clinical characteristics of epilepsy related with ATP6V1A gene heterozygous variants.Methods:A case series study was conducted. The clinical data of 10 children of epilepsy associated with ATP6V1A gene variants who were admitted to the Children′s Medical Center, Peking University First Hospital from January 2019 to December 2024 was collected. The characteristics of children′ gene variation, clinical phenotype, auxiliary examination results, treatment and prognosis were analyzed.Results:Among the 10 children, there were 4 boys and 6 girls. All 10 children with ATP6V1A gene variants were de novo heterozygous variants, including 1 case of mosaic variant. A total of 9 different variants were identified and 7 variants have not been reported previously. The age at epilepsy onset was 28 (9, 48) months. Five children experienced their first seizure as a fever induction. The types of epileptic seizures included focal seizures in 6 children, epileptic spasms in 5 children, tonic spasms and atonic seizures in 1 child respectively. Three children had 2 seizure types. Global developmental delays were exhibited in 8 children, 2 of whom manifested autism spectrum disorder phenotypes. Two children showed normal development. Electroencephalography revealed slowed background activity in 5 children. Interictal epileptiform discharges were recorded in 9 cases, including hypsarrhythmia, focal, multifocal or generalized discharges. Clinical seizures were captured in 4 children. Brain magnetic resonance imaging abnormalities were found in 4 children, including frontotemporal cortical dysplasia, prominent sulci, delayed myelination of white matter, dysplasia of the corpus callosum, bilateral ventricular enlargement, and cerebral atrophy. Five children were diagnosed with developmental and epileptic encephalopathy (DEE), and 4 of them were diagnosed with infantile epileptic spasms syndrome. At the last follow-up, the age was 78 (25, 120) months. Seizures were controlled in 6 children, while 4 children had uncontrolled seizures despite treatment with ≥3 anti-seizure medications. Conclusions:All children with ATP6V1A gene related epilepsy harbored de novo heterozygous missense variants, with few showing mosaic variants. Seizure onset age ranged widely from the neonatal period to childhood. The predominant seizure types were focal seizures and epileptic spasms. The phenotypic spectrum may exhibit DEE, while a minority maintain normal development.

Objective:To investigate the characteristics of hand dysfunction and its associated factors in patients with rheumatoid arthritis (RA).Methods:A cross-sectional study. Patients with RA were recruited from January 2019 to April 2024 at the Department of Rheumatology and Immunology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Demographic and clinical data were collected, including age, gender, active smoking, disease duration, time of morning stiffness, rheumatoid factor and anti-cyclic citrullinated peptide antibody, disease activity, radiographic indicators, and hand function assessment. Hand function was assessed by grip strength measures and self-reported items related to hand function in the Stanford Health Assessment Questionnaire. Factors related to hand function were analyzed by logistic regression analyses.Results:A total of 1 079 RA patients were recruited [mean age: (53.0±12.6) years]. Overall, 72.6% (783/1 079) patients experienced a decrease in grip strength, 57.2% (617/1 079) patients experienced a decreased grip strength in both hands, with the average grip strength of the left and right hands decreasing by 16.3% and 14.1%, respectively, compared to normal values; 39.9% (430/1 079) patients had self-reported hand dysfunction. There were 185 (17.1%) older RA patients (age ≥65 years). The proportion of older RA patients with decreased grip strength [89.7% (166/185) vs. 69.0% (617/894)] and degree of decrease in grip strength compared to normal values (left hand:-35.3%±30.6% vs. -12.3%±38.6%; right hand:-32.6%±32.3% vs. -10.3%±42.1%) were significantly higher than that in young patients, and the proportion of older patients with self-reported hand dysfunction was also significantly higher [53.0% (98/185) vs. 37.1% (332/894), all P<0.001]. Multivariate logistic regression analysis showed that pain visual analogue scale ( OR=1.375, 95% CI 1.020-1.854) was independently associated with grip strength decrease in older RA patients, while the 28-joint tender joint count ( OR=1.151, 95% CI 1.063-1.246) and provider global assessment of disease activity ( OR=1.381, 95% CI 1.171-1.628) were associated with self-reported hand dysfunction. Conclusions:Hand dysfunction is common in RA patients, especially among older RA patients, which is related to pain, joint tenderness and provider global assessment of disease activity. This result implies the importance of pain management in RA patients.

Objective:To summarize the genotype and clinical phenotype characteristics of children with epilepsy associated with the SCN1B gene encoding the sodium channel β1 subunit. Methods:The genotypes and clinical phenotypes of patients with SCN1B variants among suspected genetic epilepsy cases treated at the Children′s Medical Center of Peking University First Hospital between May 2016 and July 2024 were analyzed. These variants were identified using next-generation sequencing and subsequently validated by Sanger sequencing or quantitative polymerase chain reaction methods. Results:A total of 17 patients were analyzed, including 8 males and 9 females. Ten cases of missense variations (including 2 with the same variations), 4 cases of deletion variations, and 1 case each of nonsense variations, splice site variations, and exons 4-5 deletions were identified. Among them, 6 cases had novel SCN1B variations. The variants in 11 cases were inherited from 1 parent. Eleven types of gene variants have not been reported yet. Onset of epilepsy ranged from 3 months to 5 years and 3 months old (median age: 14 months). Types of seizures included generalized tonic-clonic seizures (GTCS) in 14 cases, focal seizures in 9 cases, myoclonic seizures in 3 cases, atypical absence seizures in 2 cases and epilepsy spasms, tonic seizures and atonic seizures in 1 case each. Eleven cases had diverse seizure types. Fourteen cases (14/17) demonstrated fever sensitivity. Electroencephalography revealed focal discharges in 3 cases, coinciding with focal and generalized discharges in 3 additional cases, and multifocal discharges in 6 cases. Seizures were identified in 4 cases: 1 case of myoclonic seizures, 1 case of GTCS, 1 case of atypical absence seizures, and 1 case exhibiting both myoclonic and tonic seizures. Nine cases (9/17) were diagnosed with genetic epilepsy with febrile seizures plus, 1 case diagnosed with myoclonic epilepsy in infancy and 1 diagnosed with infant epileptic spasms syndrome. There were 2 cases of nonspecific developmental epileptic encephalopathy, while the remaining 4 cases could not be diagnosed with a specific epileptic syndrome. Effective antiseizure medications (ASMs) included valproate in 8 cases, levetiracetam in 5 cases, topiramate in 3 cases, clobazam in 2 cases, clonazepam and vigabatrin in 1 case each. Sodium channel blockers exacerbated seizures in 3 cases, specifically oxcarbazepine in 2 cases and lamotrigine in 1 case. At the last follow-up, seizures were controlled for at least 6 months in 14 patients (14/17), while seizures remained uncontrolled in 3 patients despite trialing 2 or more ASMs. Thirteen patients exhibited normal development, while 4 experienced developmental delays. Conclusions:The heterozygous variants in children with SCN1B gene-related epilepsy include missense, deletion, nonsense, splice site variants, and exon deletions. The correlation between different genetic variants and clinical phenotypes remains unclear. These variants are associated with epilepsy onset from infancy to early childhood, presenting with various seizure types, with GTCS being the most common. Phenotypic manifestations can vary significantly in severity, ranging from benign febrile seizures or febrile seizures plus to developmental epileptic encephalopathy. Valproic acid demonstrates the highest effectiveness rate, while the use of sodium channel blockers may worsen seizures in certain patients, necessitating cautious administration.

Objective:To summarize the clinical phenotype and genetic features of patients with relapsing encephalopathy with cerebellar ataxia (RECA) caused by ATP1A3 gene R756 variants. Methods:A retrospective analysis was performed on patients carrying the ATP1A3 gene R756 variants, identified by whole-exome sequencing of family members, at Capital Center for Children′s Health, Capital Medical University and Children's Medical Center, Peking University First Hospital from August 2005 to February 2024. Their clinical, laboratory, neuroimaging, electrophysiological and genetic characteristics were summarized. Results:A total of 13 RECA patients were enrolled in this study, including 8 males and 5 females. The age of onset was 8 months to 5 years, with a median age of onset of 18 months. All of 13 patients presented paroxysmal episodes of neurological decompensations triggered by fever and residual symptoms following the acute phase. During acute attack stage, ataxia was observed in all 13 cases, muscle weakness in 12 cases, dysarthria in 12 cases, altered consciousness in 10 cases, dysphagia in 10 cases, dystonic episodes in 4 cases, abnormal eye movement in 2 cases, choreoathetosis in 2 cases, and epileptic seizures in 1 case. All 13 patients had residual symptoms during the nonparoxysmal period, of whom 9 patients had ataxia, 9 patients had dysarthria, 4 patients had dystonia, 3 patients had cognitive disorders, and 1 patient had epileptic seizures. All 13 cases had ATP1A3 missense variants, and variant c.2266C>T/p.R756C was found in 6 cases, c.2267G>A/p.R756H in 5 cases, and c.2267G>T/p.R756L in 2 cases. Nine cases carried de novo variants, 4 with inherited variants. Conclusions:RECA caused by variants of ATP1A3 in residue 756 typically presents with an acute onset during infancy or early childhood, precipitated by febrile episodes and characterized by recurrent episodes of ataxia, with bulbar paralysis, muscle weakness and altered consciousness. Recurrence is common, and the most common persistent symptoms are cerebellar ataxia and dysarthria. A few patients have cognitive impairment. Three types of ATP1A3 gene variants R756C, R756H and R756L are related with RECA, and R756C is the most common variant.

Triple-negative breast cancer (TNBC) represents a distinctive subtype, characterized by the absence of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2). Due to its high inter-tumor and intra-tumor heterogeneity, TNBC poses significant chanllenges for personalized diagnosis and treatment. The advant of clustered regular interspaced short palindromic repeats (CRISPR) technology has profoundly enhanced our understanding of the structure and function of the TNBC genome, providing a powerful tool for investigating the occurrence and development of diseases. This review focuses on the application of CRISPR/Cas technology in the personalized diagnosis and treatment of TNBC. We begin by discussing the unique attributes of TNBC and the limitations of current diagnostic and treatment approaches: conventional diagnostic methods provide limited insights into TNBC, while traditional chemotherapy drugs are often associated with low efficacy and severe side effects. The CRISPR/Cas system, which activates Cas enzymes through complementary guide RNAs (gRNAs) to selectively degrade specific nucleic acids, has emerged as a robust tool for TNBC research. This technology enables precise gene editing, allowing for a deeper understanding of TNBC heterogeneity by marking and tracking diverse cell clones. Additionally, CRISPR facilitates high-throughput screening to promptly identify genes involved in TNBC growth, metastasis, and drug resistance, thus revealing new therapeutic targets and strategies. In TNBC diagnostics, CRISPR/Cas was applied to develop molecular diagnostic systems based on Cas9, Cas12, and Cas13, each employing distinct detection principles. These systems can sensitively and specifically detect a variety of TNBC biomarkers, including cell-specific DNA/RNA and circulating tumor DNA (ctDNA). In the realm of precision therapy, CRISPR/Cas has been utilized to identify key genes implicated in TNBC progression and treatment resistance. CRISPR-based screening has uncovered potential therapeutic targets, while its gene-editing capabilities have facilitated the development of combination therapies with traditional chemotherapy drugs, enhancing their efficacy. Despite its promise, the clinical translation of CRISPR/Cas technology remains in its early stages. Several clinical trials are underway to assess its safety and efficacy in the treatment of various genetic diseases and cancers. Challenges such as off-target effects, editing efficiency, and delivery methods remain to be addressed. The integration of CRISPR/Cas with other technologies, such as 3D cell culture systems, human induced pluripotent stem cells (hiPSCs), and artificial intelligence (AI), is expected to further advance precision medicine for TNBC. These technological convergences can offer deeper insights into disease mechanisms and facilitate the development of personalized treatment strategies. In conclusion, the CRISPR/Cas system holds immense potential in the precise diagnosis and treatment of TNBC. As the technology progresses and becomes more costs-effective, its clinical relevance will grow, and the translation of CRISPR/Cas system data into clinical applications will pave the way for optimal diagnosis and treatment strategies for TNBC patients. However, technical hurdles and ethical considerations require ongoing research and regulation to ensure safety and efficacy.

ObjectiveTo investigate the mechanism of action of Huangqi Guizhi Wuwutang （HGWT） against cerebral ischemia-reperfusion injury （CIRI） based on bioinformatics and experimental validation. MethodsBiological informatics methods were used to screen for active components of HGWT and their targets. The GEO database was utilized to obtain CIRI-related differentially expressed genes （DEGs）， and platforms such as GeneCards were used to identify disease targets. Venn diagram analysis was conducted to identify overlapping targets， followed by protein-protein interaction （PPI）， gene ontology （GO）， and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis， as well as immune infiltration and immune cell differential analysis. Core genes （Hub genes） were screened using LASSO regression and ROC curves， and molecular docking was used to validate the binding efficiency between the active components of the drug and the core targets. A rat CIRI model was established， with rats randomly divided into five groups （n=10）： Sham surgery group （Sham）， model group （MG）， and low-dose （LD，5.3 g·kg^-1）， medium-dose （MD，10.6 g·kg^-1）， and high-dose （HD，21.2 g·kg^-1） HGWT groups. From 3 days before modeling to 7 days after surgery， oral administration was performed daily： Sham and MG groups received physiological saline， while each drug group received the corresponding dose of HGWT. Hematoxylin-eosin （HE） staining， Nissl staining， and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL staining） were used to assess the repair effects of HGWT on neural damage. Western blot analysis was used to detect B-cell lymphoma-2 protein （Bcl-2）， Bcl-2-associated X protein （Bax）， signal transducer and activator of transcription 3 （STAT3）， phosphorylated STAT3 ［p-STAT3 （Tyr705）］， protein kinase B1 （Akt1）， and phosphorylated Akt1 ［p-Akt1 （Ser473）］， among other target proteins. ResultsAfter screening， 56 common target points of DEGs-disease-drug were obtained. GO and KEGG analyses indicated that HGWT primarily functions in pathways such as apoptosis， oxidative stress， and inflammatory responses. Immune infiltration analysis revealed a significant association between HGWT's anti-CIRI activity and immune cells such as Th17 cells and myeloid-derived suppressor cells （MDSCs） （P0.01）. LASSO-ROC analysis identified Akt1， Caspase-3， glycogen synthase kinase-3β （GSK-3β）， and STAT3 as core genes. Molecular docking confirmed that Hub genes exhibit significant binding affinity with the active components of HGWT （binding energy ≤ -5 kJ·mol^-1）（1 cal≈4.186 J）. Animal experiment results showed that compared with the sham group， the MG group exhibited significant neuronal necrosis， nuclear condensation， and vacuolar degeneration in rat brains， with a significant decrease in Nissl body density （P0.01） and increased neuronal apoptosis in rat brains as indicated by TUNEL staining （P0.01）. Compared with the MG， the LD， MD， and HD groups showed reduced neuronal necrosis， nuclear condensation， and vacuolar degeneration in rat brain neurons， increased Nissl body density， and reduced apoptosis （P0.01）， with significant differences among the drug groups （P0.01）. Western blot results showed that compared with the sham group， the MG group had reduced Bcl-2 and p-Akt1 （P0.01） and increased Bax and p-STAT3 （P0.01）. Compared with the MG group， the drug groups showed increased Bcl-2 and p-Akt1 （P0.01） and decreased Bax and p-STAT3 （P0.01）. There were no significant changes in total Akt1 and STAT3 protein levels among the groups. ConclusionBased on network pharmacology and experimental verification， HGWT may exert its neuroprotective effects by regulating the phosphorylation levels of Akt1 and STAT3， thereby alleviating cell apoptosis， inflammatory responses， and oxidative stress in rat brain tissue following CIRI. This provides theoretical support for the clinical treatment of CIRI.