Objective To evaluate the clinical utility of combining magnetic resonance imaging (MRI) findings with serum levels of cysteine-rich protein 61 (CYR61) and C-X-C motif chemokine ligand 1 (CXCL1) for early identification of acute stroke. Methods The study was conducted 205 patients with suspected acute stroke admitted to the neurology department of our hospital between April 2021 and April 2024. Based on brain CT imaging and clinical diagnosis, 119 patients were acute stroke group and 86 to non-acute stroke group. All patients underwent MRI examination. Serum levels of CYR61 and CXCL1 were measured. Receiver operating characteristic (ROC) curves were generated to assess diagnostic value, and Kappa tests were used to performed to evaluate the consistency. Results Serum level of CYR61 and CXCL1were significantly higher in the acute stroke group than in the non-acute stroke group (P<0.05). The area under the ROC curve (AUC) for serum CYR61 in diagnosing acute stroke was 0.867 (95%CI: 0.819-0.915), with diagnostic threshold of 6.02 μg/L; for CXCL1, the AUC was 0.818 (95%CI: 0.762-0.875), with a threshold of 1.28 ng/mL. The Kappa values for agreement between MRI, serum CYR61, and CXCL1 clinical diagnosis results were 0.766, 0.586, and 0.528, respectively, the Kappa value for the consistency between the combination of the three with clinical diagnosis results was 0.920 (P<0.05). The sensitivity, negative predictive value, and accuracy of the combination of MRI, serum CYR61, and CXCL1 were obviously higher than that of the individual diagnosis, and the specificity and positive predictive value were obviously higher than that of the individual diagnosis of serum CYR61 and CXCL1 (P<0.05). Conclusion The clinical diagnostic accuracy of MRI combined with serum CYR61 and CXCL1 in the diagnosis of acute stroke is high, the combined diagnosis is obviously better than the individual diagnosis of each indicator.

OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na⁺, K⁺, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. CONCLUSIONS We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

Diabetic kidney disease(DKD)is one of the serious complications of diabetes.Ferroptosis causes pathological damage and is involved in the progression of DKD.How to inhibit ferroptosis and delay DKD is a new academic hotspot.The author's team further enriched and proposed the core pathogenesis theory of"internal heat induced disease"on the basis of the traditional DKD"yin deficiency and dryness-heat"theory,and explained the general development law of DKD.In this article,"internal heat"and"Zhengjia"in the theory of DKD"internal heat induced diseases"are used as the starting point to explore the relationship of the main pathological damage of ferroptosis such as activating inflammatory response,enhancing oxidative stress,promoting fibrosis and so on with the internal mechanism of the theory of"internal heat induced diseases",further explained the scientific connotation of DKD"internal heat induced diseases",in order to provide ideas and theoretical basis for the TCM prevention and treatment of DKD.

Objective To analyze the influencing factors and action paths of frailty in elderly patients with col-orectal cancer,so as to provide a theoretical clinical intervention.Methods A convenience sampling method was adopted.A total of 254 elderly patients with colorectal cancer who were scheduled for elective surgery in a tertiary A hospital in Shanxi Province from March 2023 to September 2024 were selected as the research subjects.General information questionnaires,Fried frailty phenotype,Perceived Ageism Scale,Multidimensional Assessment Scale of In-teroceptive Awareness Version 2-Chinese,Cancer Patient Self-Management Assessment Scale,and Anderson Symptom Inventory were used for the investigation,and a structural equation model was constructed.Results The incidence of frailty in elderly colorectal cancer patients before surgery is 38.98％.Anderson Symptom Assessment and Per-ceived Ageism were positively correlated with frailty(r=0.534,0.568,P＜0.01),while interoceptive sensitivity and the self-management level were negatively correlated with frailty(r=-0.444,-0.659,P＜0.01).Self-management behavior had only a direct effect on patients' frailty(β=-0.349,P＜0.001),while Anderson Symptom Assessment,perceived age discrimination and interoceptive sensitivity had both direct and indirect effects on patients' frailty(direct effects β=0.235,0.287,-0.220;indirect effects β=0.147,0.090,-0.113;all P＜0.001).Conclusion The incidence of frailty in elderly patients with colorectal cancer before surgery is high.Attention should be paid to patients who are elderly,female and have a low level of education.Medical staff can take targeted measures to enhance patients' interocep-tive concentration and stability,so that patients can better monitor and regulate their bodies and emotions,and im-prove their health-promoting decisions and behaviors.At the same time,attention should also be paid to the prob-lem of age discrimination to create a good medical environment for patients.

Objective:To explore any effect of electrical stimulation of the right median nerve (RMNS) in modulating arousal patterns in the prefrontal cortex of a healthy adult.Methods:Twenty-three right-handed healthy participants received block-designed patterns of RMNS arousal. Functional near-infrared spectroscopy (fNIRS) was used to record any hemodynamic response in their prefrontal cortex. Changes in oxyhemoglobin (HbO), deoxyhemoglobin (HbR), and total hemoglobin (HbT) were analyzed by comparing the levels in stimulation blocks with resting-state periods.Results:fNIRS revealed significant activation of HbO signals in the left frontopolar area (channel 15) and the right frontopolar area (channels 37, 43 and 48) following RMNS arousal. There were significant differences in the HbR signals from the right frontopolar area (channel 41). There were, however, no significant differences in the HbT signals across all the measured channels.Conclusion:RMNS arousal enhances neurometabolic activity within the frontopolar sub-region of the prefrontal cortex in healthy adults.

Objective To investigate the effects of exosomal microRNA-1234-3p(miR-1234-3p)of lung cancer cells on the malignant behaviors of lung cancer cells,the polarization of tumor-associat-ed macrophages(TAMs),and the functions of T cells,as well as its potential molecular mecha-nisms.Methods Exosomes were extracted from lung cancer A549 cells,and their morphology was observed using transmission electron microscopy.The expression of exosomal marker proteins[tumor susceptibility gene 101(TSG101),CD63 and CD9]was detected by western blot.The relative ex-pression level of miR-1234-3p was measured using real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The expression of exosomal miR-1234-3p was inhibited by transfecting,the proliferation,migration,and invasion abilities of A549 cells were detected using the CCK-8 method,wound healing assay,and Transwell invasion assay,respectively.The exosomes were co-cultured with macrophages and T lymphocytes separately.The expression of TAM polarization markers CD206 and CD163 was detected by western blot.The secretion levels of cytokines[interleukin(IL)-6,IL-10,and transforming growth factor-β(TGF-β)]were measured using an enzyme-linked immu-nosorbent assay(ELISA).The proliferation and apoptosis abilities of T lymphocytes,as well as the expression of functional molecules[γ-interferon(IFN-γ)and programmed death receptor 1(PD-1)]were detected through cell experiments.The expression levels of proteins related to the phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(AKT)signaling pathway were detected by western blot.Results The isolated exosomes were round double-layer vesicles with a diameter of 50 to 200 nm and expressed TSG101,CD63 and CD9 proteins.The expression level of miR-1234-3p in A549 cells was higher than that in human normal lung epithelial cells BEAS-2B,and the expression level of miR-1234-3p in A549 cell exosomes was higher than that in parental A549 cells,with statis-tically significant differences(P＜0.001).After inhibiting exosomal miR-1234-3p,the prolifera-tion,migration,and invasion abilities of A549 cells decreased,with statistically significantdifferenc-es(P＜0.001).After inhibiting exosomal miR-1234-3p,the levels of M2-type polarization marker proteins CD206 and CD163 in macrophages decreased,the levels of cytokines IL-10 and TGF-β de-creased,and the level of IL-6 increased,with statistically significant differences(P＜0.001),indi-cating that the polarization of macrophages toward the M2 type was inhibited.In T lymphocytes,af-ter inhibiting exosomal miR-1234-3p,cell viability increased,the apoptosis rate decreased,the ex-pression level of the functional molecule IFN-γ increased,and the expression level of PD-1 de-creased,with statistically significant differences(P＜0.01 orP＜0.001).After inhibiting exosom-al miR-1234-3p,the protein levels of phosphorylated(p)-PI3K and p-AKT in A549 cells de-creased,with statistically significant differences(P＜0.001).Conclusion Exosomal miR-1234-3p of lung cancer cells may promote the malignant behaviors of lung cancer cells,the polarization of TAMs toward the M2 type,and the dysfunction of T lymphocytes by activating the PI3K/AKT signa-ling pathway,thereby regulating the immunosuppressive microenvironment and promoting the pro-gression of lung cancer.

Objective To explore the clinical characteristics and prognostic analysis of acute corpus callosum in-farction.Methods A total of 1 466 patients with acute cerebral infarction admitted to the Neurology Department of Jiangbin Hospital in Guangxi from January 2019 to October 2023 and Neurology Department of Fuwai Hospital,Chinese Academy of Medical Sciences from January 2022 to December 2022.Among them,21 patients with acute corpus callosum infarction confirmed by MRI(observation group)and 25 patients with isolated subcortical infarction at the same period(control group)were selected.By comparing clinical data and follow-up information between two groups,we summarized the clinical characteristics of acute corpus callosum infarction,and analyzed the relevant factors affecting the prognosis of the corpus callosum infarction group.Results Acute corpus callosum infarction accounted for 1.43％(21/1 466)of patients with acute cerebral infarction in the same period.The main clinical manifestations include limb paralysis,cognitive decline,and speech impairment.Compared with the control group,the group with corpus callosum infarction had a longer onset visit time(P<0.05),more cognitive impair-ment(P<0.05),a higher proportion of mild stroke(P<0.05),more worsening symptoms during the course of the disease(P<0.05),a higher proportion of cardioembolic type(P<0.05),a lower proportion of small artery occlu?sion type(P<0.05)and a higher proportion of good prognosis(mRS score≤2 points)(P<0.05).The poor prog?nosis of corpus callosum infarction was found to be related to factors such as large lesions,multiple high?risk factors(≥3),concomitant lobar infarction and worsening of symptoms during the course of the disease.Conclusions Acute corpus callosum infarction often leads to cognitive impairment and mild limb paralysis,which frequently de?lays treatment.The poor prognosis is mainly related to factors such as lobar infarction,major lesions,and worsening of symptoms during the process.

ObjectiveTo investigate the effect and mechanism of Yishen Tongluo prescription in protecting mice from oxidative stress injury in diabetic kidney disease （DKD） via the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1）/NAD（P）H quinone oxidoreductase 1 （NQO1） signaling pathway. MethodsSpecific pathogen-free （SPF） male C57BL/6 mice were assigned into a control group （n=10） and a modeling group （n=50）. The DKD model was established by intraperitoneal injection of streptozotocin. The mice in the modeling group were randomized into a model group， a semaglutide （40 μg·kg^-1） group， and high-， medium-， and low-dose （18.2， 9.1， 4.55 g·kg^-1， respectively） Yishen Tongluo prescription groups， with 10 mice in each group. The treatment lasted for 12 weeks. Blood glucose and 24-h urine protein levels were measured， and the kidney index （KI） was calculated. Serum levels of creatinine （SCr）， blood urea nitrogen （BUN）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） were assessed. The pathological changes in the renal tissue were evaluated by hematoxylin-eosin， periodic acid-Schiff， periodic acid-silver methenamine， and Masson’s trichrome staining. Enzyme-linked immunosorbent assay kits were used to measure the levels of β2-microglobulin （β2-MG）， neutrophil gelatinase-associated lipocalin （NGAL）， kidney injury molecule-1 （KIM-1）， liver fatty acid-binding protein （L-FABP）， nitric oxide synthase （NOS）， glutathione （GSH）， total antioxidant capacity （T-AOC）， and 8-hydroxy-2'-deoxyguanosine （8-OHdG）. Immunohistochemical staining was performed to examine the expression of Kelch-like ECH-associated protein 1 （Keap1） and malondialdehyde （MDA）. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR） and Western blot were employed to determine the mRNA and protein levels， respectively， of factors in the Nrf2/HO-1/NQO1 signaling pathway. ResultsCompared with the control group， the DKD model group showed rises in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with glomerular hypertrophy， renal tubular dilation， thickened basement membrane， mesangial expansion， and collagen deposition. Additionally， the model group showed elevated levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， lowered levels of GSH and T-AOC， up-regulated expression of MDA and Keap1， and down-regulated expression of Nrf2， HO-1， NQO1， and glutamate-cysteine ligase catalytic subunit （GCLC） （P<0.05）. Compared with the model group， the semaglutide group and the medium- and high-dose Yishen Tongluo prescription groups showed reductions in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with alleviated pathological injuries in the renal tissue. In addition， the three groups showed lowered levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， elevated levels of GSH and T-AOC， down-regulated expression of MDA and Keap1， and up-regulated expression of Nrf2， HO-1， NQO1， and GCLC （P<0.05）. ConclusionYishen Tongluo prescription exerts renoprotective effects in the mouse model of DKD by modulating the Nrf2/HO-1/NQO1 signaling pathway， mitigating oxidative stress， and reducing renal tubular injuries.

Objective: To investigate the mechanism of Yishen Tongluo Formula in ameliorating renal pyroptosis in diabetic nephropathy mice by regulating the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. Methods: Sixty C57BL/6 male mice were randomly divided into control (10 mice) and intervention groups (50 mice) using random number table method. The diabetes nephropathy model was established by intraperitoneally injecting streptozotocin(50 mg/kg). After modeling, the intervention group was further divided into model, semaglutide (40 μg/kg), and high-, medium-, and low-dose Yishen Tongluo Formula groups (15.6, 7.8, and 3.9 g/kg, respectively) using random number table method. The high-, medium-, and low-dose Yishen Tongluo Formula groups were administered corresponding doses of medication by gavage, the semaglutide group received a subcutaneous injection of semaglutide injection, and the control group and model groups were administered distilled water by gavage for 12 consecutive weeks. Random blood glucose levels of mice in each group were monitored, and the 24-h urinary protein content was measured using biochemical method every 4 weeks; after treatment, the serum creatinine and urea nitrogen levels were measured using biochemical method. The weight of the kidneys was measured, and the renal index was calculated. Hematoxylin and eosin, periodic acid-Schiff, periodic Schiff-methenamine, and Masson staining were used to observe the pathological changes in renal tissue. An enzyme-linked immunosorbent assay was used to detect urinary β2-microglobulin (β2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels. Western blotting and real-time fluorescence PCR were used to detect the relative protein and mRNA expression levels of nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3), Caspase-1, gasdermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in renal tissue. Immunohistochemistry was used to detect the proportion of protein staining area of the TLR4, MyD88, and NF-κB in renal tissue. Results: Compared with the control group, the random blood glucose, 24-h urinary protein, serum creatinine, urea nitrogen, and renal index of the model group increased, and the urine β2-MG, NGAL, and KIM-1 levels increased. The relative protein and mRNA expression levels of NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18 in renal tissue increased, and the proportion of TLR4, MyD88, and NF-κB protein positive staining areas increased (P<0.05). Pathological changes such as glomerular hypertrophy were observed in the renal tissue of the model group. Compared with the model group, the Yishen Tongluo Formula high-dose group showed a decrease in random blood glucose after 12 weeks of treatment (P<0.05). The Yishen Tongluo Formula high- and medium-dose groups showed a decrease in 24-h urinary protein, creatinine, urea nitrogen, and renal index, as well as decreased β2-MG, NGAL, and KIM-1 levels. NLRP3, Caspase-1, GSDMD, IL-1 β, and IL-18 relative protein and mRNA expression levels were also reduced, and the proportion of TLR4, MyD88, and NF-κB protein positive staining areas was reduced (P<0.05). Pathological damage to renal tissue was ameliorated. Conclusion Yishen Tongluo Formula may exert protective renal effects by inhibiting renal pyroptosis and alleviating tubular interstitial injury in diabetic nephropathy mice by regulating the TLR4/MyD88/NF-κB signaling pathway.