ObjectiveTo investigate the material basis of the pathogenesis of cerebral ischemic injury with blood stasis and toxin syndrome and to explore the protective effects of Huayu Jiedu prescription （HYJDP） on neutrophil extracellular trap-related cell death （NETosis） in cerebral ischemic injury following acute cerebral infarction. MethodsSeventy-two Sprague-Dawley （SD） rats were randomly divided into six groups （n=12 per group）： sham operation （Sham） group， blood stasis and toxin model （Model） group， low-， medium-， and high-dose HYJDP groups （HYJDP-L， HYJDP-M， and HYJDP-H； 9， 18， and 36 g·kg^-1， respectively）， and butylphthalide （NBP） group （0.06 g·kg^-1）. Except for the Sham group， rats in all other groups were subjected to carrageenan/dry yeast combined with a modified intraluminal filament method to establish a focal cerebral ischemia model of the middle cerebral artery with blood stasis and toxin syndrome. Neurological function was evaluated at 24 h after modeling using the Zea-Longa neurological deficit score. Cerebral infarction rate was assessed by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Pathological morphology of brain tissue was observed using hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was used to determine serum levels of interleukin-8 （IL-8）， myeloperoxidase-DNA complexes （MPO-DNA）， and citrullinated histone H3 （CitH3）. Protein expression of phosphorylated phosphatidylinositol 3-kinase （p-PI3K）， protein kinase B （p-Akt）， mammalian target of rapamycin （p-mTOR）， sequestosome 1 （p62）， and CitH3 in brain tissue was detected by Western blot. Immunofluorescence （IF） was used to detect the expression of neutrophil-specific marker Ly6G， CitH3， and neuron-specific nuclear protein （NeuN） in brain tissue. ResultsCompared with the Sham group， neurological deficit scores and cerebral infarction rates in the model group were significantly increased （P<0.01 for both）. HE staining showed varying degrees of neuronal degeneration and necrosis， characterized by blurred neuronal structures， nuclear pyknosis and fragmentation， cytoplasmic dissolution into a vacuolated reticular pattern， and mild glial cell proliferation. ELISA results showed that serum levels of IL-8， MPO-DNA， and CitH3 were significantly increased （P<0.01）. Western blot analysis demonstrated decreased expression of p-PI3K， p-Akt， p-mTOR， and p62， while CitH3 expression was significantly increased （P<0.01）. IF results showed an increased number of NETs⁺ cells and a significant decrease in NeuN⁺ cells （P<0.01）. Compared with the Model group， neurological deficit scores in the HYJDP-H group were significantly decreased （P<0.05）， and cerebral infarction rates in the HYJDP-H and NBP groups were significantly reduced （P<0.01）. HE staining showed that brain tissue damage was markedly alleviated in the HYJDP-H group. ELISA results showed that levels of IL-8， MPO-DNA， and CitH3 were significantly decreased in the HYJDP-M， HYJDP-H， and NBP groups （P<0.01）. Western blot analysis showed that expression of p-PI3K， p-Akt， p-mTOR， and p62 was significantly increased in the HYJDP-H and NBP groups， while CitH3 expression was significantly reduced in all drug intervention groups （P<0.01）. IF results showed that the number of NETs⁺ cells was significantly decreased and the number of NeuN⁺ cells was significantly increased in all drug intervention groups （P<0.01）. ConclusionNETs may be the material basis of the pathogenesis of cerebral ischemic injury characterized by blood stasis and toxin. HYJDP can regulate the PI3K/Akt/mTOR signaling pathway， reduce the release of pro-inflammatory mediators and NETosis-related products， alleviate cerebral ischemic injury caused by autophagy-dependent NETosis， and thereby exert a neuroprotective effect.

Metabolic associated fatty liver disease (MAFLD) is fundamentally driven by an imbalance in hepatic fatty-acid flux: the influx of fatty acids exceeds the liver’s capacity for disposal, resulting in excessive hepatic lipid accumulation, predominantly in the form of triglycerides (TGs). The occurrence and progression of MAFLD depend on disordered regulation across multiple metabolic steps, including fatty-acid uptake, de novo lipogenesis (DNL), fatty-acid oxidation (FAO), and very low-density lipoprotein (VLDL) export. Forkhead box protein O1 (FOXO1) is a key transcriptional regulator within the hepatic network coordinating glucose and lipid metabolism. Under metabolic stress and insulin resistance (IR), FOXO1 expression is frequently increased, whereas its inhibitory phosphorylation is reduced. These changes enhance FOXO1 nuclear localization and transcriptional activity, thereby reprogramming the expression of genes related to metabolism in the liver. Because hepatic lipid deposition is the central pathological feature of MAFLD, the functional status of FOXO1 directly influences hepatic lipid homeostasis. Growing evidence suggests that FOXO1 can exert bidirectional, environment-dependent effects on hepatic lipid accumulation; however, the molecular basis for this functional switch remains incompletely understood. This review systematically summarizes the biological functions and regulatory mechanisms of FOXO1 and its roles in hepatic lipid metabolism, with a particular focus on its crosstalk with insulin signaling. FOXO1 expression is shaped by RNA modifications and epigenetic regulation mediated by non-coding RNAs. Its transcriptional output is precisely governed by post-translational modifications—such as phosphorylation and acetylation—as well as by coordinated nucleocytoplasmic shuttling. Notably, these regulatory patterns vary markedly across nutritional states, degrees of insulin resistance, and stages of disease. In the fed state, insulin/IGF-1 signaling activates the PI3K-AKT pathway, promoting the inhibitory phosphorylation of FOXO1 and facilitating additional modifications, including acetylation, methylation, and ubiquitination. Together, these events drive FOXO1 export from the nucleus and dampen its transcriptional activity, suppressing gluconeogenesis and constraining lipogenic programs. Conversely, during fasting or when insulin signaling is weakened, FOXO1 inhibition is relieved. FOXO1 accumulates in the nucleus, binds to DNA, and regulates the transcription of downstream target genes. Mechanistically, FOXO1 can aggravate hepatic lipid accumulation by activating genes involved in TG synthesis while repressing FAO-related pathways, thereby favoring storage over oxidation. However, under specific conditions, FOXO1 may also alleviate the hepatic lipid burden by promoting TG hydrolysis and enhancing VLDL secretion, thereby reducing the net hepatic lipid load. In addition, lipotoxic signals mediated by ceramides and diacylglycerols (Cer/DAG) activate atypical protein kinase C (aPKC), further exacerbating the disruption of the AKT-FOXO1 axis. This vicious cycle ultimately produces a metabolic paradox in which increased hepatic glucose output coexists with persistent, insulin-independent lipogenesis, accelerating MAFLD progression. Importantly, FOXO1 regulation is not uniform: during early metabolic overload, insulin-mediated suppression may remain effective, whereas in advanced insulin resistance, the loss of AKT control permits sustained FOXO1 activity. Such stage-dependent dynamics may help explain why FOXO1 can either promote steatosis or, in certain contexts, support programs that facilitate lipid turnover. Accordingly, interventions should be liver-specific and tuned to the disease stage, aiming to curb maladaptive FOXO1 signaling while preserving its capacity to promote triglyceride hydrolysis and VLDL secretion when advantageous. Overall, this review offers an important perspective on MAFLD pathogenesis, emphasizing FOXO1 as a potential therapeutic target and providing a theoretical basis for developing liver-specific, disease-course-dependent precision interventions.

Objective To provide a reference for the establishment, development and application of the adverse drug reaction （ADR） automated monitoring system, through verifying and quantifying the research hotspots and advantages of the system by CiteSpace software and systematic review. Methods Literature on ADR automated monitoring up to December 2023 were retrieved and screened from CNKI and web of science databases. CiteSpace 6.4.R1 software was used to conduct co-occurrence, clustering and emergence analysis, and to visualize and comparatively analyze the research hotspots, rules and distribution in the field of automated monitoring of ADR at home and abroad. In compliance with the preferred reporting items for systematic reviews and Meta-analyses （PRISMA）, literature covering publications in English and Chinese including detection rates of ADR collected using Incident Reporting Systems （IRSs） and/or automated monitoring systems were retrieved and screened. The advantages and disadvantages of automated monitoring systems were analyzed by comparing the differences between these two systems in terms of the number of ADR reports and the types of positive signals. Results A total of 56 articles in English and 80 articles in Chinese were indexed by CiteSpace. The research hotspots in recent years included data mining, deep learning, text classification techniques, machine learning and so on. A total of seven studies compiled with the inclusion criteria for the systematic evaluation, all of which were completed between 1991 and 2021 in hospitals in four countries. 150 526 medical records were reviewed from 15 institutions. A total of 194 ADR reports were collected by IRSs. A total of 2 090 ADR reports were collected by the automated monitoring system over the same period, indicating a 977% increase in the number of ADR reports （P=0.0156） compared with the IRSs. Conclusion The ADR automatic monitoring system had significantly improved the level of drug risk identification and reduced costs, but it was necessary to optimize the algorithm, expand the data source and carry out standardization construction to overcome the current limitations.

BACKGROUND: Increasing evidence indicates that oxidative stress and interferon γ (IFNγ)-driven cellular immune responses are responsible for the pathogenesis of vitiligo. However, the connection between oxidative stress and the local production of IFNγ in early vitiligo remains unexplored. The aim of this study was to identify the mechanism underlying the production of IFNγ by mast cells and its impact on vitiligo pathogenesis. METHODS: Skin specimens from the central, marginal, and perilesional skin areas of active vitiligo lesions were collected to characterize changes of mast cells, CD8 + T cells, and IFNγ-producing cells. Cell supernatants from hydrogen peroxide (H 2 O 2 )-treated keratinocytes (KCs) were harvested to measure levels of soluble stem cell factor (sSCF) and matrix metalloproteinase (MMP)-9. A murine vitiligo model was established using Mas-related G protein-coupled receptor-B2 (MrgB2, mouse ortholog of human MrgX2) conditional knockout (MrgB2 -/- ) mice to investigate IFNγ production and inflammatory cell infiltrations in tail skin following the challenge with tyrosinase-related protein (Tyrp)-2 180 peptide. Potential interactions between the Tyrp-2 180 peptide and MrgX2 were predicted using molecular docking. The siRNAs targeting MrgX2 and the calcineurin inhibitor FK506 were also used to examine the signaling pathways involved in mast cell activation. RESULTS: IFNγ-producing mast cells were closely aligned with the recruitment of CD8 + T cells in the early phase of vitiligo skin. sSCF released by KCs through stress-enhanced MMP9-dependent proteolytic cleavage recruited mast cells into sites of inflamed skin (Perilesion vs . lesion, 13.00 ± 4.00/high-power fields [HPF] vs . 26.60 ± 5.72/HPF, P <0.05). Moreover, IFNγ-producing mast cells were also observed in mouse tail skin following challenge with Tyrp-2 180 (0 h vs . 48 h post-recall, 0/HPF vs . 3.80 ± 1.92/HPF, P <0.05). The IFNγ + mast cell and CD8 + T cell counts were lower in the skin of MrgB2 -/- mice than in those of wild-type mice (WT vs . KO 48 h post-recall, 4.20 ± 0.84/HPF vs . 0.80 ± 0.84/HPF, P <0.05). CONCLUSION Mast cells activated by MrgX2 serve as a local IFNγ producer that bridges between innate and adaptive immune responses against MCs in early vitiligo. Targeting MrgX2-mediated mast cell activation may represent a new strategy for treating vitiligo.
Vitiligo/metabolism* ; Mast Cells/immunology* ; Animals ; Interferon-gamma/metabolism* ; Mice ; Humans ; Melanocytes/metabolism* ; Receptors, G-Protein-Coupled/genetics* ; Mice, Knockout ; Mice, Inbred C57BL ; Male ; Female ; Matrix Metalloproteinase 9/metabolism* ; Stem Cell Factor/metabolism*

BACKGROUND: Diabetic foot is a complex condition with high incidence, recurrence, mortality, and disability rates. Current treatments for diabetic foot ulcers are often insufficient. This study was conducted to identify potential therapeutic targets for diabetic foot. METHODS: Datasets related to diabetic foot and diabetic skin were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified using R software. Enrichment analysis was conducted to screen for critical gene functions and pathways. A protein interaction network was constructed to identify node genes corresponding to key proteins. The DEGs and node genes were overlapped to pinpoint target genes. Plasma and chronic ulcer samples from diabetic and non-diabetic individuals were collected. Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays were performed to verify the S100 calcium binding protein A9 (S100A9), inflammatory cytokine, and related pathway protein levels. Hematoxylin and eosin staining was used to measure epidermal layer thickness. RESULTS: In total, 283 common DEGs and 42 node genes in diabetic foot ulcers were identified. Forty-three genes were differentially expressed in the skin of diabetic and non-diabetic individuals. The overlapping of the most significant DEGs and node genes led to the identification of S100A9 as a target gene. The S100A9 level was significantly higher in diabetic than in non-diabetic plasma (178.40 ± 44.65 ng/mL vs. 40.84 ± 18.86 ng/mL) and in chronic ulcers, and the wound healing time correlated positively with the plasma S100A9 level. The levels of inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1, and IL-6) and related pathway proteins (phospho-extracellular signal regulated kinase [ERK], phospho-p38, phospho-p65, and p-protein kinase B [Akt]) were also elevated. The epidermal layer was notably thinner in chronic diabetic ulcers than in non-diabetic skin (24.17 ± 25.60 μm vs. 412.00 ± 181.60 μm). CONCLUSIONS S100A9 was significantly upregulated in diabetic foot and was associated with prolonged wound healing. S100A9 may impair diabetic wound healing by disrupting local inflammatory responses and skin re-epithelialization.
Calgranulin B/therapeutic use* ; Diabetic Foot/metabolism* ; Humans ; Datasets as Topic ; Computational Biology ; Mice, Inbred C57BL ; Animals ; Mice ; Protein Interaction Maps ; Immunohistochemistry

Given that ovarian stimulation is vital for assisted reproductive technology (ART) and results in elevated serum estrogen levels, exploring the impact of elevated estrogen exposure on oocytes and embryos is necessary. We investigated the effects of various ovarian stimulation treatments on oocyte and embryo morphology and gene expression using a mouse model and estrogen-treated mouse embryonic stem cells (mESCs). Female C57BL/6J mice were subjected to two types of conventional ovarian stimulation and ovarian hyperstimulation; mice treated with only normal saline served as controls. Hyperstimulation resulted in high serum estrogen levels, enlarged ovaries, an increased number of aberrant oocytes, and decreased embryo formation. The messenger RNA (mRNA)‍-sequencing of oocytes revealed the dysregulated expression of lysine-specific demethylase 6b (Kdm6b), which may be a key factor indicating hyperstimulation-induced aberrant oocytes and embryos. In vitro, Kdm6b expression was downregulated in mESCs treated with high-dose estrogen; treatment with an estrogen receptor antagonist could reverse this downregulated expression level. Furthermore, treatment with high-dose estrogen resulted in the upregulated expression of histone H3 lysine 27 trimethylation (H3K27me3) and phosphorylated H2A histone family member X (γ-H2AX). Notably, knockdown of Kdm6b and high estrogen levels hindered the formation of embryoid bodies, with a concomitant increase in the expression of H3K27me3 and γ-H2AX. Collectively, our findings revealed that hyperstimulation-induced high-dose estrogen could impair the demethylation of H3K27me3 by reducing Kdm6b expression. Accordingly, Kdm6b could be a promising marker for clinically predicting ART outcomes in patients with ovarian hyperstimulation syndrome.
Female ; Mice ; Demethylation/drug effects* ; Embryonic Stem Cells ; Estrogens/administration & dosage* ; Gene Expression/drug effects* ; Histones/metabolism* ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Mice, Inbred C57BL ; Oocytes ; Ovary/drug effects* ; Reproductive Techniques, Assisted ; Animals

Bronchoalveolar lavage fluid (BALF) is rich in various bioactive substances such as cytokines and enzymes, making it a high-quality clinical specimen and research sample. It holds significant value in fields such as etiological analysis, proteomics, pathology, and disease diagnosis and treatment. This article systematically reviews the key technical points for the preservation of BALF samples and provides an in-depth interpretation of bronchoalveolar lavage fluid from an application perspective. The aim is to offer references for the standardization of sample preservation practices and to promote the widespread use of BALF samples in clinical research.

The objective of this study was to evaluate the toxicity and safety of novel Mycobacterium tuberculosis fusion strain protein derivatives,referred to as B/R strain active proteins.In cellular experiments,RAW264.7 cells were treated with each vaccine preparation,and apoptosis rates were measured.In subsequent animal experiments,C57BL/6 mice were immunized via subcutaneous injection,and their survival and body weight changes were monitored and recorded at 2,4,8,12,and 16 weeks.The lungs and spleens were harvested to calculate organ coefficients,and pathological examinations were conducted.At the eighth week of immunization,the mice were infected with high concentrations of BCG,and pathological changes in the lungs and spleens were observed 4 weeks post-infection.The apoptosis rate at 6 hours was significantly higher in the experimental group than the PBS group(P<0.05).At 12 and 24 hours,the apoptosis rate in the experimental group remained higher than that in the PBS group,although this difference was not statistically significant.After immunization,mice in all four groups exhibited normal growth patterns,as indicated by stable body weight changes.At 4 and 12 weeks post-immunization,the lung coefficients in the protein group were significantly higher than those in the PBS group at the same time points.Additionally,the lung coefficients in the BCG group were significantly elevated across all time periods(P<0.05).The spleen coefficients in the protein and BCG groups were significantly higher than those in the PBS group at 2,4,8,12,and 16 weeks,whereas the ICD B/R group showed higher spleen coefficients than the PBS group only at week 8(P<0.05).Pathological examination revealed normal lung and spleen tissues in the PBS group.However,during the 2-8 weeks immunization period,lung and spleen tissues in all experimental groups exhibited varying degrees of damage,which gradually diminished by 12-16 weeks.Notably,no tuberculosis nodules were observed in any experimental group.After infection with high concentrations of BCG,no overt pathological changes were observed on the surfaces of the lungs and spleens in any group.Microscopic examination revealed less severe pathological changes in the lungs and spleens of mice in the experimental groups than the PBS group.Furthermore,no statistically significant differences were observed between the protein group and the BCG group.Our findings suggested that the B/R strain active proteins'toxicity and safety profiles were comparable to those of BCG,and showed immunoprotective effects.This study provides an experimental foundation for the development of a novel tuberculosis vaccine.

Post-stroke spasticity is a series of symptoms after stroke,such as hand and foot urgency,unflexion and extension of muscles,etc.In order to deeply understand the cognition of post-stroke spasticity of ancient Chinese physicians and comb out their therapeutic thoughts,this study took the General Catalogue of Chinese Ancient Books of Traditional Chinese Medicine as a bibliographic reference,all the ancient Chinese literature on spasms after stroke was retrieved manually and by computer,and then sorted and analyzed,and classified them by longitudinal time,and extracted the description about post-stroke spasticity,including medical classics,prescriptions,clinical evidence,medical records and so on.And this paper verified and summarized the etiology,pathogenesis,functional and indications and prescription characteristics of spasticity after stroke,in order to deeply understand systematic theories and treatment ideas of the ancient medical practitioners in the bud,development and mature stages of their understanding of spasticity after stroke,and provide the theoretical basis for the later doctors to understand this disease and the modern clinical treatment of traditional Chinese medicine.

Objective To establish a scientific and feasible Chinese medicine(CM)comprehensive control and prevention program for elderly hypertension with early renal damage(EH-ERD)patients through high-level evidence-based medicine(EBM)evidence.Methods On the basis of literature research and evidence evaluation,we construct a database of specific prescriptions and implementation methods of CM comprehensive control and prevention program with EH-ERD.40 senior titled-experts were consulted in two rounds of questionnaires based on Delphi methods.We selected,evaluated,and revised specific CM comprehensive control and prevention program of EH-ERD through the analysis of multiple factors,such as expert's positive activity coefficient,authority coefficient,degree of opinion concentration and degree of coordination.Results In view of the CM appropriate intervention techniques with high-grade evidence(level Ⅰ and Ⅱ)and recommendation(level A and B),we have developed the CM comprehensive control and prevention plan items for EH-ERD.The activity coefficients in two-round consultation were 92.5%and 97.14%respectively,the overall authority coefficient(Cr)was exceeded 0.70,and the coordination coefficient was less than 0.25.With a sound activity coefficient,a relatively high Cr and a rather unified degree of opinion concentration and coordination,the CM comprehensive control and prevention program for EH-ERD was established,which included six primary indicators(CM compound preparation,acupuncture and massage,CM health education,medicinal diet therapy,health care exercise and foot bath)and seventeen secondary indicators.Conclusion The CM comprehensive control and prevention program for EH-ERD established in this study can provide a basis for the further formation of expert consensus or guidelines.Moreover,it can supply the strategy and paradigm for standardization of CM-based health management of EH-ERD.