Objective: To explore the effects of intergenerational parent-child separation on internalizing and externalizing behavioral problems among rural preschoolers aged 3-6 years, so as to provide empirical evidence for formulating targeted intervention strategies and blocking the intergenerational transmission of parent-child separation. Methods: From March to June, 2024, ten kindergartens were selected from rural areas of Nanling County, Wuhu City, Anhui Province, and 2 279 preschool children aged 3- 6 years, along with their fathers, mothers and primary caregivers, were recruited for survey by cluster convenient sampling method. Children s fathers and mothers reported their own childhood parent-child separation information separately. Information on the children s history of psychological and behavioral problems was reported by the primary caregivers. Information of internalizing and externalizing behavioral problems in preschool children was reported with Strengths and Difficulties Questionnaire (SDQ).The Chi square test was used to compare the differences in the detection rates of psychological and behavioral problems among children with different intergenerational continuity of parent-child separation. Binary Logistic regression model was applied to analyze the association between intergenerational continuity of parent-child separation and internalizing and externalizing behavioral problems in preschool children. Results: The detection rate of internalizing behavioral problems was 27.5% and externalizing behavioral problems was 19.1% among rural preschool children. There were significant differences in internalization behavior, externalization behavior, hyperactivity impulse and peer communication problems among preschool children in different types of parent-child separation groups( χ 2=14.29, 8.80, 17.26, 13.84, all P <0.05). Binary Logistic regression analysis showed that after adjusting for covariates such as children s gender, birth weight, and delivery mode, compared with children without separation, intergenerational continuity of parent-child separation group( OR =1.95), only child separation( OR =1.74) were associated with significant higher risk of conduct problems(both P ＜0.05). Parent-child separation intergenerational continuation groups( OR =1.68), only child separation( OR = 1.47) were associated with significant higher risk of peer problems(both P ＜0.05). Both types of parent-child separation experiences significantly increased the risk of children s prosocial behavior problems，among them, the intergenerational continuation group was 1.99 times that of the control group ( OR =1.99), and the childhood only separation group was 2.09 times that of the control group ( OR =2.09)(both P ＜0.05). Compared with children whose parents and children had no experience of parent-child separation, children in the intergenerational continuation group of parent-child separation had increased risks of both internalizing and externalizing behavior problems ( OR =1.53, 1.65, both P <0.05). Conclusion All types of parent-child separation are associated with internalizing and externalizing behavioral problems among rural children aged 3 to 6 years.

Objective: To explore the association between daytime outdoor physical activity (OPA) and oral health indicators among primary school students, aiming to provide evidence for developing oral health intervention strategies based on natural exposure. Methods: In October 2023, based on the "Tianchang Children s Light Exposure and Growth Development Cohort", 799 second and third grades children were recruited from two primary schools in Tianchang, Chuzhou City, Anhui Province. Physical activity intensity and light exposure were objectively monitored for 24 hours over 5 consecutive days using triaxial accelerometers synchronized with portable illuminance meters. Standardized oral examinations were performed to record dental caries, gingivitis, and malocclusion. Demographics, lifestyle variables, and household socioeconomic data were collected via questionnaires. Multiple linear regression (for the number of carious teeth) and Logistic regression (for gingivitis risk) were used to analyze the relationship between daytime outdoor moderate to vigorous physical activity (MVPA) and oral health outcomes. Results: The average daytime outdoor MVPA was (0.76±0.35)h, with (0.95±0.40)h on weekdays and (0.49±0.47)h on weekends. The detection rates for dental caries, gingivitis, and malocclusion were 31.0%, 4.6%, and 59.7%, respectively. Compared with children with good oral health, the duration of outdoor MVPA on school days was reduced in children with caries or gingivitis ( Z =-11.4, -5.01, both P <0.01). Multiple regression analysis showed that after adjusting for factors such as gender, age, body mass index, oral hygiene behaviors, an increase in daytime outdoor MVPA duration was associated with a decrease in the number of dental caries ( β=-0.64, 95%CI =-0.93 to -0.35) and a reduced risk of gingivitis ( OR= 0.58 , 95%CI =0.34-0.98) in primary school students (both P <0.05). The association was primarily observed on school days ( β=-0.72, 95%CI = -1.07 to -0.37; OR=0.42, 95%CI =0.21-0.85) (both P < 0.05). Conclusions Daytime outdoor MVPA on weekdays is significantly associated with a lower number of carious teeth and a reduced risk of gingivitis in primary school students. Increasing daytime outdoor activities on weekdays may serve as a promising and potential strategy for promoting children s oral health.

Objective To investigate the clinical features and key diagnostic points of aceruloplasminemia （ACP）， as well as the features of a novel pathogenic mutation. Methods A systematic analysis was performed for the clinical data of one patient with a confirmed diagnosis of ACP， and a literature review was performed based on related articles in China and globally. Results Based on the clinical features of this patient and the analysis of the family， it was clarified that the homozygous c.1944C>G （p.Ser648Arg） mutation in the Ceruloplasmin （CP） gene could cause ACP and was reported for the first time worldwide. Conclusion ACP is an extremely rare autosomal recessive disease due to abnormal iron metabolism caused by a significant reduction in ceruloplasmin， with the main clinical manifestations of retinopathy， diabetes， ataxia， and cognitive impairment， and genetic testing of the CPgene has a relatively high diagnostic value.
Ceruloplasmin

ObjectiveTo evaluate the comprehensive intervention effects of Huangqin Qingre Chubi Capsule （黄芩清热除痹胶囊， HQC） on self-perception of patients （SPP） and immune inflammation in patients with rheumatoid arthritis （RA）， and to explore its potential mechanisms. MethodsClinical data of 452 RA patients were retrospectively collected. Patients were divided into a control group （274 cases）， treated with conventional western medicine， and an observation group （178 cases）， treated with HQC for at least 2 weeks in addition to conventional western medicine. The treatment duration was 2 weeks for both groups. Propensity score matching （PSM） was performed at a ratio of 1∶1 to match patients between groups. SPP including the Chinese version of the short form-36 health survey （SF-36）， self-rating anxiety scale （SAS）， self-rating depression scale （SDS）， visual analog scale （VAS）， and Chinese patient-reported index for rheumatoid arthritis （CPRI-RA）， as well as immune inflammatory indicators， including erythrocyte sedimentation rate （ESR）， high-sensitivity C-reactive protein （hs-CRP）， rheumatoid factor （RF）， anti-cyclic citrullinated peptide antibody （anti-CCP）， interleukin-6 （IL-6）， immunoglobulin A （IgA）， immunoglobulin G （IgG）， immunoglobulin M （IgM）， complement C3， and complement C4， were collected before and after treatment. Spearman correlation analysis was used to assess the relationships between SPP and immune inflammatory indicators. Logistic regression， association rule analysis， and mediation analysis were performed to evaluate the effects and potential pathways of HQC on SPP and immune inflammatory indicators. Network pharmacology was applied to identify the active components and core targets of HQC in the treatment of RA， followed by molecular docking verification. In cell experiments， cells were divided into normal group， model group， 20% medicated serum group， and 80 nmol/L control group. Human synovial fibroblasts （FLS） were cultured with complete medium in the normal group， while human rheumatoid arthritis fibroblast-like synoviocytes （RA-FLS） were cultured in the model group. In the 20% medicated serum group， RA-FLS were cultured with medium containing 20% HQC-medicated serum， and in the 80 nmol/L control group， RA-FLS were cultured with complete medium containing 80 nmol/L methotrexate suspension. After 48 h of culture， cell viability was detected by cell counting kit-8 （CCK-8） assay. Levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， and interleukin-10 （IL-10） in the cell supernatant were measured by enzyme-linked immunosorbent assay （ELISA）. Protein levels of matrix metalloproteinase 9 （MMP9）， transcription factor AP-1 subunit （JUN）， vascular endothelial growth factor A （VEGFA）， and C-X-C motif chemokine ligand 8 （CXCL8） were detected by Western Blot， and cell migration ability was evaluated using Transwell assay. ResultsAfter PSM， 178 cases were included in each group. After treatment， SF-36 scores increased， while scores of SAS， SDS， VAS and CPRI-RA， levels of ESR， hs-CRP， IL-6， complement C3， and complement C4 levels decreased in both groups； IgG and IgM levels were also reduced in the observation group （P＜0.05）. Physical functioning （correlation coefficient -0.19， P＜0.05） and social functioning （correlation coefficient -0.18， P＜0.05） of SF-36 were negatively correlated with hs-CRP， while VAS score was positively correlated with hs-CRP （correlation coefficient 0.19， P＜0.05）. HQC showed high associations with improvements in multiple indicators of SPP and immune inflammatory， and acted as a protective factor for the improvement of several SPP； hs-CRP and ESR played partial mediating roles in the improvement of SPP induced by HQC （P＜0.05）. Network pharmacology analysis identified baicalein， quercetin， α1-sitosterol， β-sitosterol， stigmasterol， baicalin， and crocetin as the core active components， and JUN， IL-6， VEGFA， MMP9， IL-1β， and CXCL8 as the core targets. Molecular docking results showed strong binding affinities of quercetin with VEGFA， JUN， MMP9， IL-6， and IL-1β， of baicalin with VEGFA and MMP9， and of wogonin with CXCL8. Cell experiments demonstrated that HQC and methotrexate inhibited RA-FLS viability and migration， reduced levels of IL-1β， IL-6， and IL-8， decreased protein levels of MMP9， JUN， VEGFA， and CXCL8， and increased IL-10 levels （P＜0.05）. ConclusionHQC can improve SPP in RA by regulating immune inflammatory responses. Its mechanism may be related to multi-pathway and multi-target inhibition of synovial cell inflammation and migration.

ObjectiveTo investigate the effect of bone morphogenetic protein 3 （BMP3） on the expression of inflammatory factors and joint damage in adjuvant arthritis （AA） induced by Freund′s complete adjuvant （FCA） in rats. MethodsThe AIA model was established in SD rats by intradermal injection of FCA into the toes of the left hind limb， and BMP3 overexpressing adenovirus （Ad-BMP3） or control adenovirus （Ad-NC） was injected in situ into the knee joint cavity on day 8 after modeling. Subsequently， HE staining was used to observe the histopathological changes in the synovium， immunohistochemistry was used to detect the expression of BMP3 in the synovium， and ELISA was used to analyze the expression levels of IL-6， IL-1β and TNF-α in the serum. Primary fibroblast-like synoviocytes （FLS） were isolated from AIA rats， the expression of BMP3 in FLS was knocked down or overexpressed， and Western blot and qRT-PCR were used to detect the expression levels of BMP3 and inflammatory factors in FLS. ResultsHE staining confirmed the successful establishment of the AIA model. Compared with normal rats， AIA rats showed decreased BMP3 expression in synovial tissue. Knockdown of BMP3 promoted the protein expression of inflammatory factors （IL-6， IL-1β， IL-17A， TNF-α） and the mRNA expression of chemokines ［C-C motif chemokine ligand 2 （CCL2）， C-C motif chemokine ligand 3 （CCL3）， Vascular Cell Adhesion Molecule-1 （VCAM-1）］ in FLS. In contrast， overexpression of BMP3 suppressed the expression of these inflammatory factors and chemokines. Intra-articular injection of BMP3-overexpressing adenovirus in AIA rats upregulated BMP3 expression in synovial tissue and inhibited synovial inflammation and bone erosion. ConclusionBMP3 suppresses the production of inflammatory factors and chemokines in FLS， thereby alleviating synovial hyperplasia and bone erosion in arthritis.

ObjectiveTo investigate the role of Ras-GTPase-activating protein SH3 domain-binding protein 2 （G3BP2） in regulating the activation， proliferation， and migration of hepatic stellate cells （HSCs）. MethodsThe mouse HSCs （JS-1 cell line） were treated with 5 μg/L transforming growth factor-beta 1（TGF-β1） for 24 hours to establish an HSC activation and proliferation model. A G3BP2 knockdown system was constructed using siRNA interference technology. The experiment was divided into four groups： Control， TGF-β1 treatment， TGF-β1+si-NC， and TGF-β1+ G3BP2-siRNA. The expression levels of key fibrosis indicators， including type I collagen （Collagen I）， α-smooth muscle actin （α-SMA）， and G3BP2， were detected by Western blot and RT-qPCR. Cell proliferation activity was assessed using the CCK-8 proliferation assay kit and EdU fluorescence labeling technology. Cell migration ability was analyzed by scratch wound healing assay and Transwell migration assay. The formation level of stress granules was quantified by immunofluorescence microscopy to investigate the effects of G3BP2 on stress granule formation in activated HSCs. ResultsStimulation with TGF-β1 upregulated the expression of G3BP2 in JS-1 cells （RT-qPCR： P0.000 1； Western blot： P0.000 1）， while a downward trend in its expression was observed in the G3BP2‑silenced group （RT-qPCR： P0.01； Western blot： P0.000 1）. Compared with the control group， the TGF-β1 group exhibited increased protein expression levels of α-SMA and Collagen I （RT-qPCR： both P0.01； Western blot： P0.01 and P0.05， respectively）， concomitant with an increased number of stress granules and enhanced cell proliferation and migration capacity （all P0.001）. The experimental results demonstrated that G3BP2 knockout effectively reversed the aforementioned phenotypes， with the G3BP2-silenced group showing reduced expression of fibrotic markers （all P0.01）， decreased stress granule formation （P0.01）， and reduced cell proliferation and migration capacity （all P0.05）， compared to the negative control group. ConclusionG3BP2 enhances the activation， proliferation， and migration of HSCs by promoting the formation of stress granules， thereby accelerating the pathological progression of liver fibrosis. This suggests that stress granules may serve as important regulators in controlling the activation， proliferation， and migration of HSCs.

ObjectiveTo develop and validate a nomogram based on the albumin-bilirubin （ALBI） score for predicting the risk of severe perioperative complications in patients undergoing hepatectomy for hepatolithiasis. MethodsA retrospective analysis was conducted on the clinical data of 163 hepatolithiasis patients who underwent hepatectomy. Univariate and multivariate logistic regression analyses were used to identify independent risk factors for severe perioperative complications. A nomogram prediction model was constructed and its performance was evaluated. ResultsAmong the 163 patients， 66 and 97 were classified into the low-grade and high-grade ALBI groups， respectively. Significant intergroup differences were observed in gender， total bilirubin， albumin levels， and the incidence of severe complications （P0.05）. Severe complications occurred in 40 patients. Independent risk factors included age 60 years （OR=5.49， P0.001）， high-grade ALBI （OR=8.30， P0.001）， history of biliary surgery （OR=2.60， P=0.035）， hepatectomy （segmentectomy）≥3 （OR=2.75， P=0.028）， and open surgical approach （OR=4.00， P=0.009）. A nomogram for predicting severe perioperative complications was successfully established. Internal validation showed that the model had an area under the ROC curve （AUC） of 0.865， which outperformed traditional single predictors. The calibration curve closely aligned with the ideal curve， with a mean absolute error （MAE） of 0.027. Decision curve analysis （DCA） demonstrated a net clinical benefit when the threshold probability exceeded 10%， superior to that of traditional predictors. ConclusionThe ALBI score-based nomogram is successfully developed and validated to predict the risk of severe perioperative complications in hepatolithiasis patients undergoing hepatectomy. The model demonstrated favorable predictive performance and high clinical utility， serving as an effective tool for both preoperative risk assessment and postoperative risk stratification.

Cardiovascular diseases （CVD）， particularly atherosclerosis， represent a major global health burden. Recent studies have revealed that innate immune cells such as monocytes and macrophages can develop immune memory after an initial stimulus， a phenomenon termed “trained immunity”. Growing evidence indicates that trained immunity serves as an underlying mechanism of chronic inflammation in atherosclerotic cardiovascular diseases. This review focuses on outlining the key effector cells involved in trained immunity and their mechanisms of formation， including processes such as metabolic reprogramming and epigenetic modifications， which collectively lead to a heightened immune response upon secondary stimulation. Furthermore， this review systematically summarizes the role of trained immunity in the initiation and progression of atherosclerosis， and elaborates on various therapeutic strategies targeting trained immunity along with their application prospects.

ObjectiveTo investigate the effect of isoacteoside on the replication process of respiratory syncytial virus （RSV） and its underlying mechanism. MethodsRSV-infected HEp-2 cells （control group） were treated with isoacteoside （isoacteoside-treated group）. Real-time quantitative PCR （qPCR） was used to analyze the mRNA expression levels of the RSV fusion protein （F） gene and matrix protein （M） gene in both groups. Viral titers were determined by plaque assay to assess the inhibitory effect of isoacteoside on viral replication. The number of RSV inclusion bodies formed was determined using cell transfection and laser confocal imaging to evaluate the role of isoacteoside in the formation of inclusion bodies required for RSV replication. 18 seven-week-old female BALB/c mice were randomly divided into three groups： uninfected control group （n=6）， RSV-infected group （n=6）， and RSV-infected group treated with isoacteoside （n=6）. 5 days after infection， the mice were euthanized and lung tissues were collected for pathological analysis to assess the therapeutic effects of isoacteoside on RSV-induced lung tissue injury. ResultsCompared to the control group， the isoacteoside-treated group exhibited the reduced mRNA levels of the RSV F gene （t=17.13， P<0.001） and M gene （t=18.22， P<0.001）， as well as a decrease in viral titer （t=15.32， P<0.001）. The number of inclusion bodies was also significantly reduced （t=16.12， P<0.001）. In the RSV-infected mouse experiment， compared to the RSV-infected group， the isoacteoside-treated group showed a decrease in the levels of inflammatory cytokines IL-4 （t=14.76， P<0.01） and IL-6 （t=21.13， P<0.001） in lung tissues， as well as decreased mRNA levels of the viral load-associated genes F （t=19.52， P<0.001） and M （t=18.76， P<0.001） in lung tissues. Pathological damage to lung tissue caused by RSV infection was alleviated. ConclusionIsoacteoside inhibits RSV replication by interfering with the formation of RSV inclusion bodies and alleviates lung tissue injury induced by RSV infection in mice.

ObjectiveTo evaluate the therapeutic effects of Huanglian Jiedutang on cerebral infarction injury in a mouse model of middle cerebral artery occlusion （MCAO） and to explore its mechanism of action on oligodendrocytes， particularly its potential in myelin repair. MethodsMultiple experimental approaches were used to evaluate cerebral ischemic injury and the effects of drug intervention. Laser speckle imaging was used to detect changes in cerebral blood flow， 2，3，5-Triphenyltetrazolium chloride （TTC） staining was used to measure infarct volume， and neurological function was scored according to the Zea-Longa criteria. Brain tissues were routinely embedded in paraffin and subjected to HE and Nissl staining to observe tissue structure and neuronal damage. Animals were divided into a sham group （n=24）， model group （n=24）， Huanglian Jiedutang group （n=24）， and Ginkgo biloba extract （GBE） group （n=18）. After 1 week of acclimatization， intragastric administration was initiated. The sham and model groups received normal saline， the Huanglian Jiedutang group was administered 1.82 g·kg^-1， and the GBE group was administered 0.432 g·kg^-1 after preparation as a 2.16 g·L^-1 solution. All groups were treated for 5 consecutive days at a dose of 0.2 mL·（10 g）^-¹·d^-¹. The MCAO model was established after the final administration on day 6. Single-cell RNA sequencing was used to analyze brain tissue cellular composition and changes in oligodendrocyte subpopulations. Distinct subpopulations were identified by Uniform manifold approximation and projection （UMAP） dimensionality reduction and unsupervised clustering， and marker gene expression was analyzed. Pathway enrichment and causal inference were further performed using IPA. Finally， real-time quantitative PCR was used to verify mRNA expression changes of myelin-related genes. ResultsCompared with the sham group， the model group showed significantly increased neurological function scores （P<0.01）， significantly impaired blood flow （P<0.01）， significantly enlarged cerebral infarct area （P<0.01）， and pathological changes including disordered cortical structural arrangement， aggravated cytoplasmic vacuolization， and increased Nissl bodies. Compared with the model group， the Huanglian Jiedutang and GBE groups showed significantly decreased neurological function scores （P<0.01）， markedly restored blood flow levels （P<0.01）， significantly reduced cerebral infarct area （P<0.01）， and improvement in cortical structural disorder， alleviation of cytoplasmic vacuolization， and a reduction in Nissl bodies. Single-cell data showed that a myelin-associated oligodendrocyte （Mye-OL） subpopulation existed among oligodendrocytes， which was closely related to myelin generation. Compared with the sham group， the number of Mye-OL cells decreased in the model group. Compared with the model group， the number of Mye-OL cells increased in the Huanglian Jiedutang group. This subpopulation promoted the expression of myelin-related genes， including MOG， MBP， and MAG， via transcription factors such as OLIG1， OLIG2， NKX2-2， and SOX10， thereby regulating myelin generation， restoring cognition， and exerting therapeutic effects on acute cerebral infarction. Compared with the sham group， the mRNA expression levels of OLIG1， OLIG2， NKX2-2， and SOX10 were significantly downregulated in the model group （P<0.01）， and the mRNA expression levels of myelin-related genes， including MOG， MBP， and MAG， were also significantly downregulated （P<0.01）. In contrast， compared with the model group， the Huanglian Jiedutang and GBE groups showed significantly upregulated mRNA expression levels of OLIG1， OLIG2， NKX2-2， and SOX10 （P<0.01）， and significantly upregulated mRNA expression levels of myelin-related genes， including MOG， MBP， and MAG （P<0.01）. ConclusionHuanglian Jiedutang exerts therapeutic effects on acute cerebral infarction by regulating the OLIG1/2-NKX2-2-SOX10 signaling pathway to promote myelin generation by Mye-OL cells.