Objective: To understand the current status and associated factors of sleep problems among preschool children in Hainan Province, so as to provide scientific evidence for improving sleep health in this population. Methods: From January 2021 to June 2022, a total of 4 105 preschool children aged 3-6 years from 62 kindergartens in Hainan Province were selected using stratified cluster random sampling method. Demographic information and lifestyle habits were collected through the Hainan Province Child Growth and Development Survey Questionnaire. The Children s Sleep Habits Questionnaire (CSHQ) was employed to assess sleep status. Unconditional binary Logistic regression model was applied to investigate the associated factors of sleep problems among preschool children. Results: The overall CSHQ score for children was 58.03±18.84, with 80.95% of preschool children exhibiting sleep related issues. The top three most prevalent sleep problem domains were bedtime resistance (72.42%), sleep anxiety ( 54.88 %), and parasomnias (38.86%). Logistic regression analysis revealed that higher family annual income ( OR=0.60, 95%CI = 0.45-0.79), higher maternal education level ( OR=0.53, 95%CI =0.32-0.89), regular or daily vitamin D supplementation ( OR=0.77, 95%CI =0.60-0.99), and fully self initiated eating behavior ( OR=0.71, 95%CI =0.59-0.85) were negatively related with children s sleep problems; in addition, screen exposure ( OR=1.27, 95%CI =1.06-1.51) and picky eating ( OR= 1.47 , 95%CI =1.21-1.78) were positively related to children s sleep problems (all P <0.05). Conclusion The high detection rate of sleep problems among preschool children in Hainan Province is multifactorially associated with family environment, dietary habits, and lifestyle behaviors.

ObjectiveTo investigate the mechanism by which Anmei Dan （AMD） regulates recombinant sirtuin 3/nuclear factor erythroid 2-related factor 2（SIRT3/Nrf2） signaling to ameliorate mitochondrial oxidative stress and improve the cognitive function in aged sleep-deprived mice. MethodsSixty aged C57 mice were randomly assigned to a control group， a model group， a melatonin （1.3 mg·kg^-1·d^-1） group， and high-， medium-， and low-dose （26.26， 13.13， 6.565 g·kg^-1·d^-1， respectively） AMD groups， with ten mice per group. Continuous sleep deprivation was administered for 4 weeks via a custom-built sleep deprivation chamber. The cognitive function of mice was assessed via the Morris water maze test. Hematoxylin-eosin （HE） staining was performed to observe morphological alterations in pyramidal neurons. Biochemical assays were carried out to measure the hippocampal and serum levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， catalase （CAT）， and total antioxidant capacity （T-AOC）. Transmission electron microscopy was adopted to examine mitochondrial pathological alterations in the hippocampus. Immunohistochemical assay was conducted to examine the expression of nuclear factor E₂-related factor 2 （Nrf2）， sirtuin 3 （SIRT3）， peroxisome proliferator-activated receptor gamma coactivator-1α （PGC-1α）， mitochondrial transcription factor A （TFAM）， and brain-derived neurotrophic factor （BDNF） in the mouse hippocampus. The protein levels of Nrf2， SIRT3， heme oxygenase-1 （HO-1）， and NAD（P）H：quinone oxidoreductase 1 （NQO1） in mouse hippocampal tissue were determined by Western blot. Immunofluorescence double labeling was employed to determine the protein levels of SIRT3/Nrf2 in mouse hippocampal tissue. ResultsCompared with the control group， the model group exhibited prolonged latency to navigate， increased total swimming distance， reduced number of platform crossings， and shortened time spent in the target quadrant （P<0.05， P<0.01）， disorganized morphology and arrangement of hippocampal neurons， with increased damaged mitochondria， mitochondrial swelling， reduced cristae， and vacuolization， declined levels of SOD， GSH-Px， CAT， and T-AOC and elevated levels of MDA in the hippocampus and serum （P<0.01）， and downregulated protein levels of SIRT3， Nrf2， PGC-1α， TFAM， BDNF， HO-1， and NQO1 （P<0.01）. Compared with the model group， the melatonin group and AMD groups exhibited shortened spatial navigation latency， reduced total swimming distance， increased number of platform crossings， and prolonged activity time in the target quadrant （P<0.05， P<0.01）， reduced neuronal damage and mitochondrial damage in the hippocampal tissue， declined level of MDA and elevated levels of SOD， GSH-Px， CAT， and T-AOC in the hippocampus and serum （P<0.05， P<0.01）， and upregulated protein levels of SIRT3， Nrf2， PGC-1α， TFAM， BDNF， HO-1， and NQO1 in the hippocampus （P<0.05， P<0.01）. ConclusionAMD may improve the learning and memory in aged sleep-deprived mice by mediating mitochondrial oxidative damage through the SIRT3/Nrf2 signaling pathway.

ObjectiveTo investigate whether Anmei Dan （AMD） improves cognitive function and alleviates circadian rhythm disorders in an aged mouse model of sleep deprivation by inhibiting the erythropoietin-producing hepatocellular receptor A4 （EphA4）/Eph receptor-interacting protein A3 （EphrinA3） signaling pathway and its downstream Ras homolog family member A （RhoA）/Rho-associated coiled-coil containing protein kinase （ROCK） signaling， and to analyze its multi-target regulatory characteristics. MethodsAn aged C57 mouse model of sleep deprivation was established by the modified multiple platform water environment method. The mice were randomized into blank， model， rhynchophylline （Rhy， antagonist， 50 mg·kg^-1·d^-1）， and antagonist plus AMD （26.26 g·kg^-1·d^-1） groups. Spontaneous activity was assessed by the open field test， and circadian rhythm activity was monitored. Hematoxylin-eosin （HE） staining and Nissl staining were conducted to observe neuronal morphology in the hippocampal CA1/CA3 regions. Western blot and immunofluorescence assay were employed to determine the protein levels of EphA4， EphrinA3， Neuronal Nuclei Antigen （NEUN）， glial fibrillary acidic protein （GFAP）， phosphorylated （p）-EphA4， RhoA， Rho-associated coiled-coil containing protein kinase 1 （ROCK1）， Rho-associated coiled-coil containing protein kinase 2 （ROCK2）， brain-derived neurotrophic factor （BDNF）， synaptophysin （SYN）， postsynaptic density protein 95 （PSD95）， and growth-associated protein 43 （GAP43）. The mRNA levels of EphA4 and EphrinA3 were determined by real-time PCR. ResultsCompared with the blank group， the model group showed decreased spontaneous activity （P<0.01）， circadian rhythm disruption （P<0.01）， hippocampal neuron disarrangement and reduced Nissl bodies， upregulated protein levels of EphA4， EphrinA3， NEUN， p-EphA4， RhoA， ROCK1， ROCK2， and GFAP （P<0.01）， and downregulated protein levels of NEUN， BDNF， SYN， PSD95， and GAP43 （P<0.01）. Compared with the model group， both the antagonist group and the antagonist plus AMD group showed recovery in the above indicators （P<0.05， P<0.01）， with the combination group outperforming the antagonist group in behavioral， pathological， and molecular expression aspects （P<0.05， P<0.01）. ConclusionAMD can alleviate cognitive and rhythm disorders caused by sleep deprivation by inhibiting the EphA4/EphrinA3 signaling pathway and its downstream RhoA/ROCK signaling， and upregulating the expression of synapse-related proteins such as BDNF. Its combination with an EphA4 antagonist exhibits a synergistic effect， which suggests that the mechanism involves multi-link synergistic intervention.

ObjectiveTo investigate the effects of Anmei Dan （AMD） on the expression of proteins in the C-X3-C motif chemokine ligand 1 （CX3CL1）/C-X3-C chemokine receptor 1 （CX3CR1） signaling pathway， hippocampal inflammation， and learning and memory in an aged sleep-deprived model. MethodsSixty aged C57 mice were randomly assigned into a control group， a model group， a melatonin group （1.3 mg·kg^-1·d^-1）， and high-， medium-， and low-dose AMD groups （26.26， 13.13， 6.565 g·kg^-1·d^-1， respectively）， with 10 mice per group. Continuous sleep deprivation was administered for 4 weeks through a custom-built sleep deprivation chamber. The cognitive function of mice was assessed via the Y-maze test. Histomorphological alterations in pyramidal cells of the hippocampal CA1 and DG regions were examined by hematoxylin-eosin （HE） and Nissl staining. Synaptic morphology in the hippocampus was visualized with Golgi staining. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of inflammatory cytokines interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in the hippocampal tissue as well as the level of interleukin-10 （IL-10） in the prefrontal cortex. Real-time PCR was performed to detect markers of M1-type ［cluster of differentiation （CD） 86 and inducible nitric oxide synthase （iNOS）］ and M2-type ［CD206 and arginase 1 （Arg1）］ macrophages. Western blot was employed to quantify the protein levels of CX3CL1， CX3CR1， phosphorylated nuclear factor κB p65 subunit （p-NF-κB p65）， and phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK） in the hippocampus. Immunofluorescence double staining was performed to co-label CX3CR1 with ionized calcium-binding adapter molecule 1 （Iba-1） for observation of the co-localization of target proteins with microglia. ResultsCompared with the control group， the model group exhibited disrupted daily activity patterns with reduced spontaneous alternation rates （P<0.01）， disorganized morphology and arrangement of hippocampal neurons， accompanied by reduced numbers of Nissl bodies and dendritic spines （P<0.01）， upregulated protein levels of IL-6， IL-10， IL-1β， TNF-α， CX3CL1， CX3CR1， p-NF-κB p65， p-p38 MAPK， and Iba-1 and mRNA levels of CD86 and iNOS （P<0.01）， and downregulated mRNA levels of CD206 and Arg1 （P<0.05，P<0.01）. Compared with the model group， the melatonin group and medium- and high-dose AMD groups showed increased spontaneous alternation rates （P<0.01）， improved neuronal morphology， number， and spine density in the hippocampal CA1 and DG regions （P<0.01）， declined levels of IL-6， IL-10， IL-1β， and TNF-α （P<0.05， P<0.01）， downregulated protein levels of CX3CL1， CX3CR1， p-NF-κB p65， p-p38 MAPK， and Iba-1 and mRNA levels of CD86 and iNOS， and upregulated mRNA levels of CD206 and Arg1 （P<0.01）. ConclusionAMD may improve the learning and memory in aged sleep-deprived mice by mediating neuroinflammation through the CX3CL1/CX3CR1 signaling pathway.

OBJECTIVES: To evaluate the protective effect of Schistosoma japonicum cystatin (rSj-Cystatin) in a mouse mode of "two-hit" sepsis. METHODS: Sixty male C57BL/6 mice randomized equally into sham-operated group, protein group, "two-hit" modeling group, and protein intervention group. In the former two groups, the mice received an intraperitoneal injection of 100 μL PBS followed by exposure of the cecum and then by intraperitoneal injection of 100 μL PBS or 25 μg rSj-Cystatin 30 min later; In the latter two groups, 100 μL PBS containing LPS (5 mg/kg) was injected intraperitoneally 24 h before cecal ligation and puncture (CLP), and 100 μL PBS or 25 μg rSj-Cystatin were injected 30 min after CLP. At 12 h after rSj-Cystatin treatment, 6 mice from each group were sacrificed for detection of TNF-α, IL-6, IL-10, TGF-β, iNOS and Arg-1 in the serum, spleen, liver, lung and kidney tissues using ELISA, for examinations of liver, lung and kidney pathologies with HE staining, and for analysis of CD3⁺CD4⁺CD25⁺Foxp3⁺ T cell percentage in the spleen using flow cytometry. The remaining mice were observed for general condition and 72-h survival. RESULTS: The 72-h survival rates in the 4 groups were 100%, 100%, 0% and 20%, respectively, showing significant differences between the latter two groups. The mouse models of "two-hit" sepsis exhibited obvious tissue pathologies and significant elevations of TNF-α and IL-6 in both the serum and tissue homogenate, which were significantly ameliorated by rSj-Cystatin treatment. Treatment with rSj-Cystatin also increased IL-10 and TGF-β levels and spleen CD3⁺CD4⁺CD25⁺Foxp3⁺ T cell percentage. The septic mouse models also showed increased iNOS levels in all the detected tissues and a decreased Arg-1 level in the kidney, and these changes were obviously improved by rSj-Cystatin treatment. CONCLUSIONS rSj-Cystatin has a protective effect against "two-hit" sepsis in mice by regulating the inflammatory microenvironment.
Animals ; Mice ; Sepsis/drug therapy* ; Male ; Schistosoma japonicum/chemistry* ; Mice, Inbred C57BL ; Cystatins/therapeutic use* ; Interleukin-10/metabolism* ; Interleukin-6/blood* ; Tumor Necrosis Factor-alpha/blood* ; Disease Models, Animal ; Transforming Growth Factor beta/metabolism*

Rheumatoid arthritis (RA) is an autoimmune disease characterized by complex pathogenesis, with its development closely linked to immune dysregulation. Regulatory T cells (Tregs), as specialized immunomodulatory cells, play a pivotal role in negatively regulating immune responses and maintaining autoimmune tolerance. In recent years, extensive research has focused on the relationship between Tregs and RA pathogenesis, with the functional role of CD8⁺ Tregs as a critical area of investigation. This review summarizes the alterations in CD8⁺ Tregs during RA progression and their potential mechanisms of action. By elucidating the diversity of CD8⁺ Treg subsets and their intricate roles in modulating immune responses, this analysis provides novel insights and therapeutic strategies for RA diagnosis and treatment.
Arthritis, Rheumatoid/pathology* ; Humans ; T-Lymphocytes, Regulatory/immunology* ; CD8-Positive T-Lymphocytes/immunology* ; Animals

Objectives:To explore whether short-course radiotherapy (SCRT)-based total neoadjuvant therapy (TNT) combined with PD-1 inhibitors could further promote tumor regression and improve the prognosis.Methods:This is a prospective, multicenter, two-arm randomized controlled, seamless phase Ⅱ/Ⅲ trial for proficient mismatch repair or microsatellite stable (pMMR/MSS) locally advanced rectal cancer (LARC). Eligible patients were randomly assigned to the iTNT (TNT+PD-1) group or the TNT group. Patients in the TNT group received SCRT (5 Gy×5) followed by 4 cycles of CAPOX or 6 cycles of mFOLFOX chemotherapy, with the iTNT group receiving SCRT followed by the same regime in combination with 4 cycles of Sintilimab. Total mesorectal excision (TME) surgery or watch and wait (W&W) was performed after neoadjuvant therapy and then 2 cycles of same regimen as before were recommended. The primary endpoints are the complete response (CR) rate for phase Ⅱ trial and 3-year disease-free survival (DFS) for phase Ⅲ trial. A total of 588 patients will be enrolled for the phase Ⅱ/Ⅲ trial. Short-term efficacy and safety data from the initial 100 treated patients were analyzed as planned.Results:From 2022-8-31 to 2023-5-24 the initial 100 patients were enrolled from 10 hospitals in China, 76.0%(76/100) patients were male, and the median age was 61 years (21-74 years). More patients had tumors located in the lower rectum (78.0%, 78/100), staged T3-4 (97.0%, 97/100) and N1-2 (93.0%, 93/100), and about half of the tumors invaded the mesorectal fascia (52.0%, 52/100) and with extramural vascular invasion (51.0%, 51/100). Analyses were performed according to the per-protocal (PP) set. All patients in the iTNT group ( n=52) and the TNT group ( n=48) completed SCRT; The 4-cycle chemotherapy±Sintilimab completion rates were 86.5% and 100.0% in the iTNT and TNT groups, respectively. In the iTNT group, 82.7% (43/52), 11.5% (6/52), and 5.8% (3/52) of the patients received 4, 3, and 2 cycles of PD-1 inhibitor. After TNT, 68 patients underwent radical surgery and 15 patients achieved cCR and adopted W&W. The pathological complete response (pCR) rates were 48.5% (16/33) and 17.1% (6/35) in the iTNT and TNT groups, with CR rates of 50.0% (25/50) and 26.1% (12/46), respectively. The incidence of treatment-related grade 3-4 adverse events was 26.9% (14/52, iTNT group) and 18.8% (9/48, TNT group), with thrombocytopenia and leukopenia being the most common. Among patients receiving immunotherapy, grade 3 immunotherapy-related adverse events occurred in 2 (3.8%, 2/52) patients: one case was pancreatitis, another case was hepatitis combined with myositis and myocarditis. Conclusion:The preliminary results show that SCRT-based TNT combined with PD-1 inhibitors could further improve the CR rate for LARC without unexpected serious adverse events.

Objective:To study the effects of obesity on alveolar bone loss and gut microbiota in mice with periodontitis.Methods:Twenty-four seven-week-old female C57BL/6J mice were randomly divided into four groups based on table of random numbers ( n=6 in each group): normal-fat diet group (NFD group), high-fat diet group (HFD group), normal-fat diet and periodontitis group (NFD_PD group) and high-fat diet and periodontitis group (HFD_PD group). NFD and HFD groups were fed with normal or high-fat diet for twelve weeks respectively; NFD_PD and HFD_PD groups were induced to periodontitis by ligating the bilateral maxillary second molars with 5-0 silk thread at the fourth week after feeding with normal or high-fat diet respectively. The body weight was measured weekly. The mice were euthanized for collecting the samples at the end of the 12th week. Liver, kidneys, perirenal and retroperitoneal fat were weighed. Serum was collected to detect the level of serum lipids and inflammatory factors. The right maxilla bones were scanned by micro-CT. HE staining was performed to observe the periodontal tissue. The cecum contents were collected for gut microbiota 16S rRNA gene sequencing. Spearman correlation analysis was performed to analyze the correlation between the abundance of gut microbiota and serum inflammatory level and CT value. Results:After 12 weeks of high-fat diet fed, the body weight of HFD group [(26.52±1.96) g] was significantly higher than that of NFD group [(20.95±0.63) g] ( t=6.63, P<0.001). The body weight of HFD_PD group [(23.82±1.12) g] was significantly higher than that of NFD_PD group [(20.73±0.47) g] ( t=6.23, P=0.001). The serum levels of total cholesterol, triglyceride and low density lipoprotein in HFD group and HFD_PD group were significantly higher than those in NFD group and NFD_PD group ( P<0.01). The distance from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC) on the mesial site of maxillary second molar in HFD_PD group [(647.46±47.46) μm] was significantly higher than that in NFD_PD group [(440.48±68.08) μm] ( t=5.58, P<0.001). HE staining showed that the maxillary second molar attachment loss, collagen fiber destruction and inflammatory cell infiltration were more significant serious in HFD_PD group compared with NFD_PD group. The levels of interleukin (IL)-1β, IL-6 and monocyte chemotactic protein-1 (MCP-1) of serum in HFD_PD group [(17.11±1.92), (31.61±3.20) and (204.42±35.96) ng/L, respectively] were significantly higher than those in NFD_PD group [(10.44±1.65), (19.96±2.09) and (147.36±10.76) ng/L, respectively] ( P<0.001, P<0.001, P=0.004). The 16S rRNA gene analysis revealed that the Bacteroides/Firmicutes ratio in HFD_PD group (4.00±3.30) was significantly higher than that in NFD_PD group (0.62±0.19) ( t=2.50, P=0.030). The abundance of Oscillospira in HFD_PD group [(12.25±0.05) %] was significantly higher than that in NFD_PD group [(2.80±0.01) %] ( t=4.64, P<0.001). The abundance of Parabacteroides in HFD_PD group [(0.25±0.27)% ] was significantly lower than that in NFD_PD group [(2.04±0.02)%] ( t=2.32, P=0.043). The β-diversity analysis of gut microbiota based on Bray-Curtis distance showed that samples of HFD_PD group and NFD_PD group were obviously grouped. Correlation analysis showed that the abundance of Oscillospira was positively correlated with IL-1β, IL-6, MCP-1 concentration and CEJ-ABC value in serum significantly ( r values were 0.80, 0.79, 0.80, 0.89, P<0.05). The abundance of Parabacteroides was negatively correlated with IL-1β, IL-6 concentration and CEJ-ABC value in serum significantly ( r values were -0.71, -0.71, -0.86, -0.95, P<0.05). Conclusions:Obesity promotes alveolar bone resorption in periodontitis mice and changes the gut microbiota. Oscillospira and Parabacteroides may play a key role.

Objective To evaluate the protective effect of Schistosoma japonicum cystatin(rSj-Cystatin)in a mouse mode of"two-hit"sepsis.Methods Sixty male C57BL/6 mice randomized equally into sham-operated group,protein group,"two-hit"modeling group,and protein intervention group.In the former two groups,the mice received an intraperitoneal injection of 100 μL PBS followed by exposure of the cecum and then by intraperitoneal injection of 100 μL PBS or 25 μg rSj-Cystatin 30 min later;In the latter two groups,100 μL PBS containing LPS(5 mg/kg)was injected intraperitoneally 24 h before cecal ligation and puncture(CLP),and 100 μL PBS or 25 μg rSj-Cystatin were injected 30 min after CLP.At 12 h after rSj-Cystatin treatment,6 mice from each group were sacrificed for detection of TNF-α,IL-6,IL-10,TGF-β,iNOS and Arg-1 in the serum,spleen,liver,lung and kidney tissues using ELISA,for examinations of liver,lung and kidney pathologies with HE staining,and for analysis of CD3+CD4+CD25+Foxp3+T cell percentage in the spleen using flow cytometry.The remaining mice were observed for general condition and 72-h survival.Results The 72-h survival rates in the 4 groups were 100％,100％,0％and 20％,respectively,showing significant differences between the latter two groups.The mouse models of"two-hit"sepsis exhibited obvious tissue pathologies and significant elevations of TNF-α and IL-6 in both the serum and tissue homogenate,which were significantly ameliorated by rSj-Cystatin treatment.Treatment with rSj-Cystatin also increased IL-10 and TGF-β levels and spleen CD3+CD4+CD25+Foxp3+T cell percentage.The septic mouse models also showed increased iNOS levels in all the detected tissues and a decreased Arg-1 level in the kidney,and these changes were obviously improved by rSj-Cystatin treatment.Conclusion rSj-Cystatin has a protective effect against"two-hit"sepsis in mice by regulating the inflammatory microenvironment.

Background: Metals and their ions have been used to reduce bacterial infection risks. Among them, minerals containing rubidium (MCR), natural minerals containing metal ions, show potential as novel and tunable materials. Objective: This study aimed to investigate the antibacterial activity and mechanism of MCR. Methods: The inhibitory effect of MCR on bacteria was clarified using the growth curve method, turbidimetric method, and minimum inhibitory concentration method. Physiological and biochemical indices were employed to investigate the inhibitory mechanism of MCR. Results: The results revealed that MCR inhibited Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli with minimum inhibitory concentrations of 11.95, 2.60, and 2.60 mg/mL, respectively. The inhibitory activity of MCR was insignificant against Bacillus subtilis, Salmonella typhimurium, and Helicobacter pylori at 3.25 mg/mL. Mechanistic assessments showed that MCR affected bacterial conductivity, protein and nucleic acid levels, reducing sugar content, respiratory chain dehydrogenase activity, bacterial lipid peroxidation, intracellular adenosine triphosphate, and extracellular alkaline phosphatase. Conclusion: MCR has bacteriostatic activity and the mechanism primarily involves adhesion to bacteria, disrupting the integrity of their cell walls and membranes, and altering their permeability. This disruption leads to the release of intracellular molecules of various sizes, inhibiting cellular respiration and metabolism, and causing oxidative damage. These combined effects impair cellular functions, affecting cell growth and metabolism, or leading to cell death. These findings provide a theoretical reference for the development of MCR as a bacteriostatic agent.