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.

BackgroundWith the rapid development of the networking technologies， internet addiction has increasingly become a serious mental health issue. Previous studies have revealed the link between childhood trauma and internet addiction， while the mediating role of perceived stress in this link is not yet clear. ObjectiveTo investigate the role of medical students' perceived stress in the relationship between childhood trauma and internet addiction， so as to provide references for the intervention of internet addiction. MethodsFrom February to March 2023， a random sampling technique was used to select 1 232 undergraduate students from the School of Clinical Medical Sciences of Southwest Medical University as research subjects. The Childhood Trauma Questionnaire-Short Form （CTQ-SF）， Perceived Stress Scale （PSS）， Internet Gaming Disorder Scale （IGDS）， and Bergen Social Media Addiction Scale （BSMAS） were used for assessment. Pearson's correlation coefficients were calculated. The mediation effect of perceived stress in the relationship between childhood trauma and internet addiction was tested using Model 4 in the SPSS Process 4.1， and Bootstrapping procedure involving 5 000 replicates was employed to confirm the statistical significance. ResultsA total of 1 016 （82.47%） valid completed questionnaires were gathered. The CTQ-SF scores of medical students were positively correlated with PSS scores， IGD scores， and BSMAS scores （r=0.583， 0.474， 0.465， P<0.01）. PSS scores were positively correlated with IGD scores and BSMAS scores （r=0.369， 0.479， P<0.01）. Childhood trauma in medical students was found to positively predict perceived stress （β=0.191， P<0.01）， social media addiction （β=0.160， P<0.01）， and internet gaming disorder （β=0.106， P<0.01）. Perceived stress played a significant mediating role in the relationship between childhood trauma and internet gaming disorder， indirect effect value was 0.018 （95% CI： 0.009~0.027）， accounting for 16.98%. Perceived stress also exhibited a significant mediating role in the relationship between childhood trauma and social media addiction， indirect effect value was 0.063 （95% CI： 0.048~0.079）， accounting for 39.38%. ConclusionChildhood trauma in medical students may affect internet gaming disorder and social media addiction through perceived stress. ［Funded by 2022 Annual Research Project of Sichuan Applied Psychology Research Center，（number，CSXL-22102）］

Objective: To explore the role of the c.598G>A mutation of the ITGB3 gene in the occurrence of fetal and neonatal alloimmune thrombocytopenia (FNAIT) through its expression in vitro. Methods: The platelet antibodies in the sera of the affected neonate and her mother were detected using commercial enzyme-linked immunosorbent assay (ELISA), solid-phase agglutination, flow cytometry and the gold standard monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The common human platelet antigen (HPA) genotypes of the neonate and her parents were obtained using the HPA-SSP method. The presence of mutations was analyzed by sequencing the exons of the ITGB3 and ITGA2B genes. The target gene of ITGB3 was obtained by PCR amplification using the existing human platelet cDNA. The wild-type ITGB3 eukaryotic expression vector was constructed by TA cloning technology. The 598G>A mutant ITGB3 eukaryotic expression vector was obtained by point mutation, and the plasmid DNA was co-transfected with that of ITGA2B (αⅡb) into HEK293 cells. The transfected cells stably expressing GP Ⅱb/Ⅲa were screened and obtained. The expression of GP Ⅱb/Ⅲa in 598G>A mutant transfected cells and the presence of antibodies against this mutation in the serum of mother were detected by flow cytometry and MAIPA. Results: Antibodies against HLA-class Ⅰ and GP Ⅱb/Ⅲa glycoproteins were detected in the serum of the neonate's mother, and subsequent HLA antibody-specific testing confirmed the presence of antibodies against HLA-B 57∶01 and A 02∶05. ITGB3 sequencing showed that the neonate and her father carried the c.598G>A point mutation, which results in the change of glutamate to lysine at position 200. Antibodies against GP Ⅱb/Ⅲa glycoproteins were not detected using constructed c.598G>A mutant transfected cells reacted with the maternal serum. Conclusion: The in vitro expression and analysis of the ITGB3 c.598G>A mutation did not support a role for this mutation in the pathogenesis of FNAIT. The establishment of this method facilitates the discovery of new platelet low-frequency antigens, and provides a theoretical foundation for the detection of antibodies against platelet antigens associated with patients with adverse pregnancy and childbirth histories.

ObjectiveTo investigate the potential mechanisms of Zhimu （Anemarrhena asphodeloides）-Huangbai （Phellodendron amurense） medicinal pair in alleviating postmenopausal osteoporosis （PMOP）. MethodsSixty unpregnant female SD rats were randomly divided into five groups， blank group， model group， low-dose Zhimu-Huangbai group， high-dose Zhimu-Huangbai group， and estradiol group， with 12 rats in each group. Except for the blank group， all other groups had their ovaries removed to create PMOP rat models， while the blank group only had the fat tissue around the ovaries removed. One week after the ovarian removal， the low-dose and high-dose Zhimu-Huangbai groups received concentrated solution of Zhimu and Huangbai with 1.8， 7.2 g/（kg·d） via gavage， the estradiol group received estradiol solution 0.09 mg/（kg·d） via gavage， and the blank group and the model group received 10 ml/（kg·d） of normal saline via gavage， once daily for 12 weeks. Before sampling， the body mass of the rats was recorded， and uterine tissue was taken to calculate the uterine index. The levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in serum were detected by ELISA； micro CT was used to examine the parameters of femoral microstructure， including bone volume/tissue volume （BV/TV）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， bone mineral density （BMD）， trabecular separation （Tb.Sp）， and cortical bone area （Ct.Ar）. HE staining was used to observe pathological changes in the femur； RT-qPCR was used to detect the mRNA expression of osteogenic-adipogenic differentiation-related factors in femoral tissue， including Runt-related transcription factor 2 （Runx2）， bone morphogenetic protein 2 （BMP-2）， peroxisome proliferator-activated receptor γ （PPARγ）， chemerin and chemokine-like receptor 1 （CMKLR1）. ResultsCompared with the blank group， the model group showed a significant increase in body mass， a significant decrease in the uterine index， BV/TV， Tb.N， Tb.Th and BMD， a significant increase in Tb.Sp， and serum IL-1β， IL-6， and TNF-α levels， a significant reduction of mRNA expression of Runx2 and BMP-2 in bone tissue， and a significant increased mRNA expression of PPARγ， chemerin， and CMKLR1 （P＜0.01）. HE staining revealed that the femoral tissue showed a reduction and sparsity of trabeculae， a significant enlargement of the medullary cavity， and a large number of fat cells. Compared to the model group， the low-dose， high-dose Zhimu-Huangbai groups， and estradiol group showed significant improvements in all the above-mentioned indicators （P＜0.05 or P＜0.01）. HE staining revealed a significant increase in trabeculae， more organized arrangement， and a marked reduction in fat cells. Compared to low-dose Zhimu-Huangbai group， the high-dose Zhimu-Huangbai group exhibited a significant increase in the uterine index and BMD， and a significant reduction in body mass and PPARγ and Chemerin mRNA expression （P＜0.05 or P＜0.01）. Compared to high-dose Zhimu-Huangbai group， the estradiol group showed a decrease in uterine index， BV/TV， Tb.N， Tb.Th， BMD， and BMP-2 mRNA expression， while the levels of IL-1β， TNF-α， and IL-6， as well as Tb.Sp and the mRNA expressions of PPARγ， chemerin， and CMKLR1 increased （P＜0.05 or P＜0.01）. ConclusionThe Zhimu-Huangbai medicinal pair can alleviate PMOP bone loss， and its mechanism of action is related to reducing the levels of inflammatory factors， correcting the disorder of osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells （BMSCs）， and promoting the differentiation of BMSCs into osteoblasts.

Objective: To explore the role of the c.598G>A mutation of the ITGB3 gene in the occurrence of fetal and neonatal alloimmune thrombocytopenia (FNAIT) through its expression in vitro. Methods: The platelet antibodies in the sera of the affected neonate and her mother were detected using commercial enzyme-linked immunosorbent assay (ELISA), solid-phase agglutination, flow cytometry and the gold standard monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The common human platelet antigen (HPA) genotypes of the neonate and her parents were obtained using the HPA-SSP method. The presence of mutations was analyzed by sequencing the exons of the ITGB3 and ITGA2B genes. The target gene of ITGB3 was obtained by PCR amplification using the existing human platelet cDNA. The wild-type ITGB3 eukaryotic expression vector was constructed by TA cloning technology. The 598G>A mutant ITGB3 eukaryotic expression vector was obtained by point mutation, and the plasmid DNA was co-transfected with that of ITGA2B (αⅡb) into HEK293 cells. The transfected cells stably expressing GP Ⅱb/Ⅲa were screened and obtained. The expression of GP Ⅱb/Ⅲa in 598G>A mutant transfected cells and the presence of antibodies against this mutation in the serum of mother were detected by flow cytometry and MAIPA. Results: Antibodies against HLA-class Ⅰ and GP Ⅱb/Ⅲa glycoproteins were detected in the serum of the neonate's mother, and subsequent HLA antibody-specific testing confirmed the presence of antibodies against HLA-B 57∶01 and A 02∶05. ITGB3 sequencing showed that the neonate and her father carried the c.598G>A point mutation, which results in the change of glutamate to lysine at position 200. Antibodies against GP Ⅱb/Ⅲa glycoproteins were not detected using constructed c.598G>A mutant transfected cells reacted with the maternal serum. Conclusion: The in vitro expression and analysis of the ITGB3 c.598G>A mutation did not support a role for this mutation in the pathogenesis of FNAIT. The establishment of this method facilitates the discovery of new platelet low-frequency antigens, and provides a theoretical foundation for the detection of antibodies against platelet antigens associated with patients with adverse pregnancy and childbirth histories.

Objective: To explore the relationship between beverage dependence and sleep quality among college students, providing empirical evidence for improving their sleep quality. Methods: From December 2024 to January 2025, a convenience sampling method was used to conduct a questionnaire survey among 3 974 college students from four universities in Anhui Province. The Beverage Addiction Scale for College Students (BASCS) was used to assess beverage dependence, and the Self rating Scale of Sleep(SRSS) was used to evaluate sleep quality. A multivariate Logistic regression model was employed to analyze the relationship between beverage dependence and sleep quality, and a restricted cubic spline model was used to examine the dose response relationship between the two. Results: The positive rate of beverage dependence symptoms among college students was 7.6%, with positive rates of 9.6%, 13.8%, and 7.4% for the withdrawal symptoms, health effects, and dependence symptoms dimensions, respectively. The detection rate of sleep disorders was 23.6%. Multivariate Logistic regression analysis showed that after adjusting for covariates such as grade, gender, and body mass index, compared with the no beverage dependence group, students with positive beverage dependence symptoms had a higher risk of sleep disorders( OR =3.71, 95% CI =2.87-4.80, P <0.01). The OR (95% CI ) for sleep disorders among students with positive symptoms in the withdrawal symptoms, health effects, and dependence symptoms dimensions were 2.80(2.22-3.53), 2.38(1.95-2.91), and 2.45(1.89-3.18)(all P <0.01). Further analysis using a restricted cubic spline model revealed that the overall beverage dependence score and its three dimensional scores were approximately linearly related to the risk of sleep disorders among college students (all nonlinear P >0.05). Conclusions Beverage dependence is associated with sleep quality among college students. Schools should take multiple approaches, such as health education on beverage awareness, to improve students sleep quality.

Objective To analyze the epidemiological characteristics of mumps in Guangxi from 2012 to 2024, and to provide a scientific basis for formulating prevention and control strategies. Methods Descriptive epidemiological methods were used to analyze the incidence data of mumps in Guangxi from 2012 to 2024. Results A total of 159 873 mumps cases were reported from 2012 to 2024 in Guangxi, with an average annual reported incidence of 25.41/100 000, and no death. Mumps occurred every month, with the peak incidence mainly concentrated in April to July and October to January of the next year. There were 96,118 male cases (29.43 /100 000), and 63 755 female cases (21.07 /100 000). The male to female ratio was 1.40:1, and the difference between male and female was significant (χ²=4 321.276，P＜0.05). The annual incidence of mumps showed a certain periodic change, with the incidence peak and trough alternating every 4 - 5 years. The majority of patients were under 15 years old, accounting for 85.32% of the total number of cases. The patients mainly included students, preschool children and scattered children. The highest average incidence was in Nanning City with 40 231 cases (42.08/100 000), and the lowest was in Qinzhou City with 3 466 cases (8.16/100 000). From 2012 to 2024, a total of 210 mumps outbreaks with 4 483 cases were reported in Guangxi. Conclusion The incidence of mumps in Guangxi from 2012 to 2024 shows a periodic change and obvious seasonality. People under 15 years old are the key group at risk of mumps. The prevention and control of the epidemic of mumps in schools and kindergartens should be strengthened. It is suggested to carry out long-term monitoring of mumps as well as immune effect research, and continue to maintain a high vaccination rate of 2 doses of mumps-containing vaccines.

BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*