ObjectiveTo investigate the mechanisms by which Mahuang Lianqiao Chixiaodoutang （MLC） improves obesity-type polycystic ovary syndrome （PCOS） through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. MethodsThirty-six female Sprague-Dawley （SD） rats were randomly divided into a blank control group （Con） and an obesity-type PCOS model preparation group. The model was induced by gavage with letrozole （1 mg·kg^-1） combined with a high-fat diet （HFD）. After model establishment， the obesity-type PCOS model preparation group was further divided into the model group （Mod， normal saline）， metformin group （Met， 0.3 g·kg^-1）， low-dose MLC group （MLC-L， 4.3 g·kg^-1）， medium-dose MLC group （MLC-M， 8.6 g·kg^-1）， and high-dose MLC group （MLC-H， 17.2 g·kg^-1）. Active components of MLC and targets of obesity-type PCOS were screened from databases， a protein-protein interaction （PPI） network was constructed， and gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was performed. The gut microbiota structure was analyzed based on 16S rRNA sequencing and correlated with network pharmacology pathways. Body weight and estrous cycle were dynamically monitored. Ovarian morphology was observed by hematoxylin-eosin （HE） staining. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL）. Enzyme-linked immunosorbent assay （ELISA） was used to detect levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， anti-Müllerian hormone （AMH）， testosterone （T）， and estradiol （E₂）. Western blot was used to detect the protein expression levels of phosphorylated PI3K/PI3K （p-PI3K/PI3K）， phosphorylated Akt/Akt （p-Akt/Akt）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. ResultsNetwork pharmacology screening identified 124 active components of MLC and 408 overlapping targets between the herbal formula and the disease. Core targets such as Akt1 and Bcl-2 were revealed. As indicated by 16S rRNA sequencing， the abundances of Lachnospiraceae， Lachnoclostridium， and Dorea were increased in the MLC groups （P<0.05）， while the abundance of Veillonella was decreased （P<0.05）. KEGG correlation analysis integrating network pharmacology and gut microbiota data showed significant enrichment of the PI3K/Akt signaling pathway. Animal experiments showed that， compared with the Mod group， body weight decreased to normal levels in the Met， MLC-M， and MLC-H groups. The estrous cycle became regular. The number of corpora lutea increased and cystic follicles decreased. Serum levels of T， FSH， and LH/FSH were reduced （P<0.05， P<0.01）， while the E₂ level was increased （P<0.01）. Ovarian cell apoptosis was reduced （P<0.01）， and the protein expression levels of p-PI3K/PI3K， p-Akt/Akt， and Bcl-2 in ovarian tissue were significantly increased， whereas Bax protein expression was significantly decreased （P<0.05， P<0.01）. ConclusionMLC can regulate gut microbiota structure， effectively improve ovarian pathology in rats with obesity-type PCOS， and inhibit ovarian granulosa cell apoptosis. The mechanism may be associated with upregulation of the PI3K/Akt signaling pathway.

ObjectiveTo investigate the mechanisms by which Mahuang Lianqiao Chixiaodoutang （MLC） improves obesity-type polycystic ovary syndrome （PCOS） through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. MethodsThirty-six female Sprague-Dawley （SD） rats were randomly divided into a blank control group （Con） and an obesity-type PCOS model preparation group. The model was induced by gavage with letrozole （1 mg·kg^-1） combined with a high-fat diet （HFD）. After model establishment， the obesity-type PCOS model preparation group was further divided into the model group （Mod， normal saline）， metformin group （Met， 0.3 g·kg^-1）， low-dose MLC group （MLC-L， 4.3 g·kg^-1）， medium-dose MLC group （MLC-M， 8.6 g·kg^-1）， and high-dose MLC group （MLC-H， 17.2 g·kg^-1）. Active components of MLC and targets of obesity-type PCOS were screened from databases， a protein-protein interaction （PPI） network was constructed， and gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was performed. The gut microbiota structure was analyzed based on 16S rRNA sequencing and correlated with network pharmacology pathways. Body weight and estrous cycle were dynamically monitored. Ovarian morphology was observed by hematoxylin-eosin （HE） staining. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL）. Enzyme-linked immunosorbent assay （ELISA） was used to detect levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， anti-Müllerian hormone （AMH）， testosterone （T）， and estradiol （E₂）. Western blot was used to detect the protein expression levels of phosphorylated PI3K/PI3K （p-PI3K/PI3K）， phosphorylated Akt/Akt （p-Akt/Akt）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. ResultsNetwork pharmacology screening identified 124 active components of MLC and 408 overlapping targets between the herbal formula and the disease. Core targets such as Akt1 and Bcl-2 were revealed. As indicated by 16S rRNA sequencing， the abundances of Lachnospiraceae， Lachnoclostridium， and Dorea were increased in the MLC groups （P<0.05）， while the abundance of Veillonella was decreased （P<0.05）. KEGG correlation analysis integrating network pharmacology and gut microbiota data showed significant enrichment of the PI3K/Akt signaling pathway. Animal experiments showed that， compared with the Mod group， body weight decreased to normal levels in the Met， MLC-M， and MLC-H groups. The estrous cycle became regular. The number of corpora lutea increased and cystic follicles decreased. Serum levels of T， FSH， and LH/FSH were reduced （P<0.05， P<0.01）， while the E₂ level was increased （P<0.01）. Ovarian cell apoptosis was reduced （P<0.01）， and the protein expression levels of p-PI3K/PI3K， p-Akt/Akt， and Bcl-2 in ovarian tissue were significantly increased， whereas Bax protein expression was significantly decreased （P<0.05， P<0.01）. ConclusionMLC can regulate gut microbiota structure， effectively improve ovarian pathology in rats with obesity-type PCOS， and inhibit ovarian granulosa cell apoptosis. The mechanism may be associated with upregulation of the PI3K/Akt signaling pathway.

Objective: To systematically analyze the stability and decay kinetics of coagulation factors in thawed cryoprecipitate stored at different temperatures under the new storage guidelines, and to provide an evidence basis for optimizing storage strategies. Methods: A total of 30 bags of 2U cryoprecipitate prepared at our blood center from January to June 2025 were selected. After thawing in a 37℃ water bath and thorough mixing, each bag was equally divided into two portions and stored at room temperature (20-24℃) or under refrigerated temperature (2~6℃), respectively. Dynamic sampling was performed at 0, 4, 6, 24, 30, and 120 h after thawing to measure fibrinogen (FIB) content, coagulation factor Ⅷ(FⅧ) activity, and von Willebrand factor (vWF) activity. Two-way repeated measures ANOVA was used to evaluate the effects of storage temperature, time, and their interaction. A one-phase exponential decay model was applied to fit the decay kinetics of FⅧ activity and vWF activity. Results: After thawing, FⅧ activity and vWF activity in cryoprecipitate showed a decreasing trend with prolonged storage time, and a time×temperature interaction was observed for both factors (all P< 0.001). The decay rates of FⅧ activity and vWF activity were lower under room temperature storage than under refrigerated storage, with statistically significant differences between the two groups starting from 24 h. Kinetic fitting revealed that the half-life of FⅧ activity in the room temperature group (19.83 h) was 3.4 times longer than that in the refrigerated group (5.85 h); the half-life of vWF activity in the room temperature group (204.6 h) was 5.9 times longer than that in the refrigerated group (34.6 h). FIB content remained stable throughout the observation period under both storage temperatures, with no statistically significant differences. Conclusion: Storage of thawed cryoprecipitate at room temperature effectively delays the loss of FⅧ activity and vWF activity and reduces cryoprecipitate formation induced by low temperature, providing experimental evidence for optimizing transfusion strategies based on the decay kinetics of different coagulation factors and improving the utilization efficiency of cryoprecipitate resources.

ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

3CL protease (3CLpro) of SARS-CoV-2 is a pivotal enzyme required in coronavirus replication and transcription. Its highly conserved structure and the absence of homologous proteins in the host make it an ideal target for broad-spectrum anti-coronavirus drug development. In this work, we systematically investigated and compared the binding modes and dynamic properties of the four stereoisomers of a pyridyl-urea diyne ester (PyDU) molecule with two chiral centers within the 3CLpro active site. Through molecular docking, MD simulations, MM/GBSA binding free-energy calculations, and DCCM analysis, all four stereoisomers were stabilized primarily by hydrophobic packing. Among them, the (R,S) isomer exhibited the best overall performance, including docking score, binding free-energy components, and key residue interactions. The (R,S) and (S,R) isomers also enhanced the cooperative motions around the binding pocket, while the (R,S) isomer further modulated the flexibility of domain III, which may influence 3CLpro dimerization. Conversely, the (S,S) isomer exhibited the weakest affinity due to insufficient hydrophobic contact. By innovatively integrating chirality, binding energy and protein dynamical features, we revealed the dual role of chirality in modulating affinity and dynamic responses, which provides a theoretical basis for the chiral-guided design of coronavirus inhibitors.

BACKGROUND:Human periodontal ligament stem cells are potential functional cells for periodontal tissue engineering.However,long-term in vitro culture may lead to reduced stemness and replicative senescence of periodontal ligament stem cells,which may impair the therapeutic effect of human periodontal ligament stem cells. OBJECTIVE:To investigate the effect of oxymatrine on the stemness maintenance and osteogenic differentiation of periodontal ligament stem cells in vitro,and to explore the potential mechanism. METHODS:Periodontal ligament stem cells were isolated from human periodontal ligament tissues by tissue explant enzyme digestion and cultured.The surface markers of mesenchymal cells were identified by flow cytometry.Periodontal ligament stem cells were incubated with 0,2.5,5,and 10 μg/mL oxymatrine.The effect of oxymatrine on the proliferation activity of periodontal ligament stem cells was detected by CCK8 assay.The appropriate drug concentration for subsequent experiments was screened.Western blot assay was used to detect the expression of stem cell non-specific proteins SOX2 and OCT4 in periodontal ligament stem cells.qRT-PCR and western blot assay were used to detect the expression levels of related osteogenic genes and proteins in periodontal ligament stem cells. RESULTS AND CONCLUSION:(1)The results of CCK8 assay showed that 2.5 μg/mL oxymatrine significantly enhanced the proliferative activity of periodontal stem cells,and the subsequent experiment selected 2.5 μg/mL oxymatrine to intervene.(2)Compared with the blank control group,the protein expression level of SOX2,a stem marker of periodontal ligament stem cells in the oxymatrine group did not change significantly(P>0.05),and the expression of OCT4 was significantly up-regulated(P<0.05).(3)Compared with the osteogenic induction group,the osteogenic genes ALP,RUNX2 mRNA expression and their osteogenic associated protein ALP protein expression of periodontal ligament stem cells were significantly down-regulated in the oxymatrine+osteogenic induction group(P<0.05).(4)The oxymatrine up-regulated the expression of stemness markers of periodontal ligament stem cells and inhibited the bone differentiation of periodontal ligament stem cells,and the results of high-throughput sequencing showed that it may be associated with WNT2,WNT16,COMP,and BMP6.

Cannabidiol(CBD),a natural,non-toxic,non-psy-choactive cannabinoid extracted from Cannabis sativa L,has a wide range of pharmacological activities,including anti-inflam-matory,antioxidant,immunomodulatory,analgesic,anti-tumor,anti-convulsant,anxiolytic effects.Recent studies have indica-ted that CBD may have potential neuroprotective effects in vari-ous models of central nervous system diseases.This paper re-views the experimental research progress of CBD in central nerv-ous system diseases and its possible neuroprotective mecha-nisms,aiming to provide references for its clinical application and new drug development.

Objective:Based on multivariate analysis of the influencing factors of fecal pollution after radical re-section in children with congenital megacolon,a risk prediction model was constructed and evaluated.Methods:A total of 200 children with Hirschsprung's disease who underwent radical surgery at Nanyang Central Hospital of Henan Province from June 2020 to June 2023 were selected,and all children were followed up for 1 year.According to the occurrence of postoperative fecal incontinence,the children were divided into the non-occurrence group and the oc-currence group.Univariate Logistic regression analysis was performed on all possible influencing factors,and the back-ward stepwise regression method was used to screen out the relevant factors affecting postoperative fecal inconti-nence in children with Hirschsprung's disease after radical surgery.A risk prediction model was constructed based on these relevant factors,and its efficacy was verified using calibration curves,the Hosmer-Lemeshow test,and ROC curves.Results:Postoperative fecal incontinence occurred in 19.50%(39/200)of the children.A statistically signifi-cant difference was observed between the non-occurrence group and the occurrence group in terms of the following data(P＜0.05):age,preoperative nutritional support,need for auxiliary defecation before surgery,length of the dis-eased intestinal segment,educational level of caregivers,preoperative hypoproteinemia,resection range,postoperative diet compliance of the children,postoperative defecation training of the children,postoperative enterocolitis,and post-operative anal dilatation therapy.Multivariate Logistic regression analysis showed that preoperative nutritional support,need for auxiliary defecation before surgery,length of the diseased intestinal segment,educational level of caregivers,postoperative diet compliance of the children,postoperative defecation training of the children,postoperative entero-colitis,and postoperative anal dilatation therapy were all independent influencing factors for postoperative fecal inconti-nence in children with Hirschsprung's Disease after radical surgery(P＜0.05).Among these factors,the educational level of caregivers was the strongest predictor of postoperative fecal incontinence in children with Hirschsprung's dis-ease after radical surgery.Results of the calibration curve showed that the concordance index of the risk prediction model for postoperative fecal incontinence in children with Hirschsprung's Disease after radical surgery was 0.837.The Hosmer-Lemeshow test results indicated no statistically significant difference between the predicted values and the actual values of risk prediction(P＜0.05),suggesting that the model had a good fit and high prediction accuracy.Re-sults of the ROC curve showed that the area under the curve(AUC)of the risk prediction model for predicting postop-erative fecal incontinence in children with Hirschsprung's Disease after radical surgery was 0.887(95%CI:0.762～1.000,P＜0.05),with a sensitivity of 0.914,a specificity of 0.763,and a Youden index of 0.677.These findings indicate that the risk prediction model has moderate discriminative ability and good predictive performance.Conclusion:Preop-erative nutritional support,need for auxiliary defecation before surgery,length of the diseased intestinal segment,edu-cational level of caregivers,postoperative diet compliance of children,postoperative defecation training of children,postoperative enterocolitis,and postoperative anal dilatation therapy are all independent influencing factors for postop-erative fecal incontinence in children with Hirschsprung's disease after radical surgery.The risk prediction model for postoperative fecal incontinence in children with Hirschsprung's disease after radical surgery,established based on multivariate analysis,is helpful for early identification of high-risk groups and timely implementation of individualized in-tervention measures.

Objective To investigae microbial community structure of lower respiratory tract in severe hospital-acquired pneumonia(SHAP)patients in intensive care unit(ICU)using metagenomic next-generation sequencing(mNGS).Methods mNGS was performed on bronchoalveolar lavage fluid(BALF)of 84 patients with SHAP.Patients were grouped based on age,smoking status,underlying diseases and duration of artificial airway.The differences in α diversity,β diversity,microbial composition and community structure of airway microbiota were compared between different groups.The differential airway microbiota associated with artificial airway were screened,and microbial co-occurrence networks was constructed to observe the interaction in microorganisms.Results Results of α diversity analysis revealed that diversity and evenness of the microbial community were higher in young adults compared to those of middle-aged patients,while microbial diversity and evenness were significantly reduced in patients with comorbid stroke.In the group of artificial airway treatment,the diversity and uniformity of microorganisms decreased as the duration of artificial airway treatment increased.The diversity was the lowest when the artificial airway treatment lasted for more than 3 days.β diversity analysis confirmed that there were significant differences in the distinct microbial community structures between the>3 days support group and the non-intubated and≤3 days support cohorts.Acinetobacter baumannii was dominant in all groups.The bacterial diversity was significantly higher in the middle-aged group,the non-smoking group,the group without artificial airway therapy,the group without diabetes mellitus,the group with artificial airway therapy≥3 days and the group with chronic lung disease than those of other groups.In particular,pseudomonas aeruginosa,corynebacterium striatum and veillonella parvula were enriched in these groups.Difference analysis showed that there were significant differences in pseudomonas aeruginosa and corynebacterium striatum between the group with artificial airway treatment>3 days,the group without artificial airway therapy and the group with artificial airway treatment≤3 days.Network co-occurrence showed that there may be synergistic or antagonistic relationships between some microorganisms.Conclusion The microbial diversity of the lower respiratory tract in patients with SHAP significantly decreases in the elderly,those with concurrent stroke and those receiving artificial airway treatment.For these groups,rational use of antibiotics should be adopted to guide precise anti-infection treatment.

OBJECTIVE T o explore the long-term effect of multidimensional evidence-based interventions based on i-PARIHS theoretical framework on reduction of incidence of central line-associated bloodstream infections(CLABSI)in pediatric intensive care units(PICU)of pediatrics department and evaluate the impact on nurses'compliance to taking the interventions and use intensity of catheters.METHODS By means of quasi-experimental design,the multidimensional intervention system covering multidisciplinary collaboration,standardized operation procedures,information system optimization and hierarchical training was established and staged for implementa-tion of 5 years(from T0 baseline stage to T3 maintenance stage).The variations in implementation rates of cathe-ter maintenance(daily maintenance,dressings change,catheter removal)were analyzed by Chi-square test,and the change of incidence of CLABSI was monitored with the use of statistical process control U chart.RESULTS The nurses'compliance to operations was remarkably improved(P＜0.05)o The implementation rate of dressings change continuously increased from 52.91％in T0 to81.62％in T3(x2=72.444,P＜0.001),the implementa-tion rate of catheter removal increased from 48.72％to 79.31％(x2=8.179,P=0.042).The incidence rate of CLABSI decreased from 1.92％0 in 2019 to 0.5％0 in 2022,and the control chart showed that most of the months fluctuated within control limits.CONCLUSIONS The multidimensional evidence-based interventions can achieve a long term control of CLABSI by raising the nurses' compliance to operations.The information monitoring and closed-loop management are crucial to maintenance of the interventional effect,and the risk early warning system should be optimized with the combination of artificial intelligence technology.