AIM To investigate the mechanism of Jiedu Yizhi Formula on cognitive function and neuroinflammation in a mouse model of Alzheimer's disease(AD).METHODS 50 APP/PS1 double transgenic mice were randomly divided into the model group,the donepezil group,and the low-dose,moderate-dose,and high-dose Jiedu Yizhi Formula group(1.78,3.56 and 7.12 g/kg),with 10 mice in each group,in contrast to the 10 WT mice of the blank group.Following anesthesia administration and 8-week oral gavage regimen with respective drugs,all mice underwent final tissue sample collection.The mice had their learning and memory ability assessed by Morris water maze and nesting behavior scores;their pathology of brain tissue and Aβ expression observed using HE,Nissl and IHC staining;their polarization of microglia and the expression of inflammatory factors in hippocampal tissue detected by IF and ELISA;their hippocampal expression of PI3K/Akt/mTOR signaling pathway detected by RT-qPCR and Western blot.RESULTS Compared with the blank group,the model group had lower scores in total swimming distance,frequency in crossing the platform,residence time in the target quadrant,and nesting behavior scores(P＜0.05,P＜0.01);prolonged evasion latency(P＜0.01);more disorganized arrangement of pyramidal cells,solidification and deep staining,unclear demarcation,irregular cell shapes,reduction of Nyctinidia,and increased Aβ deposition in the brain tissue(P＜0.01);elevated expression of hippocampal microglia M1-type markers CD16/32 and lba-1(P＜0.01);decreased levels of M2-type marker CD206(P＜0.05);elevated levels of TNF-α and IL-1β(P＜0.01);decreased expressions of IL-13 and IL-4(P＜0.01);and decreased levels of PI3K,Akt and mTOR mRNA,and reduced p-PI3K,p-Akt and p-mTOR protein expressions(P＜0.01).Compared to the model group,the donepezil group and the Jiedu Yizhi Formula groups showed statistically significant improvements in the aforementioned indexes(P＜0.05,P＜0.01),with the magnitude of improvement being higher in the high-dose Jiedu Yizhi Formula group.CONCLUSION Jiedu Yizhi Formula suppresses microglia Ml-type polarization while enhancing M2-type polarization via activation the PI3K/Akt/mTOR signaling pathway,which subsequently reduces inflammatory cytokine secretion.This mechanism attenuates Aβ deposition in brain tissues and ameliorates cognitive dysfunction in AD mouse models.

Objective To investigate the characteristics of a novel FANCL mutation identified in a patient with premature ovarian insufficiency(POI)and to explore its potential functional impacts in vitro.Methods A novel FANCL heterozygous mutation c.1033G>A(p.Glu345Lys)was screened in a patient with POI using whole exome sequencing(WES),which was found to be inherited from a mother who had undergone early menopause.The authenticity of the mutation was identified by Sanger sequencing and the conserved nature of the mutation site was predicted by software.Overexpressing FANCL mutant and wildtype plasmids were constructed and transiently transfected into HEK293T cell lines,and the effect of the mutation was detected by qPCR,immunofluorescence and Western blot.Results The mutation site of FANCL was located within the Ring domain of FANCL,which was highly conserved across multiple species.The mutant showed no significant change in mRNA expression level,while the protein expression level was significantly down-regulated.In vitro cellular experiments further revealed that the mutation leads to decreased expression levels by reducing protein stability.Conclusion A FANCL c.1033G>A mutation was found and it may cause disease in the POI patient due to decreased protein stability.

Aim To identify the underlying target of bullatine A(BA)against rheumatoid arthritis(RA)u-sing limited proteolysis-small molecule mapping(LiP-SMap)drug target proteomics and to provide a scientif-ic basis for clinical application of Aconiti brachypodi Radix in the treatment of RA.Methods LiP-SMap drug target proteomics was employed to perform bioin-formatics analysis for comparing and validating the dif-ferential protein expression after BA intervention.A collagen-induced arthritis(CIA)model was estab-lished in DBA/1 mice using bovine type Ⅱ collagen.The mice were then divided into the CIA model group,methotrexate-positive control group(MTX group),and BA groups(10 mg·kg-1 and 20 mg·kg-1)based on their clinical scores.After drug intervention,the thera-peutic efficacy against RA was assessed by joint index scores and foot thickness measurements.Histopatholog-ical changes in the arthritic joints of CIA mice were e-valuated using hematoxylin and eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was employed to detect inflammatory cytokines interleukin-17(IL-17)and total IgG and IgG3 anti-collagen-spe-cific antibodies levels from the serum of CIA mice.Flow cytometry was used to detect the expression levels of intracellular Th17 cells(IL-17+CD4+T cells)and Th1 cells(IFN-γ+CD4+T cells).Fluorescent quanti-tative PCR was performed to detect the expression of genes related to differential proteins.Results The proteomic analysis identified Serpinb1a as a protein with strong binding affinity to BA,and KEGG enrich-ment analysis indicated IL-17 signaling pathway was a crucial pathway of BA in against RA.BA treatment significantly reduced clinical scores and foot thickness,improved local arthritis symptoms in CIA mice,and al-leviated inflammatory cell infiltration into arthritic joints(P＜0.05).Differential protein validation re-sults showed that BA had strong affinity with Serpinb1a(-5.92 kJ·mol-1)and downregulated the expres-sion of Serpinb1a mRNA.Furthermore,the administra-tion of BA markedly reduced serum IL-17 A levels from CIA mice,inhibited the expression of intracellular IL-17 A and IFN-γ cytokines in splenic CD4+T cells(P＜0.05),and significantly downregulated the transcrip-tional expression of IL-17F(P＜0.05).Conclusion BA exhibits therapeutic effects on collagen-induced arthritis,and its mechanism of action may involve the regulation of Serpinb1a and the IL-17 signaling path-way.

Objective To explore the mechanism of honey-processed Hedysari Radix in the regulation of intestinal immunity in rats with spleen qi deficiency,which was based on G protein-coupled receptor 41(GPR41)/GPR43-mediated mitogen-activated protein kinase(MAPK)signaling pathway.Methods The three-factor composite modeling method of eating disorder,diarrhea and fatigue was used to establish a model of spleen qi deficiency,and the rats were randomly divided into model,honey-processed Hedysari Radix,probiotics and blank groups with 15 rats per group.The honey-processed Hedysari Radix group was given by gavage 12.6 g·kg-1 aqueous extract of honey-processed Hedysari Radix.The probiotics group was given 0.625 g·kg-1 bifidobacterium triple viable solution by gavage.The blank and model groups were given the same dose of distilled water by gavage.Four groups were treated for 15 d with once a day.The expression levels of GPR41,GPR43,P38 MAPK,c-Jun N-terminal kinase(JNK)and extracellular regulatory protein kinase 1/2(ERK1/2)in colon tissues were detected by Western blotting.Results The relative expression levels of GPR41 in the blank,model,honey-processed Hedysari Radix and probiotics groups were 0.95±0.07,0.45±0.03,0.84±0.19 and 0.86±0.20;the relative expression levels of GPR43 were 1.17±0.11,0.41±0.06,0.66±0.03 and 0.57±0.01;the phosphorylated ERK1/2/ERK1/2 ratios were 0.16±0.01,0.43±0.01,0.39±0.01 and 0.36±0.02;the phosphorylated JNK/JNK ratios were 0.58±0.05,1.47±0.10,0.90±0.11 and 0.90±0.11;the phosphorylated P38 MAPK/P38 MAPK ratios were 1.77±0.33,3.19±0.03,2.01±0.17 and 2.23±0.59,respectively.Compared with the model group,the differences of above indexes were statistically significant in the honey-processed Hedysari Radix and probiotics groups(P＜0.05,P＜0.01).Conclusion The mechanism of honey-processed Hedysari Radix regulating intestinal immunity in rats with spleen qi deficiency is related to the regulation of GPR41/GPR43 mediated MAPK signaling pathway.

Nucleic acid detection technology has been widely applied in fields such as pathogen detection due to its characteristics of rapidity,sensitivity,and specificity.With the numerous nucleic acid markers related to diseases,the demand for multiplex nucleic acid detection is gradually increasing.Multiplex polymerase chain reaction(PCR)can simultaneously amplify multiple targets,but there are problems such as easy contamination when opening the tube during the analysis process after amplification and high technical requirements.With the continuous advancement of detection technology,a series of simple,re-liable single-tube multiplex PCR detection technologies that do not require opening the tube have emerged successively.A common technology is the single-closed tube multiplex PCR detection method based on fluorescent probes,which mainly uses different fluorescent labels to distinguish multiple targets.Com-bined with different specific enzymatic digestion reactions,it can achieve multiplex detection of rare tumor mutations and single nucleotide-specific genotyping.In addition,the monochromatic melting curve analysis method based on differences in melting temperatures enables parallel detection of multiple targets within a single fluorescence channel.When performed within multiple fluorescence channels,it is called the multicolor melting curve analysis method,which can increase the number of detected targets to doz-ens,greatly breaking through the limitation of the number of fluorescence channels on the multiplexity of detection.At the same time,the fluorescence coding method using different combinations of fluorescent labels also provides new ideas for single-closed tube multiplex PCR detection.These include encoding the sequence of signals generated by different fluorescent labels corresponding to the same target,using a combination of two fluorescent labels to identify specific targets,and controlling the amplitude of fluores-cent signals of different targets,all of which can also improve the multiplexity of detection.This article summarizes and prospects the research progress of single-closed tube multiplex PCR detection technology in recent years from multiple dimensions such as principles,applications,and the advantages and disad-vantages of the methods,providing valuable references for subsequent scientific research exploration and application.

Fucoidan(FUC)is a natural seaweed-derived drug.Previously,our experiments have shown that FUC can significantly inhibit the cell viability of human osteosarcoma 143B cells and induce cell death,but the mechanism remains unclear.Ferroptosis,a novel form of cell death,has emerged as an important target for tumor therapy.This study aims to investigate whether FUC induces ferroptosis of 143B cells and elucidate its underlying molecular mechanisms.CCK-8 and LDH assays result showed that FUC(10,100,400 μg/mL)significantly reduced cell viability of 143B cells and induced cell death.Calce-in-AM staining,FeRhoNox-1 staining,and C11 BODIPY 581/591 staining indicated that FUC obviously increased the levels of labile iron pool(LIP),Fe2+,and lipid reactive oxygen species(Lip ROS)in 143B cells.Chemical colorimetric analysis revealed that FUC markedly decreased intracellular Glutathi-one(GSH)contents.Real-time quantitative PCR showed that FUC dramatically reduced the mRNA lev-els of ferroptosis-related factors solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4),while increasing the mRNA levels of prostaglandin endoperoxide synthase 2(PTGS2)and acyl-CoA synthetase long-chain family member 4(ACSL4).Western blotting analysis demonstrated that FUC significantly reduced the protein levels of SLC7A11 and GPX4,and the ratios of p-PI3K/PI3K,p-AktSer473/Akt,and p-AktThr308/Akt,but increased the protein level of ACSL4.Immunofluorescence staining showed that FUC obviously inhibited the nuclear translocation of p-AktSer473.The ferroptosis in-hibitor ferrostatin-1(Fer-1)and iron chelator deferoxamine(DFO)remarkably suppressed cell death in-duced by FUC in 143B cells.Additionally,the PI3K/Akt pathway activator 740Y-P significantly inhibi-ted FUC-induced iron overload and lipid peroxidation in 143B cells,and restored the protein levels of SLC7A11 and GPX4.In conclusion,FUC can induce ferroptosis of 143B cells by inhibiting the PI3K/Akt signaling pathway,which may be a potential target for the prevention and treatment of osteosarcoma.

Purpose To assess the utility of echocardiographic parameters in predicting adverse cardiovascular in patients with connective tissue disease.Materials and Methods A retrospective analysis was conducted on the clinical and echocardiographic records of patients with connective tissue disease from the Affiliated Hospital of Inner Mongolia Medical University(June 1st,2020 to June 1st,2023)who had complete medical data and at least twelve months of follow-up.Variables were screened based on univariate analysis,combined with clinical expertise and XGBoost feature weight analysis;this information was used to construct a Cox proportional hazards regression model designed to predict composite endpoint events of adverse cardiovascular outcomes.Internal validation was performed using the Bootstrap resampling method,and the model's performance was evaluated.Results The study included 123 participants.The incidence of positive events reached 39.02%(48/123).Mitral valve early diastolic annular velocity(reflecting left heart function)(HR=0.79,P=0.041)and tricuspid annular plane systolic excursion(reflecting right heart function)(HR=0.92,P=0.044)emerged as significant predictors for adverse cardiovascular outcomes.Compared with the clinical model,the model combined with left heart function parameters showed significant improvements in both risk classification and absolute accuracy for short-term and medium-term adverse prognosis(NRI365=0.054,IDI365=0.060,NRI730=0.064,IDI730=0.079,all P<0.05)and optimized risk classification for long-term adverse prognosis(NRI1 095=0.256,P<0.05).In contrast,the model combined with right heart function parameters improved risk classification at all time points(NRI365=0.054,NRI730=0.000,NRI1 095=0.135,all P<0.05).Conclusion Mitral valve early diastolic annular velocity and tricuspid annular plane systolic excursion,which reflect cardiac function,are factors for predicting adverse cardiovascular outcomes among individuals diagnosed with connective tissue disease.Multi-parameter combined models incorporating echocardiographic variables can provide incremental predictive value compared with clinical models.

Purpose To assess the utility of echocardiographic parameters in predicting adverse cardiovascular in patients with connective tissue disease.Materials and Methods A retrospective analysis was conducted on the clinical and echocardiographic records of patients with connective tissue disease from the Affiliated Hospital of Inner Mongolia Medical University(June 1st,2020 to June 1st,2023)who had complete medical data and at least twelve months of follow-up.Variables were screened based on univariate analysis,combined with clinical expertise and XGBoost feature weight analysis;this information was used to construct a Cox proportional hazards regression model designed to predict composite endpoint events of adverse cardiovascular outcomes.Internal validation was performed using the Bootstrap resampling method,and the model's performance was evaluated.Results The study included 123 participants.The incidence of positive events reached 39.02%(48/123).Mitral valve early diastolic annular velocity(reflecting left heart function)(HR=0.79,P=0.041)and tricuspid annular plane systolic excursion(reflecting right heart function)(HR=0.92,P=0.044)emerged as significant predictors for adverse cardiovascular outcomes.Compared with the clinical model,the model combined with left heart function parameters showed significant improvements in both risk classification and absolute accuracy for short-term and medium-term adverse prognosis(NRI365=0.054,IDI365=0.060,NRI730=0.064,IDI730=0.079,all P<0.05)and optimized risk classification for long-term adverse prognosis(NRI1 095=0.256,P<0.05).In contrast,the model combined with right heart function parameters improved risk classification at all time points(NRI365=0.054,NRI730=0.000,NRI1 095=0.135,all P<0.05).Conclusion Mitral valve early diastolic annular velocity and tricuspid annular plane systolic excursion,which reflect cardiac function,are factors for predicting adverse cardiovascular outcomes among individuals diagnosed with connective tissue disease.Multi-parameter combined models incorporating echocardiographic variables can provide incremental predictive value compared with clinical models.

Objective:To explore the clinical features and risk factors for pediatric severe Mycoplasma pneumoniae pneumonia (SMPP) complicated with pulmonary embolism. Methods:SMPP patients admitted to Department of Pediatrics, Jiaxing First Hospital and Pediatric Intensive Care Unit, Shanghai Children′s Hospital from December 2019 to December 2023 were included in this retrospective case-control study.According to whether they were complicated with pulmonary embolism, SMPP patients were divided into a pulmonary embolism group and a non-pulmonary embolism group.Clinical characteristics of the two groups, including general data, laboratory examination and imaging data were compared and analyzed.The t-test and Mann-Whitney rank-sum test were used to compare the measurement data, and the χ2 test was used to compare the count data.The risk factors of SMPP patients developing pulmonary embolism were analyzed by the univariate method. Results:There were 10 out of 62 SMPP children developing pulmonary embolism, showing an incidence of 16.13%.In the pulmonary embolism group, there were 5 boys and 5 girls, with a median age of 7.50 (5.75, 9.25) years.There were 52 children in the non-pulmonary embolism group, including 29 boys and 23 girls, with a median age of 6.50(5.00, 8.00)years.The hospitalization time, body temperature, total white blood cell count, neutrophil count, C-reactive protein levels, lactate dehydrogenase levels, prothrombin time, activated partial thromboplastin time, D-dimer (D-D) levels, fibrinogen degradation product levels, pleural effusion and atelectasis rates in the pulmonary embolism group were significantly higher than those in the non-pulmonary embolism group (all P<0.05). Fibrinogen levels in the pulmonary embolism group were significantly lower than those in the non-pulmonary embolism group ( P<0.05). Univariate Logistic regression analysis showed that the D-D level was a risk factor for SMPP patient developing pulmonary embolism.The receiver operating characteristic curve analysis revealed that the D-D level had the largest area under the curve for predicting pulmonary embolism of 0.990(95% CI: 0.972-1.000, P<0.001), with a sensitivity of 100%, a specificity of 92%, and a cut-off value of 4.63 mg/L. Conclusions:SMPP children complicated with pulmonary embolism are prone to high inflammation and impaired coagulation function.The increase of D-D levels is a risk factor for the development of pulmonary embolism in SMPP.

Fucoidan(FUC)is a natural seaweed-derived drug.Previously,our experiments have shown that FUC can significantly inhibit the cell viability of human osteosarcoma 143B cells and induce cell death,but the mechanism remains unclear.Ferroptosis,a novel form of cell death,has emerged as an important target for tumor therapy.This study aims to investigate whether FUC induces ferroptosis of 143B cells and elucidate its underlying molecular mechanisms.CCK-8 and LDH assays result showed that FUC(10,100,400 μg/mL)significantly reduced cell viability of 143B cells and induced cell death.Calce-in-AM staining,FeRhoNox-1 staining,and C11 BODIPY 581/591 staining indicated that FUC obviously increased the levels of labile iron pool(LIP),Fe2+,and lipid reactive oxygen species(Lip ROS)in 143B cells.Chemical colorimetric analysis revealed that FUC markedly decreased intracellular Glutathi-one(GSH)contents.Real-time quantitative PCR showed that FUC dramatically reduced the mRNA lev-els of ferroptosis-related factors solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4),while increasing the mRNA levels of prostaglandin endoperoxide synthase 2(PTGS2)and acyl-CoA synthetase long-chain family member 4(ACSL4).Western blotting analysis demonstrated that FUC significantly reduced the protein levels of SLC7A11 and GPX4,and the ratios of p-PI3K/PI3K,p-AktSer473/Akt,and p-AktThr308/Akt,but increased the protein level of ACSL4.Immunofluorescence staining showed that FUC obviously inhibited the nuclear translocation of p-AktSer473.The ferroptosis in-hibitor ferrostatin-1(Fer-1)and iron chelator deferoxamine(DFO)remarkably suppressed cell death in-duced by FUC in 143B cells.Additionally,the PI3K/Akt pathway activator 740Y-P significantly inhibi-ted FUC-induced iron overload and lipid peroxidation in 143B cells,and restored the protein levels of SLC7A11 and GPX4.In conclusion,FUC can induce ferroptosis of 143B cells by inhibiting the PI3K/Akt signaling pathway,which may be a potential target for the prevention and treatment of osteosarcoma.