Objective To investigate the expression level and clinical significance of circular RNAs(circRNAs)in serum extracellular vesicles(EVs)of patients with recurrent spontaneous abortion(RSA).Methods The serum samples from 3 RSA patients and 3 nor-mal pregnant women(controls)visited our hospital from May 2021 to March 2023 were collected and the gene expression profiling chips were used to screen for differentially expressed circRNAs in serum EVs.Ten RSA patients and 10 normal pregnant women were enrolled in a training set and 80 RSA patients and 64 normal pregnant women were enrolled in a validation set.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the levels of differentially expressed circRNAs between the two groups.The predictive value of differentially expressed circRNAs for RSA was evaluated by the receiver operating characteristic(ROC)curve.Results A total of 57 563 circRNAs in serum EVs were detected by the gene expression profiling chips.Compared with the control group,3 516 circRNAs were differentially expressed in RSA patients,including 2 377 up-regulated and 1 139 down-regulated.The de-tection results of qRT-PCR in the training set and validation set showed that the expression levels of hsa＿circ＿0054403 in serum EVs of RSA patients(2.07[1.00,6.68])was significantly higher than that in the control group(1.00[0.42,1.46],U=1 239,P＜0.01),while those of hsa＿circ＿0020897(0.33[0.11,1.40]vs 1.00[0.46,3.66],U=1 712,P＜0.01)and hsa＿circ＿0072745(0.49[0.22,1.60]vs 1.00[0.51,7.93],U=1 714,P＜0.01)were significantly lower than that in the control group.The ROC curve showed that the hsa＿circ＿0054403,hsa＿circ＿0020897,and hsa＿circ＿0072745 in serum EVs had high predictive value for RSA.Their AUCROC were 0.758,0.666,and 0.664,respectively,and the corresponding sensitivity and specificity were 47.5% and 100.0%,50.0% and 81.2%,and 62.5% and 70.3%,respectively.When the three circRNAs were combined,it's AUCROC,sensitivity,and specificity were 0.842,73.7%,and 79.7%,respectively.Conclusion The hsa＿circ＿0054403,hsa＿circ＿0020897,and hsa＿circ＿0072745 in serum EVs of RSA patients are abnormally expressed,which may serve as the potential markers for predicting RSA.

Objective:To investigate and analyze the predictive value of inhibin A (INHA), galectin-13 (Gal-13), leucine rich alpha-2-glycoprotein 1 (LRG1) in serum for adverse pregnancy outcomes in pregnant women with gestational diabetes mellitus (GDM) .Methods:From Jan. 2022 to Dec. 2023, 87 GDM pregnant women admitted to Obstetrics Department of Shanxi Children’s Hospital were included as the study group, and were assigned into a good outcome group ( n=54) and an adverse outcome group ( n=33) based on pregnancy outcomes. Meantime, another 87 healthy pregnant women who underwent normal prenatal examinations at our hospital and had no complications were selected as the control group. ELISA method was applied to detect serum levels of INHA, LRG1, and Gal-13. Multiple factor Logistic regression model was constructed to analyze the factors affecting adverse pregnancy outcomes in GDM pregnant women. Receiver operating characteristic (ROC) curves were applied to evaluate the efficacy of the three methods in predicting adverse pregnancy outcomes in GDM pregnant women. Results:Compared with the control group, the levels of INHA and LRG1 in the serum of pregnant women in the study group were obviously higher, and the level of Gal-13 in the serum was obviously lower ( P<0.05). Compared with the good outcome group, the adverse outcome group showed an increase in serum INHA and LRG1 levels and a decrease in serum Gal-13 level ( P<0.05). Elevated levels of serum INHA and LRG1 were risk factors for adverse pregnancy outcomes in GDM pregnant women, while elevated level of serum Gal-13 was a protective factor ( P<0.05). The AUC values for predicting adverse pregnancy outcomes in GDM pregnant women based solely on serum INHA, Gal-13, and LRG1 levels were 0.859, 0.850, and 0.841, respectively. The AUC predicted by the combination of the three factors was 0.978, which was better than the individual predictions of serum INHA, Gal-13, and LRG1 ( Zcombination-HA=2.378, Z combination-Gal-13=3.193, Zcombination-LRG1=3.050, P=0.017, 0.001, 0.002) . Conclusions:Serum levels of INHA and LRG1 are elevated in GDM pregnant women, while serum level of Gal-13 is decreased. All three are potential factors that affect the pregnancy outcomes of GDM pregnant women, and the combination of the three shows higher efficacy in predicting adverse pregnancy outcomes in GDM pregnant women.

Objective To investigate the role of lupeol in mitigating chondrocyte senescence in osteoarthritis(OA)by regulating autophagy through the sirtuin 3(SIRT3)/mechanistic target of rapamycin kinase(mTOR)pathway.Methods Knee articular chondrocytes from primary-generation mice were isolated and divided into different groups,including a control group,a lupeol group(given 2.5,5,10,20,and 40 μmol/L lupeol),a tert-butyl hydrogen peroxide(TBHP)group(receiving 50 μmol/L TBHP),TBHP+lupeol group,TBHP+lupeol+chloroquine(CQ)group(receiving 20 μmol/L CQ,an autophagy inhibitor),TBHP+lupeol+si-NC group,and TBHP+lupeol+si-SIRT3 group.Cell proliferation,reactive oxygen species(ROS)levels,and apoptosis were determined by CCK-8,DCFH-DA probe,and flow cytometry.Cell senescence was evaluated by β-gal staining.Western blot was used to determine the expressions of SIRT3,mTOR,senescence marker proteins(p21 and p16),extracellular matrix(ECM)degradation-related proteins(aggrecan,collagen Ⅱ,ADAMTS5,and MMP13),and autophagy-related proteins(LC3B Ⅰ,LC3BⅡ,and P62).RT-qPCR was used to determine the mRNA levels of senescence-associated secretory phenotypes(SASP),including IL-6,Cxcl10,MCP1,and MMP3.The expression of LC3 was detected by immunofluorescence.Autophagosomes were observed by transmission electron microscopy.A total of 30 male wild-type C57BL/6 mice were divided into different groups(n=10),including a Sham group,an OA group,and an OA+lupeol group receiving 50 mg/(kg·d)lupeol via gastric gavage.Cartilage damage was evaluated by safranin O-fast green staining.Results Based on the results of cell viability assay,20 μmol/L lupeol treatment for 24 h was identified as the optimal intervention concentration and duration.Compared with that in the TBHP group,cell viability was elevated in the TBHP+lupeol group(P＜0.05);ROS production,the proportion of β-gal-positive cells,the protein expression levels of p21 and p16,and the mRNA levels of SASP were decreased(P＜0.05);the protein levels of aggrecan and collagen Ⅱ were elevated and the protein levels of ADAMTS5 and MMP13 were decreased(P＜0.05);apoptosis was reduced(P＜0.05);P62 protein levels were reduced and the LC3B Ⅱ/LC3B Ⅰ ratio,the intensity of LC3B fluorescence spots,and the number of autophagosomes were increased(P＜0.05);the expression level of SIRT3 was elevated and the level of mTOR phosphorylation was reduced(P＜0.05)in the TBHP+Lupeol group.CQ treatment effectively abolished the promotion effects of lupeol on cell viability and autophagy,and the inhibitory effects of lupeol on ROS level,cell senescence,ECM degradation,and apoptosis(P＜0.05).Silencing of SIRT3 reversed the inhibitory effect of lupeol on mTOR phosphorylation level and the promotion effect of lupeol on autophagy(P＜0.05).In the in vivo experiment,compared with the OA group,the OA+lupeol group showed reduced cartilage degeneration and lower scores for the Osteoarthritis Research Society International grading system(P＜0.05).The OA+lupeol group also showed up-regulated SIRT3 expression,reduced mTOR phosphorylation level,increased LC3B Ⅱ/LC3B Ⅰ ratio,reduced MMP13 protein level,and reduced mRNA level of SASP(P＜0.05).Conclusion Lupeol alleviates chondrocyte senescence in osteoarthritis by regulating autophagy through the SIRT3/mTOR pathway.

Objective To identify genetic regulators of ionizing radiation(IR)sensitivity through clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome-wide screening.Methods A specialized single guide RNA(sgRNA)library was developed targeting 987 stress-response regulatory genes identified through Kyoto Encyclopedia of Genes and Genomes(KEGG),Reactome and gene ontology(GO)databases,followed by construction of sgRNA plasmids and packaging into a lentiviral library.Using colon adenocarcinoma Caco-2 cells as a model system,the Cas9-stable transgenic line was established via lentiviral transduction and antibiotic selection.Library-transduced cells underwent puromycin selection,followed by γ-irradiation(dose optimized via preliminary experiments).Post-irradiation phenotypic selection was conducted after 14 days,with subsequent next-generation sequencing of surviving cell populations to identify enriched/depleted sgRNAs.Candidate genes were functionally validated through orthogonal assays:cell counting kit-8(CCK-8)proliferation analysis,clonogenic survival assays and flow cytometric quantification of reactive oxygen species(ROS)and apoptotic markers.Results The optimized screening platform identified 281 radiation response genes(165 radiosensitive and 116 radioprotective candidates).Functional validation revealed Bcl2-associated athanogene 3(BAG3)knockdown significantly enhanced radioresistance.Proliferation was increased 1.2-1.5 fold,clonogenic survival improved 1.5-2.0 fold,and ROS was reduced by 13％-25％coupled with 32％-73％apoptosis attenuation compared to control groups.Conclusion The integrated CRISPR/Cas9 screening platform effectively identifies novel radiation response regulators,as evidenced by the discovery of BAG3 as a critical radiosensitivity determinant.This high-throughput functional genomics approach provides a robust methodology for systematically mapping molecular determinants of cellular radiation response,with potential applications in both mechanistic studies and therapeutic target discovery.

Objective:To observe any effect of combining repeated transcranial magnetic stimulation (rTMS) with electro-acupuncture in treating post-stroke cognitive impairment (PISC).Methods:Three groups of PISC patients were formed through random selection: an rTMS group, an electro-acupuncture group, and a combined group. In addition to routine medication and conventional rehabilitation training, the rTMS and electro-acupuncture groups received rTMS and electro-acupuncture treatment, while the combined group underwent both for six weeks. Before the treatment, immediately afterward and 3, 6, and 12 months later, everyone′s cognitive functioning was assessed using the Montreal Cognitive Assessment (MoCA). The modified Barthel Index (MBI), the 17-item Hamilton Depression Rating Scale (HAMD-17), and the National Institutes of Health Stroke Scale (NIHSS) were also applied. Transcranial Doppler ultrasound (TCD) was employed to measure the mean cerebral blood flow velocity (Vm), pulsatility index (PI), and breath-holding index (BHI) in the subjects′ bilateral middle cerebral arteries (MCAs). The total effectiveness rates and the incidence of adverse reactions were compared among the three groups.Results:The MoCA scores, MBI scores, HAMD-17 scores, NIHSS scores, and Vm of the MCA had improved significantly in all three groups right after the treatment. There was further significant improvement in the average MoCA scores 12 months later. The combined group showed significantly higher MoCA scores than the other two groups at each time point after the treatment. That group also had superior MBI, HAMD-17 and NIHSS scores and a better BHI compared to the other 2 groups, on average. Its total effectiveness rate was significantly higher too. There was no significant difference in the incidence of adverse reactions among the groups.Conclusions:Combining rTMS with electro-acupuncture significantly improves cognition, ADL ability, depression and neurological functioning after a stroke. The combined treatment is worthy of wider clinical application.

Objective To investigate the feasibility and accuracy of convolutional neural networks for automatically delineating the pancreatic cancer gross target volume(GTV)in pancreatic enhanced CT.Methods The localizable enhanced CT images of 114 patients with pancreatic cancer were retrospectively selected,in which the GTV was manually delineated using AccuContour.The imaging data were then import to AccuLearning and randomly divided as the training set,validation set and test set at a ratio of 8:1:1.Flex and Segresnet were used to train the automatic segmentation model,with each network structure trained continuously 3 times using fixed training parameters.The model was evaluated in terms of Dice similarity coefficient(DSC),95%Hausdorff distance(HD95),average symmetric surface distance(ASSD)and relative volume difference(RVD).Results In the model training phase,Flex-3 test results in Flex group were the worst,with a minimum DSC of 0.14%and an average DSC of 56.30%,while Flex-1 performed well,achieving a minimum DSC of 47.90%and an average DSC of 67.35%.Meanwhile,Segresnet-2 in Segresnet group had the worst test results,with a minimum DSC of 0.00%and an average DSC of 42.46%,while Segresnet-3 test results were better,with a minimum DSC of 42.65%and an average DSC of 63.28%.In the fixed testing phase,the best results among all were as follows:average DSC and RVD values of 63.88%and 29.41%in Segresnet-3 group,average ASSD value of 4.43 mm in Segresnet-2 group,and average HD95 value of 12.87 mm in Segresnet-1 group.Conclusion Both Flex and Segresnet architectures of convolutional neural network can be used for the automatic pancreatic tumor GTV segmentation training,with Segresnet performing better in comprehensive evaluation.

Objective:To investigate and analyze the predictive value of inhibin A (INHA), galectin-13 (Gal-13), leucine rich alpha-2-glycoprotein 1 (LRG1) in serum for adverse pregnancy outcomes in pregnant women with gestational diabetes mellitus (GDM) .Methods:From Jan. 2022 to Dec. 2023, 87 GDM pregnant women admitted to Obstetrics Department of Shanxi Children’s Hospital were included as the study group, and were assigned into a good outcome group ( n=54) and an adverse outcome group ( n=33) based on pregnancy outcomes. Meantime, another 87 healthy pregnant women who underwent normal prenatal examinations at our hospital and had no complications were selected as the control group. ELISA method was applied to detect serum levels of INHA, LRG1, and Gal-13. Multiple factor Logistic regression model was constructed to analyze the factors affecting adverse pregnancy outcomes in GDM pregnant women. Receiver operating characteristic (ROC) curves were applied to evaluate the efficacy of the three methods in predicting adverse pregnancy outcomes in GDM pregnant women. Results:Compared with the control group, the levels of INHA and LRG1 in the serum of pregnant women in the study group were obviously higher, and the level of Gal-13 in the serum was obviously lower ( P<0.05). Compared with the good outcome group, the adverse outcome group showed an increase in serum INHA and LRG1 levels and a decrease in serum Gal-13 level ( P<0.05). Elevated levels of serum INHA and LRG1 were risk factors for adverse pregnancy outcomes in GDM pregnant women, while elevated level of serum Gal-13 was a protective factor ( P<0.05). The AUC values for predicting adverse pregnancy outcomes in GDM pregnant women based solely on serum INHA, Gal-13, and LRG1 levels were 0.859, 0.850, and 0.841, respectively. The AUC predicted by the combination of the three factors was 0.978, which was better than the individual predictions of serum INHA, Gal-13, and LRG1 ( Zcombination-HA=2.378, Z combination-Gal-13=3.193, Zcombination-LRG1=3.050, P=0.017, 0.001, 0.002) . Conclusions:Serum levels of INHA and LRG1 are elevated in GDM pregnant women, while serum level of Gal-13 is decreased. All three are potential factors that affect the pregnancy outcomes of GDM pregnant women, and the combination of the three shows higher efficacy in predicting adverse pregnancy outcomes in GDM pregnant women.

Objective To investigate the effects and molecular mechanisms of ginsenoside Rg1(GSRG1)on the proliferation,mi-gration,and inflammatory cytokine expression of human gingival fibroblasts(HGFs)induced by lipopolysaccharide(LPS).Methods HGFs were isolated and cultured using the tissue block method.The effects of different concentrations of LPS(0,1,5,10,20μg/mL)on inflammatory cytokines in HGFs were detected by ELISA.Cells were divided into three groups:control group(no treat-ment),LPS group(20 μg/mL LPS),and LPS+GSRG1 group(20 μg/mL LPS and 100 mg/L GSRG1).Cell proliferation was detec-ted by cell counting kit-8(CCK-8);cell migration was assessed by Transwell assay;intracellular reactive oxygen species(ROS)lev-els were compared using flow cytometry;and the expression levels of TNF-α,IL-6,NLRP3,p-p38,and p38 proteins were detected by Western blot.Results LPS concentrations of 5,10,and 20 μg/mL significantly increased the expression of IL-6 and TNF-α in HGFs(P＜0.05),with 20 μg/mL showing the strongest pro-inflammatory effect.Compared with the control group,there was no notable difference in the proliferation of HGFs in the LPS group at Day 1 and 2.However,on Day 3,4 and 5,decreased cell proliferation,re-duced migration,significantly increased ROS levels(P＜0.001),elevated protein expression of TNF-α,IL-6,and NLRP3(P＜0.001),and reduced p-p38 protein expression(P＜0.001)were exhibited.Compared with the LPS group,the LPS+GSRG1 group showed significantly enhanced cell proliferation and migration(P＜0.05),reduced ROS levels,decreased protein expression of TNF-α,IL-6,and NLRP3,and increased p-p38 protein expression(P＜0.001).There was no significant change in p38 expression among the three groups.Conclusion GSRG1 can alleviate the inhibitory effects of LPS on the proliferation and migration of HGFs and inhibit the inflammatory response,potentially through mechanisms involving p-p38 protein regulation.

Objective To investigate the therapeutic effect,underlying mechanism,and key genes involved in tetramethylpyrazine-pretreated umbilical cord mesenchymal stem cell (ucMSC) transplantation in a rat model of ischemic stroke. Methods The rat MCAO model was established,and umbilical cord-derived mesenchymal stem cells (ucMSCs) pretreated with or without tetramethylpyrazine were transplanted via the tail vein. Neurological function scores,TTC staining,and infarct rates were assessed. Localization of ucMSCs in brain tissue was observed. Experimental groups were analyzed using chip technology,and sample data were standardized. Bioinfor-matics analysis was employed to identify differential genes,which were subsequently validated by PCR. Results The treatment effect in ucMSCs pretreated with tetramethylpyrazine group was significantly superior to that of the untreated group,as evidenced by a significant reduction in neurological function score,infarct rate,and infarct area observed through TTC staining. Moreover,the treated group exhibited a significantly higher number of ucMSCs located within brain injury tissues compared to the untreated group. Subsequently,2905 differential mRNA were screened based on predetermined criteria,including 1754 up-regulated and 1151 down-regulated genes. Among these differentially expressed genes related to the chemokine signaling pathway (identified using a multiple change value ≥ 2.0 and P value ≤ 0.05),we identified 27 genes of interest. Notably,our analysis revealed activation of four genes closely associated with cell migration:Ccr6,Ccr3,Cxcr1 and Ccl6 respectively. Random verification experiments further confirmed a significant increase in gene expression for both Ccr3 and Cxcr1. Conclusions Pretreatment of umbilical cord-derived mesenchymal stem cells (ucMSCs) with tetramethylpyrazine significantly augmented the therapeutic efficacy in a rat model of ischemic stroke. Following pretreatment,there was a substantial increase in the migration of ucMSCs towards the site of brain injury. Our analysis suggests that this effect may be attributed to the activation of multiple chemokines,including Ccr6,Ccr3,Cxcr1,and Ccl6,by tetramethylpyrazine.

Surface plasmon resonance （SPR） sensor is an optical detection technique enables real-time and dynamic monitoring of biological samples. SPR-based biosensors have remarkable characteristics such as label-free detection and high sensitivity, making them important tools for studying molecular interactions. The recognition element, which plays a critical role in SPR sensors,which could specifically identify and capture of target analytes, closely influencing the selectivity performance of the sensor. The progress on SPR sensors in pharmaceutical research were reviewed, which focused on the application of recognition elements such as antibodies, aptamers, molecularly imprinted polymers, and metal nanoparticles.