Abstract:Objective To investigate the molecular mechanism by which veratric acid(VA)ameliorates oxidative stress injury and intestinal barrier dysfunction in mice with dextran sulfate sodium(DSS)-induced colitis.Methods Thirty male C57BL/6 mice were randomized equally into control group,DSS model group,and VA treatment group.The mice were assessed for changes in body weight,disease activity index(DAI),colon length,and colonic histopathology.Colonic expressions of TNF-α,IL-6,and IL-10 and oxidative stress markers(SOD,GSH,MDA,and COX-2)were determined using ELISA,and the expressions of tight junction proteins(ZO-1 and claudin-1)and Nrf2/HO-1 pathway proteins were detected using immunofluorescence staining and Western blotting.In Caco-2 cells with H₂O₂-induced oxidative stress,ROS accumulation was examined using flow cytometry and a DCFH-DA probe,and Nrf2 inhibitor(ML385)was used to validate the mechanism of VA for ameliorating oxidative stress.Results VA treatment significantly alleviated DSS-induced body weight loss,colon shortening and the increase of DAI score of the mice,resulting also in improved crypt structure and increased expressions of ZO-1 and claudin-1 and the number of goblet cells.VA obviously reduced colonic levels of TNF-α and IL-6,increased the level of IL-10,and reversed DSS-induced decreases in SOD and GSH activity and increases in MDA and COX-2 levels.In H₂O₂-treated Caco-2 cells,VA decreased ROS-positive cell rate and intracellular ROS accumulation,and increased cellular expressions of claudin-1 and ZO-1.Mechanistically,VA promoted the expressions of Nrf2 and the downstream HO-1 protein,and ML385 partially reversed ROS-reducing effect of VA.Conclusion VA enhances antioxidant defense,inhibits inflammation,and repairs intestinal barrier function in mice with DSS-induced colitis by activating the Nrf2/HO-1 pathway,suggesting a novel strategy for treatment of inflammatory bowel disease.

Abstract:Objective To investigate the therapeutic mechanism of curcumin for osteoporosis induced by type 2 diabetes mellitus(T2DM).Methods In cultured MC3T3-E1 osteoblasts exposed to high glucose(HG),the effects of curcumin treatment on osteogenic differentiation and mineralization were examined by assessing alkaline phosphatase(ALP)activity and using alizarin red S(ARS)staining.RNA-seq sequencing was used to analyze the transcriptional characteristics of MC3T3-E1 osteoblasts in HG culture after curcumin treatment.In a mouse model of T2DM established by high-fat diet feeding and streptococcal injection,the effects of solvent vehicle,vehicle+RANBP3L knockdown,and RANBP3L knockdown+curcumin treatment on blood lipids,bone microstructure and calcium deposition was evaluated using ELISA,HE staining,and Alizarin red S staining.Osteogenic differentiation ability of mouse femoral tissues were assessed by detecting ALP,RANBP3L,OCN and RANKL expressions using immunohistochemical staining,RT-qPCR and Western blotting,and the changes in the TNF-α/NF-κB pathway were detected with Western blotting.Results Curcumin obviously promoted proliferation and osteogenic differentiation of MC3T3-E1 cells in HG culture and down-regulated cellular expression of RANBP3L protein.In the diabetic mouse models,RANBP3L knockdown significantly improved bone microstructure and blood lipid balance,increased expression levels of osteogenic markers,decreased blood glucose level,and caused inhibition of the TNF-α/NF-κB signaling pathway.The combined treatment with curcumin further reduced RANBP3L expression levels in the bone tissue and significantly enhanced the therapeutic effect.Conclusion Curcumin promotes osteogenesis,alleviates glucose and lipid metabolism disorders,and improves osteoporosis in type 2 diabetic mice possibly by inhibiting TNF-α/NF-κB signaling pathway via suppressing RANBP3L expression.

Abstract:Objective To investigate the expression of membrane protein palmitoylated 6(MPP6)in hepatocellular carcinoma(HCC)and analyze its correlation with clinicopathological features and patient prognosis and its impact on biological behaviors of HCC cells.Methods We examined MPP6 expressions using immunohistochemistry in tumor and adjacent tissues from 118 HCC patients undergoing surgeries in our hospital from January,2017 to December,2019,and analyzed the correlation of MPP6 expression levels with clinicopathological data and overall survival(OS)of the patients using Cox regression analysis.In human hepatoma Hep3B cells,the effects of lentivirus-mediated MPP6 knockdown on biological behaviors of the cells were evaluated using colony formation assay,wound-healing assay,Transwell assay and flow cytometry.Results Compared with adjacent tissues,HCC tissues showed significant overexpression of MPP6(P<0.001),whose expression levels was correlated with liver cirrhosis(P=0.028)and baseline albumin level(P=0.035)of the patients.Survival analysis suggested that HCC patients with a high expression of MPP6 had a worse prognosis(P<0.001);Cox regression analysis indicated that MPP6 expression(P=0.012,HR:2.335,95%CI:1.502-3.629)was an independent predictor of OS of HCC patients following hepatectomy.In Hep3B cells,MPP6 knockdown obviously inhibited cell proliferation,migration and invasion(P<0.05)and caused cell cycle arrest in G0/G1 phase(P<0.001).Conclusion MPP6 is highly expressed in HCC tissues and correlates with important clinicopathological features and unfavorable prognosis of the patients.MPP6 knockdown inhibits proliferation,migration and invasion and blocks cell cycle progression in G0/G1 phase.

Abstract:Objective To develop an cost-effective and convenient method for culturing human nasal organoids to establish an in vitro Epstein-Barr virus(EBV)infection model.Methods Nasal polyp tissue obtained from surgery was routinely washed,cut,digested and filtered to obtain cell clusters.The cell clusters were then expanded into undifferentiated nasal organoids through dynamic suspension culture under matrix-free conditions,followed by a 14-day differentiation induction treatment to obtain differentiated nasal organoids.The major cellular components of the organoids were identified by immunofluorescence staining and immunohistochemistry.The nasal organoids were infected by EBV in vitro,and viral replication was verified by detecting the expressions of the virus-specific genes EBNA1 and BALF5 using RT-qPCR and immunofluorescence staining.Results Nasal organoids consisting of basal cells,mucous cells,and ciliated cells in a martigel-free system were obtained successfully by dynamic suspension culture.The differentiated nasal organoids expressed high levels of EBV-associated receptors EphA2,NRP1,and NMHCII-A.Both the undifferentiated and differentiated nasal organoids could be infected by EBV.Viral replication in the organoids increased with the viral exposure load,and the differentiated organoids appeared more permissive to viral replication.Conclusion The matrigel-free dynamic suspension culture method is economical and simple for constructing nasal organoids,which can be used as a model of EBV infection for studies of epithelial EBV infection.

Abstract:Objective To investigate the therapeutic effect of verbenalin(VE)on Crohn's disease(CD)-like colitis and the underlying molecular mechanism.Methods Fifty C57BL/6 mice were randomly divided into control group,TNBS group,and low-,medium-,and high-dose VE treatment groups(n=10).Mouse models of CD-like colitis were established in all but the control group by enema with 25 mg/L TNBS dissolved in ethanol,and the mice in VE treatment groups received daily intraperitoneal injections of VE at 5,10,or 20 mg/kg for 7 days.Cultured colon organoids derived from mouse crypts were exposed to 100 μg/mL lipopolysaccharide(LPS)for 24 h and treated with 5,10,or 20 μmol/L VE.The therapeutic effects of VE in the mouse models were evaluated by assessing changes in disease activity index(DAI),histopathological scores,and spleen index.In both colonic mucosa of the mouse models and the colon organoids,the levels of inflammatory cytokines,expressions of tight junction proteins,and changes in PI3K-AKT pathway proteins were analyzed,and the regulatory mechanism of VE was verified using the PI3K-AKT agonist 740 Y-P.Results In TNBS-treated mice,VE treatment significantly reduced DAI,histopathological scores,and spleen index,and mitigated weight loss,colon shortening and bacterial translocation.VE obviously lowered the expression of pro-inflammatory cytokines in colonic mucosa of the mice and the colon organoids,upregulated ZO-1 and claudin-1 expressions,and reduced bacterial translocation.VE significantly downregulated p-PI3K and p-AKT protein expressions,which was reversed by treatment with 740 Y-P.Conclusion VE inhibits intestinal inflammation and protects intestinal barrier function in mice with CD-like colitis by modulating the PI3K-AKT signaling pathway.

Abstract:Objective To investigate the mechanism of Radix codonopsis combined with Poria(CRP)for improving cognitive impairment induced by permanent unilateral common carotid artery ligation(UCCA)in rats.Methods UPLC-Q-TOF-MS/MS and network pharmacology analysis were used to analyze the components of CRP in the blood and brain of rats medicated with CRP.Rat models of UCCA were treated with CRP gavage for 1 month,and Morris water maze,HE staining,and NeuN staining were used to assess the therapeutic efficacy.Metabolomics analysis,ELISA,immunohistochemistry,and Western blotting were employed to explore and validate the therapeutic mechanism of CRP.Results A total of 125 chemical constituents were identified in the brain tissue(89 Radix codonopsis components and 36 Poria components)and 126 in blood samples(85 Radix codonopsis components and 41 Poria components)of medicated rats.Network pharmacology analysis revealed that the therapeutic mechanism of CRP on dementia involved primarily the PI3K/Akt signaling pathway,regulated by the brain-targeting constituents of CRP.In UCCA rats,CRP treatment significantly improved their performance in Morris water maze test,alleviated neuronal damage in the hippocampus and cortex,and increased expression level of NeuN.Metabolomics analysis revealed significant enrichment of the estrogen and GABAergic synapse signaling pathways in CRP-treated rats.CRP obviously reduced the brain levels of glutamate and GABA/glutamate in UCCA rats,and downregulated the proteins expressions of ERα,p-PI3K/PI3K and p-Akt/Akt.Conclusion CRP improves learning and memory impairment in UCCA rats possibly by modulating the ERα/PI3K/Akt signaling pathway in the brain.

Abstract:Objective To explore the impact of different missing data handling methods on AC1 coefficient estimation through simulation studies.Methods Monte Carlo simulation was used to generate evaluation data under different missing mechanisms.The parameters generated included the number of raters,categories,sample size,disease prevalence,random rating probability,and missing proportion.Four missing data handling methods,by excluding subjects with zero ratings,excluding subjects with incomplete ratings,rater mode imputation,and subject mode imputation,were compared using bias and mean squared error(MSE)as metrics.Results When disease prevalence was balanced or the missing data mechanism was missing completely at random(MCAR)or at random(MAR),excluding subjects with zero ratings showed the best performance,with bias and MSE close to zero at a missing proportion below 30%.Under skewed prevalence and missing not at random(MNAR),subject mode imputation was superior for AC1 coefficient estimation,resulting in a bias within±0.10 and an MSE below 0.09;for a sufficient sample size and a missing proportion≤30%,the MSE of this method was nearly zero.Rater mode imputation showed the worst performance across all these scenarios.Excluding subjects with incomplete ratings resulted in an acceptable error only in relatively simple settings(two raters and two categories)with low a missing proportion under MCAR/MAR,but showed a poor stability in other scenarios.Conclusion No universally optimal method exists for handling missing data in AC1 estimation.We recommend excluding subjects with zero ratings for balanced prevalence or MCAR/MAR,and subject mode imputation for skewed prevalence under MNAR.Researchers should report AC1 estimates from multiple methods to allow assessment of result sensitivity.

Abstract:Objective To explore the molecular mechanisms by which quercetin regulates proliferation,migration,and apoptosis of gastric cancer cells.Methods Bioinformatics analysis was conducted to examine the differential expression of Hippo pathway protein yes-associated protein(YAP)between gastric cancer and normal tissues and its correlation with patient survival rates.CCK-8 assay was used to evaluate the effect of quercetin on viability of gastric cancer HGC-27 cells.The expression levels of YAP and phosphorylated YAP(p-YAP)in gastric cancer and normal gastric tissues were detected using Western blotting.In HGC-27 cells with YAP knockdown,the effects of quercetin treatment on cell proliferation,migration and apoptosis were assessed using EdU assay,colony formation assay,Transwell assay,and flow cytometry;the localization of YAP was determined using immunofluorescence staining.Rescue experiments were performed by overexpressing YAP in HGC-27 cells.Results YAP expression was markedly lower in gastric cancer tissues than in normal tissues,and its expression level was negatively correlated with patient survival rates.Quercetin significantly inhibited the survival of HGC-27 cells,promoted MST1 and LATS1 expression,and activated the Hippo signaling pathway,leading to phosphorylation-mediated inactivation of YAP,thereby reducing its nuclear translocation.Both YAP knockdown and quercetin treatment significantly suppressed proliferation and migration and promoted apoptosis of HGC-27 cells.Overexpression of YAP substantially attenuated the inhibitory effects of quercetin in HGC-27 cells.Conclusion Quercetin inhibits proliferation and migration and promotes apoptosis of gastric cancer cells by promoting phosphorylation-mediated inactivation of YAP.

Abstract:Objective To explore the mechanism of Lactobacillus plantarum(L.plantarum)for alleviating learning and memory impairments caused by lead exposure.Methods Twenty-four C57BL/6J mice were randomized equally into control group,lead exposure(100 mg/L)group,and L.plantarum treatment group with daily gavage of L.plantarum(109 CFU)for 10 weeks.Learning and memory abilities of the mice were evaluated using Morris water maze test,and blood lead level and bile acid levels were determined using inductively coupled plasma mass spectrometry(ICP-MS)and liquid chromatography-mass spectrometry(LC-MS),respectively.Hippocampal pathologies of the mice were examined with HE staining,and immunohistochemistry was used to observe morphological changes and microglia activation in the hippocampus.Serum levels of TNF-α and IL-1β of the mice were determined with ELISA,and hippocampal expression levels of NLRP3 and TGR5 proteins were detected using Western blotting.Results Compared with those in lead exposure group,the mice receiving L.plantarum intervention showed significantly increased exploration time,swimming distance,and target platform crossings with reduced latency period to find the hidden platforms in Morris water maze test.L.plantarum intervention partially reversed lead exposure induced reduction of serum levels of deoxycholic acid,3-hydroxydeoxycholic acid,3-hydroxyursodeoxycholic acid,3-thiodeoxycholic acid,and 3-thio-α-methylcholic acid.The treatment also significantly reduced hippocampus pathologies in mice with lead exposure,and reduced their serum levels of IL-1β and TNF-α and hippocampal NLRP3 protein expression levels.Conclusion L.plantarum can alleviate learning and memory impairments caused by lead exposure in mice possibly by regulating bile acid metabolism and inhibiting hippocampal expression of NLRP3 protein.

Abstract:Objective To explore the therapeutic mechanism of Qingjie Fuzheng granules(QFG)for alleviating 5-fluorouracil(5-FU)-induced skeletal muscle atrophy.Methods Male BALB/c mice bearing subcutaneous colorectal cancer CT26 cell xenografts were randomized into control group,model group,and treatment group.The mice in model and treatment groups were given intraperitoneal 5-FU injections every 3 days and treated with daily gavage of saline and QFG for 21 days,respectively;the mice in the control group and normally fed mice were given only saline gavage.Gripping test and hanging test of the mice were performed before and after the treatment,and on day 21,tumor weight and gastrocnemius muscle weight were measured,and histopathology and cell apoptosis in the gastrocnemius muscle were examined with HE staining,transmission electron microscopy and TUNEL assay.ATP content in the muscle was measured,and protein expressions of AMPK,PGC-1α,Cyt c,AIF,Apaf-1,Smac,Bcl-2,Bax,cleaved caspase-3 and cleaved caspase-9 were determined with immunohistochemistry.Results The tumor-bearing mice in the control group showed significantly decreased gastrocnemius muscle weight and grip and suspension test scores.The gastrocnemius muscle showed ultrastructure injuries with lowered ATP content,obvious cell apoptosis,decreased expressions of AMPK,PGC-1 α,and Bcl-2,and increased expressions of Bax,Cyto C,AIF,Apaf-1,Smac,cleaved caspase-3 and cleaved caspase-9.These changes were obviously worsened in 5-FU-treated mice,while QFG treatment significantly increased gastrocnemius muscle weight and strength,ameliorated its ultrastructural injuries,reduced cell apoptosis,and reversed the abnormal protein expressions.Conclusion QFG alleviates 5-FU-induced skeletal muscle fatigue in tumor-bearing mice by activating the AMPK/PGC-1α pathway and inhibiting mitochondria-dependent apoptosis in the gastrocnemius muscle.

Abstract:Objective To enhance the accuracy and reliability of 12-lead electrocardiogram(ECG)automatic diagnosis.Methods Herein we propose a 12-lead ECG automatic diagnosis model based on deep feature fusion(MRHL-ECGNet),which consists of a multi-scale feature extraction front-end,ResNet-34,a global feature mixing module,and a time-series analysis module.The Hyena Hierarchy Convolution Operator was applied to the 12-lead ECG automatic diagnosis task for more efficient capture of long-range dependencies while reducing computational complexity.Integrated Gradients(IG)-based interpretability analysis technology was used to achieve visualization of the decision-making basis of MRHL-ECGNet.The CPSC2018 dataset was used to train and test MRHL-ECGNet,and its performance was assessed using multiple quantitative evaluation indicators and evaluation experiments.Results In the 9-class ECG classification task on the test set,MRHL-ECGNet achieved an accuracy of 0.972,an AUC of 0.983,an F1 score of 0.864,a precision of 0.873,and a recall of 0.857,all surpassing other comparative models.This model only took 0.007 s to output a diagnosis for a single sample on a GPU and 0.156 s on a CPU,with a memory footprint of 67.196 MB.Conclusion The proposed MRHL-ECGNet model demonstrates excellent classification performance in 12-lead ECG automatic diagnosis with a lightweight design and good interpretability,and thus has great potential for clinical application in ECG-aided diagnosis.

Abstract:Objective To explore the therapeutic mechanism of Jiangzhi Quban Recipe(JZQBR)for type 2 diabetes mellitus(T2DM)complicated with hyperlipidemia and validate its clinical efficacy and safety.Methods The active components and disease targets of JZQBR were screened using TCMSP and GeneCards databases,followed by protein-protein interaction analysis and GO and KEGG enrichment analyses.In the animal experiments,ApoE-/-mice were randomized into blank control,model,simvastatin treatment,and low-and high-dose JZQBR groups.In the latter 4 groups,the mice were fed a high-fat diet for 24 weeks with corresponding treatments from Weeks 9 to 24.The changes in body weight,blood glucose,lipids,liver pathology,and inflammatory cytokine expressions of the mice were examined.In the clinical study,72 T2DM patients with hyperlipidemia were randomized equally into control group for treatment with metformin plus empagliflozin and JZQBR group with additional JZQBR for 12 consecutive weeks.Results Network pharmacology identified 65 potential targets,with quercetin,kaempferol,and luteolin as the core components and IL-6,IL-1β,and TNF-α as the key targets.The targets were enriched mainly in the pathways involving inflammatory responses and diabetic complications.In the ApoE-/-mouse models,JZQBR treatment dose-dependently improved body weight,blood glucose,and blood lipid profiles,and high-dose JZQBR produced a stronger effect than simvastatin for improving hepatic steatosis and significantly reduced inflammatory cytokine levels.In the clinical trial,29 patients in JZQBR group and 31 in the control group completed the trial.The patients in JZQBR group showed significant improvements in body weight,FBG,TG,HbA1c,and liver enzymes with significantly lower fasting blood glucose level than the control group.The total effective rates were comparable between the two groups.Conclusion JZQBR improves T2DM complicated with hyperlipidemia possibly by multi-target regulation of the inflammation-metabolism network.

Abstract:Objective To explore the role of miR-221-3p in mediating the positive effects of aerobic exercise on macrophage polarization in the adipose tissues and insulin resistance(IR).Methods Sixteen normal C57BL/6J mice and 16 mice with IR induced by high-fat diet(HFD)feeding for 12 weeks were both randomized into sedentary group and exercise group with aerobic exercise training on a treadmill(5 times per week for 8 consecutive weeks).All the mice were examined for changes in body weight,body composition,fasting blood glucose,blood lipid levels,insulin levels,miR-221-3p expression level,mRNA levels of Socs1,Tnf-α and Arg-1,and protein levels of SOCS1,JAK1,p-STAT1,and p-STAT3 in the adipose tissues,and the targeting relationship between miR-221-3p and SOCS1 was validated using dual-luciferase reporter gene assay.In RAW264.7 macrophages,the effects of transfection with miR-221-3p mimic or inhibitor on macrophage polarization were observed.Results In mice with normal feeding,aerobic exercise significantly decreased body weight,fat mass,fat percent,fasting blood glucose,serum insulin level,HOMA-IR,and TC and TG levels,and reduced miR-221-3p levels in both the plasma and the adipose tissues.The sedentary IR mice showed significantly increased miR-221-3p levels in both the plasma and adipose tissue,increased protein levels of iNOS,JAK1,and p-STAT1/STAT1,and decreased protein levels of Arg-1,SOCS1 and p-STAT3/STAT3,which were significantly reversed after aerobic exercise intervention.Dual-luciferase reporter gene assays validated the targeting relationship between miR-221-3p and SOCS1.In RAW264.7 macrophages,miR-221-3p overexpression significantly reduced Socs1 and Arg-1 mRNA expression,whereas miR-221-3p inhibition obviously promoted M2 polarization of the macrophages.Conclusion Aerobic exercise improves HFD-induced IR in mice possibly by inhibiting miR-221-3p to activate the SOCS1 and JAK/STAT signaling pathway,thereby promoting macrophage M2 polarization and alleviating chronic inflammation in the adipose tissue.

Abstract:Objective To investigate the mechanism of poricoic acid A(PAA)for alleviating dextran sulfate sodium(DSS)-induced colitis in mice.Methods Eighteen C57BL/6 mice were randomly divided into control group,DSS-induced colitis model group,and PAA intervention(10 mg/kg)group.The changes in body weight,colon length,disease activity index(DAI),and histopathological scores of the mice were evaluated.In a DSS-induced Caco-2 cell model,the changes in expressions of ZO-1,claudin-1,Bcl-2,Bax,cleaved caspase-3,LC3-II/I,and P62 were detected.Molecular docking and Western blotting were used to analyze the mechanisms underlying the ameliorating effect of PAA on DSS-induced colitis.Results In the mouse models of DSS-induced colitis,PAA significantly ameliorated DSS-induced weight loss,colon shortening,and elevation of DAI scores while reducing colonic IL-1β and TNF-α levels.HE staining showed that PAA obviously alleviated colonic crypt damage,reduced inflammatory cell infiltration,and lowered histopathological scores of the colon.AB-PAS staining revealed significantly increased goblet cell counts in PAA-treated mice compared to those in DSS group.In DSS-induced Caco-2 cells,PAA treatment effectively inhibited DSS-induced downregulation of the tight junction proteins,reduced Bax and cleaved caspase-3 expressions,increased Bcl-2 expression and the LC3-II/I ratio,and decreased P62 expression.Mechanistic study suggested that PAA targeted the AMPK/mTOR pathway to activate autophagy and suppress cell apoptosis.Conclusion PAA protects intestinal barrier function and alleviates DSS-induced colitis in mice by activating AMPK/mTOR-mediated autophagy and inhibiting intestinal epithelial cell apoptosis.

Abstract:Objective To establish a pelvic active bone marrow(ABM)segmentation method based on diffusion cycle-consistent generative adversarial networks for improving individualized precision of conventional anatomical atlas-based methods.Methods We collected pelvic PET-CT data from 253 patients and constructed a 3-stage cascaded cross-modal learning framework for precise individualized ABM identification from CT images.The framework used cycle-consistent generative adversarial networks for bidirectional CT-PET mapping,conditional diffusion modules with 1000-step Markov chains for progressive denoising,and multi-scale progressive feature pyramid fusion networks for segmentation.The peak signal-to-noise ratio(PSNR),structural similarity index(SSIM),normalized mean square error(NMSE),Dice similarity coefficient(DSC),and average symmetric surface distance(ASSD)were used for evaluation of the model performance for ABM segmentation.Results The proposed method outperformed the existing methods with a PSNR of 26.42±0.63 dB,an SSIM of 0.894±0.011,and an NMSE of 0.0235±0.0026.For ABM segmentation,the average Dice coefficient of the model reached 0.777±0.023 with an ASSD of 3.52±0.41 mm.Conclusion Compared with the conventional methods,the propose method significantly improves individualized segmentation accuracy of the ABM and is thus suitable use in individualized bone marrow protection radiotherapy for rectal cancer.

Abstract:Objective To explore the transcriptomic characteristics of the endplate cartilage of the cervical intervertebral disc in cervical spondylosis(CS)and the effects of acupotomy on expressions of fibroblast growth factor 18(FGF18)/protein kinase B(Akt)axis key molecules.Methods Transcriptomic analyses were performed using bioinformatics methods based on the GEO database.In the animal experiment,24 New Zealand rabbits were randomized equally into control,CS model,and acupotomy groups.In the latter two groups,CS models were established followed 7 days later by acupotomy intervention(once a week for 3 weeks)or no particular treatment.Histopathological changes and cell apoptosis in the intervertebral discs were examined with HE staining and TUNEL assay,and the mRNA expressions of FGF18,FGFR3,and Akt in the intervertebral discs were detected using RT-qPCR;the localization and expressions of FGF18,p-Akt,and Akt proteins were examined with immunohistochemistry.Results The endplate cartilage in CS exhibited numerous differentially expressed genes enriched in the PI3K-Akt signaling,calcium signaling,and Rap1 signaling pathways.CS rabbits showed obvious changes in the cervical spine curvature and joint degenerations,changes in cervical intervertebral disc texture,thinning of the annulus fibrosus,shrinkage or even absence of the nucleus pulposus,and increased apoptotic cells in the endplate cartilage,which were all obviously alleviated after acupotomy.No significant differences were found in the mRNA expressions of FGF18,FGFR3,and Akt in the cervical intervertebral discs among the 3 groups.Acupotomy significantly increased FGF18 and p-Akt protein expressions and reduced the Akt/p-Akt ratio in the cervical endplate cartilage of CS rabbits.Conclusion The cervical endplate cartilage of CS show numerous differentially expressed genes.Acupotomy may delay degenerative changes of the intervertebral discs and improves CS by activating the FGF18/Akt axis to reduce apoptosis of endplate cartilage cells.

Abstract:Objective To analyze the expression pattern of Exoribonuclease Family Member 3(ERI3)in hepatocellular carcinoma(HCC)tissues and its influences on long-term prognosis and cancer cell metastasis.Methods Based on the TCGA-LIHC dataset(including 377 HCC and 50 adjacent normal tissues),the differential expression of ERI3 was analyzed using DESeq2,and the results were validated using immunohistochemical data from the HPA database.A protein-protein interaction network was constructed using STRING and GeneMANIA.The prognostic value of ERI3 was assessed by Cox regression and Kaplan-Meier(KM)survival analyses,its diagnostic efficacy evaluated by ROC curve analysis,and its correlation with immune infiltration analyzed with ssGSEA algorithm.A nomogram prognostic model was established using multivariate Cox regression.For functional validation of ERI3 in vitro,a human HCC cell line SMMC-7721 with ERI3 knockdown was constructed,and the changes in cell proliferation,migration,and invasion were assessed using CCK-8,colony formation,wound healing,and Transwell assays.Results ERI3 was significantly overexpressed in HCC tissues(P<0.001)and its expression levels increased progressively with advanced TNM stages(T1-T4:P<0.001).In HCC patients,high ERI3 expressions were correlated with a reduced overall survival(HR=2.86,95%CI:1.68-4.88;P<0.001),disease-specific survival(HR=2.27,P=0.013),and progression-free interval(HR=1.83,P=0.012).Diagnostic efficacy analysis revealed an AUC of 0.955(95%CI:0.931-0.978)for ERI3.Immune infiltration studies demonstrated a positive correlation of ERI3 expression level with Th2 cells(r=0.340,P<0.001)and a negative correlation with Th17 cells(r=-0.284,P<0.001).Multivariate Cox regression analysis identified ERI3 as an independent prognostic factor for HCC(HR=1.987,P=0.003),and the constructed nomogram showed a good predictive accuracy(C-index=0.668).In SMMC-7721 cells,ERI3 knockdown significantly suppressed cell proliferation,migration,and invasion.Conclusion ERI3 overexpression promotes HCC cell proliferation,migration,and invasion and is strongly linked to a poor prognosis of the patients.

Abstract:Objective To evaluate the effect of exosomes derived from folic acid(FA)-treated infrapatellar fat pad mesenchymal stem cells(IPFP-MSCs)on M1 and M2 polarization of macrophages in vitro.Methods Infrapatellar fat pad tissues were obtained from surgical patients without knee osteoarthritis to isolate IPFP-MSCs.The exosomes were extracted from the cell cultures with or without FA treatment and identified by transmission electron microscopy,TEM,NTA and Western blotting.RAW264.7 cells were induced with lipopolysaccharide(LPS)and incubated with exosomes from FA-treated or untreated IPFP-MSCs for 12 h,and Exos uptake was observed using confocal microscopy.The changes in expression levels of IL-1β,IL-6,TNF-α,i NOS,ARG1,MRC1,and CD206 in the macrophages were detected using qRT-PCR,ELISA,flow cytometry and immunofluorescence staining.Results The exosomes derived from IPFP-MSCs showed a typical cup shape,were positive for CD9 and CD81,and could be uptaken by macrophages.In LPS-induced macrophages,incubation with exosomes from FA-treated IPFP-MSCs significantly decreased the expressions of IL-1β,IL-6,TNF-α,and NOS2,and increased the expressions of ARG1 and MRC1.Treatment of the macrophages with exosomes from FA-treated IPFP-MSCs significantly lowered CD86-positive cell percentage,increased CD206-positive cells and the CD206/CD86 ratio,lowered cellular expression of iNOS,and enhanced the expression of CD206.Conclusion Exosomes from FA-treated IPFP-MSCs promotes M2 polarization of macrophages more effectively than exosomes from unmodified IPFP-MSCs,suggesting a new exosome modification strategy for targeted treatment of knee osteoarthritis.

Abstract:Objective To investigate the protective effects of Poria cocos polysaccharide(PCP)against cyclophosphamide(CTX)-induced intestinal mucosal injury and its impact on gut flora and their metabolites in mice.Methods Adult BALB/C mice were randomized into normal control group,CTX model group,glutamine(positive control)group,and low-,medium-and high-dose PCP treatment groups.In all but the normal control group,the mice were subjected to modeling of CTX-induced intestinal mucosal injury by intraperitoneal CTX injections for 3 days,followed by treatment with gavage of normal saline,glutamine(300 mg/kg),or PCP at 75,150,or 300 mg/kg for 7 consecutive days.The colonic expressions of tight junction proteins(occludin and ZO-1),serum endotoxin,D-lactate,and DAO levels,intestinal permeability,colon injury,and colonic cytokine levels(IL-4,IL-22,IL-17A,and IFN-γ mRNA)were assessed.Gut microbiota,short-chain fatty acids(SCFAs;mainly acetates and propionates)and colonic GPR41 expression were analyzed using 16S rRNA sequencing,GC-MS,and Western blotting,respectively.Fecal microbiota transplantation(FMT)experiment was conducted to validate the role of gut microbes in PCP-mediated repair of intestinal injuries.Results Compared with those in the model group,the mice treated with PCP showed significantly increased colonic occludin and ZO-1 expressions,reduced serum endotoxin,D-lactate and DAO levels,and lowered intestinal permeability with increased colonic expressions of IL-4,IL-22,IL-17A,and IFN-γ mRNA.PCP treatment obviously increased the abundance of Muribaculaceae,decreased Lactobacillus and Bacteroides,increased the contents of acetate and propionate in the colon,and upregulated colonic GPR41 expression.The results of FMT experiment confirmed the crucial role of gut microbes in PCP-mediated repair of CTX-induced intestinal injuries in mice.Conclusion PCP can protect against CTX-induced intestinal mucosal injury in mice possibly by modulating gut flora and SCFAs metabolism to enhance intestinal defense capacity.

Abstract:Objective To improve the accuracy of machine learning models for preoperative prediction of high-intensity focused ultrasound(HIFU)ablation efficacy for uterine fibroids by correcting class imbalance in small sample datasets using undersampling methods.Methods Clinical and imaging data were collected from 140 patients with uterine fibroids undergoing HIFU treatment at Foshan Women and Children Hospital,including 104 with high ablation rates and 36 with low ablation rates.Radiomic features were extracted from MRI T2-weighted images(T2WI)of the patients,and machine learning models were constructed to predict HIFU treatment outcomes.Four machine learning algorithms,including k-Nearest Neighbors(KNN),Random Forest(RF),Support Vector Machine(SVM),and Multilayer Perceptron(MLP),were coupled with 7 undersampling methods,namely Random Undersampling(RUS),Repeated Edited Nearest Neighbors(RENN),All k-Nearest Neighbors(AllKNN),Neighborhood Cleaning Rule-3(NM),Condensed Nearest Neighbor(CNN),Neighborhood Cleaning Rule(NCR),and Instance Hardness Threshold(IHT),for handling class imbalance in the datasets.The 28 prediction models were evaluated using 5-fold cross-validation for areas under the receiver operating characteristic curve(AUC),accuracy,recall,and specificity.Results The best combinations of undersampling methods and machine learning models CNN-RF,NM-SVM,CNN-KNN,and NM-MLP had AUCs of 0.772(95%CI:0.566-0.942),0.797(95%CI:0.600-0.950),0.822(95%CI:0.635-0.964),and 0.822(95%CI:0.632-0.960),respectively.The AUCs of the machine learning models significantly increased after coupling with undersampling methods,with the MLP model showing the most pronounced improvement.The recall rates of the 4 combined models also improved significantly(by 0.389 for CNN-RF,0.836 for NM-SVM,0.532 for CNN-KNN,and 0.372 for NM-MLP).Conclusion The use of undersampling methods can effectively correct class imbalance in small sample datasets to improve the accuracy of machine learning models for predicting the efficacy of HIFU ablation for uterine fibroids.