BACKGROUND:Bone morphogenetic protein 2 is vital in embryonic development,bone formation,and regeneration,but its high-dose application is linked to cancer.Bone morphogenetic protein 2 osteogenic peptide L20 reduces adverse effects like cancer and boosts bone tissue regeneration.OBJECTIVE:To graft bone morphogenetic protein 2 active peptide segments onto mesopores and surfaces through a peptide mimicry strategy inspired by oysters,and explore its impact on osteogenic properties of tissue-engineered bone.METHODS:(1)Mesoporous bioactive glass was synthesized using a template method.Bone morphogenetic protein 2 osteogenic peptide L20 was loaded onto mesoporous bioactive glass using a one-step synthesis method to characterize the morphology and in vitro sustained release properties of mesoporous active glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20.(2)Bone marrow mesenchymal stem cells were isolated and extracted from SD rats.After two generations,they were co-cultured with PBS(blank group),mesoporous bioactive glass nanoparticles(control group),and mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20(experimental group).Cell live/dead fluorescence staining and CCK-8 assay were used to detect cytotoxicity and cell proliferation.Scanning electron microscopy was used to observe cell adhesion.After osteogenic induction and differentiation,alkaline phosphatase staining,Alizarin red S staining,and osteogenesis-related gene expression were detected.(3)Fifteen SD rats were selected to establish bilateral femoral condyle defect models and divided into three groups using a random number table method:the blank group(n=5)was not implanted with any material;the control group(n=5)was implanted with mesoporous bioactive glass nanoparticles,and the experimental group(n=5)was implanted with mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20.Eight weeks after surgery,femoral Micro-CT scanning and tissue morphology observation were performed.RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the mesoporous bioactive glass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20 were spherical and monodisperse particles.Transmission electron microscopy showed their porous structure with an average particle size of(268.10±0.58)nm,which could release L20 in vitro.(2)Mesoporous bioglass nanoparticles loaded with bone morphogenetic protein 2 osteogenic active peptide L20 were non-cytotoxic and could promote the proliferation and adhesion of bone marrow mesenchymal stem cells.Compared with the blank group and the control group,the alkaline phosphatase activity and extracellular matrix mineralization capacity of the experimental group were increased(P<0.05),and the mRNA expression levels of alkaline phosphatase,Runx2,and osteocalcin were increased(P<0.05).(3)The results of femoral Micro-CT scanning showed that compared with the blank group and the control group,the new bone mass and bone density of the experimental group were increased(P<0.05).The results of hematoxylin-eosin and Masson staining showed that compared with the blank group and the control group,the new bone formation and collagen fibers of the experimental group were increased.(4)These findings indicate that mesoporous bioactive glass loaded with bone morphogenetic protein 2 active peptide L20 exhibits excellent biocompatibility and in vitro and in vivo osteogenic properties,promoting regeneration and repair of SD rat femoral condyle defects.

This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.

AIM:To investigate the antitumor activity and targets of baicalein(Bai)in pancreatic cancer using network pharmacology combined with in vitro and in vivo experiments.METHODS:The targets of Bai and pancreatic can-cer were analyzed via multi-data screening.A protein-protein interaction network was constructed using STRING,and core targets were identified via Cytoscape.Functional enrichment was analyzed by Gene Ontology(GO)and Kyoto Ency-clopedia of Genes and Genomes(KEGG).Antitumor effects of Bai were assessed in pancreatic cancer cells Aspc-1 and Bxpc-3 using MTT and colony formation assays for proliferation,flow cytometry for apoptosis and cell cycle analysis,and Transwell assays for migration and invasion.A xenograft tumor model was established to evaluate tumor proliferation,im-munohistochemistry was performed to detect the protein expression of AKT in tumor tissues,and Western blot was used to analyze the expression levels of AKT,β-catenin,N-cadherin and Slug.RESULTS:A total of 108 overlapping targets were identified between Bai and pancreatic cancer.Among these,7 core targets were recognized,including proto-onco-gene tyrosine-protein kinase Src,heat shock protein 90 alpha family class A member 1(HSP90AA1),estrogen receptor 1(ESR1),tumor protein p53(TP53),epidermal growth factor receptor(EGFR),AKT1,and mitogen-activated protein ki-nase 3(MAPK3).The GO analysis revealed significant enrichment in oxidative stress response,protein phosphorylation,and serine/threonine kinase activity.The KEGG analysis primarily enriched the PI3K/AKT,MAPK and Ras signaling pathways.The MTT and colony formation assays showed that Bai inhibited the viability of Aspc-1 and Bxpc-3 cells in a dose-dependent manner(72 h IC50 values were 73.6 μmol/L and 83.4 μmol/L,respectively)and reduced cell colony for-mation(P<0.05 or P<0.01).Flow cytometry confirmed that Bai induced apoptosis of Aspc-1 and Bxpc-3 cells(P<0.01)and blocked the cell cycle at the G0/G1 phase(P<0.05 or P<0.01).Transwell experiments indicated that Bai inhibited the migration and invasion of Aspc-1 and Bxpc-3 cells(P<0.05 or P<0.01).In vivo,Bai significantly inhibited the growth of Aspc-1 cell xenograft tumors(P<0.01).Immunohistochemistry revealed a significant reduction in AKT expression in tu-mor tissues(P<0.01),and Western blot showed decreased expression of AKT,β-catenin,N-cadherin and Slug in both Aspc-1 and Bxpc-3 cells(P<0.01).CONCLUSION:Baicalein inhibits pancreatic cancer cell proliferation,migration,and invasion,potentially through down-regulation of AKT,β-catenin,N-cadherin,and Slug expression.

AIM To investigate the effects of Heixiaoyao Powder on neuroinflammation in APP/PS1 transgenic mice.METHODS The 16-week-old male APP/PS1 transgenic mice were randomly divided into the model group,the BBG group(P2X7R specific antagonist,30 mg/kg)and high,medium and low dose Heixiaoyao Powder groups of(22.10,11.05,5.53 g/kg),in contrast to the male C57BL/6J mice of the same age and the same strain of the blank groups,with 12 mice in each group.When normal saline by gavage was dosed upon the blank group and the model group,and the other groups were treated with corresponding drug by gavage.After 90 days of administration,the mice had their learning and memory ability detected by Morris water maze test;their hippocampal pathological changes observed with HE staining;their MyD88 expression detected by immunofluorescence staining;their hippocampal levels of pro-inflammatory factors(TNF-α,IL-6),anti-inflammatory factors(IL-10),ATP and amyloid protein β(Aβ)detected by ELISA;their hippocampal mRNA expressions of P2X7R,TLR4,MyD88 and NF-κB-P65 detected by RT-qPCR method;and their hippocampal protein expressions of P2X7R,TLR4,MyD88,NF-κB-P65 and p-NF-κB-P65 detected by Western blot.RESULTS Compared with the blank group,the model group demonstrated prolonged escape latency and reduced frequency in crossing platform(P＜0.01);decreased number of hippocampal neurons,deranged neurons,and darker cytoplasm staining;increased immunofluorescence expression of hippocampal MyD88(P＜0.01);decreased IL-10 level(P＜0.01);increased levels of TNF-α,IL-6,ATP and Aβ(P＜0.01);increased mRNA and protein expressions ofP2X7R,TLR4 and MyD88(P＜0.01),and increased protein expression of p-NF-κB-P65(P＜0.01).Compared with the model group,the groups intervened with Heixiaoyao Powder or BBG demonstrated shortened escape latency and increased frequency in crossing platform(P＜0.01);more number of neatly arranged hippocampal neurons;increased hippocampal IL-10 level(P＜0.01),decreased levels of TNF-α,IL-6,ATP and Aβ(P＜0.05,P＜0.01);decreased mRNA and protein expressions of P2X7R,TLR4 and MyD88(P＜0.05,P＜0.01);and decreased protein expression of p-NF-κB-P65(P＜0.05,P＜0.01).Except the low dose Heixiaoyao Powder group,the other treatment groups shared decreased immunofluorescence expression of MyD88(P＜0.05,P＜0.01).CONCLUSION Heixiaoyao Powder can significantly improve the learning and memory ability of APP/PS1 model mice,and its mechanism may lie in its function in alleviating cerebral neuroinflammation by reducing the abnormal Aβ aggregation via inhibiting the activation of ATP/P2X7R/NF-κB signaling pathway.

AIM To investigate the effects of Heixiaoyao Powder on neuroinflammation in APP/PS1 transgenic mice.METHODS The 16-week-old male APP/PS1 transgenic mice were randomly divided into the model group,the BBG group(P2X7R specific antagonist,30 mg/kg)and high,medium and low dose Heixiaoyao Powder groups of(22.10,11.05,5.53 g/kg),in contrast to the male C57BL/6J mice of the same age and the same strain of the blank groups,with 12 mice in each group.When normal saline by gavage was dosed upon the blank group and the model group,and the other groups were treated with corresponding drug by gavage.After 90 days of administration,the mice had their learning and memory ability detected by Morris water maze test;their hippocampal pathological changes observed with HE staining;their MyD88 expression detected by immunofluorescence staining;their hippocampal levels of pro-inflammatory factors(TNF-α,IL-6),anti-inflammatory factors(IL-10),ATP and amyloid protein β(Aβ)detected by ELISA;their hippocampal mRNA expressions of P2X7R,TLR4,MyD88 and NF-κB-P65 detected by RT-qPCR method;and their hippocampal protein expressions of P2X7R,TLR4,MyD88,NF-κB-P65 and p-NF-κB-P65 detected by Western blot.RESULTS Compared with the blank group,the model group demonstrated prolonged escape latency and reduced frequency in crossing platform(P＜0.01);decreased number of hippocampal neurons,deranged neurons,and darker cytoplasm staining;increased immunofluorescence expression of hippocampal MyD88(P＜0.01);decreased IL-10 level(P＜0.01);increased levels of TNF-α,IL-6,ATP and Aβ(P＜0.01);increased mRNA and protein expressions ofP2X7R,TLR4 and MyD88(P＜0.01),and increased protein expression of p-NF-κB-P65(P＜0.01).Compared with the model group,the groups intervened with Heixiaoyao Powder or BBG demonstrated shortened escape latency and increased frequency in crossing platform(P＜0.01);more number of neatly arranged hippocampal neurons;increased hippocampal IL-10 level(P＜0.01),decreased levels of TNF-α,IL-6,ATP and Aβ(P＜0.05,P＜0.01);decreased mRNA and protein expressions of P2X7R,TLR4 and MyD88(P＜0.05,P＜0.01);and decreased protein expression of p-NF-κB-P65(P＜0.05,P＜0.01).Except the low dose Heixiaoyao Powder group,the other treatment groups shared decreased immunofluorescence expression of MyD88(P＜0.05,P＜0.01).CONCLUSION Heixiaoyao Powder can significantly improve the learning and memory ability of APP/PS1 model mice,and its mechanism may lie in its function in alleviating cerebral neuroinflammation by reducing the abnormal Aβ aggregation via inhibiting the activation of ATP/P2X7R/NF-κB signaling pathway.

Aim To investigate the protective effect of Gualou Guizhi granules(GLGZG)on neuronal injury induced by LPS-activated microglia based on Notch signaling pathway.Methods LPS-activated microglia were co-cultured with neurons to construct neuron inju-ry models,and the cells were divided into the control group,model group,Notch inhibitor(DAPT)group,GLGZG(50,100,200 mg·L-1)group,DAPT+100 mg·L-1GLGZG group.After intervention,the activity of HT22 cells was detected by CCK-8 method,and rel-ative mRNA expression was detected by real-time PCR.The relative protein expression was detected by Western blot.Results Compared with the model group,after GLGZG intervention,the cell activity was significantly improved,GLGZG decreased IL-6,IL-12,Bax,Notch 1,caspase-3,Delta-1,NICD,RBPSUH,HES1 expression,and increased Bcl-2 expression(P＜0.05).Compared with the model group,the NICD,RBPSUH and HES1 mRNA and protein expressions significantly decreased after DAPT treatment(P＜0.05),and there was no superposition effect with GLG-ZG.Conclusion GLGZG may play a neuroprotective role by inhibiting inflammatory factors and apoptosis,and inhibiting Notch signaling pathway.

Objective:To explore the risk factors of suicidal ideation in community residents in Shandong province,China,and to establish a prediction model and a nomogram for the risk of suicidal ideation.Methods:To-tally 11 087 community residents in Jining City,Zoucheng City,and Weifang City,Shandong Province,were recrui-ted as the study subjects.The general information questionnaire,Composite International Diagnostic Interview-3.0,General Anxiety Disorder-7,were used to collect demographic information,mental health status,family information,and lifestyle using a computer-assisted interview system to conduct face-to-face interview.LASSO regression and binary logistic regression analyses were used to develop a nomogram model of the risk of suicidal ideation.Results:Young people,female,smoking,non-new town residents,no physical exercise,difficulty approaching people,anxiety symptoms and depression were effective predictors of suicidal ideation.The area under the curve(AUC)of the training set(AUC)was 0.89(95％CI:0.85-0.93)and AUC of the validation set(AUC)was 0.83(95％CI:0.75-0.92).Conclusion:The nomogram prediction model constructed in this study on suicidal ideation in com-munity residents has a high degree of differentiation and accuracy.It could be used to screen people at high risk of suicide and provide intervention to suicidal ideation in community residents.

This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.

AIM:To investigate the antitumor activity and targets of baicalein(Bai)in pancreatic cancer using network pharmacology combined with in vitro and in vivo experiments.METHODS:The targets of Bai and pancreatic can-cer were analyzed via multi-data screening.A protein-protein interaction network was constructed using STRING,and core targets were identified via Cytoscape.Functional enrichment was analyzed by Gene Ontology(GO)and Kyoto Ency-clopedia of Genes and Genomes(KEGG).Antitumor effects of Bai were assessed in pancreatic cancer cells Aspc-1 and Bxpc-3 using MTT and colony formation assays for proliferation,flow cytometry for apoptosis and cell cycle analysis,and Transwell assays for migration and invasion.A xenograft tumor model was established to evaluate tumor proliferation,im-munohistochemistry was performed to detect the protein expression of AKT in tumor tissues,and Western blot was used to analyze the expression levels of AKT,β-catenin,N-cadherin and Slug.RESULTS:A total of 108 overlapping targets were identified between Bai and pancreatic cancer.Among these,7 core targets were recognized,including proto-onco-gene tyrosine-protein kinase Src,heat shock protein 90 alpha family class A member 1(HSP90AA1),estrogen receptor 1(ESR1),tumor protein p53(TP53),epidermal growth factor receptor(EGFR),AKT1,and mitogen-activated protein ki-nase 3(MAPK3).The GO analysis revealed significant enrichment in oxidative stress response,protein phosphorylation,and serine/threonine kinase activity.The KEGG analysis primarily enriched the PI3K/AKT,MAPK and Ras signaling pathways.The MTT and colony formation assays showed that Bai inhibited the viability of Aspc-1 and Bxpc-3 cells in a dose-dependent manner(72 h IC50 values were 73.6 μmol/L and 83.4 μmol/L,respectively)and reduced cell colony for-mation(P<0.05 or P<0.01).Flow cytometry confirmed that Bai induced apoptosis of Aspc-1 and Bxpc-3 cells(P<0.01)and blocked the cell cycle at the G0/G1 phase(P<0.05 or P<0.01).Transwell experiments indicated that Bai inhibited the migration and invasion of Aspc-1 and Bxpc-3 cells(P<0.05 or P<0.01).In vivo,Bai significantly inhibited the growth of Aspc-1 cell xenograft tumors(P<0.01).Immunohistochemistry revealed a significant reduction in AKT expression in tu-mor tissues(P<0.01),and Western blot showed decreased expression of AKT,β-catenin,N-cadherin and Slug in both Aspc-1 and Bxpc-3 cells(P<0.01).CONCLUSION:Baicalein inhibits pancreatic cancer cell proliferation,migration,and invasion,potentially through down-regulation of AKT,β-catenin,N-cadherin,and Slug expression.

Adipose-derived stem cells(ASCs)have become important seed cells in tissue engineering,because of their unique advantages.Extensive research has been conducted to determine the ideal method of cryopreservation for ASCs.In this review,we summarize the basic cryobiology and cryopreservation protocols for ASCs,and look forward to future research into cryopreservation protocols for ASCs.