Danhong injection （DHI）, a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is widely used in China for treating acute isch-emic stroke. In the pres...Danhong injection （DHI）, a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is widely used in China for treating acute isch-emic stroke. In the present study, we explored the neuroprotective efficacy of DHI in a rat model of temporary middle cerebral artery ocdusion, and evaluated the potential mechanisms under-lying its effects. Pretreatment with DHI （0.9 and 1.8 mL/kg） resulted in a significantly smaller infarct volume and better neurological scores than pretreatment with saline. Furthermore, DHI significantly reduced the permeability of the blood-brain barrier, increased occludin protein expression and decreased neutrophil infiltration, as well as profoundly suppressing the upreg-ulation of matrix metallopeptidase-9 expression seen in rats that had received vehicle. Matrix metallopeptidase-2 expression was not affected by ischemia or DHI. Moreover, DHI （1.8 mL/kg） administered 3 hours after the onset of ischemia also improved neurological scores and reduced infarct size. Our results indicate that the neuroprotective efficacy of DHI in a rat model of cerebral ischemia-reperfusion injury is mediated by a protective effect on the blood-brain barrier and the reversal of neutrophil infiltration.展开更多
AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） tra...AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notc展开更多
High myopia has long been highly prevalent worldwide with a largely yet unexplained genetic contribution.To identify novel susceptibility genes for axial length(AL)in highly myopic eyes,a genome-wide association study...High myopia has long been highly prevalent worldwide with a largely yet unexplained genetic contribution.To identify novel susceptibility genes for axial length(AL)in highly myopic eyes,a genome-wide association study(GWAS)was performed using the genomic dataset of 350 deep whole-genome sequencing data from highly myopic patients.Top single nucleotide polymorphisms(SNPs)were functionally annotated.Immunofluorescence staining,quantitative polymerase chain reaction,and western blot were performed using neural retina of form-deprived myopic mice.Enrichment analyses were further performed.We identified the four top SNPs and found that ADAM Metallopeptidase With Thrombospondin Type 1 Motif 16(ADAMTS16)and Phosphatidylinositol Glycan Anchor Biosynthesis Class Z(PIGZ)had the potential of clinical signifi-cance.Animal experiments confirmed that PIGZ expression could be observed and showed higher expression level in form-deprived mice,especially in the ganglion cell layer.The messenger RNA(mRNA)levels of both ADAMTS16 and PIGZ were significantly higher in the neural retina of form-deprived eyes(p=0.005 and 0.007 respectively),and both proteins showed significantly upregulated expression in the neural retina of deprived eyes(p=0.004 and 0.042,respectively).Enrichment analysis revealed a significant role of cellular adhesion and signal transduction in AL,and also several AL-related pathways including circadian entrainment and inflammatory mediator regulation of transient receptor potential channels were proposed.In conclusion,the current study identified four novel SNPs associated with AL in highly myopic eyes and confirmed that the expression of ADAMTS16 and PIGZ was significantly upregulated in neural retina of deprived eyes.Enrichment analyses provided novel insight into the etiology of high myopia and opened avenues for future research interest.展开更多
OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect th...OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.展开更多
BACKGROUND The objectives of this study were to identify hub genes and biological pathways involved in lung adenocarcinoma(LUAD)via bioinformatics analysis,and investigate potential therapeutic targets.AIM To determin...BACKGROUND The objectives of this study were to identify hub genes and biological pathways involved in lung adenocarcinoma(LUAD)via bioinformatics analysis,and investigate potential therapeutic targets.AIM To determine reliable prognostic biomarkers for early diagnosis and treatment of LUAD.METHODS To identify potential therapeutic targets for LUAD,two microarray datasets derived from the Gene Expression Omnibus(GEO)database were analyzed,GSE3116959 and GSE118370.Differentially expressed genes(DEGs)in LUAD and normal tissues were identified using the GEO2R tool.The Hiplot database was then used to generate a volcanic map of the DEGs.Weighted gene co-expression network analysis was conducted to cluster the genes in GSE116959 and GSE-118370 into different modules,and identify immune genes shared between them.A protein-protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes database,then the CytoNCA and CytoHubba components of Cytoscape software were used to visualize the genes.Hub genes with high scores and co-expression were identified,and the Database for Annotation,Visualization and Integrated Discovery was used to perform enrichment analysis of these genes.The diagnostic and prognostic values of the hub genes were calculated using receiver operating characteristic curves and Kaplan-Meier survival analysis,and gene-set enrichment analysis was conducted.The University of Alabama at Birmingham Cancer data analysis portal was used to analyze relationships between the hub genes and normal specimens,as well as their expression during tumor progression.Lastly,validation of protein expression was conducted on the identified hub genes via the Human Protein Atlas database.RESULTS Three hub genes with high connectivity were identified;cellular retinoic acid binding protein 2(CRABP2),matrix metallopeptidase 12(MMP12),and DNA topoisomerase II alpha(TOP2A).High expression of these genes was associated with a poor LUAD prognosis,and the genes exhibited high diagnostic value.CON展开更多
Matrix metallopeptidase 3 is a zinc-containing proteinase that participates in tissue remodeling and immune responses. In this study, a cDNA encoding matrix metallopeptidase 3 was isolated and characterized from the A...Matrix metallopeptidase 3 is a zinc-containing proteinase that participates in tissue remodeling and immune responses. In this study, a cDNA encoding matrix metallopeptidase 3 was isolated and characterized from the Asian yellow pond turtle Mauremys mutica（designated as MaMMP3）. The MaMMP3 cDNA is 1805 bp and consists of a 5＇-untranslated region（UTR） of 56 bp, a 3＇-UTR of 243 bp, and an open reading frame（ORF） of 1506 bp encoding 481 amino acids. Homology analysis of MaMMP3 revealed that the MaMMP3 shared 25%–63% similarity to other known MMP3 sequences. The genomic sequence covers 6007 bp. Comparative analysis of the cDNA sequence revealed that the Asian yellow pond turtle MMP3 has eight exons and seven introns. The phylogenetic tree showed that the MaMMP3 is closely related to Gallus gallus MMP3 and Taeniopygia guttata MMP3. The mRNA expression of the MaMMP3 in normal group without any bacterial challenge could be detected in all studied tissues including kidney, heart, live and spleen, with the highest level in the spleen. The results of immune challenge showed that the expression level of MaMMP3 was up-regulated in the spleen and liver. These results provided an important information for studying the roles of Asian yellow pond turtle MMP3 in immunity further.展开更多
Summary： Recently, several studies showed that gastrointestinal tract may be associated with patho- physiology of Parkinson＇s disease （PD）. Intestine tight junction protein zonula occluden-1 （ZO-1） is an importa...Summary： Recently, several studies showed that gastrointestinal tract may be associated with patho- physiology of Parkinson＇s disease （PD）. Intestine tight junction protein zonula occluden-1 （ZO-1） is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 （MMP-9）. In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine （MPTP）-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-I expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvas- tatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that＇pass across the intestinal barrier.展开更多
AIM:To solidify the involvement of Saa-related pathway in corneal neovascularization(CorNV).The pathogenesis of inflammatory CorNV is not fully understood yet,and our previous study implicated that serum amyloid A(Saa...AIM:To solidify the involvement of Saa-related pathway in corneal neovascularization(CorNV).The pathogenesis of inflammatory CorNV is not fully understood yet,and our previous study implicated that serum amyloid A(Saa)1(Saa1)and Saa3 were among the genes up-regulated upon CorNV induction in mice.METHODS:Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members(Saa1-4),six reported SAA receptors(formyl peptide receptor 2,Tlr2,Tlr4,Cd36,Scarb1,P2rx7)and seven matrix metallopeptidases(Mmp)1a,1b,2,3,9,10,13reportedly to be expressed upon SAA pathway activation.The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods.CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea.At desired time points,the corneas were harvested for histology examination or for extraction of mRNA and protein.The mRNA levels of Saa1,Saa3,Fpr2,Mmp2and Mmp3 in corneas were detected using quantitative reverse transcription-PCR,and SAA3 protein in tissues detected using immunohistochemistry or western blotting.RESULTS:Microarray data analysis revealed that Saa1,Saa3,Fpr2,Mmp2,Mmp3 messengers were readily detected in normal corneas and significantly upregulated upon CorNV induction.The changes of these five genes were confirmed with real-time PCR assay.Onthe contrary,other SAA members(Saa2,Saa4),other SAA receptors(Tlr2,Tlr4,Cd36,P2rx7,etc),or other Mmps(Mmp1a,Mmp1b,Mmp9,Mmp10,Mmp13)did not show consistent changes.Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their upregulation in corneas with CorNV.CONCLUSION:SAA-FPR2 pathway composing genes were expressed in normal murine corneas and,upon inflammatory stimuli challenge to the corneas,their expressions were up-regulated,suggesting their roles in 展开更多
Background： Dendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopep...Background： Dendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 （MMP- 13）, a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells （DCs）. The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis. Methods： Bone marrow-derived dendritic cells were obtained fiom C57BL/6 mice. One short-interfering RNA specific for MMP- 13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry. Results： Compared to the control DCs, MMP- 13-silenced DCs increased expression of anti-apoptosis-related genes, BAGl （control group vs. MMP-13-silenced group： 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008）, BCL-2 （control group vs. MMP-13-silenced group： 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036）, and TP73 （control group vs. MMP- 13-silenced group： 4.33 ± 0.29 vs. 5.60 ±0.32, P = 0.001 ） and decreased apoptosis-related genes, CASPI （control group vs. MMP- 13-silenced group： 3.79±0.67 vs. 2.54±0.39, P - 0.019）, LTBR （control group vs. MMP- 13-silenced group： 9.23 ±1.25 vs. 6.24 ± 1.15, P = 0.012）, and CASP4 （control group vs. MMP-13-silenced group： 2.07 ± 0.56 vs. 0.35 ±0.35, P = 0.002）. Protein levels confirmed the same expression pattern. MMP- 13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability. Conclusion： These results indicate that MMP-13 inhibition 展开更多
Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic human pathogen that can lead to severe diseases in immunocompromised patients. The insulin-cleaving membrane protease (ICMP) of P. aeruginosa plays a vi...Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic human pathogen that can lead to severe diseases in immunocompromised patients. The insulin-cleaving membrane protease (ICMP) of P. aeruginosa plays a vital role in the pathogenesis of the bacterium and is therefore characterized as an important bacterial virulence factor. In addition, ICMP also serves as a founding member of the M75 peptidase family and represents a prototype of the imelysin/imelysin-like proteins. Despite of its functional importance in the pathogenesis of P. aeruginosa and of a root position as the prototypic imelysin/imelysin-like member, the structural features of the protein remain uninvestigated. Since preparation of homogeneous and crystallizable protein species is the prerequisite for structural studies by crystallography, we reported the successful expression, purification, and crystallization of P. aeruginosa ICMP in this study. The protein was over-expressed in Escherichia coli as a GST-fusion protein, cleaved to remove the fusion tag, and then purified to homogeneity. Diffractable crystals were obtained using the sitting-drop vapour-diffusion method. The crystals diffracted to 2.5 Å?resolution and belonged to space group P212121, with unit-cell parameters a = 54.47, b = 158.98, c = 162.84 Å, α = β = γ = 90°. Preliminary analysis of the diffraction data revealed high-quality crystallographic statistics with a Matthews coefficient of about 2.61 Å3.Da-1 and a solvent content of about 52.58%, indicating the presence of three ICMP molecules in the asymmetric unit. The current work therefore paved the way for future studies aiming to delineate the characteristics of ICMP at the atomic level.展开更多
BACKGROUND A growing amount of evidence provides support for the hypothesis that acute myocardial infarction(AMI)patients should go through cardiopulmonary exercise testing(CPET)about 3-5 d after AMI is diagnosed,make...BACKGROUND A growing amount of evidence provides support for the hypothesis that acute myocardial infarction(AMI)patients should go through cardiopulmonary exercise testing(CPET)about 3-5 d after AMI is diagnosed,make reasonable exercising prescription,and conduct exercise training under guidance.AIM To investigate the effect of exercise training(ET)on left ventricular systolic function and left ventricular remodeling(LVRM)and to study the possible mechanisms of LVRM by the changes of matrix metallopeptidase 9(MMP-9)and tissue inhibitor of metalloproteinases 1(TIMP-1)in patients with acute STsegment elevation myocardial infarction(STEMI).METHODS Sixty patients with first STEMI undergoing direct percutaneous coronary intervention from February 2008 to October 2008 were randomly assigned to an exercise group(n=30)and a control group(n=30).The levels of MMP-9 and TIMP-1 were measured in all patients at 1 d,10-14 d,30 d,and 6 mo after admission.Two-dimensional echocardiography and cardiopulmonary exercise testing were done in patients at 10-14 d and 6 mo after admission.RESULTS There was no significant difference in CPET at baseline between the exercise group and the control group.At 6 mo,the time of exercise,peak and anaerobic threshold values of O2 uptake,and metabolic equivalents increased in both groups,but markedly increased in the exercise group.At baseline,there were no significant differences in left ventricular ejection fraction(LVEF)between the two groups.At 6 mo,LVEF increased in the exercise group,but not in the control group.At 6 mo,the percentage of patients with positive result of LVRM was 26.6%in the exercise group and 52.6%in the control group(P<0.05).The levels of plasma MMP-9 and TIMP-1 and the ratio of MMP-9 to TIMP-1 in both groups had no significant difference at 1 d and 10-14 d after AMI,but at 30 d and 6 mo,the levels of plasma MMP-9 and TIMP-1 in the exercise group were significantly lower than those in the control group;the ratio of MMP-9 to TIMP-1 in the exercise group was significantly hig展开更多
BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an im...BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced展开更多
基金supported by the National Natural Science Foundation of China,No.81173592National Science and Technology Major Project of the Ministry of Science and Technology of China,No.2011ZX09201-201,2012ZX09101201-004,2012ZX09101202,NCET-130935,2013ZX09201020+1 种基金Tianjin Municipal Applied Basic Research and Cutting-Edge Technology Research Scheme of China,No.14JCYBJC28900Program for Innovation Team Training in Universities in Tianjin,No.TD12-5035
文摘Danhong injection （DHI）, a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is widely used in China for treating acute isch-emic stroke. In the present study, we explored the neuroprotective efficacy of DHI in a rat model of temporary middle cerebral artery ocdusion, and evaluated the potential mechanisms under-lying its effects. Pretreatment with DHI （0.9 and 1.8 mL/kg） resulted in a significantly smaller infarct volume and better neurological scores than pretreatment with saline. Furthermore, DHI significantly reduced the permeability of the blood-brain barrier, increased occludin protein expression and decreased neutrophil infiltration, as well as profoundly suppressing the upreg-ulation of matrix metallopeptidase-9 expression seen in rats that had received vehicle. Matrix metallopeptidase-2 expression was not affected by ischemia or DHI. Moreover, DHI （1.8 mL/kg） administered 3 hours after the onset of ischemia also improved neurological scores and reduced infarct size. Our results indicate that the neuroprotective efficacy of DHI in a rat model of cerebral ischemia-reperfusion injury is mediated by a protective effect on the blood-brain barrier and the reversal of neutrophil infiltration.
基金Supported by The National Natural Science Foundation of China,No. 30870364Fund for Key Laboratory of Digestive System Tumors,Gansu Province,No. lzujbky-2011-t03
文摘AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notc
基金Funding was provided by the National Natural Science Foundation of China,82122017:Xiangjia Zhu,81970780:Yi Lu,81870642:Xiangjia Zhu,81670835:Yi LuScience and Technology Innovation Plan of Shanghai Science and Technology Commission,19441900700:Xiangjia Zhu,21S31904900:Xiangjia Zhu+3 种基金Shanghai Hospital Development Center,SHDC12019X08:Xiangjia ZhuShanghai Shenkang Hospital Development Center,SHDC2020CR4078:Xiangjia ZhuDouble-E Plan of Eye&ENT Hospital,SYA202006:Xiangjia ZhuShanghai Municipal Key Clinical Specialty Program,shslczdzk01901:Xiangjia Zhu.
文摘High myopia has long been highly prevalent worldwide with a largely yet unexplained genetic contribution.To identify novel susceptibility genes for axial length(AL)in highly myopic eyes,a genome-wide association study(GWAS)was performed using the genomic dataset of 350 deep whole-genome sequencing data from highly myopic patients.Top single nucleotide polymorphisms(SNPs)were functionally annotated.Immunofluorescence staining,quantitative polymerase chain reaction,and western blot were performed using neural retina of form-deprived myopic mice.Enrichment analyses were further performed.We identified the four top SNPs and found that ADAM Metallopeptidase With Thrombospondin Type 1 Motif 16(ADAMTS16)and Phosphatidylinositol Glycan Anchor Biosynthesis Class Z(PIGZ)had the potential of clinical signifi-cance.Animal experiments confirmed that PIGZ expression could be observed and showed higher expression level in form-deprived mice,especially in the ganglion cell layer.The messenger RNA(mRNA)levels of both ADAMTS16 and PIGZ were significantly higher in the neural retina of form-deprived eyes(p=0.005 and 0.007 respectively),and both proteins showed significantly upregulated expression in the neural retina of deprived eyes(p=0.004 and 0.042,respectively).Enrichment analysis revealed a significant role of cellular adhesion and signal transduction in AL,and also several AL-related pathways including circadian entrainment and inflammatory mediator regulation of transient receptor potential channels were proposed.In conclusion,the current study identified four novel SNPs associated with AL in highly myopic eyes and confirmed that the expression of ADAMTS16 and PIGZ was significantly upregulated in neural retina of deprived eyes.Enrichment analyses provided novel insight into the etiology of high myopia and opened avenues for future research interest.
文摘OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.
文摘BACKGROUND The objectives of this study were to identify hub genes and biological pathways involved in lung adenocarcinoma(LUAD)via bioinformatics analysis,and investigate potential therapeutic targets.AIM To determine reliable prognostic biomarkers for early diagnosis and treatment of LUAD.METHODS To identify potential therapeutic targets for LUAD,two microarray datasets derived from the Gene Expression Omnibus(GEO)database were analyzed,GSE3116959 and GSE118370.Differentially expressed genes(DEGs)in LUAD and normal tissues were identified using the GEO2R tool.The Hiplot database was then used to generate a volcanic map of the DEGs.Weighted gene co-expression network analysis was conducted to cluster the genes in GSE116959 and GSE-118370 into different modules,and identify immune genes shared between them.A protein-protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes database,then the CytoNCA and CytoHubba components of Cytoscape software were used to visualize the genes.Hub genes with high scores and co-expression were identified,and the Database for Annotation,Visualization and Integrated Discovery was used to perform enrichment analysis of these genes.The diagnostic and prognostic values of the hub genes were calculated using receiver operating characteristic curves and Kaplan-Meier survival analysis,and gene-set enrichment analysis was conducted.The University of Alabama at Birmingham Cancer data analysis portal was used to analyze relationships between the hub genes and normal specimens,as well as their expression during tumor progression.Lastly,validation of protein expression was conducted on the identified hub genes via the Human Protein Atlas database.RESULTS Three hub genes with high connectivity were identified;cellular retinoic acid binding protein 2(CRABP2),matrix metallopeptidase 12(MMP12),and DNA topoisomerase II alpha(TOP2A).High expression of these genes was associated with a poor LUAD prognosis,and the genes exhibited high diagnostic value.CON
基金supported by the Ministry of Science and Technology funds (No. 2011GB23260021)Major State Basic Research Development Program of China (No. 2004CB117401)+1 种基金Science and Technology New Star from Guangzhou City (No. 2012089)Fundamental Research Funds from CAFS (No. 2012A0403)
文摘Matrix metallopeptidase 3 is a zinc-containing proteinase that participates in tissue remodeling and immune responses. In this study, a cDNA encoding matrix metallopeptidase 3 was isolated and characterized from the Asian yellow pond turtle Mauremys mutica（designated as MaMMP3）. The MaMMP3 cDNA is 1805 bp and consists of a 5＇-untranslated region（UTR） of 56 bp, a 3＇-UTR of 243 bp, and an open reading frame（ORF） of 1506 bp encoding 481 amino acids. Homology analysis of MaMMP3 revealed that the MaMMP3 shared 25%–63% similarity to other known MMP3 sequences. The genomic sequence covers 6007 bp. Comparative analysis of the cDNA sequence revealed that the Asian yellow pond turtle MMP3 has eight exons and seven introns. The phylogenetic tree showed that the MaMMP3 is closely related to Gallus gallus MMP3 and Taeniopygia guttata MMP3. The mRNA expression of the MaMMP3 in normal group without any bacterial challenge could be detected in all studied tissues including kidney, heart, live and spleen, with the highest level in the spleen. The results of immune challenge showed that the expression level of MaMMP3 was up-regulated in the spleen and liver. These results provided an important information for studying the roles of Asian yellow pond turtle MMP3 in immunity further.
基金supported by grants from the Natural Science Foundation of Jiangxi Province(No.20142BAB205092)Foundation of Jiangxi Educational Committee for Youths(No.GJJ13030)Jiangxi Province Science Foundation for Youths(No.20132BAB215037)
文摘Summary： Recently, several studies showed that gastrointestinal tract may be associated with patho- physiology of Parkinson＇s disease （PD）. Intestine tight junction protein zonula occluden-1 （ZO-1） is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 （MMP-9）. In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine （MPTP）-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-I expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvas- tatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that＇pass across the intestinal barrier.
基金Supported by National Natural Science Foundation of China(No.8120066481271050)
文摘AIM:To solidify the involvement of Saa-related pathway in corneal neovascularization(CorNV).The pathogenesis of inflammatory CorNV is not fully understood yet,and our previous study implicated that serum amyloid A(Saa)1(Saa1)and Saa3 were among the genes up-regulated upon CorNV induction in mice.METHODS:Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members(Saa1-4),six reported SAA receptors(formyl peptide receptor 2,Tlr2,Tlr4,Cd36,Scarb1,P2rx7)and seven matrix metallopeptidases(Mmp)1a,1b,2,3,9,10,13reportedly to be expressed upon SAA pathway activation.The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods.CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea.At desired time points,the corneas were harvested for histology examination or for extraction of mRNA and protein.The mRNA levels of Saa1,Saa3,Fpr2,Mmp2and Mmp3 in corneas were detected using quantitative reverse transcription-PCR,and SAA3 protein in tissues detected using immunohistochemistry or western blotting.RESULTS:Microarray data analysis revealed that Saa1,Saa3,Fpr2,Mmp2,Mmp3 messengers were readily detected in normal corneas and significantly upregulated upon CorNV induction.The changes of these five genes were confirmed with real-time PCR assay.Onthe contrary,other SAA members(Saa2,Saa4),other SAA receptors(Tlr2,Tlr4,Cd36,P2rx7,etc),or other Mmps(Mmp1a,Mmp1b,Mmp9,Mmp10,Mmp13)did not show consistent changes.Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their upregulation in corneas with CorNV.CONCLUSION:SAA-FPR2 pathway composing genes were expressed in normal murine corneas and,upon inflammatory stimuli challenge to the corneas,their expressions were up-regulated,suggesting their roles in
文摘Background： Dendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 （MMP- 13）, a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells （DCs）. The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis. Methods： Bone marrow-derived dendritic cells were obtained fiom C57BL/6 mice. One short-interfering RNA specific for MMP- 13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry. Results： Compared to the control DCs, MMP- 13-silenced DCs increased expression of anti-apoptosis-related genes, BAGl （control group vs. MMP-13-silenced group： 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008）, BCL-2 （control group vs. MMP-13-silenced group： 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036）, and TP73 （control group vs. MMP- 13-silenced group： 4.33 ± 0.29 vs. 5.60 ±0.32, P = 0.001 ） and decreased apoptosis-related genes, CASPI （control group vs. MMP- 13-silenced group： 3.79±0.67 vs. 2.54±0.39, P - 0.019）, LTBR （control group vs. MMP- 13-silenced group： 9.23 ±1.25 vs. 6.24 ± 1.15, P = 0.012）, and CASP4 （control group vs. MMP-13-silenced group： 2.07 ± 0.56 vs. 0.35 ±0.35, P = 0.002）. Protein levels confirmed the same expression pattern. MMP- 13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability. Conclusion： These results indicate that MMP-13 inhibition
文摘Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic human pathogen that can lead to severe diseases in immunocompromised patients. The insulin-cleaving membrane protease (ICMP) of P. aeruginosa plays a vital role in the pathogenesis of the bacterium and is therefore characterized as an important bacterial virulence factor. In addition, ICMP also serves as a founding member of the M75 peptidase family and represents a prototype of the imelysin/imelysin-like proteins. Despite of its functional importance in the pathogenesis of P. aeruginosa and of a root position as the prototypic imelysin/imelysin-like member, the structural features of the protein remain uninvestigated. Since preparation of homogeneous and crystallizable protein species is the prerequisite for structural studies by crystallography, we reported the successful expression, purification, and crystallization of P. aeruginosa ICMP in this study. The protein was over-expressed in Escherichia coli as a GST-fusion protein, cleaved to remove the fusion tag, and then purified to homogeneity. Diffractable crystals were obtained using the sitting-drop vapour-diffusion method. The crystals diffracted to 2.5 Å?resolution and belonged to space group P212121, with unit-cell parameters a = 54.47, b = 158.98, c = 162.84 Å, α = β = γ = 90°. Preliminary analysis of the diffraction data revealed high-quality crystallographic statistics with a Matthews coefficient of about 2.61 Å3.Da-1 and a solvent content of about 52.58%, indicating the presence of three ICMP molecules in the asymmetric unit. The current work therefore paved the way for future studies aiming to delineate the characteristics of ICMP at the atomic level.
基金Supported by Beijing Hospitals Authority Incubating Program,No.PZ2021007Beijing Hospitals Authority Youth Program,No.QML20200604Beijing Municipal Health Commission(No.17-3)and the Beijing Natural Science Foundation,No.7184205.
文摘BACKGROUND A growing amount of evidence provides support for the hypothesis that acute myocardial infarction(AMI)patients should go through cardiopulmonary exercise testing(CPET)about 3-5 d after AMI is diagnosed,make reasonable exercising prescription,and conduct exercise training under guidance.AIM To investigate the effect of exercise training(ET)on left ventricular systolic function and left ventricular remodeling(LVRM)and to study the possible mechanisms of LVRM by the changes of matrix metallopeptidase 9(MMP-9)and tissue inhibitor of metalloproteinases 1(TIMP-1)in patients with acute STsegment elevation myocardial infarction(STEMI).METHODS Sixty patients with first STEMI undergoing direct percutaneous coronary intervention from February 2008 to October 2008 were randomly assigned to an exercise group(n=30)and a control group(n=30).The levels of MMP-9 and TIMP-1 were measured in all patients at 1 d,10-14 d,30 d,and 6 mo after admission.Two-dimensional echocardiography and cardiopulmonary exercise testing were done in patients at 10-14 d and 6 mo after admission.RESULTS There was no significant difference in CPET at baseline between the exercise group and the control group.At 6 mo,the time of exercise,peak and anaerobic threshold values of O2 uptake,and metabolic equivalents increased in both groups,but markedly increased in the exercise group.At baseline,there were no significant differences in left ventricular ejection fraction(LVEF)between the two groups.At 6 mo,LVEF increased in the exercise group,but not in the control group.At 6 mo,the percentage of patients with positive result of LVRM was 26.6%in the exercise group and 52.6%in the control group(P<0.05).The levels of plasma MMP-9 and TIMP-1 and the ratio of MMP-9 to TIMP-1 in both groups had no significant difference at 1 d and 10-14 d after AMI,but at 30 d and 6 mo,the levels of plasma MMP-9 and TIMP-1 in the exercise group were significantly lower than those in the control group;the ratio of MMP-9 to TIMP-1 in the exercise group was significantly hig
基金Ministry of Science and Technology,Taiwan,No.MOST 108-2320-B-255-002-MY3 and No.MOST 110-2635-B-255-001Chang Gung Medical Research Foundation,Taoyuan,Taiwan,No.CMRPF1I0031,No.CMRPF1L0081,No.CMRPF1L0021,No.CMRPF1L0041,and No.CMRPF1I0042Chang Gung University of Science and Technology,Taoyuan,Taiwan,No.ZRRPF3K0111 and No.ZRRPF3L0091。
文摘BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced
