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Background and Objectives@#Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction ofarticular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. @*Methods@#and Results: Real-time reverse transcription–polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3’-untranscribed region. @*Conclusions@#These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.
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Objective To explore the molecular mechanisms of the effect of eluting stent drug rapamycin for injuring human coronary artery endothelial cells(HCAECs)by using the proteomics method.Methods HCAECs were treated with rapamycin,and the differentially expressed proteins were analyzed by two dimension fluorescence differential gel electrophoresis(2D-DIGE).The changed proteins were identified by MALDI-ToF-ToF.Results At least 85 differential protein spots were found,including 49 up-regulated and 36 down-regulated protein spots.Twenty-six proteins were identified by MALDI-ToF-ToF,including the endoplasmic reticulum protein,mitochondrial protein,molecular chaperones,ubiquitin system related protein,structural protein and oxidative stress related proteins,etc.Conclusion The changes of specific proteins of HCAECs injury induced by rapamycin are investigated by the proteomic method.
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<p><b>OBJECTIVE</b>To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.</p><p><b>METHODS</b>The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.</p><p><b>RESULTS</b>The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.</p><p><b>CONCLUSION</b>The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.</p>
Subject(s)
Animals , Male , Mice , Rats , Antibodies, Helminth , Blood , Antigens, Helminth , Allergy and Immunology , Calmodulin , Allergy and Immunology , Clonorchiasis , Allergy and Immunology , Clonorchis sinensis , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Gene Library , Immunoglobulin G , Blood , Inflammation , Liver Cirrhosis , Parasitology , Recombinant Proteins , Allergy and ImmunologyABSTRACT
AIM:To identify the serum proteins that might serve as biomarkers for predicting mucosal healing ( MH) in the patients with Crohn’ s disease ( CD) treated with infliximab ( IFX) .METHODS:We collected serum sam-ples before treatment (0 week, group A) and 14 weeks after treatment (group B) from 7 CD patients with IFX treatment who had achieved MH, as well as the serum samples from 7 CD patients who had not achieved MH (0 week, group C;14 weeks, group D) .Two-dimensional fluorescence difference gel electrophoresis was applied to analyze and compare the re-sults of serum profiles between groups A and B, C and D, A and C, B and D.Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and bioinformatics tools were utilized to preliminarily identify and figure out the dif-ferentially expressed proteins.RESULTS:(1) In total, there were 44 differentially expressed spots, 36, 3, 10 and 31 differentially expressed spots were detected while comparing A with B, C with D, A with C and B with D, respectively. (2) Among those spots, 17, 2, 2 and 15 proteins were identified, respectively.In total, there were 19 differentially ex-pressed proteins, including apolipoprotein E, apolipoprotein A-I, complement factor H, and so on.(3) Protein functional association networks were carried out based on STRING database.CONCLUSION: The serum protein profiles obviously change after IFX treatment in MH CD patients, and the serum protein profiles of MH patients are different from that of non-MH patients after IFX treatment.The 19 proteins we identified may serve as potential biomarkers for predicting MH in CD patients with IFX treatment.
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Objective To screen the sensitive plasma molecular markers in diabetic nephropathy ( DN) patients with kidney yang deficiency. Methods Two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was used to analyze the plasma which was sampled from 4 early DN patients with kidney yang deficiency and 4 healthy adult volunteers. Proteins that showed differential expression with an over 1.5-fold change were analyzed by MALDI-TOF/TOF mass spectrometry. Results Two-dimensional electrophoresis (2-DE) maps of plasma proteins in DN patients with kidney yang deficiency and healthy adults were established successfully. We validated 9 differentially expressed proteins, including complements C3 and C4, apolipoprotein E and ubiquitination factor. Conclusion Proteomic analysis by 2-D DIGE can objectively reveal the differences of plasma protein expression in DN patents with kidney yang deficiency and healthy adult volunteers. The obtained 9 proteins have potential to provide plasma molecular markers for the early clinical diagnosis and for the research of traditional Chinese medical patterns of DN.
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Objective To explore the sensitive blood plasma molecular markers in diabetic nephropathy (DN) .Methods Two-di-mensional fluorescence difference gel electrophoresis (2D-DIGE) was used to analyze early DN patients plasma (n=8) and normal plasma ,proteins that showed differential expression of a 1 .5 fold change were analyzed by MALDI-TOF/TOF mass spectrometry . Results 2D-DIGE maps of plasma proteins in patients with DN and normal plasma were established successfully .We validated 13 differentially expressed proteins detected by 2D-DIGE ,including complement component C3、complement component C4、apolipopro-tein E ,etc .Conclusion Proteomic analysis can objectively revealed the differences of protein expression between the two kinds of blood plasma .The 13 proteins might be the potential plasma molecular markers for the clinical diagnosis of diabetic nephropathy .
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[Objective]To apply an acetonitrile-pretreatment method for proteomic analysis of serum of patient with hepatocellular carcinoma(HCC),searching for the proteins that might be related to occurrence and development of HCC.[Methods]Serum samples were recruited from 12 patients with primary HCC and 12 health adult.Firsdy,the sera were pretreated with acetonitrile (ACN),and the high-abundance proteins were removed,then two-dimensional electrophoreaia(2-DE)was performed to screen the differentially expressed proteins between the patient with HCC and health control.Finally,the differentially expressed proteins in the 2-DE gel were identified by a MALDI-TOF/TOF mass spectrometry.[Results]After the sera were pretreated with 0%,20% and 30% ACN,2-DE analysis showed that the number of the protein spots in the gels were 532±96,623±102,and 674±123,respectively.By the proteomic analysis of the ACN-pretreatment sera of patient with HCC and health control,we found that tetraiodo-L-thyronine and proapolipoproteinin were up-regulated,vitamin D-binding protein precurser and transferrin were downregulated in the HCC serum.[Conclusion]Proteomic analysis of serum using the ACN-pretreatment method can improve the detection of proteins which were non-specifically binding to the albumin.Abnormal expression of tetraiodo-L-thyronine,proapolipoproteinin,vitamin D-binding protein precursor and transferrin in the serum are related to HCC.