OBJECTIVE: To identify protein markers that may be associated with ulcerative colitis (UC) by analyzing differential proteins in the salivary exosomes from newly diagnosed patients with active UC and healthy controls (HC), and to investigate the function of salivary exosome-specific high-expression proteins in UC patients and their potential role in the pathogenesis of UC. METHODS: All patients and healthy controls were recruited from Peking University People' s Hospital. Whole saliva was obtained from 37 patients with newly diagnosed active ulcerative colitis (n=37) and apparently healthy controls (n=10). Salivary exosomes were extracted from samples, and the proteins within the exosomes were identified by liquid chromatograph-mass spectrometer (LC-MS/MS). The differentially expressed protein genes underwent gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis using the DAVID tool. In vitro, macrophages were co-cultured with salivary exosomes from UC group and those from HC group, respectively, and real-time quantitative polymerase chain reaction (qPCR) was used to detect levels of CD80⁺ and CD86⁺. Additionally, ELISA was performed to measure secretion levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) in the cell supernatant. RESULTS: A total of 259 proteins were co-expressed in saliva exosomes from UC group and HC group, among which 11 proteins were highly expressed in the UC group, including PDIA4, A2M, EEF2, C3, PSMA2, PSMB6, PSMA1, IGHG1, IGHG3, IGHG4 and SERPING1, while 4 proteins were lowly expressed in UC group, including TCN1, SLPI and SERPING. Functional analysis of these 15 proteins, along with 129 specific proteins found only in the UC patients and 69 specific proteins found only in HC patients, respectively, was conducted using GO/KEGG. The results revealed that in the UC group, proteasome-related proteins such as PSMA1, PSMA2 and PSMB6 expressions were increased in salivary exosomes while many key molecules involved in complement cascade pathways, such as C3 were up-regu-lated. In vitro co-culture experiments demonstrated that compared with healthy controls, the salivary exosomes of the UC patients in active stage could play a pro-inflammatory role by promoting the transformation of macrophages into M1 type cells that secrete inflammatory factors IL-1β, IL-6 and TNF-α. CONCLUSION Salivary exosomes in the UC patients may have the function of promoting inflammation. Analysis of protein levels in the saliva of the UC patients and healthy controls revealed significant differences in the expression levels of 15 co-expressed proteins between the two groups. Among them, C3, PSMA2, PSMB6 and PSMA1 were found to be mainly related to immune and inflammatory reactions in the UC group. These findings suggest that proteins with high specific expression in salivary exosomes of the UC patients have the potential to be used as a disease marker for UC diagnosis and may contribute to the pathogenesis of UC.
Humans ; Colitis, Ulcerative/metabolism* ; Exosomes/metabolism* ; Saliva/metabolism* ; Biomarkers/analysis* ; Male ; Female ; Adult ; Case-Control Studies ; Interleukin-6/metabolism* ; Middle Aged

Background Ocular alkali burns leads to corneal ulcer and angiogenesis and even corneal opacity.There is still no ideal treatment method.Studies showed that mesenchymal stem cells (MSCs) can repair corneal wound in vivo,but the specific mechanism is still not clear.Objective This study aimed to observe the histopathological change after the early transplatation of bone marrow MSCs (BMSCs) for corneal alkali burn model in rabbits and explore the anti-inflammatory effects of MSCs after corneal alkali burn.Methods Bone marrow of 4 ml was collected from 2-3 month-old Japanese rabbit.BMSCs were isolated and cultured from the bone marrow of rabbits,and the third generation of cells were used in this study.Cultured cells were identified by morphology and the expressions of surface markers.Corneal alkali burn models were extablished in the right eyes of 24 rabbits by attaching the filter paper with 0.1％ NaOH at the central cornea for 30 seconds,and then the models were randomized into 2 groups.BMSCs suspension of 300 μl (concentration 5×l06/μl) was subconjunctivally injected 1 hour after modeling in the BMSCs group,and equal volume of PBS was used in the same way in the PBS group.Corneal opacification was scored under the slim lamp microscope in 3,14 and 28 days after injection.The polymorphonuclear neutrophils (PMNs) were counted by histopathological examination,and the expression of matrix metalloproteinase-2 (MMP-2) in the corneal tissue was evaluated by immunochemistry in various time points.The use and care of the rabbits followed the statement of ARVO.Results The rabbit BMSCs were plastic-adherent cells that exhibited a fibroblastlike shape.Cultrued cells highly expressed surface adhesion molecular markers CD29 and CD90 (99.18％ and 97.94％) and lowly expressed hematopoietic cell markers CD34 and CD31 (0.74％ and 0.15％).Opacification of cornea,defect of corneal epithelium,stromal edema and neovascularization appeared after modeling.In 14 days and 28 days after modeling,the opacification scores in the BMSCs group were 2.37±0.52 and 2.25±0.50,which were significantly lower than 3.00±0.53 and 3.25 ±0.50 in the PBS group (t =2.376,2.828,both at P＜0.05).After subconjunctival injection,the number of PMNs was (34.17 ±1.85) /12 fields and (25.64 ±3.86)/12 fields in the BMSCs group,showing significant decrease in comparison with (42.70 ±1.54) /12 fields and (32.67 ±1.42)/12 fields in the PBS group (t=10.021,4.832,both at P=0.000).The expression levels of MMP-2 (A value) in cornea were 0.388±0.016 and 0.384±0.006 in the BMSCs group,with considerable decreases in comparison with 0.438± 0.006 and 0.412± 0.005 in the PBS group (t=10.205,13.514,both at P=0.000).Conclusions Early transplantation of BMSCs can arrest the occurrance of corneal ulcer by suppressing the infiltration of PMNs,alleviateing the inflammation reaction,downregulating the expression of MMP-2 in cornea and inhibiting the degradation of stromal collagen fibers.

Objective To observe the effects of serum with drug Penthorum chinense Pursh extractum on transforming growth factor (TGF)-β1 and collagen I secretions of activated hepatic stellate cells. Methods Twenty male SD rats were divided into 2 groups, and were administered with 0.9% sodium chloride solution and Penthorum chinense Pursh extraetum via gastrogavage for 3 days respectively and then sacrificed. Serum samples of these rats were collected. HSC-T6 cells were divided into the normal group and the treatment group. The cells of the normal group were incubated in Dulbecco's modified eagle medium (DMEM)with sera of normal rats, while those of the treatment group were incubated in DMEM with sera from Penthorum chinense Pursh extractum treated rats. The HSC-T6 viability was observed by AlamarBlue assay, while the toxicity of Penthorum ehinense Pursh extractum was measured by 3-(4, 5-Dimethyhhiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The expression of collagen I and TGF-β1 mRNA were determined by real timepolymerase chain reaction (real time PCR). The protein expressions of collagen I and TGF-β1 were analyzed by Western blotting. The data were analyzed by single factor analysis of variance and pairwise comparison was done by q test. Results Different concentrations of sera from Penthorum chinense Pursh extractum treated rats could all inhibit HSC-T6 proliferation, especially when the sera concentration were 10% and the HSC-T6 cells were incubated for 24 h (P＜0.01 ). MTT assay indicated that sera from Penthorum chinense Pursh extractum treated rats showed no obvious toxicity to HSC-T6 compared with those from normal rats (P ＞0.05). After 24 h incubation, 10% sera from Penthorum chinense Pursh extractum treated rats could significantly down-regulate mRNA expression of TGF-β1 and collagen I compared with normal group (TGF-β1 2.790±0.174 vs 9. 827 ± 1.429, P＜0.01 ; collagen I 1.213 ± 0.099 vs 4.053 ± 1.005, P＜0.01 ). Mcanwhile, the protein expressions of TGF-β1 and collagen I were also obviously inhibited in drug treated group compared with normal group (P＜0.01). Conclusions Serum from Penthorum chinense Pursh extractum treated rats can significantly decrease TGF-β1 and collagen I secretions of activated hepatic stellate cells, which provides the experimental evidence for liver fibrosis treatment.