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Background Chronic excessive exposure to fluoride can cause damage to the central nervous system and a certain degree of learning and memory impairment. However, the associated mechanism is not yet clear and further exploration is needed. Objective Using 4D unlabelled quantitative proteomics techniques to explore differentially expressed proteins and their potential mechanisms of action in chronic excessive fluoride exposure induced brain injury. Methods Twenty-four SPF-grade adult SD rats, half male and half male, were selected and divided into a control group and a fluoride group by random number table method, with 12 rats in each group. Among them, the control group drank tap water (fluorine content<1 mg·L−1), the fluoride group drank sodium fluoride solution (fluorine content 10 mg·L−1), and both groups were fed with ordinary mouse feed (fluoride content<0.6 mg·kg−1). After 180 d of feeding, the SD rats were weighed, and then part of the brain tissue was sampled for pathological examination by hematoxylin-eosin (HE) staining and Nissl staining. The rest of the brain tissue was frozen and stored at −80 ℃. Three brain tissue samples from each group were randomly selected for proteomics detection. Differentially expressed proteins were screened and subcellular localization analysis was performed, followed by Gene Ontology (GO) function analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, cluster analysis, and protein-protein interaction analysis. Finally, Western blotting was used to detect the expression levels of key proteins extracted from the brain tissue samples. Results After 180 d of feeding, the average weight of the rats in the fluoride group was significantly lower than that in the control group (P<0.05). The brain tissue stained with HE showed no significant morphological changes in the cerebral cortex of the fluoride treated rats, and neuron loss, irregular arrangement of neurons, eosinophilic changes, and cell body pyknosis were observed in the hippocampus. The Nissl staining results showed that the staining of neurons in the cerebral cortex and hippocampus of rats exposed to fluoride decreased (Nissl bodies decreased). The proteomics results showed that a total of 6927 proteins were identified. After screening, 206 differentially expressed proteins were obtained between the control group and the fluoride group, including 96 up-regulated proteins and 110 down-regulated proteins. The differential proteins were mainly located in cytoplasm (30.6%), nucleus (27.2%), mitochondria (13.6%), plasma membrane (13.6%), and extracellular domain (11.7%). The GO analysis results showed that differentially expressed proteins mainly participated in biological processes such as iron ion transport, regulation of dopamine neuron differentiation, and negative regulation of respiratory burst in inflammatory response, exercised molecular functions such as ferrous binding, iron oxidase activity, and cytokine activity, and were located in the smooth endoplasmic reticulum membrane, fixed components of the membrane, chloride channel complexes, and other cellular components. The KEGG significantly enriched pathways included biosynthesis of secondary metabolites, carbon metabolism, and microbial metabolism in diverse environments. The results of differential protein-protein interaction analysis showed that the highest connectivity was found in glucose-6-phosphate isomerase (Gpi). The expression level of Gpi in the brain tissue of the rats in the fluoride group was lower than that in the control group by Western blotting (P<0.05). Conclusion Multiple differentially expressed proteins are present in the brain tissue of rats with chronic fluorosis, and their functions are related to biosynthesis of secondary metabolites, carbon metabolism, and microbial metabolism in diverse environments; Gpi may be involved in cerebral neurological damage caused by chronic overdose fluoride exposure.
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Objective:To explore the effect of the synthetic immunomodulator CH 2b with a thiazolidin-4-one ring on the pathogenesis of rheumatoid arthritis (RA) mice induced by glucose-6-phosphate isomerase(GPI) mixed peptides.Methods:hGPI325-339 ,hGPI469-483 peptide fragments were mixed with complete freund′s adjuvant fully ,DBA/1 mice were givien subcutaneous injection of the emulsifiers,pertussis toxin to strengthen immunity.And then RA mice were intervened with α-GalCer and CH2b,the changes of body weight ,ankle joint symptoms scores were observed .The joint tissues stained with hematoxylin and eosin ( HE) was used to evaluate the inflammatory cells.Fluorescence-activated cell sorting ( FACS) was used to detect the frequency changes of iNKT cells .Cytometric bead array(CBA) was used to analyze the levels of serum cytokines TNF-α,IL-6,IL-4,IFN-γ.Results: Compared with the model group,α-GalCer,CH2b could reduce the inflammation of the model mice ,significantly improve the body weight growth and the joint swelling(P0.05).Conclusion:Immunomodulator CH2b by activating iNKT cells affect the secretion of inflammatory cytokines ,and it relieved the GPI induced arthritis .
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[ ABSTRACT] AIM:To establish an animal model of rheumatoid arthritis ( RA) in DBA/1 mice induced by im-munodominant mixed peptides derived from glucose-6-phosphate isomerase (GPI).METHODS: The DBA/1 mice were immunized with emulsified mixed peptide fragments of hGPI 325-339+hGPI469-483 or single peptide hGPI325-339 in com-plete Freund′s adjuvant by subcutaneous injection to induce the model of RA .Body weight , ankle joint symptom scores , the pathological change of the ankle joint , the levels of CD4 +T cells in the spleen and peripheral blood , the proportion of iNKT cells in the peripheral blood , and the levels of TNF-αand IL-6 in serum were detected to evaluate and analyze the model.RESULTS:The hind paw of the model mice appeared red swelling on the 8th day, and then aggravated gradually to the limbs.The red swelling reached peak on the 14th day, and then relieved gradually .Inflammation response dominated by lymphocytes and monocytes was observed in the ankle joint .The inflammatory effect of mixed peptides was more obvious than that of the single one (P<0.05).Compared with control group and the mice treated with single peptide , the weight gain was slow, the amount of CD4 +T cells in the peripheral blood and spleen were increased , the proportion of peripheral iNKT cells in the inflammatory peak was decreased (P<0.05), and the serum level of TNF-αwas increased significantly ( P<0.05) in the mice treated with mixed peptide fragments .CONCLUSION: The immunological characteristics of RA model induced by mixed GPI peptides in DBA/1 mice is closer to that in RA patients , especially in the immunopathology of iNKT cells.Therefore, this model can be used as a new tool for studying the mechanism and immunological intervention of RA.
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Objective This study was performed to investigate the effect of hypoxia on glucose-6-phosphate isomerase (G6PI) expression and cell cycle of fibroblast-like synoviocytes from synovium of rheumatoid arthritis (RA) and osteoarthritis (OA) under hypoxia or normoxia.Methods Fibroblast-like synoviocytes were cultured with either of hypoxia (3％ oxygen) or normoxia (21％ oxygen) for 24 hours.The mRNA expression of G6PI and HIF-1α was tested by PCR quantification,while the protein levels of G6PI and HIF-1α were measured by western blot.Cell cycle was performed by FACS.T-test and Mann-Whitney U were used for statistical analysis.Results The expression levels of G6PI mRNA under hypoxia in RA were higher than those of OA (2.6±0.4 vs 1.5±0.4,P＜0.05).The protein levels of G6PI in RA were higher than those of OA (P＜0.05).The expression levels of HIF-1α mRNA under hypoxia in RA were higher than those of OA (2.9±0.8vs 1.4 ±0.4,P＜0.05).The protein levels of HIF-1α in RA were higher than those of OA (P＜0.05).The G1 phase ratio of cell cycle was decreased significantly under hypoxia than those of normoxia in RA ELs (t=1 1.31,P＜0.05).The S and G2 phase ratio of cell cycle were increased.Conclusion Hypoxia upregulates G6PI and HIF-1α expression and improves proliferation in fibroblast-like synoviocytes.
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Objective To establish the rheumatoid arthritis (RA) mouse model induced by glucose-6-phosphate isomerase (GPI),and explore the mechanism of GPI in RA.Methods Totally 36 DBA/1 mice were randomly divided into three groups:test group (injection GPI),positive group (injection of bovine collagen Ⅱ),and negative group (saline).The rates and changes of weight were observed.The score of the arthritis,the ankle histopathological changes and serum GPI content were detected.Results Toes swollen slightly,joint swelling,deformity and accompanied by block were appeared at 35th day in the test group.Compared to the control group,the rates and changes of weight in test group showed a significant difference (P ＜ 0.05).The score of arthritis was showed by x ± s.Compared to the negative group,the test group and positive group were showed significant difference (P ＜ 0.05).A lot of lower synovial lining exudate macrophages,fibroblasts,and other inflammatory cells were increased in the test group.The GPI content in the test group [(0.39 ±0.11)μg/ml] was significantly higher than the negative group [(0.10± 0.06) μg/ml,P ＜ 0.05].Conclusions GPI could induce rheumatoid arthritis in mice.It provides the experimental basis to diagnose RA.
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Objective To investigate the detection of anti-cyclic citrullinated peptide(anti-CCP)antibody,alkaline phosphatase (ALP),glucose-6-phosphate isomerase(G6PI)and rheumatoid factor(RF)in the diagnosis of rheumatoid arthritis(RA).Methods Serum RF,anti-CCP body,ALP and G6PI were detected in 206 cases of RA,68 cases of other autoimmune diseases and 48 persons with the healthy physical examination,at the same time the erythrocyte sedimentation rate(ESR)was measured;the sensitivity and specificity of each indicator and the relationship between each index and RA′s diagnosis.Results The serum level of each index in the RA group was significantly higher than that in the other two groups(P <0.05).The sensitivity of RF,anti-CCP antibody and G6PI for diagnosing RA is similar;the specificity of anti-CCP body was highest(94.8%),RF was similar to G6PI;ALP with rela-tively lower sensitivity and specificity also reflected the potential of RA to some extent.Conclusion The simultaneous detection of serum RF,anti-CCP body,G6PI and ALP can improve the accuracy of early diagnosis of RA,reduce the missed diagnosis and guide the clinical treatment.
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ObjectiveTo detect glucose-6-phosphate isomerase (GPI) in knee joint synovial fluid of active rheumatoid arthritis (RA) and explore the correlation between GPI and anti-cyclic citrulline peptide antibody (anti-CCP).MethodsGPI and anti-CCP in the synovial fluid were measured by enzyme linked immunosorbent assay(ELISA) from 22 patients with active RA and 37 patients with active osteoarthritis (OA).Student's t-test was used for intergroup comparison and Spearman's analysis was used for correlation analysis.ResultsThe level of GPI and anti-CCP in the synovial of active RA [ (9.6±8.4) μg/ml,( 14.61 ±18.64) U/ml] was significantly higher than that of active OA[ (0.9±1.8) μg/ml,(1.42±0.09) U/ml)].There was positive correlation between GPI and anti-CCP (r=0.447,P=0.037).ConclusionHigh expression of GPI is shown in active RA synovial fluid.It is suggested that GPI as an antigen that may participate in chronic synovitis,bone destruction and joint malformation.Both GPI and anti-CCP may be the laboratory markers for the diagnosis of RA.
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Objective To investigate the diagnostic value of gluocose-6-phosphate isomerase (G6PI) detected by an enzy-me linked immunosorbent assay (ELISA) in patients with rheumatoid arthritis (RA). Methods The G6PI was detected by ELISA in serum samples from 106 patients with RA, 53 non-RA controls with various rheumatic diseases, and healthy individuals. The level of rheumatoid factor (RF), anti-CCP antibodies and AKA were also assessed in RA patients. The correlation analysis beween G6PI and anti-CCP, IgM-RF. G6PI, anti-CCP, IgM-RF and AKA were carried out between patients with erosion and with non-erosion diseases . Results ① G6PI serum level of patients with RA was (1.61 ±1.20) μg/ml, and was (0.11 ±0.17) in patients with other rheumatic diseases, and (0.06±0.07) μg/ml in healthy individuals. There was statistical significant difference between RA patients and patients with other rheumatic diseases (P<0.05). Receiver operator curve analysis (ROC) showed an opitium cut off level for C6PI at 0.225 μl/ml. The sensitivity of G6PI was 0.868, the specificity was 0.853 in RA. C6PI was associated with RF, but was not associated with anti -CCP. C6PI ws not associated with disease activity index by Spearman' s correlation analysis. The association between above parameters with bone erosion was not detected, however. Conclusion C6PI is abnormally increased in some RA so it may be a new diagnostic marker for RA. G6PI has a reasonable sensitivity (86.8%) and with high specificity(85.3%) to RA and it is valuable for RA diagnosis. C6PI is associated with RF, but not completely overlaps. C6PI is not associated with diseases activity. No association is found between G6PI and bone erosions.
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Objective To assess the levels of glucose-6-phosphate isomerase(GPI) mRNA in peripheral blood monocytes and serum GPI levels in patients with rheumatoid arthritis, and analyze the association of serum GPI with MCV antibody, CCP antibody and RF of RA. Methods Fluorogenic quantitative real-time polymerase chain reaction (FQ-RT-PCR) was used to examine mRNA expression on peripheral blood monocytes in 60 RA patients (28 case in active stage,32 cases in stable stage) ,30 patients with other rheumatic diseases, and 30 healthy controls. ELISA was used to detect the levels of serum GPI, anti-MCV antibodies, anti-CCP antibodies and RF in each group. Results The levels of GPI mRNA in RA group [△Ct=4.21 (3.04-7.23)] were significantly higher than those in patients with other rheumatic diseases [△Ct=8.42 (5.16-9.98),P<0.01] and healthy controls [△Ct=8.66 (4.90-10.01), P<0.01]. There were statistically significant differences of GPI mRNA levels between active RA [△Ct=3.78 (1.28-6.09)] and inactive RA[△Ct =5.88(3.23-8.94),H=11.760,P<0.01)]. The RA group serum GPI levels [3.02 (2.02-8.39) mg/L] were higher than those of other rheumatic diseases [0.20 (0.11-0.32) mg/L] and healthy controls [0.18(0.08-0.30) mg/L]. There were significant differences of serum GPI levels between active RA group [4.84(2.81-10.38) mg/L] and inactive RA group[2.12 (1.26-4.34) mg/L] (H=9.830, P<0.01). The sensitivities of GPI, anti-MCV and anti-CCP were 68% (41/60) ,57% (34/60),58% (35/60), respectively and specificities were 95% (57/60), 92% (55/60) and 93% (56/60), respectively. Conclusions The high expression of GPI mRNA in RA patients shows that it may play a pathological role in the development of RA, and it may be correlated with the activity of RA. It may be a valuable diagnostic parameter for RA, because of its high sensitivity and specificity.
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Objective To investigate the level of five auto-antibodies including MCV and GPI in the serum of RA patients and assess the application value of five auto-antibodies in RA diagnosis.Methods The five auto-antibodies were detected by ELISA in serum samples of 150 patients with RA and 40 healthy controls,32 patients of SLE,30 patients of OA,20 patients of AS,20 patients of SS,20 patients of CTD.Results The positive rates of these five auto-antibodies in RA patients were significantly higher than in other group(X2=88.5,76.0,279.2,88.2,94.8,P＜0.05).Except anti-AKA,there was no the differences in the level of other antibodies among groups(X2=21.9,9.4,20.2,43.2,41.6,P＞0.05).Anti-MCV and anti-GPI has the highest sensitivity(78.0% and 83.3%),while anti-CCP has the highest specificity(97.1%)and anti-AKA has good specificity(96.1%)and lowest sensitivity(49.4%).When two antibodies were detected together,the sensitivity and specificity of MCV/CCP were highest(92.7% and 96.9%).When RF/GPI/CCP were detected together,the sensitivity and specificity were 90.7% and 96.9%,respectively.When RF/MCv/CCP were detected together,the sensitivity and specificity were 94.0% and 96.9%.Conclusions Anti-MCV and anti-GPI has the hishest sensitivity in laboratory diagnosis of RA,while anti-CCP has the highest specificity and anti-AKA have good specificity and lowest sensitivity.The combination detection can decrease the amount of missed diagnosis caused by single test. The combination detection of RF/GPI/CCP and RF/MCV/CCP will improve sensitivity and specificity for diagnosis to the RA patients.
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We have used a surface plasmon resonance biosensor (SPR, BIACORE 2000) to detect antibodies against glucose 6-phosphate isomerase (GPI) in synovial fluids of rheumatoid arthritis (RA) and osteoarthritis (OA). Recombinant human GPI proteins fused with or without NusA were expressed in E. coli, purified to homogeneity and immobilized in flow cells of CM5 sensor chips. The flow cells immobilized with NusA protein or bovine serum albumin were used to monitor non-specific binding. Synovial fluid samples from RA patients showed a significantly higher level of binding to recombinant GPI proteins than samples from OA patients. Proteins which bound to the recombinant GPI proteins were confirmed to be immunoglobulin through the administration of anti-human immunoglobulin. NusA fusion protein was excellent for this assay because of a low background binding activity in the SPR analysis and its advantage of increased solubility in recombinant protein production. These results suggested a useful utilization of recombinant NusA-GPI fusion protein for the detection of autoantibodies against GPI in RA patients.