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【Objective】 To investigate the mechanism of pristane inducing autophagy in macrophages. 【Methods】 Pristane was used to stimulate NR8383， a rat macrophage cell line. The changes in signaling pathways of AMPK， mTOR， and endoplasmic reticulum （ER） stress pathways including eIF2α and IRE1α in the cell model， as well as the expression of transcriptional factor TFEB and its translocation to the nucleus， were detected by using Western blotting. ER stress pathways were intervened by using an inducer DTT or an inhibitor 4-PBA to determin its effect on mTOR expression and autophay. 【Results】 In pristane-stimulated NR8383 cell model， ER stress pathway eIF2α was activated at 0.5 h after stimulation， and then mTOR expression was decreased at 1 and 3 h after stimulation. There was no change for AMPK and IRE1α pathways. With 4-PBA treatment， pristane-reduced mTOR expression and increased LC3-II were reversed， while with DTT treatment， mTOR expression decreased and LC3-II expression increased even more. Pristane induced the expression and activation of TFEB in NR8383 cells. 【Conclusion】 Pristane induces ER stress and leads to autophagy enhancement in rat macrophages.
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Introduction: Regulatory T cells’ (Tregs’) role remains unclear in the pathogenesis ofsystemic lupus erythematosus (SLE). This study was aimed at monitoring the percentage of Tregswithin 32 weeks and monitoring its relationship with the percentage of other T helper (Th) cellsubsets and the levels of autoantibodies and pro-inflammatory cytokines in a murine SLE modelinduced by pristane.Methods: Forty-eight female BALB/c mice were divided into a healthy control (HC) and apristine-induced (PI) group. SLE was induced by a single 0.5 cc pristane intraperitoneal injection.Six from each group were sacrificed every eight weeks until 32 weeks post-pristane injection. Treg,Th1, Th2 and Th17 percentages from the spleen were measured using flowcytometry. ANA, IL-6 andIFN-α levels were measured from serum using ELISA.Results: The Treg percentage from the PI group increased significantly at 16 weekscompared to the HC group, while Th1, Th2 and Th17 percentages decreased. Tregs in the PIgroup began to reduce from the 24th to 32nd weeks, followed by an elevation of the Th1, Th2and Th17 percentages. Tregs were negatively correlated with Th1 and Th2. Tregs in the PI grouphad a negative correlation with ANA and IFN-α levels from serum, whereas Tregs had a positivecorrelation with IL-6 levels.Conclusion: The compensation of Tregs observed at 16 weeks after pristane injectionfailed, marked by a decreasing number of Tregs, followed by an increase of Th subsets, proinflammatorycytokines and autoantibodies. This compensatory failure of Tregs could be affectedby pro-inflammatory cytokines, such as IFN-α and IL-6.
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Objective To investigate the protective effects of iguratimod on systemic lupus erythematosus model mice. Methods A total of 30 female BALB/c mice were randomly divided into blank control group,model control group and igurati-mod drug intervention group,with 10 mice in each group.The blank control group was given saline by intraperitoneal injection in-tervention group.The model control group and iguratimod intervention group were given 0.5 mL of pristane, Then the drug inter-vention group began to be fed with iguratimod 6.5 mg·kg-1·d-1from the next day.After 7 months of feeding,the serum autoanti-body(anti nuclear antibody,anti ds-DNA antibody,anti RNP/sm antibody),the level of urine protein,serum urea nitrogen and serum creatinine,as well as the renal pathological changes of the three groups were detected and compared. Results Serum an-ti nuclear antibody,anti ds-DNA antibody and anti RNP/sm antibody levels of the drug intervention group were significantly lower than those of the model control group (P<0.05);Positive rate of urine protein,serum urea nitrogen,serum creatinine of the drug intervention group were significantly lower than those of the model control group(P<0.05).while the renal pathological change of this group is not obvious. Conclusion Iguratimod can inhibit the occurrence of serum antibody in a certain extent,improve the urine protein,serum urea and serum creatinine level of mouse model with systemic lupus erythematosus,which has protective effects on systemic lupus erythematosus.
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The purpose of this study was to investigate the effects of salvianolic acid A (SAA) in systemic lupus erythematosus (SLE) induced by pristane in BALB/c mice. Lupus mice were established by confirming elevated levels of autoantibodies and IL-6 after intraperitoneal injection of pristane. Mice were then treated with daily oral doses of SAA for 5 months in parallel with mice treated with prednisone and aspirin as positive controls. The levels of autoantibodies were monitored at monthly intervals and nephritic symptoms observed by hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. Western blot analysis of renal tissue was also employed. SAA treatment caused a significant reduction in the levels of anti-Sm autoantibodies and reduced renal histopathological changes and pathological effects. SAA treatment also significantly inhibited the phosphorylation of IKK, IB and NFB in renal tissues of lupus mice. In conclusion, the results suggest that SAA alleviates renal injury in pristane-induced SLE in BALB/c mice through inhibition of phosphorylation of IKK, IB and NFB.
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Objective To establish the systemic lupus erythematosus (SLE) mouse model through pristaneip injection and validate the model comprehensively.Methods Female BALB/c mice of 6-8 weeks were randomly divided into two groups.Animals in model group were injected with 0.5 mL pristane by ip injection while in control group with 0.5 mL normal saline.Anti-systemic lupus erythematosus antibodies (anti-SLE) and anti-double strand DNA antibodies (anti-dsDNA) were checked before injection and monthly thereafter.Proteinuria was detected before injection and every month thereafter.All mice were bled to death 6 months after injection.Kidneys were excised to observe the histopathologic evidence of glomerulonephritis.Results The concentration of anti-dsDNA and anti-SLE antibody in sera was higher of model group than that of control group two months after pristane injection,and the concentration of antibody gradually increased within 6 months.At the sixth months,the protein concentration of urine in most model group mice was ++++.The histopathology and imunoflorescence of kidney sections indicated typical evidence of glomerulonephritis in model group.Conclusion The murine lupus model can be successfully established in female BALB/e mouse with a single ip injection of 0.5mL ofpristane.
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Objective:On the basis of the use of chemical methods to establish mouse model of lupus nephritis and its biological identification , we investigate the reverse effect of pathological lesions of B 7-1 human-mouse chimeric antibody blockade against B7/D28 signaling pathway in mice with lupus nephritis model.Methods:Pristane was injected intraperitoneally to 6-week-old female C57BL/6J mice at dose of 0.5 ml per mouse in one go,and urine protein,ANA and renal pathological changes were detected on a monthly basis.Mice whose urine protein content reached ++and ANA fluorescence intensity reached ++were randomly devided into three groups ,five each.Antibody intervention group was sequentially injected with B 7-1-mouse chimeric antibody by orbital venous , positive control group was injected with immunosuppressant CTX , negative control group was injected with isotype control IgG.Urine protein and ANA were also detected on a monthly basis.Mice were sacrificed three months after intervention was executed.Kidney was used for H&E dying , IC detection and electric microscope observation.Results: After four-month Pristane induction , urine protein content of 80%mice reached +-+++,meanwhile,serum ANA fluorescence intensity reached ++-+++.Glomerulonephritis infiltrating cells were observed Mice with urine protein and ANA , glomerular inflammatory cell infiltration , tubular epithelial cell degeneration visible edema ,vascular congestion significantly ,fibrosis.After antibody intervention ,urine protein content in antibody intervention group gradually reduced from ++-+++to ±-+++,ANA ++-+++to +-++,and were significantly different from that in the negative control group ( P<0.01 ).Analysis of kidney H&E dying showed that antibody glomerular infiltration of inflammatory cells in the intervention group and tubular congestion and other symptoms were improved significantly.Immunofluorescence staining indicated that fluorescence intensity of IC was significantly reduced in the antibody intervention group.Electron dense deposits reduction and glomerular basement membrane uniformity were observed in antibody intervention group by electric microscope when compared with the negative control group.Conclusion:B7-1 antibodies could downregulate immune response through inhibiting B 7-1/CD28 signaling pathway , reducing the production of autoantibodies and reversing pathological damage caused by autoimmune response .
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Introdução: O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune multissistêmica de etiologia complexa que envolve fatores ambientais, genéticos e hormonais. É caracterizada pela produção de autoanticorpos e mediadores inflamatórios, ativação e proliferação de células T autorreativas e perda da autotolerância imunológica. Em pacientes com LES, a expressão do receptor primário de ativação CD69 é aumentada e a de células T supressoras/reguladoras (Treg) CD4+CD25+FoxP3+ é reduzida. O CD69 é essencial para ativação de células T CD4 autorreativas enquanto que as células Treg são importantes na manutenção da autotolerância. Desta forma, células T tem um papel central na patogênese do LES, mas os mecanismos implicados na falência da autotolerância ainda não são elucidados, destacando a importância de estudos em modelos experimentais da doença, como o de LES-induzido por pristane. Objetivo: Quantificar células T CD4+CD69+ ativadas e Treg CD4+CD25+FoxP3+ no sangue, baço e LP de camundongos Balb/c LESinduzido por pristane no sentido de avaliar a falência de autotolerância neste modelo. Métodos: Analisamos 84 camundongos Balb/c fêmeas: 52 receberam por via intraperitoneal uma dose única de 0,5 ml de pristane e 32 a mesma dose de salina. Amostras de sangue, baço e LP dos camundongos eutanasiados foram coletadas 90, 120, 180 e 300 (T90, T120, T180 e T300) dias após a inoculação de pristane ou salina. Células mononucleares do sangue periférico (CMSP), do LP (CMLP) e esplenócitos foram obtidos por lise das hemácias seguida de lavagens com RPMI medium 1640 e centrifugação, e posteriormente criopreservadas até a avaliação por citometria de fluxo usando o aparelho Guava EasyCyteTM HT (Millipore). Para esta etapa, as células foram descongeladas, lavadas com RPMI medium 1640 e incubadas com anticorpos monoclonais dirigidos contra CD3, CD4, CD25, CD28, CD69, CTLA-4, FoxP3, CD14 e Ly6C (BD PharmingenTM). Os resultados foram expressos como média ± DP e teste de...
Introduction: Systemic Lupus Erythematosus (SLE) is multisystemic autoimmune disease with complex etiology that involves environmental, genetic and hormonal factors. Is characterized by auto-antibodies and inflammatory mediators production, autoreactive T cells activation and proliferation and loss of immunogenic autotolerance. In patients with SLE, expression of CD69 activation primary receptor is increased and the CD4+CD25+FoxP3+ suppressor/regulatory T cell (Treg) is reduced. CD69 is essential for activation of autoreactive CD4 T cells while Treg cells are important in autotolerance maintenance. In this way, T cells have a central role in the pathogenesis of SLE however, the mechanisms implied in the autotolerance failure are still not elucidated, highlighting the importance of studies in this disease's experimental models, such as pristane-induced SLE. Objective: Quantify activated CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg in blood, spleen and peritoneal lavage (PL) of Balb/c mice with pristane-induced SLE in order to evaluate autotolerance failure in this model. Methods: 84 female Balb/c mice were analyzed: 52 received a single intraperitoneal 0,5 ml dose of pristane and 32 the same dose of saline. Euthanized mice samples of blood, spleen and peritoneal lavage were collected 90, 120, 180 and 300 (T90, T120, T180 and T300) days after inoculation of pristane or saline. Mononuclear cells from peripheral blood (PBMC), PL (PLMC) and splenocytes were obtained by lysis of erythrocytes followed by washings with RPMI medium 1640 and centrifugation, subsequently criopreserved until evaluation by flow cytometry using the appliance GuavaEasyCyteTM HT (Millipore). For this step, cells were unfrozen, washed with RPMI medium 1640 and incubated with monoclonal antibodies against CD3, CD4, CD25, CD28, CD69, CTLA-4, FoxP3, CD14 and Ly6C (BD PharmingenTM). The results were expressed as mean ± SD and Mann-Whitney 11 test was used for statistical analysis, being considered...