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Objective To investigate the effect of high mobility group box chromosomal protein 1 (HMGB1) knockdown on improving renal function and decreasing cell proliferation of glomeruli in lupus nephritis (LN) MRL/Faslpr mice.Methods Twenty-four MRL/Faslpr mice were randomly divided into 3 groups:LN model group,shHMGB1 group and empty plasmid group.Besides,eight MRL/MpJ mice,age and mass matched to the MRL/Faslpr mice,were chosen as normal control group (shNC group).Electroporation technology was used for in vivo transfection in treatment group.shHMGB1 group and empty plasmid group were transfected by electroporation technology for shHMGB1 plasmids and empty plasmid,LN model group and normal control group were transfected only with saline.Automatic biochemical analyzer was used to detect serum urea nitrogen (BUN) and creatinine (Scr) levels and 24 h urinary protein (UP) was tested.HE staining was used to detect the pathological change of renal tissues; real-time PCR,immunofluorence staining and Western blotting were used to detect the mRNA and protein expression of HMGB1 and PCNA.Results (1) The HMGB1 mRNA and protein expression in LN group increased compared with those in control group,HMGB1 mRNA and protein expression in shHMGB1 group reduced compared with those in LN model group (all P ＜ 0.05).(2) 24 h UP of MRL/Faslpr mice in shHMGB1 group significantly reduced compared with those in LN group (P ＜ 0.05).(3) Immunofluorence and Western blotting showed that positive signal of proliferating cell nuclear antigen (PCNA) was mainly located in nuclei,PCNA mRNA and protein in glomeruli of LN model group increased compared with those of control mice (P ＜ 0.05).Interestingly,PCNA expression in glomeruli of shHMGB1 group remarkably reduced (P ＜ 0.05).Conclusions shHMGB1 significantly improves renal function and decreases cell proliferation of glomeruli in LN MRL/Faslpr mice.
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Objective: To investigate whether high mobility group box chromosomal protein 1 (HMGB1) can activate macrophages to secrete IL-18. Methods: Mouse peritoneal macrophages were cultured with HMGB1 for indicated period, then the mRNA and protein expression of IL-18 and IL-18 receptors were examined by real-time PCR, ELISA, and flow cytometric analysis. Toll-like receptor (TLR) 2 and 4 agonists (1 μg/ml Pam3Cys or 100 ng/ml LPS) were used as parallel controls. Results: Two hours after culture with HMGB1(100 ng/ml), IL-18 mRNA expression was greatly increased. The expression of IL-18 protein was also greatly increased 24 h after culture with HMGB1, and the expression of IL-18R was slightly up-regulated. Pam3Cys (1 μg/ml) and LPS(100 ng/ml) could promote the expression of IL-18 at both mRNA and protein levels in the macrophages, and could up-regulate IL-18 receptors. Conclusion: HMGB1 can directly activate macrophages to secrete IL-18 and to express IL-18 receptor, suggesting that IL-18 may mediate the proinflammatory activity of HMGB1.
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Objective To establish ulcerative colitis(UC)model in BALB/c mice and to investigate the role of high mobility group box chromosomal protein 1(HMGBI)in pathogenesis of UC.Methods Thirty-two BALB/c mice were randomly divided into UC group(n=24,which were fed with 3%dextran sulfate 80dium solution)and control group(n=8,which were fed with water).The animals were sacrificed at 24.96 and 1 68 hours,respectively,to collect samples of colon and blood.The sernm level of HMGB1 was measured with ELISA and the expression of HMGB1 in colon was determined by Western blotting analysis.Results Histological scoring increased with the induction of the model,and manifestation of colon mucosa at 168h was similar with that of UC in human.The serum level of HMGB1 was slightly higer at 24 h than that of control(5.09±0.61 μg/L vs 4.49±0.53μg/L,P＞0.05),and reached a peak at 96 h (14.74±0.60 μg/L,P＜0.01),decreased at 168 h(9.03±0.78μg/L,P＜0.01).The expression 0.05).significandy increased at 96h(0.76±0.03,P＜0.05)and at 168 h(0.77±0.04,P＜0.05).Conclusion HMGB1 might be involved in pathologic changes of UC at a later stage.
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Objective To investigate the effect and possible mechanisms of high mobility group box (HMGB) 1 on the proliferation of RSC-364 synoviocytes. Methods ① RSC-364 cells stimulated by 10 μg/L TNF-α and cells of the normal control groups were collected at 6, 12, 24 h respectively in vitro. HMGB1mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC); ②RSC-364 cells induced by 10 μg/L HMGB1 were collected in 6, 12, 24 h respectively, so did normal control group cells in vitro. The expression of signal transducer and activator of transcription (STAT) mRNA 1 was detected by RT-PCR. The expression of STATland SOCSI proteins were detected by ICC and flow cytometry analysis (FCM). The expression of PCNA was detected by ICC. Results ① Compared with the control group, TNF-α markedly up-regulated HMGBI mRNA at 6, 12, 24 h respectively [0.86, 0.92, 1.06 vs 0.70, P<0.01 ], as well as protein expression level. Positive signal of HMGB1 proteins was not only expressed in nuclear but also in cytoplasm after stimulation. ② Compared with normal group, HMGBI increased the expression of P-STAT1 mRNA and protein at 6, 12, 24 h respectively [0.30, 0.69, 1.05 vs 0.24, P<0.01 ] and [1.34±0.09，1.55±0.16，1.74±0.13 vs 1.00±0.15，P<0.01]. The expression of SOCSI protein increased significantly in HMGB1 group at 6 and 12 hours ( 1.43±0.10 vs 1.58±0.05), but it decreased at 24 hours (1.24±0.15). ③The expression of p-STATI protein was negatively correlated with that of SOCS1 protein. Conclusion HMGB1 appears to be an important mediator in the proliferation of RSC-364 cells, partly by up-regulating the expression and aetivity of p-STAT1.
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Objective To investigate the effect and possible mechanism of arsenic trioxid(As2O3)on proliferation of RSC-364 synoviocyte lines stimulated with TNF-?.Methods RSC-364 synoviocytes were cultured with standard medium as control group or medium supplemented with 10 ?g/LTNF-? and different concentrations of As2O3 respectively. MTT assay were carried out to study cell proliferation. Proliferation index (PI) and cell cycle were detected by flow cytometry (FCM). RT-PCR was used to detect the mRNA expression of High mobility group box chromosomal protein (HMGB)1. HMGB-1 and proliferation cell nuclear antigen (PCNA) proteins were detected by immunocytochemistry and FCM. Results (1)As2O3 inhibited proliferation of cell lines stimulated by TNF-? time-dependently and dose-dependently. (2)Compared with normal group, TNF-? up-regulated HMGB-1 protein and mRNA as well as PCNA protein. HMGB-1 protein was not only in nuclear but also in cytoplasm by immunocy-tochemistry. As2O3 down-regulated mRNA and protein of HMGB-1 in a dose-dependent manner; so did PCNA proteins (P
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Objective To demonstrate high mobility group box chromosomal protein 1(HMGBI) expression in synovium and joint,and to identify the role of HMGB1 in the pathogenesis of synovitis and joint destruction in adjuvant-induced arthritis(AA).Methods AA of 15 male rats were induced in SD rats by intradermal injection of 100?l Freud's complete adjuvant in the foot pad of the left hind paw.All rats were killed at the 18th day.Synovium and joints were collected for histopathology studies and determining the expression of HMGB1 by immunohistochemistry,and serum was collected for determining the expression of HMGB1 by western blotting analysis.Results Immunostaining of specimens from normal rats showed that HMGB1 was primarily confined to the nucleus of synoviocytes with occasional cytoplasmic staining.In contrast, inflammatory synovial tissues from AA rats showed a distinctly different HMGB1 staining pattern.Nuclear HMGBI expression was accompanied by a cytoplasmic staining in many mononuclear cells.The cytoplasmic HMGB1 expression in synovium of AA rats is significantly higher than that of normal rats.Additionally,HMGBI was highly expressed in the nuclei and cytoplasm of the subchondral chondrocytes and inflammatory cells in bone erosion in AA rats(P＜0.01),while fewer positive cytoplasmic staining of HMGB1 was found in chondrocytes and fewer positive nuclear staining was found in bone cells in normal rats.HMGB1 concentration was significantly higher in serum of AA rats than that in normal rats(P＜0.001).Conclusion The cytoplasmic HMGBI expression in synovium and joints is greatly upregulated;the level of HMGB1 in serum is increased in AA rats which suggests a patbogenetic role of HMGB1 in synovitis and bone destruction of adjuvant-induced arthritis.
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HMGB-1, an ubiquitously expressed 25-KD nucleoprotein among mammals, belongs to the HMG family. Recent studies have identified that HMGB-1 is secreted by activated monocytes/macrophages via a non-classical ,vesicle-mediated secretory pathway. Extracellular HMGB-1 is an important proinflammatory cytokine. It participates in the pathogenesis of many diseases such as rheumatoid arthritis, sepsis , acute lung injury, and even leads animals death. Further studies of the mechanism and function of extracellular HMGB-1 may provide a novel strategy for the diagnosis and treatment of these diseases.
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High mobility group box chromosomal protein (HMGB1), an abundant eukaryotic nonhistone chromosomal protein, is previously known as a nuclear DNA-binding protein that stabilizes the structure and function of chromatin, regulates gene transcription. Recent studies identify that extracellular HMGB1 as a late mediator of endotoxemia and sepsis.HMGB1 is released by activated macrophages,induces the release of other proinflammatory mediators,and mediates lethality when overexpressed. It may also be a key signal for eliciting immune responses to cellular injury and death.Moreover,the late kinetics of HMGB1,in compared with other proinflammatory cytokines such as TNF and IL-1,suggest that targeting HMGB1 may provide a wide and clinically accessible therapeutic window.Three independent strategies to inhibit HMGB1 release and action are now available:anti-HMGB1 antibodies,A box,and ethyl pyruvate. This review covers the general features of HMGB1 and progress in research on its newly role as a cytokine participating in the development of sepsis.