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Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on cytokine expression in splenic dendritic cell (DCs). Method DCs isolated from the spleens of male Wistar rats were seed-ed on 96-well (1×10s cells/well) cell culture plates, and the cells were stimulated with HMGB1 for various length of time or in different concentrations. (1) The time-dependent response between HMGB1 and tumor necrosis factor-α(TNF-α) as well as interleukin-12 (IL-12) gene/protein expressions: 24 wells of DCs were dividedly into six groups including 24 h-normal controls (n=4), 48 h-normal controls (n=4), 72 h-normal controls (n=4), and 24 h-HMGB1 treated group (n=4), 48 h-HMGB1 treated group (n=4) as well as 72 h-HMGB1 treated group (n=4), respectively. Among three HMGB1-treated groups, DCs were stiraulated by 1 μg/mL HMGB1. DCs were denatured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvested to determine Il-12 as well as TNF-α protein levels. (2) The dose-dependent response between HMGB1 and TNF-α as well as IL-12 gene/protein expressions: 16 wells of DCs were dividedly into four groups in-cludingnormal controls (n=4), 0.1 μg/mL HMGB1 treated group (n=4), 1 μg/mL HMGB1 treated group (n =4), and 10 μg/mL HMGB1 treated group (n=4), respectively. After stimulated for 48 h, DCs were dena-tured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvest-ed to determine IL-12 as well as TNF-α protein levels. Total RNA was extracted from cells using the single-step technique of acid guanidinium thiocyanate-chloroform extraction according to the manufacturer' s instruction (Promega, Madison, WI). mRNA for TNF-α and IL-12 were quantified by SYBR Green two-step, real-time re-verse transeription-polymerase chain reaction taking glyceraldehyde-3-phesphate dehydrogenase (GAPDH) as an internal standard. Levels of IL-12 and TNF-α in cell culture supernatants were determined with ELISA, strictly fol-lowing the protocols provided by the manufacturer. Data were analyzed with a one-way ANOVA. A P-values <0.05 were considered statistically significant. Results After stimulation with 1 μg/mL HMGB1, IL-12 and TNF-α protein and gene expressions in rat splenic DCs were markedly up-regulated at 24 h to 72 h (P<0.05 or P<0.01), and the expression levels of IL-12 and TNF-α peaked at 48 h (P<0.01). When DCs were cultured in the presence of 0.1 μg/mL, 1 μg/mL, and 10 μg/ml HMGBI for 48 h, expressions of IL-12 and TNF-α were also significantly up-regulated (P<0.01), and values of these cytokines were highest in 1 μg/mL HMGB1-treated group (P<0.01). Conclusions These data suggest that HMGB1 appears to be a potential immunostimulatory signal that induced DC maturation, and HMGB1 stimulation can result in marked up-regulation of IL-12 as well as TNF-α synthesis and release in splenic DCs.
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Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on inter-leukin-2 (1L-2) and interleukin-2 receptor α (IL-2α) expressions in human T lymphocytes and its potential regulat-ing mechanism in vitro. Method Human T lymphocytes were isolated and suspended, the cells were cultured with 20 μg/mL phytohemagglutinin (PHA) in 5% CO_2 at 37 ℃, recombinant human HMGB1 (rhHMGB1, 0, 10, 100, 1000 ng/mL) was added with the PHA and cultures were centrifuged at 12 and 48 hours for cell collect-ing. Reverse transcription polymerase chain reaction (RT-PCR) amplification was perfomed to determine gene ex-pressions of IL-2, IL-2Rα. IL-2, sIL-2R protein levels in cell culture supematants were measured by ELIZA. Re-sults After coincubated with rhHMGB1 (10, 100, and 1000 ng/mL) for 12 hours, IL-2 levels in cell culture su-pernatants respectively were 0 . 064 ± 0. 017 μg/L, 0.076±0.033 μg/L, and 0.061 ±0.02 μg/L, which were significantly higher compared with the untreated cells (0.045±0.011 μg/L, P < 0.05 or P < 0.01). Mean-while, IL-2 mRNA expression was markedly up-regulated following rhHMGB1 stimulation in various doses (F = 4.6872, P < 0.01). At 48 bourn, however, both IL-2 mRNA expression and protein production tended to de-crease along with an increased dose of dd-IMGB1 stimulationn. IL-2/sIL-2R ratio in 1000 ng/mL rhHMGB1 was markedly lower than that in 10 ng/ml rhHMGB1 (0.036±0.015 vs.0.055±0.017, P <0.05), together with down-regulation of IL-2Rα mRNA expression(P <0.01). Conclusions These data indieated that HMGBI could marked influence the IL-2/IL-2R expression in human T lymphocytes. With the increase in stimulating doses and prolongation of time, HMGBI might down-regulate T cell-mediated immune response of human lymphocytes.
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Objective:To investigate the clinical characteristics of renal injury caused by Guanxinsuhe pill(冠心苏合丸,including slander dutchanspipe root(青木香)).Methods:The clinical data of 27 patients with renal injury caused by Guanxinsuhe pill were studied.The clinical characteristics of the renal damage caused by Guanxinsuhe pill and the relationships between duration of illness,duration of taking Guanxinsuhe pill,anemia,renal atrophy and the progression of renal injury respectively were also analyzed.Results:Twenty-seven patients all taken Guanxinsuhe pill for suspicious coronary heart disease(CHD) and were diagnosed as chronic tubulointerstitial nephropathy accompanied with renal tubular acidosis,renal glucosuria and hyposthenuria after admission.Anemia was of more severity than renal dysfunction.Atrophic kidney occurred in 26 patients.The main complains were nonspecific symptoms including fatigue,anorexia,nausea,vomiting,etc.and accompanied with different degrees of chronic renal dysfunction.Twentyfour hours urinary protein excretions were 120-900 mg((490?250) mg).There were few red and white cells in uroscopy.Sixteen patients discontinuously took the Guanxinsuhe pill(one capsule,2-3 times a day) according to the pharmacopoeia for a long period.Ten patients took a dosage(two capsules,2-3 times a day) higher than that limited by the pharmacopoeia.Twenty-six patients took Guanxinsuhe pill discontinuously.One patient continuously took Guanxinsuhe pill until got renal dysfunction.The duration of taking Guanxinsuhe pill was 1-120 months(37.38?31.58),and the amount of 50-43 800 pills(4 576.00?8 830.54) were used,but the amount of pills of 3 cases was uncertain.The renal injury occurred within 30-216 months(95.41?56.74) after initiation of the drug taken.The renal injury obviously correlated with the anemia and the duration of taking Guanxinsuhe pill.Conclusion:Taking Guanxinsuhe pill can cause renal injury.The Guanxinsuhe pill dose indicated in pharmacopoeia also causes renal damage.The onset of renal injury is insidious.Most of the patients have the history of CHD and when the chronic nephropathy in most of such patients is detected,it is advanced to stage 5.The renal injury induced by Guanxinsuhe pill usually presents as chronic tubulointerstitial nephropathy,renal tubular dysfunction and anemia.Anemia is of more severity than renal dysfunction.The 24 hours urinary protein excretions of all the patients are lower than 1 000 mg daily.It is suggested to avoid taking Guanxinsuhe pill,adjust the prescription of Guanxinsuhe pill and do not use slander dutchanspipe root as an ingredient.
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Objective To observe the expression law of cytokine signaling suppressors (SOCSs) mRNA in burn rats with Staphylococcus aureus sepsis and investigate their potential role in the pathogenesis of postburn sepsis. Methods Wistar rats were inflicted with 20% TBSA Ⅲ? scald, followed by Staphylococcus aureus challenge. Then, the expressions of SOCS1, SOCS2 and SOCS3 mRNA and interferon-? (IFN-?) levels in the liver and lungs were determined. Results With Staphylococcus aureus challenge after burn, IFN-? levels in the liver and lungs were significantly elevated and reached peak at the 0.5th and 6th hours, respectively (P