Coactivator p300-induced H3K27 acetylation mediates lipopolysaccharide-induced inflammatory mediator synthesis / 南方医科大学学报

OBJECTIVE@#To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)- induced inflammatory mediator synthesis and its molecular mechanism.@*METHODS@#Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq).@*RESULTS@#Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P < 0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P < 0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P < 0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P < 0.05) without affecting p300 binding or H3K27 acetylation level.@*CONCLUSION@#LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.

Treament of CRRT combined with low-flow ECMO for canines with ARDS / 医学研究生学报

Objective Single mechanical ventilation for curing severe acute respiratory distress syndrome (ARDS) is sometimes not effective in improving oxygenation and removing excess CO2 in the blood. The purpose of this study is to observe the effect of continuous renal replacement therapy（CRRT）combined with low-flow extracorporeal membrane oxygenation （ECMO）of V-V mode on clearance of inflammatory mediators，improving oxygenation and reducing PaCO2 in canines with ARDS and hypercapnia. Methods 30 healthy adult canines were divided into 3 groups (n=10 each) using a random number table method sham group, ECMO (EC) group and CRRT+ECMO（CR+EC）group. A canine model of ARDS with hypercapnia were prepared by injected intravenously oleic acid. Sham group was only treated with invasive mechanical ventilation. EC group was treated with invasive mechanical ventilation and ECMO. CR+EC group was treated with CRRT combined with low-flow ECMO of V-V mode besides invasive mechanical ventilation. The heart rate(HR)，mean artery pressure (MAP)，cardiac output(CO) and concentration of serum TNF-α and IL-6 were examined at the beginning of modeling(T0)，after 1，3，6 and 9 (T1, T3, T6, T9) hours of mechanical treatment respectively. Oxygenation index（OI），PaCO2, temperature（T）were examined at the beginning of modeling，after 3, 6, 9 hours of mechanical treatment，and at the time point of 3 hours(T12) after ECMO treatment (EC group) and CRRT+ECMO treatment(CR+EC group)，respectively. The differences were compared between groups involved in above-mentioned indexes at different time points. Results Compared with the sham group, the HR decreased at T6 and T9 in the EC group and CR+EC group (P<0.05), and the concentration of IL-6 and TNF-α decreased at T3, T6 and T9 in the CR+EC group (P<0.05). Compared with T0, HR decreased at T6 and T9 in the EC group (P<0.05). Compared with T0, the concentration of IL-6 and TNF-α decreased at T3, T6 and T9 in the CR+EC group (P<0.05). Compared with the EC group, the HR decreased at T6 and T9 in the CR+EC group (P<0.05). Compared with the EC group, the concentration of IL-6 and TNF-α decreased at T3, T6 and T9 in the CR+EC group (P<0.05). Compared with the sham group, the OI decreased at T9 and T12 in the CR+EC group (P<0.05). Compared with the sham group, PaCO2 decreased at T3, T6 and T9 in the EC group and CR+EC group (P<0.05). Compared with the EC group, the OI decreased at T9 and T12 in the CR+EC group (P<0.05). Compared with T0, the OI decreased at T6 and T12 in the CR+EC group (P<0.05). Conclusion The CRRT combined with low-flow ECMO of V-V mode has positive effects on clearance of inflammatory mediators，reducing hypercapnia and improving oxygenation in the canines with ARDS and hypercapnia.