The role of high mobility group protein 1 mediated the endoplasmic reticulum stress in cerebral ischemia/reperfusion injury / 中华危重病急救医学

ABSTRACT

Objective To explore the mechanism of high mobility group protein 1 (HMGB1) involved in endoplasmic reticulum stress (ERS) induced by brain ischemia/reperfusion (I/R),based on I/R-HMGB1-ERS as the breakthrough point.Methods The brain of rats birthed 1-3 days was harvested,and the brain cells were cultured in vitro,which were used in the experiment when the cells were in the third passage.The cells were divided into two groupscells in blank control group were cultured under the normal conditions without any treatment,and the cells in hypoxia/reoxygenation group were cultured with 99.9％ nitrogen for 60 minutes (hypoxia) followed by opening the bottle neck for reoxygenation 120 minutes to simulate I/R model.The HMGB1 gene was silenced by using small interfering RNA (siRNA,siRNA and transfection reagent Lipofectamine 2000 mixture gradient was transfected into the cultured cells) as HMGB1-siRNA transfection group,and blank control (without any treatment) and negative control group (transfected with control siRNA) served as controls.The mRNA and protein expressions of HMGB1 and ERS related molecules were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.Results ① In cells of hypoxia/reoxygenation group,the mRNA and protein expressions of HMGB1 and ESR related proteins,including glucose regulating protein 78 (GRP78),C/EBP homologous protein (CHOP) and caspase-12,were significantly higher than those of blank control group with statistical difference (the value in blank control group was served as baseline 1,HMGB1 mRNA3.19±0.48 vs.1,t =2.183,P =0.008;GRP78 mRNA2.07±0.33 vs.1,t =3.292,P =0.016;CHOP mRNA1.93±0.28 vs.1,t =2.573,P =0.021;caspase-12 mRNA2.42±0.42 vs.1,t =2.261,P =0.027HMGB1 protein2.28±0.36 vs.1,t =2.042,P =0.009;GRP78 protein1.33±0.24 vs.1,t =2.781,P =0.016;CHOP protein1.67±0.34 vs.1,t =2.174,P =0.021;easpase-12 protein1.36±0.44 vs.1,t =3.192,P =0.008).It was indicated that ERS related molecules involved in cell hypoxia/reoxygenation process.2② After HMGB1 gene was silenced by siRNA,the cells after hypoxia/reoxygenation showed a decrease in the mRNA and protein expressions of HMGB1 and ERS related moleculars as compared with those of blank control group and negative control group (served the value in blank control group as baseline 1,HMGB1 mRNA0.27±0.12 vs.1,1.02 ± 0.04;GRP78 mRNA0.16 ± 0.13 vs.1,0.96 ± 0.04;CHOP mRNA0.47 ± 0.09 vs.1,0.98 ± 0.07;caspase-12 mRNA0.31 ±0.11 vs.1,1.05±0.02;HMGBI protein0.23±0.04 vs.1,1.08±0.01;GRP78 protein0.14±0.09 vs.1,1.35±0.03;CHOP protein0.32±0.10 vs.1,0.93±0.06;caspase-12 protein0.27±0.09 vs.1,0.97±0.08;P ＜ 0.05 or P ＜ 0.01).It was indicated that HMGB1 involved in ERS related with GPR7,CHOP,caspase-12.Conclusion Hypoxia/reoxygenation brain intracellular HMGB1 and ERS related molecules expression levels were significantly up-regulated,and silencing HMGB1 gene can significantly inhibit the expression levels of these molecules,and I/R-HMGB 1-ERS pathway may participate in the mechanism of brain I/R injury.