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 groups:cells 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 mRNA:3.19±0.48 vs.1,t =2.183,P =0.008;GRP78 mRNA:2.07±0.33 vs.1,t =3.292,P =0.016;CHOP mRNA:1.93±0.28 vs.1,t =2.573,P =0.021;caspase-12 mRNA:2.42±0.42 vs.1,t =2.261,P =0.027:HMGB1 protein:2.28±0.36 vs.1,t =2.042,P =0.009;GRP78 protein:1.33±0.24 vs.1,t =2.781,P =0.016;CHOP protein:1.67±0.34 vs.1,t =2.174,P =0.021;easpase-12 protein:1.36±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 mRNA:0.27±0.12 vs.1,1.02 ± 0.04;GRP78 mRNA:0.16 ± 0.13 vs.1,0.96 ± 0.04;CHOP mRNA:0.47 ± 0.09 vs.1,0.98 ± 0.07;caspase-12 mRNA:0.31 ±0.11 vs.1,1.05±0.02;HMGBI protein:0.23±0.04 vs.1,1.08±0.01;GRP78 protein:0.14±0.09 vs.1,1.35±0.03;CHOP protein:0.32±0.10 vs.1,0.93±0.06;caspase-12 protein:0.27±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.
ABSTRACT
Objective To investigate the expression of serum microRNA-210 (miR-210) in patients with acute cerebral infarction(ACI),and to evaluate its clinical significance. Methods A retrospective study was conducted. Eighty patients with ACI admitted to Tianjin Hospital from January 2011 to March 2014 within 48 hours of onset were enrolled,and 30 healthy volunteers served as controls. The peripheral blood was collected,and the expression of serum miR-210 was determined by reverse transcription-polymerase chain reaction(RT-PCR). The receiver operating characteristic curve(ROC)was drawn to analyze the role of miR-210 in the diagnosis of ACI. According to the pathological and physiological characteristics of patients receiving treatment,the relationship between miR-210 and clinical physiology index was analyzed. Results The expression of miR-210 in serum of patients with ACI was significantly lower than that of the healthy control group(2-ΔΔCt:1.349±0.043 vs. 1.923±0.107,t=6.567, P60 years:1.32±0.12 and 1.31±0.09,t=2.351,P=0.264;large infarction,small infarction,lacunar infarction:respectively 1.31±0.02, 1.33±0.11, 1.31±0.06, F=1.236, P=0.087), or with the severity of cerebral infarction,and there was a tendency in lowering of expression of miR-210(2-ΔΔCt,light,medium,severe:1.53±0.11, 1.33±0.11,1.08±0.04,F=5.394,P=0.014).Conclusion The serum level of miR-210 in ACI was significantly lower than that in normal healthy persons,and it may be an important new serological marker in screening and diagnosis of ACI.