[HMGB1抑制对大鼠心肌缺血/再灌注损伤后内质网应激的影响]
[Effects of HMGB1 inhibition on endoplasmic reticulum stress after myocardial ischemia/reperfusion injury in rats].
作者信息
E Lusha, Nan Jinglong
机构信息
Department of Cardiology, the Inner Mongolia Autonomous Region People's Hospital, Hohhot 010017, Inner Mongolia, China. Corresponding author: Nan Jinglong, Email:
出版信息
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2017 Oct;29(10):916-920. doi: 10.3760/cma.j.issn.2095-4352.2017.10.011.
OBJECTIVE
To investigate the effect of high mobility group protein B1 (HMGB1) inhibition on endoplasmic reticulum stress (ERS) after myocardial ischemia/reperfusion (I/R) in rats.
METHODS
Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 10): sham operation group, I/R model group, Gene silencing (HMGB1-siRNA) group, and empty vector (Scrambled-siRNA) group. Coronary blood flow of the rats were ligated for 30 minutes, relaxed the ligament line for 2 hours, to establish I/R injury model; not ligation with the sham operation group. Each group was injected 1 mL phosphate buffer (PBS), HMGB1-siRNA mixture or Scrambled-siRNA mixture preoperative by tail vein 0 hour, 12 hours, and 24 hours before surgery. After 2 hours reperfusion, the levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-8) in the serum were detected by enzyme linked immunosorbent assay (ELISA); the expression of HMGB1 protein in myocardium was detected by immunohistochemistry; the protein and mRNA expressions of HMGB1, GRP78, CHOP and caspase-12 in myocardium were detected by Western Blot and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS
Compared with sham operation group, the levels of serum inflammatory factor, HMGB1 positive cells, and the protein and mRNA expressions of GRP78, CHOP, caspase-12 were significantly increased in I/R model group. The levels of serum inflammatory factor in HMGB1-siRNA group were significantly lower than those in the I/R model group [TNF-α (ng/L): 783.4±203.4 vs. 963.9±214.1, IL-6 (ng/L): 358.8±94.8 vs. 452.3±103.7, IL-8 (ng/L): 180.5±73.6 vs. 347.3±90.3, all P < 0.05], HMGB1 positive cells, and the protein and mRNA expressions of GRP78, CHOP, caspase-12 in HMGB1-siRNA group were significantly lower than I/R model group (HMGB1 protein: 1.59±0.26 vs. 3.21±0.40, GRP78 protein: 2.59±0.28 vs. 4.21±0.42, CHOP protein: 2.01±0.23 vs. 3.21±0.43, caspase-12 protein: 1.48±0.22 vs. 3.01±0.48; HMGB1 mRNA: 2.35±0.26 vs. 4.67±0.45, GRP78 mRNA: 6.59±0.26 vs. 11.21±0.40, CHOP mRNA: 2.01±0.43 vs. 5.21±0.63, caspase-12 mRNA: 4.48±0.32 vs. 8.41±0.52, all P < 0.05). There was no significant difference between the Scrambled-siRNA group and the I/R model group.
CONCLUSIONS
HMGB1 may be involved in the activation of ERS in myocardial I/R injury and increase the damage of myocardial cells.
目的
探讨抑制高迁移率族蛋白B1（HMGB1）对大鼠心肌缺血/再灌注（I/R）后内质网应激（ERS）的影响。
方法
将40只雄性Sprague-Dawley（SD）大鼠随机分为四组（n = 10）：假手术组、I/R模型组、基因沉默（HMGB1-siRNA）组和空载体（Scrambled-siRNA）组。结扎大鼠冠状动脉血流30分钟，松开结扎线2小时，建立I/R损伤模型；假手术组不结扎。每组在术前0小时、12小时和24小时经尾静脉注射1 mL磷酸盐缓冲液（PBS）、HMGB1-siRNA混合物或Scrambled-siRNA混合物。再灌注2小时后，采用酶联免疫吸附测定（ELISA）法检测血清中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-6、IL-8）水平；采用免疫组织化学法检测心肌中HMGB1蛋白表达；采用蛋白质印迹法和实时荧光定量逆转录-聚合酶链反应（RT-PCR）检测心肌中HMGB1、葡萄糖调节蛋白78（GRP78）/、C/EBP同源蛋白（CHOP）和半胱天冬酶12（caspase-12）的蛋白及mRNA表达。
结果
与假手术组比较，I/R模型组血清炎症因子水平、HMGB1阳性细胞数及GRP78、CHOP、caspase-12蛋白及mRNA表达均显著升高。HMGB1-siRNA组血清炎症因子水平显著低于I/R模型组[TNF-α（ng/L）：783.4±203.4比963.9±214.1，IL-6（ng/L）：358.8±94.8比452.3±103.7，IL-8（ng/L）：180.5±73.6比347.3±90.3，均P < 0.05]，HMGB1-siRNA组HMGB1阳性细胞数及GRP78、CHOP、caspase-12蛋白及mRNA表达均显著低于I/R模型组（HMGB1蛋白：1.59±0.26比3.21±0.40，GRP78蛋白：2.59±0.28比4.21±0.42，CHOP蛋白：2.01±0.23比3.21±0.43，caspase-12蛋白：1.48±0.22比3.01±0.48；HMGBmRNA：2.35±0.26比4.67±0.45，GRP78mRNA：6.59±0.26比11.21±0.40，CHOPmRNA：2.01±0.43比5.21±0.63，caspase-12mRNA：4.48±0.32比8.41±0.52，均P < 0.05）。Scrambled-siRNA组与I/R模型组比较差异无统计学意义。
结论
HMGB1可能参与心肌I/R损伤时ERS的激活，加重心肌细胞损伤。
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