热休克蛋白27对内毒素血症所致心功能不全的保护作用

Effect of heat shock protein ：27 on the cardiac dysfunction induced by endotoxemia

目的探讨心肌特异性高表达热休克蛋白27（Hsp27）对内毒素血症所致心功能不全的影响，并初步探讨其机制。方法所有实验均在南京医科大学第一附属医院老年医学科研究室和南京大学模式动物研究所完成。（1）心肌特异性高表达Hsp27转基因鼠（Hsp27Tg）基因型和表达鉴定：分别采用PCFI法和westernblot法；（2）心功能测定：Hsp27Tg和野生型（WT）对照鼠均被腹腔注射内毒素（LPS，10mg／kg），24h后以超声心动图测定心功能，n=6／组；（3）死亡率：Hsp27Tg和wr鼠腹腔注射内毒素（LPS，20mg／kg），密切监测70h内的累积死亡率，n=37／WT，n=27／Hsp27Tg；（4）NF-κB活性测定：心肌组织样本采用凝胶迁移电泳法，细胞样本采用双报告基因法，n=4／组；（5）多组间的两两比较采用ANOVA和Scheffe检验，生存曲线分析采用log—rank检验，P〈0．05设为相差显著性界值。结果（1）Hsp27Tg小鼠心肌有丰富的Hsp27表达，WT则没有；（2）LPS使WT和Hsp27Tg鼠心功能均显著下降，但与WT比较，Hsp27Tg的心功能显著改善（P〈0．01或P〈0．05），其中EF增加27．3％，FS增加37．1％；（3）至LPS注射后70h，WT和Hsp27Tg小鼠死亡率分别为37．84％和11．11％，与WT鼠比较，Hsp27Tg生存率显著提高（P〈0．05）；（4）Hsp27显著抑制LPS诱导的NF-κB激活（P〈0．01或P〈0．05）。结论Hsp27心肌特异性高表达显著抑制内毒素血症所致的心功能不全，改善生存率，其机制可能与Hsp27下调LPS诱导的NF-κB激活有关。

Objective To study the effects of heat shock proetin 27 （ Hsp 27） on the cardiac dysfunction induced by endotoxemia. Method All experiments were performed in the geriatric lab of the First Affiliated Hospital Of Nanjing Medical University, and in the Animal Model Center of Nanjing University. The genotyping of the transgenic mice with cardiac-specific overexpression of Hsp27 （Hsp27 Tg） was assayed by PCR and the expression of Hsp27 was determined by western blot. Hsp27 Tg and its wild type httermates （WT） were intraperitoneally injected with LPS （10 mg/kg）, and 24 hours later, cardiac function was measttred by echocardiography （ n = 6/ group）. The accttmulated mice mortality was recorded within 70 hours after intraperitoneal injection of LPS （20 mg/kg） （ n = 37/WT, n = 27/Hsp27Tg）. The NF-κB activity for cardiac-tissue samples was analyzed by electrophoretic mobility shift assay （EMSA） and for cell cuhure samples by dual-reporter gene assay （n = 4/group）. The comparison of multiple groups was performed by one-way analysis of variance （ANOVA）, and comparison of two groups was performed by Seheffe-test. Survival curves were analyzed by the log-rank test. P 〈 0.05 was considered to be significant. Results The high expression of Hsp27 exhibited in myocardium of Hsp27 Tg, whereas not in myocardium of WT. LPS significantly reduced the cardiac function both in Hsp27 Tg and WT. However, compared with LPS-treated WT, cardiac function was more significantly improved as evidenced by the increases of EF by 27.33% and FS by 37.09% （P 〈0.01 or P 〈 0.05）. Seventy hours after LPS injection, the mortality was 11.11% in Hsp27 Tg and 57.84% in WT. Compared with WT, the survival rate of Hsp27 Tg significantly increased （P 〈 0.05）. The NF-κB activation was significantly inhibited by Hsp27 （P 〈 0.01 or P 〈 0.05）. Conclusions The high cardiac-specific expression of Hsp27 significantly inhibits cardiac dysfunction induced by endotoxemia, and at the same time improve the survival rate. The mechanism may be connected with Hsp27 downregulating NF-κB-activation induced by LPS.