热休克蛋白27改善小鼠内毒素血症心功能不全的机制

目的 探讨Hsp27的保护作用是否与激活P13K/Akt信号通路、减轻炎症反应有关.方法 (1)诱导小鼠内毒素血症心肌特异性高表达Hsp27转基因鼠(Hsp 27 Tg)和野生型对照鼠(WT)均腹腔注射内毒素(LPS,10ms/ks);(2)心功能测定 LPS注射后6 h,以心脏超声评价,n:6;(3)P13K/Akt信号通路活性测定 LPS注射后1 h收集心脏,以Western blot法测定Akt及Gsk-3(的磷酸化水平,n=4;(4)NF-B炎症通路活性检测 LPS注射后1 h以Western blot测定IκBα水平,n=4;同时在大鼠心肌细胞中进行相同实验,n:3;(5)心肌细胞凋亡LPS注射后24 h,以TUNEL法检测,,n=4.结果 (1)Hsp27显著改善LPS所致的心功能不全.与基础值相比,LPS处理使WT,Hsp27 Tg均出现心功能不全,但Hsp 27 Tg的心功能不全程度显著轻于WT(P<0.01或P<0.05).(2)Hsp27显著抑制LPS所致的IκBα降解.与WT比较,高表达Hsp27显著减轻了LPS诱导的小鼠心肌IκBα降解(41.43±24.10)%vs.(72.92±9.20)%,P<0.05],培养的大鼠心肌细胞实验结果与之类似.(3)Hsp27可增强激活P13K/Akt信号通路.LPS处理后,WT和Hsp27Tg鼠心肌组织中磷酸化Akt水平分别为(3.11±0.83)和(5.13±0.73),磷酸化GSK-30水平分别为(3.19±1.04)和(5.71±1.20).与WT比较,Hsp27Tg心肌组织中的磷酸化Akt和GSK-3β水平均显著提高(P<0.05).类似结果在体外培养的细胞实验中也被证实.(4)Hsp27显著抑制LPS所致的心肌细胞凋亡.LPS处理后24 h,WT和Hsp27Tg心肌细胞凋亡率分别为(6.46±1.74)%和(2.88±0.91)%,与WT比较,心肌细胞凋亡在Hsp27Tg中被显著减轻(P<0.01).结论 高表达Hsp27对小鼠内毒素血症心功能不全有显著改善作用,其保护作用与激活P13K/Akt信号通路、抑制NFκB;依赖性炎症反应有关.

The mechanism of cardiac protection of Hsp27 against cardiac dysfunction during endotoxema in mice

Objective To investigate the cardiac protection of Hsp27 against endotoxic cardiac depression mediated by activation of PI3K/Akt pathway and the suppression of NFκB-mediated inflammatory response in mice. Method (1) Transgenic mice with cardiac specific overexpression of Hsp27 (Hsp27 Tg) and wild littermate controls (WT) were given 10 mg/kg LPS injected intraperitoneally to induce endotoxemia, (2) The cardiac function measurement in mice was performed by using echocardiography 6 hours after LPS treatment (n = 6), (3) The activity of PBK/Akt pathway was evaluated by Western blot for hosphor-Akt (p-Akt) and phosphor-Gsk-3β (p-Gsk-3β) one hour after LPS administration ( n = 4)], (4) Activity of inflammatory response was evaluated by protein degradation of IκBα (n = 4), (5) The apoptosis of myocardial cells was determined by TUNEL assay on the paraffin section of cardiac tissue 24 hours after LPS exposure (n = 4). Results (1) Hsp27 attenuated cardiac dysfunction significantly following LPS treatment. Compared with the primary value, LPS induced the depression of cardiac function both in WT rats and Hsp27Tg rats. However, the cardiac dysfunction was attenuated significantly in Hsp27Tg rats compared with that in WT rats ( P < 0.01 or 0.05) . (2) Hsp27 attenuated IκBα degradation after LPS administration. Compared with the primary value, LPS led to LκBα degradation by (72.92 + 9.20) % in WT rats and by (41.43 + 24.10) % in Hsp27Tg rats. The overexpression of Hsp27 lessened the IκBα degradation significantly (P < 0.05). The similar results were obtained in rat myocardial cell culture of experiments. (3) Hsp27 enhanced the activation of PI3K/Akt signaling following LPS exposure. One hour after LPS administration, the relative levels of p-Akt and p-GSK-30 were (3.11 + 0.83) and (3.19 + 1.04), respectively in WT rats, and (5.13 + 0.73) and (5.71 + 1.20) in Hsp27Tg rats, respectively. Compared with WT rats, the levels of p-Akt and p-GSK-3β were significantly higher in Hsp27Tg rats (P < 0.05). (4) The Hsp27 lessened LPS-induced the apopto-sis of myocardial cells. Twenty-four hours after LPS treatment, the percentages of myocardial cell apoptosis were (6.46+ 1.74)% in WT rats and (2.88 + 0.91)% in Hsp27Tg rats. Compared with WT rats, LPS-induced apoptosis in myocardial cells was significantly decreased in Hsp27Tg rats (P < 0.01). Conclusions The overexpression ofHsp27 attenuates cardiac dysfunction significantly during endotoxemia, and the mechanisms may be attributed to the activation of PDK/Akt signaling pathway.