依布硒啉对大鼠心肌缺血再灌注损伤后磷酸肌醇-3激酶/蛋白激酶B信号通路的影响

doi:10.3877/cma.j.issn.1674-6880.2016.06.002

目的

探讨依布硒啉预处理对大鼠心肌缺血再灌注后磷酸肌醇-3激酶/蛋白激酶B（P13K/Akt）信号及其内质网应激的影响。

方法

将30只大鼠分成对照组，缺血再灌注组及依布硒啉组，每组10只。对照组大鼠冠状动脉左室支左心耳下缘约0.5 cm处只穿线，不结扎，常规腹腔注射生理盐水2 ml；缺血再灌注组大鼠缺血再灌注前30 min腹腔注射生理盐水2 ml；依布硒啉组大鼠缺血再灌注前30 min腹腔注射依布硒啉溶液5 mg/kg。每组各取8只大鼠，采用酶联免疫吸附试验（ELISA）检测各组血清炎症因子高迁移率族蛋白1（HMGB1）、丙二醛、超氧化物歧化酶（SOD）、天冬氨酸转氨酶（AST）及乳酸脱氢酶（LDH）的表达，Tunel法检测缺血心肌细胞凋亡指数，Western-blotting检测各组心肌心肌葡萄糖调节蛋白78（GRP78）蛋白表达及P13K/Akt通路磷酸化水平。

结果

三组大鼠间HMGB1、丙二醛、SOD、AST及LDH水平比较，差异均有统计学意义（F = 63.755、73.023、99.220、110.439、120.557，P均< 0.05），进一步比较发现，缺血再灌注组及依布硒啉组的HMGB1[（9.2 ± 2.7）、（5.5 ± 1.1）、（2.2 ± 0.3）U/L]、丙二醛[（7.2 ± 0.4）、（5.6 ± 0.7）、（4.1 ± 0.9）μmol/L]、AST [（1 011 ± 226）、（813 ± 82）、（671 ± 60）U/L]及LDH [（2 783 ± 674）、（2 043 ± 489）、（1 528 ± 524）U/L]水平较对照组均明显升高，SOD水平[（249 ± 28）、（149 ± 10）、（172 ± 17）kU/L]较对照组明显降低（P均< 0.05）。三组大鼠间的凋亡指数（F = 139.942，P < 0.001）、GRP78蛋白表达（F = 177.846，P < 0.001）及P13K/Akt磷酸化水平（F = 86.286，P < 0.001）比较差异均存在统计学意义，进一步比较发现，缺血再灌注组和依布硒啉组凋亡指数[（38.1 ± 4.6）、（25.4 ± 3.9）、（8.2 ± 1.5）%]明显高于对照组，且I/R组更高（P均< 0.05）。

结论

依布硒啉预处理可以抑制大鼠内质网应激，可能与调节P13K/Akt信号通路磷酸化水平有关。

关键词: 依布硒啉, 再灌注损伤, 磷酸肌醇-3激酶/蛋白激酶B

Objective

To explore the effect of ebselen pretreatment on phosphoinositide 3-kinase/protein kinase B (P13K/Akt) signal pathway and the endoplasmic reticulum stress following ischemia reperfusion (I/R) injury in rats.

Methods

A total of 30 rats were divided into the control group, model group and ebselen group, 10 rats in each group. Rats in the control group were only threaded about 0.5 cm left lower limb at left ventricle coronary artery and recevied 2 ml normal saline intraperitoneal injection. Rats in the model group and ebselen group all established I/R models, and rats in the model group were injected 2 ml normal saline intraperitoneally on 30 min before I/R, while rats in the ebselen group were injected 5 mg/kg ebselen intraperitoneally on 30 min before I/R. Eight rats in each group were used in experiments. The levels of high mobility group box-1 protein (HMGB1), malonaldehyde, superoxide dismutase (SOD), aspartate transaminase (AST) and lactic dehydrogenase (LDH) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic index was measured by Tunel method. The expression of glucose regulated protein 78 (GRP78) protein and phosphorylation of P13K/Akt pathway were detected by Western-blotting.

Results

The levels of HMGB1, malonaldehyde, SOD, AST and LDH all showed significant differences among the three groups (F = 63.755, 73.023, 99.220, 110.439, 120.557, all P< 0.05), and the levels of HMGB1 [(9.2 ± 2.7), (5.5 ± 1.1), (2.2 ± 0.3) U/L], malonaldehyde [(7.2 ± 0.4), (5.6 ± 0.7), (4.1 ± 0.9) μmol/L], AST [(1 011 ± 226), (813 ± 82), (671 ± 60) U/L], and LDH [(2 783 ± 674), (2 043 ± 489), (1 528 ± 524) U/L] in the model group and ebselen group were higher, however the SOD [(249 ± 28), (149 ± 10), (172 ± 17) kU/L] were lower than those in the control group (all P< 0.05). Meanwhile, the apoptostic index (F = 139.942, P < 0.001), GRP78 protein (F = 177.846, P < 0.001) and P13K/Akt phosphorylation (F = 86.286, P < 0.001) also had significant differences among the three groups. And the apoptotic index [(38.1 ± 4.6), (25.4 ± 3.9), (8.2 ± 1.5)%] in the model group and ebselen group were higher than that in the control group, and was the highest in the model group (all P < 0.05).

Conclusion

Ebselen pretreatment can inhibit endoplasmic reticulum stress in rats, which may be related to the regulation of P13K/Akt signaling pathway phosphorylation.

Key words: Ebselen, Reperfusion injury, Phosphoinositide 3-kinase/protein kinase B

表1 各组大鼠HMGB1、丙二醛、SOD、AST、LDH的表达（±s）

组别	例数	HMGB1(U/L)	丙二醛(μmol/L)	SOD(kU/L)	AST(U/L)	LDH(U/L)
对照组	8	2.2 ± 0.3	4.1 ± 0.9	249 ± 28	671 ± 60	1528 ± 524
缺血再灌注组	8	9.2 ± 2.7^a	7.2 ± 0.4^a	149 ± 10^a	1011 ± 226^a	2783 ± 674^a
依布硒啉组	8	5.5 ± 1.1^ab	5.6 ± 0.7^ab	172 ± 17^ab	813 ± 82^ab	2043 ± 489^ab
F值	?	63.755	73.023	99.220	110.439	120.557
P值	?	< 0.05	< 0.05	< 0.05	< 0.05	< 0.05

表1 各组大鼠HMGB1、丙二醛、SOD、AST、LDH的表达（±s）

组别	例数	HMGB1(U/L)	丙二醛(μmol/L)	SOD(kU/L)	AST(U/L)	LDH(U/L)
对照组	8	2.2 ± 0.3	4.1 ± 0.9	249 ± 28	671 ± 60	1528 ± 524
缺血再灌注组	8	9.2 ± 2.7^a	7.2 ± 0.4^a	149 ± 10^a	1011 ± 226^a	2783 ± 674^a
依布硒啉组	8	5.5 ± 1.1^ab	5.6 ± 0.7^ab	172 ± 17^ab	813 ± 82^ab	2043 ± 489^ab
F值	?	63.755	73.023	99.220	110.439	120.557
P值	?	< 0.05	< 0.05	< 0.05	< 0.05	< 0.05

图1 三组大鼠心肌细胞凋亡图。注：a图为各组大鼠心肌细胞凋亡指数的比较，与对照组比较，^*P< 0.05，与缺血再灌注组比较，^#P< 0.05（n = 8）；b~d图分别为对照组、缺血再灌注组、依布硒啉组大鼠心肌细胞凋亡情况（Tunel法染色× 400）

图2 三组大鼠GRP78蛋白表达图（n = 8）。注：a图为各组大鼠GRP78蛋白表达比较，与对照组比较，^*P< 0.05，与缺血再灌注组比较，^#P< 0.05；b图为Western-blotting检测各组大鼠心肌GRP78蛋白表达情况；GRP78：葡萄糖调节蛋白78（glucoseregulated protein 78）；GAPDH：3-磷酸甘油醛脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）

图3 三组大鼠P13K/Akt信号通路磷酸化图。注：a图为各组大鼠P13K/Akt信号通路磷酸化水平比较，与对照组比较，^*P< 0.05，与缺血再灌注组比较，^#P< 0.05（n = 8）；b图为Western-blotting检测各组大鼠心肌P13K/Akt信号通路磷酸化情况；P13K/Akt：磷酸肌醇-3激酶/蛋白激酶B（phosphoinositide 3-kinase/protein kinase B）；GAPDH：3-磷酸甘油醛脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）

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Effect of ebselen on phosphoinositide 3-kinase/protein kinase B pathway following myocardial ischemia reperfusion injury in rats

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Effect of ebselen on phosphoinositide 3-kinase/protein kinase B pathway following myocardial ischemia reperfusion injury in rats