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亚低温通过磷脂酰肌醇3-激酶/蛋白激酶B信号通路对肝缺血再灌注损伤大鼠模型的保护作用
Mild hypothermia exerts a protective effect against hepatic ischemia-reperfusion injury in rats by activating the PI3K/Akt signaling pathway
文章发布日期:2019年03月07日  来源:  作者:刘剑,李立,王晓川,等  点击次数:286次  下载次数:67次

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【摘要】:目的探讨亚低温预处理肝缺血再灌注损伤(HIRI)大鼠模型的保护作用机理。方法将40只SD大鼠分为4组:假手术(Sham)组、HIRI组、亚低温处理(MH)组和MH+LY294002处理(MH+LY)组,每组10只,分别于肝缺血再灌注3、6、12、24 h后收集大鼠的血清及肝组织标本。采用Western Blot检测大鼠肝组织中磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)和磷酸化Akt(p-Akt,Ser308)蛋白的表达水平。采用TUNEL法和Western Blot检测分析肝组织中细胞凋亡和凋亡相关蛋白的表达水平。采用ELISA试剂盒检测肝组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平。采用全自动生化分析仪检测大鼠血清ALT、AST活性。计量资料多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果HIRI组p-PI3K、p-Akt的相对表达水平明显低于Sham组,差异均有统计学意义(q值分别为5217、5456,P值均<0.01);经亚低温处理后大鼠肝组织中p-PI3K、p-Akt的相对表达水平明显高于HIRI组,差异均有统计学意义(q值分别为10434、14116,P值均<0.01)。HIRI组、MH组和MH+LY组大鼠再灌注后3、6、12、24 h的血清AST、ALT活性明显高于Sham组,差异均有统计学意义(P值均<0.001)。TUNEL染色结果显示,HIRI组大鼠的细胞凋亡百分比[(42.25±3.50)%]明显高于Sham组[(3.21±0.5)%],差异均有统计学意义(q=10.187,P<0.01);相比于HIRI组,MH组明显下调了细胞凋亡百分比(1242±13)%,差异有统计学意义(q=7.784,P<0.01),且HIRI组与MH+LY组的细胞凋亡百分比[(38.19±2.8)%]比较差异无统计学意义(q=1059,P>0.05)。Western Blot检测结果显示,与Sham组相比,HIRI组中抗凋亡蛋白Bcl-2表达明显下调,差异有统计学意义(q=2.101,P<0.05),促凋亡蛋白Bax、fas和fasl表达明显上调,差异均有统计学意义(q值分别为11016、4735、10201,P值均<001),亚低温处理之后可明显下调HIRI对凋亡相关蛋白的调控作用。氧化指标检测结果显示,HIRI组、MH组和MH+LY组大鼠肝组织中SOD、GSH-Px的表达水平均明显低于Sham组,差异均有统计学意义(P值均<0.05),而MDA在HIRI组、MH组和MH+LY组大鼠肝组织中的表达水平均明显高于Sham组,差异均有统计学意义(P值均<0.05);MH组大鼠肝组织中SOD、GSH-Px的表达水平明显高于HIRI组,差异均有统计学意义(q值分别为2.894、2.731,P值均<0.05),但MDA的表达水平明显低于HIRI组,差异有统计学意义(q=5.888,P<0.05)。此外,亚低温+LY294002处理明显下调了MH组对肝组织中SOD、MDA和GSH-Px表达的调控作用,差异均有统计学意义(q值分别为2999、2944、2620,P值均<0.05)。结论亚低温对HIRI大鼠具有保护作用,其机制可能是通过激活PI3K/Akt通路发挥下调肝细胞凋亡和增强体内抗氧化作用。
【Abstract】:ObjectiveTo investigate the protective mechanism of mild hypothermia pretreatment against hepatic ischemia-reperfusion injury in rats. MethodsA total of 40 male Sprague-Dawley rats were randomly divided into sham-operation group (Sham group), hepatic ischemia-reperfusion injury group (HIRI group), mild hypothermia group (MH group), and mild hypothermia+LY294002 group (MH+LY group), with 10 rats in each group. Serum and liver tissue samples were collected at 3, 6, 12, and 24 hours of hepatic ischemia-reperfusion. Western blotting was used to measure the protein expression of phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase-B (Akt), and phosphorylated Akt (p-Akt, Ser308) in liver tissue. TUNEL and Western blotting were used to measure cell apoptosis and expression of apoptosis-related proteins in liver tissue. ELISA was used to measure the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in liver tissue. An automatic biochemical analyzer was used to measure the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. A one-way analysis of variance was used for comparison between multiple groups, and the SNK-q test was used for further comparison between two groups. ResultsThe HIRI group had significantly lower relative expression levels of p-PI3K and p-Akt than the Sham group (q=5.217 and 5456, both P<0.01), and the MH group had significantly higher relative expression levels of p-PI3K and p-Akt in liver tissue than the HIRI group (q=10.434 and 14.116, both P<0.01). At 3, 6, 12, and 24 hours of hepatic ischemia-reperfusion, the HIRI group, the MH group, and the MH+LY group had significantly higher activities of AST and ALT in serum than the Sham group (all P<0.001). TUNEL staining showed that the HIRI group had a significantly higher proportion of apoptotic cells in liver tissue than the Sham group (4225%±3.50% vs 3.21%±0.5%, q=10.187, P<0.01). Compared with the HIRI group, the MH group had a significant reduction in the proportion of apoptotic cells (q=7.784, P<0.01), while there was no significant difference in the proportion of apoptotic cells between the HIRI group and the MH+LY group (42.25%±3.50% vs 38.19%±2.8%, q=1.059, P>0.05). Western blotting showed that compared with the Sham group, the HIRI group had a significant reduction in the expression of the anti-apoptotic protein Bcl-2 (q=2.101, P<0.05) and significant increases in the expression of the pro-apoptotic proteins Bax, Fas, and Fasl (q=11016、4735, and 10201, all P<0.01), and the regulatory effect of HIRI on apoptotic-related proteins was downregulated by mild hypothermia treatment. According to the results of oxidative indices, the HIRI group, the MH group, and the MH+LY group had significantly lower expression of SOD and GSH-Px and significantly higher expression of MDA in liver tissue than the Sham group (all P<0.05); compared with the HIRI group, the MH group had significantly higher expression of SOD and GSH-Px (q=2.894 and 2.731, both P<005) and significantly lower expression of MDA (q=5.888, P<0.05) in liver tissue. In addition, mild hypothermia+LY294002 treatment significantly reduced the regulatory effect on the expression of SOD, MDA, and GSH-Px in liver tissue in the MH group (q=2.999, 2.944, and 2.620, all P<0.05). ConclusionMH exerts a protective effect against HIRI in rats, possibly by activating the PI3K/Akt pathway to downregulate hepatocyte apoptosis and enhance antioxidant capacity in vivo.
【关键字】:肝疾病; 再灌注损伤; 1-磷脂酰肌醇3-激酶; 蛋白激酶类; 大鼠, Sprague-Dawley
【Key words】:liver diseases; reperfusion injury; 1-phosphatidylinositol 3-kinase; protein kinases; rats, Sprague-Dawley
【引证本文】:LIU J, LI L, WANG XC, et al. Mild hypothermia exerts a protective effect against hepatic ischemia-reperfusion injury in rats by activating the PI3K/Akt signaling pathway[J]. J Clin Hepatol, 2019, 35(4): 846-851. (in Chinese)
刘剑, 李立, 王晓川, 等. 亚低温通过磷脂酰肌醇3-激酶/蛋白激酶B信号通路对肝缺血再灌注损伤大鼠模型的保护作用[J]. 临床肝胆病杂志, 2019, 35(4): 846-851.

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