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

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。

Objective To investigate the protective mechanism of mild hypothermia pretreatment against hepatic ischemia-reperfusion injury in rats.Methods A 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(PI3 K),phosphorylated PI3 K(p-PI3 K),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.Results The HIRI group had significantly lower relative expression levels of p-PI3 K and p-Akt than the Sham group(q=5.217 and5.456,both P<0.01),and the MH group had significantly higher relative expression levels of p-PI3 K 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,and10.201,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).Conclusion MH exerts a protective effect against HIRI in rats,possibly by activating the PI3 K/Akt pathway to downregulate hepatocyte apoptosis and enhance antioxidant capacity in vivo.