鞘氨醇激酶及1-磷酸鞘氨醇信号参与大鼠肝窦微循环保护

The effects of sphingosine kinase/1-phosphate sphingosine signaling on hepatic ischemia-reperfusion injury in rats

目的:探讨鞘氨醇激酶/1-磷酸鞘氨醇(SphK/S1P)信号通路在肝脏缺血再灌注损伤(IRI)中肝窦微循环的保护作用。方法:2015年9月至2019年9月,将40只适龄雄性大鼠(购自常州卡文斯实验动物有限公司),信封法随机分4组包括,假手术(Sham)组,IRI+等体积生理盐水组(IRI组),IRI+SphK激动剂组(PMA组)和IRI+SphK阻断剂组(DMS组)。建立大鼠IRI模型,通过对SphK/S1P信号激动和阻滞后,应用苏木精-伊红(HE)和细胞凋亡染色原位缺口末端标记法(TUNEL)技术观察肝窦间隙、肝窦内皮细胞(LSEC)和肝细胞的形态学改变及其细胞凋亡程度,酶联免疫吸附(ELISA)和蛋白质印迹法(Western blot)对细胞间黏附分子(ICAM-1)和髓过氧化物酶(MPO)的表达进行评价。应用SPSS 17.0统计软件行χ2检验和单因素方差分析。结果:HE染色显示SphK激动剂(PMA)可改善缺血肝脏损伤,减轻肝窦异常扩张,LSEC损伤程度得到改善,其阻断剂反而加重LSEC的损伤。与Sham组(3.46±0.27)比较,IRI组大鼠血清S1P升高(4.71±0.42,t=4.336,P<0.05);与IRI组比较,PMA组血清S1P显著升高(36.01±10.13,t=5.347,P<0.01),DMS组血清S1P显著降低(15.83±3.14,t=6.080,P<0.05)。与Sham组[(10.09±1.05)%]比较,IRI组肝脏细胞凋亡率显著升高(28.83±2.06)%,t=14.040,P<0.05];与IRI组比较,PMA组肝脏细胞凋亡率显著降低[(13.59±3.02)%,t=7.221,P<0.05],而DMS组肝细胞凋亡率显著升高[(61.92±7.72)%,t=7.173,P<0.01]。与IRI组比较,PMA组肝脏S1PR的蛋白表达升高(S1PR:1.65±0.12比2.50±0.09,t=9.930,P<0.05),MPO、ICAM-1的蛋白表达降低(ICAM-1:3.21±0.39比1.52±0.26,t=6.245,P<0.05;MPO:2.51±0.20比1.09±0.13,t=10.310,P<0.05);而DMS组肝脏S1PR的蛋白表达降低(S1PR:1.65±0.12比1.31±0.09,t=3.926,P<0.05),MPO、ICAM-1蛋白表达升高(ICAM-1:3.21±0.395.92±0.22,t=7.855,P<0.05;MPO:2.51±0.20比5.21±0.43,t=9.861,P<0.05)。结论:SphK/S1P信号通过减少细胞凋亡,减少MPO和ICAM-1表达,减少白细胞和LSEC的黏附,从而改善肝窦微循环。

Objective To explore the protective effect of sphingosine kinase/sphingosine-1-phosphate(SphK/S1P)signaling pathway in hepatic sinus microcirculation during hepatic ischemia-reperfusion injury(IRI)in rats.Methods Forty adult Wistar rats were randomly divided into 4 groups(n=10 each group):IRI group,sham operation group(Sham group),SphK agonist group(PMA group)and SphK blockers group(DMS group).A model of hepatic IRI was established.The morphological changes of liver sinusoidal endothelial cells(LSECs),the degree of apoptosis of hepatocytes,and the different levels of related signaling proteins and intercellular adhesion molecules were observed.All values were expressed as Mean±SD,and were analyzed using SPSS 17.0.Statistical significance was obtained by ANOVA and chi-square analysis.A P value<0.05 was considered statistically significant.Results Hematoxylin-eosin(HE)staining showed that PMA can improve hepatic IRI,reduce abnormal expansion of hepatic sinus,and improve the degree of damage to LSEC,while DMS aggravate LSEC damage.Compared with the Sham group(3.46±0.27),enzyme linked immunosorbent assay(ELISA)showed a significant increase serum S1P in the IRI group(4.71±0.42,t=4.336,P<0.05),while a increased serum S1P significantly in the DMS group(15.83±3.14,t=6.080,P<0.05).Compared with the Sham group,TdT-mediated dUTP nick end labeling(TUNEL)staining showed a significant increase in the apoptosis rate of liver tissue in the IRI group[(28.83±2.06)%,t=14.040,P<0.05];a significant decrease in the apoptosis rate of liver tissue in the PMA group[(13.59±3.02)%,t=7.221,P<0.05];the rate increased significantly[(61.92±7.72)%,t=7.173,P<0.01]in the DMS group.Compared with the IRI group,the protein of S1PR in the PMA group was increased(S1PR:1.65±0.12 vs.2.50±0.09,t=9.930,P<0.05)and myeloperoxidase(MPO)and serum intercellular adhesion molecule-1(ICAM-1)were reduced(ICAM-1:3.21±0.39 vs.1.52±0.26,t=6.245,P<0.05;MPO:2.51±0.20 vs.1.09±0.13,t=10.310,P<0.05);while in the DMS group,the protein expression of S1PR was reduced(S1PR:1.65±0.12 vs.1.31±0.09,t=3.926,P<0.05)and MPO and ICAM-1 were increased(ICAM-1:3.21±0.39 vs.5.92±0.22,t=7.855,P<0.05;MPO:2.51±0.20 vs.5.21±0.43,t=9.861,P<0.05).Conclusion SphK/S1P signaling pathway is involved in hepatic IRI and has an protective effect on hepatic sinus microcirculation in rats.