YAP在肝脏缺血-再灌注损伤中的作用及其机制

Effect and mechanism of YAP in hepatic ischemia-reperfusion injury

目的探究Yes相关蛋白(YAP)在小鼠肝脏缺血-再灌注损伤(IRI)中的作用及机制。方法雄性C57BL/6小鼠40只,按随机数字表法分为假手术组(Sham组)、溶血磷脂酸(LPA)+Sham组、IRI组、LPA+IRI组,每组10只。缺血-再灌注6 h后,收集肝组织和血清标本。检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的水平,苏木素-伊红(HE)染色、免疫组织化学(免疫组化)染色检测肝组织病理学改变和巨噬细胞浸润情况,蛋白印迹法分析肝组织YAP的蛋白表达水平,逆转录聚合酶链反应(RT-PCR)法评估炎症因子肿瘤坏死因子(TNF)-α、诱生型一氧化氮合酶(iNOS)、白细胞介素(IL)-1和IL-6的信使核糖核酸(mRNA)表达水平。结果蛋白印迹结果显示,LPA+IRI组YAP的蛋白表达水平较IRI组明显升高。与Sham组相比,IRI组ALT和AST显著增高(均为P<0.05);LPA+IRI组较IRI组血清ALT和AST显著降低(均为P<0.05)。HE染色显示,Sham组和LPA+Sham组肝细胞形态正常;LPA+IRI组和IRI组出现肝脏淤血、肝细胞肿胀和肝小叶结构异常等病理改变;LPA+IRI组相较于IRI组病理改变程度减轻。RT-PCR提示,LPA+IRI组中炎症因子TNF-α、iNOS、IL-1和IL-6的mRNA表达水平较IRI组降低(均为P<0.05)。免疫组化显示,LPA部分抑制了IRI后缺血组织巨噬细胞浸润。结论YAP能明显缓解肝脏IRI,其作用机制与调控巨噬细胞募集和活化相关。

Objective To explore the effect and mechanism of Yes-associated protein(YAP)in hepatic ischemiareperfusion injury(IRI)of mice.Methods Forty male C57BL/6 mice were randomly divided into the sham operation group(Sham group),lysophosphatidic acid(LPA)+Sham group,IRI group and LPA+IRI group,10 mice in each group.Liver tissue and serum samples were collected at 6 h after ischemia-reperfusion.The levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected.Histopathological changes and macrophage infiltration of liver tissues were detected by hematoxylin-eosin(HE)staining and immunohistochemical staining.The protein expression level of YAP was detected by Western blot.The messenger ribonucleic acid(mRNA)expression levels of inflammatory cytokines including tumor necrosis factor(TNF)-α,inducible nitric oxide synthase(iNOS),interleukin(IL)-1 and IL-6 were quantitatively measured by reverse transcription polymerase chain reaction(RT-PCR).Results Western blot results demonstrated that the protein expression level of YAP in the LPA+IRI group was significantly upregulated than that in the IRI group.Compared with the Sham group,the ALT and AST were significantly higher in the IRI group(both P<0.05).The serum levels of ALT and AST in the LPA+IRI group were significantly lower than those in the IRI group(both P<0.05).HE staining revealed that the morphology of hepatocytes was normal in the Sham group and LPA+Sham group.Pathological changes,such as liver congestion,liver cell swelling and structural abnormalities of hepatic lobule,occurred in the LPA+IRI group and IRI group.Compared with the IRI group,pathological changes were alleviated in the LPA+IRI group.RT-PCR indicated that the mRNA expression levels of TNF-α,iNOS,IL-1 and IL-6 in the LPA+IRI group were lower than those in the IRI group(all P<0.05).Immunohistochemical demonstrated that LPA partially inhibited macrophage infiltration in ischemic tissues after IRI.Conclusions YAP can significantly mitigate hepatic IRI.The mechanism is associated with the regulation of macrophage recruitment and activation.