利多卡因调节Hippo-YAP信号通路对原位肝移植大鼠缺血再灌注损伤的影响
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新疆医科大学第一附属医院

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国家自然科学基金委员会地区科学基金项目


Effect of lidocaine regulating Hippo-YAP signal pathway on ischemia-reperfusion injury in orthotopic liver transplantation rats
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The First Affiliated Hospital of Xinjiang Medical University

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    摘要:

    目的:探究利多卡因(LID)对原位肝移植(OLT)大鼠缺血再灌注损伤的影响,并分析其作用机制。方法:将60只大鼠随机分为对照组、模型组、LID低剂量组、LID中剂量组、LID高剂量组、维替泊芬(Verteporfin)组,每组10只,除对照组外,其余大鼠构建OLT模型。苏木素-伊红(HE)染色观察肝组织病理变化,全自动生化分析仪检测血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、总胆红素(TBIL)、乳酸脱氢酶(LDH)活性,酶联免疫吸附试验(ELISA)检测肝组织炎症因子肿瘤坏死因子-α(TNF-α)、白介素(IL)-6、IL-1β和IL-10水平,荧光探针法检测活性氧(ROS),硫代巴比妥酸显色法检测丙二醛(MDA),氮蓝四唑显色法检测超氧化物歧化酶(SOD),分光光度计法检测谷胱甘肽过氧化物酶(GSH-Px),原位末端标记法(TUNEL)检测肝组织细胞凋亡,蛋白印迹法(WB)检测Hippo-YAP信号通路相关蛋白哺乳动物STE20样蛋白激酶(MST1)、磷酸化(p)-MST1、大肿瘤抑制因子1(LATS1)、p-LATS1、Yes相关蛋白(YAP)、p-YAP以及凋亡相关蛋白B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达。结果:与对照组相比,模型组大鼠肝组织出现损伤,肝细胞坏死且大量炎性细胞浸润,细胞凋亡率、血清AST、ALT、TBIL、LDH活性、肝组织TNF-α、IL-6、IL-1β、MDA、ROS、Bax水平显著升高,肝组织IL-10、SOD、GSH-Px及Bcl-2、p-MST1/MST1、p-LATS1/LATS1、p-YAP/YAP蛋白表达水平显著降低(P<0.05);与模型组相比,LID低、中、高剂量组肝组织损伤减轻,细胞凋亡率、血清AST、ALT、TBIL、LDH活性、肝组织TNF-α、IL-6、IL-1β、MDA、ROS、Bax水平显著降低,肝组织IL-10、SOD、GSH-Px及Bcl-2、p-MST1/MST1、p-LATS1/LATS1、p-YAP/YAP蛋白表达水平显著升高(P<0.05);Hippo-YAP信号通路抑制剂Verteporfin逆转了LID对OLT大鼠缺血再灌注损伤的改善作用(P<0.05)。结论:LID可能通过激活Hippo-YAP通路,减少炎症反应、氧化应激和肝细胞凋亡,对OLT大鼠肝缺血再灌注损伤发挥改善作用。

    Abstract:

    Objective: To explore the effect of lidocaine (LID) on ischemia-reperfusion injury in orthotopic liver transplantation (OLT) rats and to analyze its mechanism of action. Methods: 60 rats were randomly divided into control group, model group, low-dose LID group, medium-dose LID group, high-dose LID group, and Verteporfin group, with 10 rats in each group, except for the control group, and other rats were used to construct OLT models. The pathological changes in liver tissue was observed by hematoxylin-eosin (HE) staining, the serum aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin (TBIL) and lactate dehydrogenase (LDH) activities were detected by automatic biochemical analyzer, the liver tissue inflammatory factor tumor necrosis factor-α (TNF-α), interleukin (IL) -6, IL-1β and IL-10 levels were detected by enzyme-linked immunosorbent assay (ELISA), the reactive oxygen species (ROS) was detected by fluorescence probe method, the malondialdehyde (MDA) was detected by thiobarbituric acid colorimetric method, the superoxide dismutase (SOD) was detected by nitrogen blue tetrazole colorimetry, the glutathione peroxidase (GSH-Px) was detected by spectrophotometer method, the apoptosis of liver histiocyte was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) , and the expression of mammalian STE20 like protein kinase (MST1), phosphorylation (p) - MST1, large tumor suppressor factor 1 (LATS1), p-LATS1, Yes associated protein (YAP), p-YAP, as well as apoptosis related proteins B-cell lymphoma 2 (Bcl-2) and Bcl-2 related X protein (Bax) were detected by western blotting (WB). Results: Compared with control group, the liver tissue in model group rats showed injury, liver cells necrosis and a large number of inflammatory cells infiltration, the cell apoptosis rate, serum AST, ALT, TBIL, LDH activity, liver tissue TNF-α, IL-6, IL-1β, MDA, ROS, and Bax levels significantly increased, the liver tissue IL-10, SOD, GSH-Px, Bcl-2, p-MST1/MST1, p-LATS1/LATS1 and p-YAP/YAP proteins expression levels were significantly reduced (P<0.05). Compared with model group, the liver tissue injury was reduced in low-dose LID group, medium-dose LID group, and high-dose LID group, the cell apoptosis rate, serum AST, ALT, TBIL, LDH activity, liver tissue TNF-α, IL-6, IL-1β, MDA, ROS and Bax levels were significantly reduced, the liver tissue IL-10, SOD, GSH-Px, Bcl-2, p-MST1/MST1, p-LATS1/LATS1, and p-YAP/YAP proteins expression levels were significantly increased (P<0.05). Hippo-YAP signaling pathway inhibitor Verteporfin reversed the improvement effect of LID on ischemia-reperfusion injury in OLT rats (P<0.05). Conclusion: LID may activate the Hippo-YAP pathway, which reduce inflammatory response, oxidative stress, and liver cell apoptosis, and improve liver ischemia-reperfusion injury in OLT rats.

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  • 收稿日期:2023-07-05
  • 最后修改日期:2023-11-07
  • 录用日期:2024-01-26
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