内源性小分子肽N -乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸调控库普弗细胞功能影响慢性肝损伤修复的作用

Chronic liver injury leads to pathological liver repair which is characterized by excessive extracellular matrix（ECM） deposition. Currently Kupffer cells were recognized as the essential mediators of excessive ECM deposition. N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide normally present in the plasma and organs of mammalian. Recent studies showed a preventive effect of AcSDKP on excessive ECM depositoin in carbon tetrachloride induced chronic liver injury, however, the molecular and cellular mechanisms remains to be elucidated. Based on these background and experimental findings, we hypothesize that endogenous AcSDKP modulates Kupffer cells function playing a key role in regulating liver repair in chronically injured liver. To test this hypothesis, a rat model of chronic liver injury induced by bile duct ligation and cultured Kupffer cells will be used. Changes in endogenous level of and regulating factors for AcSDKP in cholestatic liver injury will be studied. Impacts of preserved AcSDKP on ECM deposition, biological functions of Kupffer cells and liver repair will be investigated. The direct effects of AcSDKP on cultured Kupffer cells will be examined. This project is aimed to uncover the essential effect of AcSDP on regulation of Kupffer cell function and ECM deposition during liver repair in chronically injuried liver. Our finding will provide further insight into understanding of mechamism in modulation of Kupffer cells function and ECM deposition by AcSDKP, and experimental finding for AcSDKP as a promising antifibrogenic remedy.

慢性肝损伤病理性修复特征之一是细胞外基质（ECM）过度沉积，目前认为该过程由库普弗细胞介导。N-乙酰-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）是天然存在于哺乳动物体内的四肽，既往研究发现维持肝内AcSDKP生理浓度可预防大鼠四氯化碳肝损伤时ECM过度沉积，但细胞分子机制有待阐明。结合上述背景与工作基础，我们提出"内源性AcSDKP调控库普弗细胞功能影响ECM沉积和慢性肝损伤修复"假说，并将以大鼠胆管结扎慢性肝损伤模型为研究对象，结合库普弗细胞培养，观察胆汁淤积慢性肝损伤时肝内AcSDKP调控变化，维持肝内AcSDKP浓度对ECM沉积、库普弗细胞功能和肝修复影响，及AcSDKP对培养库普弗细胞作用。目的在于明确AcSDKP调控库普弗细胞功能影响慢性肝损伤修复ECM过度沉积作用及机制，为ECM沉积调控机制认识和AcSDKP成为抗肝纤维化药物提供进一步实验依据。

本项目研究内源性小分子肽N -乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）调控库普弗细胞功能影响慢性肝损伤修复的作用。成功构建大鼠胆总管结扎肝纤维化模型，模型组肝组织AcSDKP表达水平显著降低，约为正常组的30%-40%左右。外源性AcSDKP干预可以显著改善肝纤维化情况，减轻炎症细胞浸润；和模型组相比，AcSDKP治疗组大鼠血清中AST、HA、PCIII及总胆红素水平均明显降低，肝组织中羟脯氨酸含量也较模型组显著下降； CD45阳性细胞和肥大细胞数目均有显著减少，α-SMA表达明显低于模型组。AcSDKP对小鼠RAW264.7巨噬细胞系的增殖没有影响，但可以显著抑制由脂多糖（LPS）诱导的RAW264.7巨噬细胞分泌炎症因子TNF-α、IL-1β和IL-6。LPS刺激RAW264.7巨噬细胞后细胞的吞噬活性和趋化活性较正常组显著增加，而这一作用在给予AcSDKP后显著降低。AcSDKP在10nmol/L和100nmol/L浓度时可以显著降低由LPS引起的RAW264.7细胞内ROS的水平的升高。LPS处理激活IκB/NF-κB信号通路，降低AMPK磷酸化水平，而AcSDKP可以有效阻断LPS的作用，表现为AMPK磷酸化水平的升高和IκB/NF-κB信号通路的抑制。应用AMPK特异性抑制剂compound C 和特异性小干扰RNA阻断AMPK信号分子验证了AcSDKP是通过或者部分通过AMPK/IκB/NF-κB信号通路来发挥对RAW264.7细胞作用的。本研究证实外源性AcSDKP可以预防胆总管结扎大鼠肝纤维化，这一作用与降低α-SMA表达和抑制HSC活化有关；同时，AcSDKP可显著抑制巨噬细胞活化，表现为降RAW264.7巨噬细胞炎症因子分泌，抑制其吞噬和趋化活性和减少细胞内ROS的水平，而这一作与升高AMPK磷酸化水平以及阻断IκB/NF-κB信号通路有关。AcSDKP可能成为抑制巨噬细胞诱导的过度炎症的肝脏疾病的重要内源性因子，如肝纤维化和脂肪肝。