激活α7nAChR抗肝脏缺血再灌注损伤作用及其机制

The principal neurotransmitter released by the vagus nerve can activate α7 nicotinic acetylcholine receptor (α7nAChR). Activation of α7nAChR inhibits hepatocyte injury under various pathological conditions, including hepatic ischemia-reperfusion (HIR) injury. Kupffer cells (KCs) are likely to be a major determinant influencing HIR injury. We hypothesize that hepatic vagus nerve attenuates ischemia reperfusion induced injury in mouse liver via α7nAChR on KCs. HIR models are prepared by using hepatic vagotomy,KCs eliminated and α7nAChR-/- mice, and primary KCs and hepatocytes are isolated. This work is designed to demonstrate: 1) activation of α7nAChR attenuates HIR injury; 2) α7nAChR-/- aggravates HIR injury; 3) hepatic vagotomy exacerbates HIR injury, which is prevented by pretreatment with selective α7nAChR agonist; 4) removal of KCs diminishes HIR injury; 5) possible mechanisms in HIR injury mediated by α7nAChR on KCs. The expected results are: 1) the hepatic vagus nerve plays an important role during the development of HIR injury through α7nAChR on KCs; 2) α7nAChR on KCs is a potential pharmacological target in resisting against HIR injury. HIR injury is relevant clinically in hepatic surgery and liver transplanation, our results may therefore provide a new strategy for prevention of liver injury during ischemia reperfusion.

迷走神经递质乙酰胆碱(ACh)能够激活α7尼古丁乙酰胆碱受体(α7nAChR)；激活α7nAChR能抑制多因素所致的肝损伤；枯否细胞功能影响肝缺血再灌注 (HIR) 损伤。我们假设"肝迷走神经递质ACh经激活枯否细胞α7nAChR减轻HIR损伤"。利用切除肝迷走神经小鼠、清除枯否细胞小鼠、α7nAChR-/- 小鼠制作HIR模型，分离培养小鼠枯否细胞和肝细胞，拟从以下5个方面进行验证：1)激活α7nAChR减轻HIR损伤；2) α7nAChR-/-加重HIR损伤；3) 肝迷走神经切除加重HIR损伤，预激活α7nAChR阻止这一损伤；4) 清除枯否细胞减轻HIR损伤；5) 枯否细胞α7nAChR介导抗HIR损伤可能机制。该项目试图明确肝迷走神经在抗HIR损伤的作用，并探索枯否细胞α7nAChR作为抗HIR损伤靶点的可能性，从而为临床HIR损伤的预防提供新思路。

目的：肝脏缺血再灌注损伤是肝外科手术的并发症之一。大约有50%的肝细胞在再灌注24小时内经历凋亡。迷走神经释放的递质乙酰胆碱能够激活巨噬细胞(肝脏的巨噬细胞即为枯否氏细胞)α7尼古丁乙酰胆碱受体(α7nAChR)。枯否氏细胞(KCs)功能影响肝脏缺血再灌注损伤。我们假设：肝脏迷走神经通过激活KCs表面的α7nAChR，能够减轻肝脏缺血再灌注所致的损伤。.方法：利用肝脏迷走神经切除和完整的C57小鼠、肝脏巨噬细胞清除和保留的C57小鼠、以及α7nAChR基因敲除和其野生型小鼠，制作肝脏缺血再灌注模型；细胞水平则利用原代分离的KCs和肝细胞进行缺氧/复氧单独培养，或者共培养。对肝脏损伤指标、肝细胞凋亡情况、ROS和可溶性CD163等指标进行观察和测定。.结果：和对照组比较，肝脏迷走神经切除和α7nAChR基因敲除均可使缺血再灌注诱导的血清ALT水平、肝脏Caspase-3和8活性显著升高；激活α7nAChR可在野生型小鼠减轻这些变化，在该基因敲除小鼠则无效；此外，激活α7nAChR可减轻肝脏迷走神经切除小鼠因缺血再灌注所致的肝脏损伤和肝细胞凋亡。在体外，激活α7nAChR能够减轻缺氧/复氧导致的肝细胞和KCs共培养体系中的肝细胞凋亡；激活α7nAChR和过氧化氢酶均能够抑制缺氧/复氧导致的KCs产生过量ROS和H2O2；α7nAChR激动剂和过氧化氢酶预处理的KCs上清液，能够减轻缺氧/复氧导致的肝细胞凋亡。KCs清除降低缺血再灌注所致的小鼠肝组织H2O2含量。无论在缺血再灌注小鼠，还是在缺氧/复氧的KCs，激活α7nAChR均导致可溶性CD163显著下降。.结论：迷走神经递质乙酰胆碱通过激活肝脏KCs的α7nAChR，能够减轻肝脏缺血再灌注损伤，其机制与抑制过量ROS产生相关。