南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (04): 394-.doi: 10.12122/j.issn.1673-4254.2019.04.03

• • 上一篇    下一篇

三七总皂苷对大鼠肝移植排斥反应的影响及相关机制

张先兵，李勋，熊平，易传超，陈曦   

  • 出版日期:2019-04-20 发布日期:2019-04-20

Effects of Panax notoginseng saponins on liver graft rejection in rats and the mechanisms

  • Online:2019-04-20 Published:2019-04-20

HTML

PDF

1671

摘要： 目的探讨三七总皂苷（PNS）对Kupffer细胞（KCs）功能状态、肝移植术后免疫环境的影响，及其相关机制。方法分离大 鼠KCs，吞墨台盼蓝鉴定KCs活性，细胞实验分LPS（-）组、LPS（+）+ PNS（0 μmol）组、LPS（+）+ PNS（10 μmol）组，、LPS（+）+ PNS（20 μmol）组，Western blot、ELISA检测各组细胞及上清液炎症因子和氧化应激产物的表达，免疫荧光检测KCs CD206的表 达，Western blot 检测NF-κB和Keap1-Nrf2-ARE通路蛋白表达。建立大鼠移植模型，分为SHAM组、LT（PBS处理）组和PNS （200 mg/kg）组，术后检测各组肝功能、炎症因子、肝组织病例、凋亡情况，并观察大鼠生存时间。结果随着PNS浓度的增加， KCs分泌促炎因子和氧化应激产物MDA水平逐渐降低，明显低于PNS（0 μmol）组，而抗炎因子IL-10以及抗氧化应激产物SOD 水平逐渐升高，明显高于PNS（0 μmol）组，且差异具有统计学意义（P<0.05）。免疫荧光示PNS提高了KCs表型CD206的表达。 同时，PNS减少了KCs IRAK4、p-IKKα、p-IκBα、p-p65、Keap1蛋白表达，而Nrf2、ARE蛋白表达水平逐渐升高，且与低PNS浓度 组比具有统计学上的差异（P<0.05）。与LT组比，PNS组的大鼠肝移植后肝功能明显改善，促炎因子表达水平降低，肝细胞凋亡 减少，肝组织急性排斥病理改变减轻，大鼠生存时间明显增高（P<0.05）。另外，大鼠注射GdCl3封闭KCs功能发现，PNS组出现 严重的急性排斥反应，与LT组比较无统计学上差异（P>0.05）。结论PNS能够通过抑制NF-κB和Keap1-Nrf2-ARE通路减少活 化KCs的炎症和氧化应激反应，促进KCs向免疫耐受的M2型极化，提高大鼠移植术后的生存时间。

Abstract: Objective To investigate the effects of Panax notoginseng saponins (PNS) on the functional status of Kupffer cells (KCs) and immune environment after liver transplantation and explore the possible mechanisms. Methods KCs were isolated from rats and assessed for phagocytic activity and viability using ink and Trypan blue staining. The cells were exposed to lipopolysaccharide (LPS) alone or in combination with PNS treatment at 0, 10 or 20 μmol/L. The expressions of the inflammatory factors and the oxidative stress products in the cells and the supernatant were assayed with Western blotting and ELISA; the expression of CD206 was detected using immunofluorescence assay, and the expressions of NF-κB and Keap1-Nrf2-ARE pathway proteins were detected using Western blotting. We established an orthotopic liver transplantation (LT) model in rats and assessed the effect of 200 mg/kg PNS on the graft function, inflammatory factors, pathology of the liver tissue, hepatocyte apoptosis and survival time of the rats in comparison with those in rats receiving a sham operation or PBS treatment following LT. Results Treatment with PNS significantly lowered the levels of inflammatory factors and oxidative stress products and increased the levels of interleukin-10 (IL-10) and SOD in a concentration-dependent manner in the KCs (P< 0.05). Immunofluorescence assay showed that PNS treatment obviously increased the expression of CD206 in the KCs. PNS treatment also significantly reduced the expressions of IRAK4, p-IKKα, p-IκBα, p-p65 and Keap1 proteins and increased the expression levels of Nrf2 and ARE proteins in the KCs (P<0.05). In the rat models of LT, PNS treatment significantly improved the liver graft function, lowered the expression of the pro-inflammatory factors, and reduced hepatocyte apoptosis as compared with PBS treatment. PNS treatment obviously alleviated pathological changes in the liver graft and significantly prolonged the survival time of the rats following LT (P<0.05). In addition, injection of GdCl3 to block KC function resulted in severe acute graft rejection in the rats regardless of PNS treatment (P>0.05). Conclusion PNS can reduce inflammatory response and oxidative stress in activated KCs by inhibiting NF-κB and Keap1-Nrf2-ARE pathways and promote the polarization of KCs into M2 phenotype to prolong the survival time of rats after LT.