陈斌 彭杰 张涛 朱文芳 张茜茜 丁秀丽 黎运芳

〔摘要〕 目的 研究清溫并用法对慢加急性肝衰竭（acute-on-chronic liver failure， ACLF）大鼠结肠组织Treg/Th17相关型细胞因子表达的影响，探讨其抗ACLF肠源性内毒素血症（intestinal endotoxemia， IETM）的可能调控机制。方法 140只SD大鼠随机分为正常组、模型组、清法组、温法组及清温并用法组。牛血清白蛋白致敏法建立免疫性肝纤维化大鼠模型，腹腔注射D-氨基半乳糖+脂多糖急性攻击建立ACLF大鼠模型，各组予以相应干预，观察24 h内各组大鼠外周血内毒素、外周血Treg/Th17细胞比值、结肠组织Treg/Th17型细胞因子变化。结果 与正常组比较，模型组大鼠在各时间点内毒素明显升高（P<0.01）、Treg/Th17型细胞因子明显升高（P<0.01）、Treg/Th17比值明显降低（P<0.01）;与模型组比较，清法、温法、清温并用法组在1、12、24 h各个时间点内毒素、Treg/Th17相关细胞因子表达均降低（P<0.05），以清温并用法组下降最显著（分别与清法组、温法组比较，P<0.05）;各组与模型组比较，仅清温并用法组Treg/Th17比值明显升高（P<0.05）。结论 清温并用法抗肝衰竭IETM的机制可能与调节结肠组织Treg/Th17型细胞因子失衡，促使Treg/Th17细胞恢复平衡有关。

〔关键词〕 清温并用法;慢加急性肝衰竭;Treg/Th17型细胞因子

〔中图分类号〕R285.5;R575.4       〔文献标志码〕A       〔文章编号〕doi：10.3969/j.issn.1674-070X.2019.05.003

〔Abstract〕 Objective To investigate the effects of clearing and warming simultaneously on the expression of Treg/Th17 associated cytokines in colon tissue of rats with acute and chronic liver failure （ACLF）， and to explore its possible regulation mechanism of ACLF against intestinal endotoxemia （IETM）. Methods A total of 140 SD rats were randomly divided into a normal group， a model group， a clearing group， a warming group， and a clearing and warming simultaneously group. The rats model of immune hepatic fibrosis were established by sensitization with bovine serum albumin， and then ACLF rats were induced by intraperitoneal injection of D-galactose and lipopolysaccharide （LPS）. The changes of endotoxin in peripheral blood， ratio of Treg/Th17 cells in peripheral blood and Treg/Th17 cytokines in colonic tissue were observed within 24 hours. Results Compared with the normal group， the toxins in the model group were significantly increased at each time point （P<0.01）， the Treg/Th17 cytokines were significantly increased （P<0.01）， and the Treg/Th17 ratio was significantly lower （P<0.01）. Compared with the model group， the expression of toxin and Treg/Th17-related cytokines were decreased in the clearing group， the warming group， the clearing and warming simultaneously group at 1 h， 12 h， and 24 h （P<0.05）. The decrease in the clearing and warming simultaneously group was the most significant （compared with the clearing group， the warming group， P<0.05）. Compared with the model group， the ratio of Treg/Th17 was significantly increased in the clearing and warming simultaneously group than in the model group （P<0.05） Conclusion The mechanism of treating acute-on-chronic liver failure IETM in using clearing and warming simultaneously is related to the regulation of Treg/Th17 cytokine in colonic tissue and promoting the balance of Treg/Th17 cells

〔Keywords〕 clearing and warming simultaneously; acute-on-chronic liver failure; Treg/Th17 cytokines

慢加急性肝衰竭（acute-on-chronic liver failure， ACLF）是在慢性肝病基础上出现的急性肝功能失代偿[1]，其中、后期病理机制以肠源性内毒素血症（intestinal endotoxemia， IETM）为主，发生率高达93.3%～100%[2]，是引起肝衰竭患者病情恶化和死亡的重要原因。近年来，Treg/Th17细胞成为炎症反应调控研究的热点[3]，研究显示肝衰竭患者Treg/Th17细胞频数及相关细胞因子水平明显升高，表明终末期肝病中存在Treg/Th17细胞比例及其炎症因子失衡，且对病情的进展发挥重要作用[4]。课题组长期临床实践及前期研究证实清温并用法对肝衰竭肠源性内毒素血症有效[5-6]。本实验以Treg/Th17细胞比值及相关细胞因子为主要研究指标，探讨清温并用法减轻慢加急性肝衰竭IETM炎症反应的可能免疫学机制。

1 材料与方法

1.1  动物

140只健康雄性SD大鼠，体质量（140±15） g，所有大鼠皆从湖南斯莱克景达实验动物有限公司购得，许可证号：SCXK（湘）2016-0002。……