目的 探究柚皮苷对急性肺损伤(acute lung injury,ALI)小鼠的治疗作用及机制,考察柚皮苷的药效作用是否通过肠道菌群代谢产物介导产生。方法 以脂多糖(lipopolysaccharide,LPS)诱导的ALI小鼠为动物模型,以小鼠存活率、炎症因子表达、肺组织形态等为指标,明确柚皮苷抗ALI的药效作用;采用Western blotting检测小鼠肺组织中腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)/沉默信息调节因子1(silent information regulator 1,SIRT1)/核因子-κB(nuclear factor-κB,NF-κB)通路与闭锁小带蛋白-1(zonula occluding-1,ZO-1)、Occludin蛋白表达。将正常小鼠肠道菌液与柚皮苷共孵育,采用UPLC-Q TOF MS/MS法检测柚皮苷经肠道菌转化的代谢产物。体外构建LPS诱导的肺上皮细胞炎症损伤模型,给予柚皮苷及其肠道代谢产物柚皮素与对羟基苯丙酸[3-(4′-hydroxyphenyl) propanoic acid,HPPA]干预后,采用ELISA法检测细胞培养上清液炎症因子水平,Western blotting检测AMPK/SIRT1/NF-κB通路相关蛋白的表达。结果 与模型组比较,给予柚皮苷干预后,ALI小鼠血清和肺组织中炎症因子表达显著降低(P＜0.05、0.01),肺功能得到明显改善(P＜0.05、0.01),肺组织中p-AMPK/AMPK值、SIRT1、ZO-1和Occludin蛋白表达水平显著升高(P＜0.01),p-NF-κB/NF-κB值显著降低(P＜0.05)。柚皮苷与小鼠肠道菌群共孵育24 h后部分被转化为柚皮素与HPPA。在细胞炎症模型中,100 μmol/L柚皮素、HPPA均能显著提高细胞存活率(P＜0.01),降低细胞培养上清中炎症因子水平(P＜0.05、0.01),激活AMPK/SIRT1通路(P＜0.05、0.01),降低p-NF-κB/NF-κB值(P＜0.01),而柚皮苷未表现出上述作用。结论 柚皮苷通过肠道菌群生物转化产生的代谢物柚皮素与HPPA激活AMPK/SIRT1通路,抑制NF-κB表达,从而改善LPS诱导的小鼠ALI。

Objective To investigate the therapeutic effect and mechanism of naringin (NG) on acute lung injury (ALI) in mice, as well as to determine whether the pharmacological action of NG is mediated by its intestinal flora-derived metabolites. Methods Lipopolysaccharide (LPS)-induced ALI mice was used as the animal model, the therapeutic effect of NG against ALI was determined by survival rate of mice, inflammatory factors expressions and lung tissue morphology. Western blotting was used to detect the protein expressions of adenosine monophosphate-activated protein kinase (AMPK)/silent information regulator 1 (SIRT1)/nuclear factor-κB (NF-κB) pathway and zonula occluding-1 (ZO-1) and Occludin in lung tissue of mice. Normal mouse intestinal flora was co-incubated with NG, and the metabolic products of NG transformed by intestinal flora were detected by UPLC-Q TOF MS/MS. An LPS-induced lung epithelial cell inflammatory injury model was constructed in vitro, NG and its intestinal metabolic products naringenin (NE) and 3-(4′-hydroxyphenyl) propanoic acid (HPPA) were given for intervention, the levels of inflammatory factors in cell culture supernatant were detected by ELISA, and the protein expressions related to AMPK/SIRT1/NF-κB pathway were detected by Western blotting. Results Compared with model group, after intervention with NG, the expressions of inflammatory factors in serum and lung tissue of ALI mice were significantly reduced (P < 0.05, 0.01), lung function was significantly improved (P < 0.05, 0.01), the value of p-AMPK/AMPK and protein expression levels of SIRT1, ZO-1 and Occludin were significantly increased (P < 0.01), the value of p-NF-κB/NF-κB was significantly decreased (P < 0.05). After co-incubation with mouse intestinal flora for 24 h, part of NG was converted into NE and HPPA. In the cell inflammation model, 100 μmol/L NE and HPPA could significantly increase cell survival rate (P < 0.01), reduce the levels of inflammatory factors in cell culture supernatant (P < 0.05, 0.01), activate AMPK/SIRT1 pathway (P < 0.05, 0.01), and decrease the value of p-NF-κB/NF-κB (P < 0.01), while NG did not show the above effects. Conclusion Naringin activates AMPK/SIRT1 pathway and inhibits NF-κB through metabolites NE and HPPA produced by biotransformation of intestinal flora, thereby improving LPS-induced ALI in mice.

R285.5

天津市科技计划项目（25JCLMJC00170，25JCLZJC00100）；国家自然科学基金重点项目（81830112）