目的 探究柚皮苷對急性肺損傷(acute lung injury,ALI)小鼠的治療作用及機(jī)制,考察柚皮苷的藥效作用是否通過腸道菌群代謝產(chǎn)物介導(dǎo)產(chǎn)生。方法 以脂多糖(lipopolysaccharide,LPS)誘導(dǎo)的ALI小鼠為動物模型,以小鼠存活率、炎癥因子表達(dá)、肺組織形態(tài)等為指標(biāo),明確柚皮苷抗ALI的藥效作用;采用Western blotting檢測小鼠肺組織中腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)/沉默信息調(diào)節(jié)因子1(silent information regulator 1,SIRT1)/核因子-κB(nuclear factor-κB,NF-κB)通路與閉鎖小帶蛋白-1(zonula occluding-1,ZO-1)、Occludin蛋白表達(dá)。將正常小鼠腸道菌液與柚皮苷共孵育,采用UPLC-Q TOF MS/MS法檢測柚皮苷經(jīng)腸道菌轉(zhuǎn)化的代謝產(chǎn)物。體外構(gòu)建LPS誘導(dǎo)的肺上皮細(xì)胞炎癥損傷模型,給予柚皮苷及其腸道代謝產(chǎn)物柚皮素與對羥基苯丙酸[3-(4′-hydroxyphenyl) propanoic acid,HPPA]干預(yù)后,采用ELISA法檢測細(xì)胞培養(yǎng)上清液炎癥因子水平,Western blotting檢測AMPK/SIRT1/NF-κB通路相關(guān)蛋白的表達(dá)。結(jié)果 與模型組比較,給予柚皮苷干預(yù)后,ALI小鼠血清和肺組織中炎癥因子表達(dá)顯著降低(P＜0.05、0.01),肺功能得到明顯改善(P＜0.05、0.01),肺組織中p-AMPK/AMPK值、SIRT1、ZO-1和Occludin蛋白表達(dá)水平顯著升高(P＜0.01),p-NF-κB/NF-κB值顯著降低(P＜0.05)。柚皮苷與小鼠腸道菌群共孵育24 h后部分被轉(zhuǎn)化為柚皮素與HPPA。在細(xì)胞炎癥模型中,100 μmol/L柚皮素、HPPA均能顯著提高細(xì)胞存活率(P＜0.01),降低細(xì)胞培養(yǎng)上清中炎癥因子水平(P＜0.05、0.01),激活A(yù)MPK/SIRT1通路(P＜0.05、0.01),降低p-NF-κB/NF-κB值(P＜0.01),而柚皮苷未表現(xiàn)出上述作用。結(jié)論 柚皮苷通過腸道菌群生物轉(zhuǎn)化產(chǎn)生的代謝物柚皮素與HPPA激活A(yù)MPK/SIRT1通路,抑制NF-κB表達(dá),從而改善LPS誘導(dǎo)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

天津市科技計(jì)劃項(xiàng)目（25JCLMJC00170，25JCLZJC00100）；國家自然科學(xué)基金重點(diǎn)項(xiàng)目（81830112）