缺氧诱导因子1α介导单羧酸转运蛋白1表达参与短链脂肪酸对肠道缺氧保护作用的研究

doi:10.3877/cma.j.issn.1674-0793.2023.01.004

目的

明确缺氧条件下缺氧诱导因子1α（HIF-1α）对肠上皮细胞单羧酸转运蛋白1（MCT-1）的表达调控，并初步探究短链脂肪酸（SCFAs）在缺氧条件下对肠屏障的保护作用。

方法

取健康回肠组织及肠缺血坏死患者的回肠组织，进行免疫组织化学染色，检测肠上皮MCT-1表达情况。正常C57小鼠、肠上皮特异性HIF-1α敲除鼠（HIF-1α^ΔIEC）以及对照小鼠（HIF-1α^flox/flox）构建小肠缺血再灌注（I/R）损伤模型，随机分为假手术组（Sham组）、I/R组，I/R+丁酸组，取回肠末段的组织行免疫组织化学染色及肠液行SCFAs检测，透射电镜观察小鼠回肠超微结构变化。培养肠上皮细胞系Caco-2，建立缺氧模型，用Western blotting法分别检测常氧组、缺氧组、缺氧+siHIF-1α组、丁酸+缺氧组、siMCT-1+丁酸+缺氧组MCT-1、HIF-1α和肠上皮紧密连接蛋白Claudin1、Occludin的表达情况；跨上皮电阻（TER）测定评估各组肠上皮屏障功能变化。

结果

与假手术组相比，I/R组肠道内SCFAs无明显变化。缺血、缺氧均可以诱导肠上皮细胞高表达MCT-1。HIF-1α^ΔIEC小鼠较对照小鼠肠道低表达MCT-1，且对I/R损伤更为敏感。丁酸在HIF-1α、MCT-1正常表达时可以介导肠屏障保护和上调Claudin1、Occludin表达。

结论

缺氧条件下，HIF-1α介导肠上皮细胞表达MCT-1，参与SCFAs对肠屏障的保护。

关键词: 缺氧诱导因子1α, 缺血再灌注损伤, 短链脂肪酸, 单羧酸转运蛋白1, 肠屏障

Objective

To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α)on monocarboxylate transporter-1 (MCT-1) expression in intestinal epithelial cells under hypoxia, and the protective effects of short-chain fatty acids (SCFAs) on intestinal barrier under hypoxia.

Methods

Normal ileum and ischemic ileum were resected and stained by immunohistochemistry targeting MCT-1. Normal C57 mice, intestinal epithelial-specific HIF-1α^ΔIEC mice and HIF-1α^flox/flox controlled mice were randomly divided into Sham group, ischemia reperfusion (I/R) group, I/R plus butyrate group. The distal ileum tissues were collected for immunohistochemical staining and the intestinal fluid in the intestinal lumen was detected for SCFAs. Intestinal epithelial cell line Caco-2 was cultured and hypoxia model was established. The cells were divided into control group, hypoxia group, hypoxia+siHIF-1α group, hypoxia+butyrate group and siMCT-1+hypoxia+butyrate group. Expression of MCT-1, HIF-1α and tight junction protein were detected by Western blotting. Changes in intestinal epithelial barrier function were assessed by transepithelial electrical resistance (TER).

Results

Compared with Sham group, intestinal SCFAs in I/R group did not change significantly. Both ischemia and hypoxia could induce high expression of MCT-1 in intestinal epithelial cells. HIF-1α^ΔIEC mice had lower intestinal MCT-1 expression than the control group and were more sensitive to I/R injury. Butyrate mediated intestinal barrier protection and up-regulated tight junction proteins Claudin1 and Occludin when HIF-1α and MCT-1 were normally expressed.

Conclusion

HIF-1α mediates the expression of MCT-1 in intestinal epithelial cells during hypoxia and participates in the protection of intestinal barrier by SCFAs.

Key words: Hypoxia-inducible factor-1α, Ischemia reperfusion injury, Short-chain fatty acids, Monocarboxylate transporter-1, Intestinal barrier

图1 假手术组与缺血再灌注组回肠肠腔内短链脂肪酸质量摩尔浓度检测　A为乙酸；B为丙酸；C为丁酸；D为戊酸

图2 人体缺血坏死肠道组织和小鼠缺血再灌注（I/R）肠道组织MCT-1蛋白表达情况　健康（A）和缺血坏死（B）回肠表达MCT-1情况和免疫组织化学定量结果（×100，C）；假手术组（Sham组，D）和缺血再灌注组（I/R组，E）回肠表达MCT-1情况和免疫组织化学定量结果（×100，F）

图3 HIF-1α介导MCT-1在体内、体外表达情况　阴性对照HIF-1α^flox/flox小鼠（A）和肠上皮特异性HIF-1α敲除鼠HIF-1α^ΔIEC小鼠（B）正常条件下回肠表达MCT-1情况和免疫组织化学定量结果（×200，C）；Caco-2细胞在常氧、缺氧以及缺氧+siHIF-1α条件下HIF-1α和MCT-1表达情况（D）

图4 丁酸对肠道、肠上皮屏障缺血缺氧的保护情况HIF-1α^flox/flox、HIF-1α^ΔIEC小鼠回肠缺血再灌注苏木精-伊红染色（×40，A、B）和透射电镜观察紧密连接蛋白情况（×25 000，E、F）；HIF-1α^flox/flox、HIF-1α^ΔIEC小鼠丁酸预处理后回肠缺血再灌注苏木精-伊红染色（×40，C、D）和透射电镜观察紧密连接蛋白情况（×25 000，G、H）；Caco-2细胞在常氧、缺氧、缺氧+丁酸、缺氧+丁酸+siMCT-1条件下紧密连接蛋白Claudin1、Occludin表达情况（I）和跨上皮电阻测定值（J）

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Hypoxia-inducible factor-1α mediating the expression of monocarboxylate transporter-1 and participating in the protective effect of short-chain fatty acids on intestinal hypoxia

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Hypoxia-inducible factor-1α mediating the expression of monocarboxylate transporter-1 and participating in the protective effect of short-chain fatty acids on intestinal hypoxia