Deng Yiyun, Zhang Zhe, Yang Hui, Wang Jing, Feng Lijuan, Su Yong, Xu Dujuan
The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
School of Pharmacy, Anhui Medical University, Hefei, Anhui, China.
Oxid Med Cell Longev. 2022 Jul 5;2022:4195699. doi: 10.1155/2022/4195699. eCollection 2022.
Mycophenolic acid (MPA) may cause gastrointestinal adverse effects by damaging the intestinal epithelial barrier, the underlying mechanisms remain elusive. Studies have demonstrated that oxidative stress caused by reactive oxygen species (ROS) is linked to tight junction (TJ) proteins and apoptosis, both of which cause abnormalities in intestinal barrier function. Mitochondria, one of the main sources of ROS and abnormally high levels of ROS are linked to mitochondrial dysfunction. The aim of this study was to investigate whether MPA induces intestinal barrier dysfunction through regulation of the mitochondrial ROS. MPA-induced intestinal injury model in Kunming mice and Caco-2 cells. The effect of MPA on Caco-2 cell viability was measured by MTT; tissue diamine oxidase and endotoxin expression were determined by ELISA; expression of total proteins of ZO-1, occludin, Bax, Bcl-2, and mitochondrial proteins of Cytochrome C and Bax was measured by Western blot; and the localization of Cytochrome C with MitoTraker was observed by immunofluorescence staining. Caco-2 cell apoptosis, ROS levels, and mitochondrial membrane potential were detected by flow cytometry, while intramitochondrial ROS levels were observed by MitoSOX fluorescence staining. The results showed that MPA increased intracellular and mitochondrial ROS production to promote oxidative stress and the antioxidant NAC effectively restored ZO-1 and occludin expressions, reduced apoptosis in intestinal epithelial cells. Furthermore, we found that low concentrations of MPA caused mitochondrial damage, induced hyperpolarization of the mitochondrial membrane potential and the translocation of Cytochrome C and Bax proteins from the cytoplasm to the mitochondria. The mitochondrial protectant SS-31 reduces intracellular and intramitochondrial ROS, upregulates TJ, and reduces apoptosis. Our studies suggest that MPA-induced intestinal barrier dysfunction and is mediated, at least in part, by impairing mitochondrial function and promoting oxidative stress.
霉酚酸（MPA）可能通过破坏肠道上皮屏障而导致胃肠道不良反应，但其潜在机制仍不清楚。研究表明，活性氧（ROS）引起的氧化应激与紧密连接（TJ）蛋白和细胞凋亡有关，这两者都会导致肠道屏障功能异常。线粒体是ROS的主要来源之一，而异常高水平的ROS与线粒体功能障碍有关。本研究的目的是探讨MPA是否通过调节线粒体ROS来诱导肠道屏障功能障碍。建立MPA诱导的昆明小鼠和Caco-2细胞肠道损伤模型。用MTT法检测MPA对Caco-2细胞活力的影响；用ELISA法测定组织二胺氧化酶和内毒素表达；用蛋白质免疫印迹法检测ZO-1、闭合蛋白、Bax、Bcl-2的总蛋白表达以及细胞色素C和Bax的线粒体蛋白表达；用免疫荧光染色法观察细胞色素C与线粒体示踪剂的定位。通过流式细胞术检测Caco-2细胞凋亡、ROS水平和线粒体膜电位，同时用MitoSOX荧光染色法观察线粒体内ROS水平。结果表明，MPA增加细胞内和线粒体内ROS的产生以促进氧化应激，抗氧化剂NAC有效恢复ZO-1和闭合蛋白的表达，减少肠道上皮细胞凋亡。此外，我们发现低浓度的MPA会导致线粒体损伤，诱导线粒体膜电位超极化以及细胞色素C和Bax蛋白从细胞质向线粒体的转位。线粒体保护剂SS-31可降低细胞内和线粒体内的ROS，上调TJ，并减少细胞凋亡。我们的研究表明，MPA诱导的肠道屏障功能障碍至少部分是由线粒体功能受损和氧化应激增加介导的。
