氧化三甲胺通过PI3K/AKT/SREBP1通路诱导肾纤维化

陈静; 黄英辉; 赵景宏

doi:10.12182/20231160106

氧化三甲胺通过PI3K/AKT/SREBP1通路诱导肾纤维化

Trimethylamine N-Oxide Induces Renal Fibrosis Through the PI3K/AKT/SREBP1 Pathway

摘要

摘要:
目的探讨尿毒症毒素氧化三甲胺（trimethylamine N-oxide, TMAO）在肾脏纤维化中的作用及其机制。
方法将20只雄性BALB/c小鼠随机平均分为对照组和TMAO组，每组10只。对照组腹腔注射生理盐水，TMAO组腹腔注射含TMAO的生理盐水〔20 mg/(kg·d) 〕，每日1次，持续8周；使用HE染色与Masson染色法观察分析小鼠肾脏切片病理及纤维化水平；采用免疫组化法检测肾脏组织中α-肌动蛋白（alpha smooth muscle actin, α-SMA）、重组人纤维连接蛋白片段（recombinant human fibronectin fragment, Fibronectin）、胆固醇调节元件结合蛋白-1（sterol-regulatory element binding protein 1, SREBP1）水平；采用Western blot检测肾脏组织中α-SMA、SREBP1、磷脂酰肌醇3激酶（phosphatidylinositol 3 kinase, PI3K）、磷酸化PI3K（phospho-PI3K, p-PI3K）、蛋白激酶B（protein kinase B, PKB，又称AKT）和磷酸化AKT（phospho-AKT, p-AKT）蛋白水平。分别用SREBP1 siRNA和PI3K/AKT抑制剂组处理HK2细胞，检测对TMAO效应的逆转作用。
结果动物实验显示，与对照组相比，腹腔注射TMAO后小鼠出现肾脏组织病理损伤和纤维化，纤维化标志物α-SMA、Fibronectin表达升高（P均<0.05）。相对于对照组，TMAO处理组小鼠肾脏中SREBP1表达升高（P<0.05），PI3K磷酸化比值、AKT磷酸化比值也发生了上调（P均<0.05）。细胞实验验证了上述结果。利用siRNA干扰肾小管上皮细胞中SREBP1表达后，纤维化指标蛋白表达下降（P<0.05)；利用PI3K-AKT通路抑制剂LY294002孵育HK2细胞后，TMAO导致的SREBP1高表达被抑制（P<0.05）。
结论 TMAO可能通过促进PI3K/AKT/SREBP1通路而诱导肾脏纤维化。

Abstract:
Objective To investigate the role and mechanism of trimethylamine N-oxide (TMAO), a uremic toxin, in renal fibrosis.
Methods A total of 20 male BALB/c mice were randomly and evenly assigned to a Control group and a TMAO group. Mice in the Control group received intraperitoneal injection of normal saline, while mice in the TMAO group received intraperitoneal injection of TMAO (20 mg/kg·d). The injection was given once a day for 8 weeks. Histopathology and fibrosis of kidney were observed by H&E staining and Masson staining. Immunohistochemistry was performed to determine the levels of alpha smooth muscle actin (α-SMA), recombinant human fibronectin fragment (Fibronectin), and sterol-regulatory element binding protein 1 (SREBP1). Western blot was performed to determine α-SMA, SREBP1, phosphatidylinositol 3 kinase (PI3K), phospho-phosphatidylinositol 3 kinase (p-PI3K), protein kinase B (PKB, also known as AKT), and phospho-AKT (p-AKT) protein levels. HK2 cells were treated with SREBP1 small interfering RNA (siRNA) and PI3K/AKT inhibitor, respectively, and the reversal of the effects of TMAO was examined.
Results Animal experiments showed that, compared with the Control group, the mice treated with TMAO experienced pathological damage and fibrosis of the kidney tissue and the expression levels of fibrosis markers, α-SMA and Fibronectin, in the kidney were increased (all P<0.05). According to the findings from further investigation, the TMAO-treatment group showed increased expression of SREBP1 and an up-regulation of PI3K phosphorylation ratio and AKT phosphorylation ratio compared with those of the Control group (all P<0.05). Cell experiments produced results similar to those of the animal experiment. After siRNA interference with SREBP1 expression, the expression levels of fibrosis marker proteins decreased (P<0.05). Besides, the high expression of SREBP1 caused by TMAO was inhibited after HK2 cells were incubated with LY294002, a PI3K-AKT pathway inhibitor (P<0.05).
Conclusion TMAO may induce renal fibrosis by promoting the PI3K/AKT/SREBP1 pathway.

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