目的 基于 Tribbles 同源蛋白 3（TRIB3）探讨大黄素对小鼠肾脏的毒性效应及潜在分子机制。方法 将 ICR 小鼠随机分为对照组、大黄素低剂量组、大黄素高剂量组，每组 6 只。大黄素低、高剂量组分别以 20 、 40 mg·kg-1剂量腹腔注射大黄素，连续 4 周。采用终浓度 10、20、40、80、100 µmol·L-1大黄素对小鼠肾小管 上皮细胞（TCMK-1）干预 24、48 h。采用苏木精-伊红（HE）、马松（Masson）、高碘酸-希夫（PAS）、天狼星红 （Sirius Red）染色法观察肾脏组织病理形态学变化；检测肾脏组织丙二醛（MDA）、还原型谷胱甘肽（GSH）水平； CCK-8 法检测 TCMK-1 细胞存活率；2'，7'-二氯二氢荧光素-二乙酸酯（DCFH-DA）及二氢乙锭（DHE）荧光探针 法检测细胞内的活性氧（ROS）水平；RT-qPCR 法检测肾脏组织及 TCMK-1 细胞中 TRIB3、α-平滑肌肌 动蛋白（α-SMA）mRNA 表达水平；Western Blot 法检测 TCMK-1 细胞中 TRIB3 蛋白表达水平；免疫组化法测定 肾脏组织中 4-羟基壬烯醛（4-HNE）、α-SMA、肾损伤分子 1（KIM-1）、TRIB3 的表达水平。结果 （1）与对照组 比较，大黄素低、高剂量组小鼠的肾脏明显萎缩，肾脏指数显著降低（P＜0.05，P＜0.01），肾脏组织 KIM-1 表 达水平显著升高（P＜0.05，P＜0.01）；大黄素高剂量组小鼠的肾脏组织可见明显肾小管上皮细胞水肿、肾小管 管腔中蛋白管型改变及空泡样变性、基底膜增厚、肾小球肿大及囊腔改变、肾间质中可见炎症细胞浸润及间质 纤维化改变。（2）与对照组小鼠比较，大黄素低、高剂量组小鼠肾小球与肾小管周围的胶原纤维沉积增多，胶原 容积分数显著上升（P＜0.01）；出现了较明显的肾小球基底膜增厚、肾小球系膜增生、肾小管空泡样改变及间 质的炎症浸润；肾脏组织中红染的Ⅰ型胶原纤维面积显著增加；大黄素高剂量组小鼠肾脏组织中 α-SMA mRNA 及蛋白表达水平均显著升高（P＜0.05，P＜0.001）。（3）与对照组比较，大黄素高剂量组小鼠肾脏组织中 MDA 含量显著增加（P＜0.01），GSH 水平显著降低（P＜0.01）；大黄素低、高剂量组小鼠肾脏组织中 4-HNE 表达 水平均显著升高（P＜0.05，P＜0.001）。（4）干预 24、48 h 后，与对照组（0 µmol·L-1 ）比较，10～100 µmol·L-1大黄 素组的 TCMK-1 细胞存活率均显著降低（P＜0.01，P＜0.001）。干预 3 h 后，20、40 µmol·L-1大黄素组 TCMK-1 细胞内绿色或红色荧光信号均显著增强，表明细胞内 ROS 积累显著增多。（5）与对照组比较，大黄素低、高剂 量组小鼠肾脏组织 TRIB3 蛋白表达显著上调（P＜0.05，P＜0.01），大黄素高剂量组小鼠肾脏组织 TRIB3 mRNA 表达明显上调（P＜0.05）；20、40 µmol·L-1 大黄素组 TCMK-1 细胞的 TRIB3 蛋白及 mRNA 表达均显著上调 （P＜0.01，P＜0.001）。结论 大黄素长期大剂量给药会对小鼠肾脏结构和功能造成损伤，其潜在毒性机制可 能与诱导 TRIB3 的异常表达，导致肾小管上皮细胞氧化应激损伤，进而引起肾脏纤维化病变有关。

Objective To investigate the toxic effects of emodin on mouse kidneys and the underlying molecular mechanisms based on Tribbles homolog 3（TRIB3）. Methods ICR mice were randomly divided into a control group，a low-dose emodin group，and a high-dose emodin group，with 6 mice in each group. The low-dose and high-dose emodin groups received intraperitoneal injections of emodin at doses of 20 mg · kg-1 and 40 mg · kg-1 ， respectively ， for 4 consecutive weeks. Mouse renal tubular epithelial cells （TCMK-1） were treated with emodin at final concentrations of 10，20，40，80，and 100 µmol·L-1 for 24 and 48 hours. Pathological and morphological changes in kidney tissue were observed by hematoxylin-eosin （HE）， Masson， periodic acid-Schiff （PAS）， and Sirius Red staining. Levels of malondialdehyde （MDA） and reduced glutathione （GSH） in kidney tissue were measured. Cell viability was detected by the CCK-8 assay. Intracellular reactive oxygen species （ROS） levels were detected using the fluorescent probes 2'， 7'-dichlorodihydrofluorescein diacetate （DCFH-DA） and dihydroethidium （DHE）. The mRNA expression levels of TRIB3 and α-smooth muscle actin （α-SMA） in kidney tissue and TCMK-1 cells were measured by RT-qPCR. The protein expression level of TRIB3 in TCMK-1 cells was detected by Western Blot. The expression levels of 4- hydroxynonenal （4-HNE），α-SMA，kidney injury molecule-1（KIM-1），and TRIB3 in kidney tissue were determined by immunohistochemistry. Results （1） Compared with the control group，the kidneys of mice in the low-dose and highdose emodin groups showed significant atrophy，with markedly decreased kidney indices （P＜0.05，P＜0.01），and significantly increased expression of KIM-1 in kidney tissue （P＜0.05， P＜0.01）. The high-dose emodin group exhibited significant pathological changes，including edema of renal tubular epithelial cells，protein cast formation and vacuolar degeneration in tubular lumens， thickened basement membranes， glomerular enlargement with capsular changes，inflammatory cell infiltration in the renal interstitium，and interstitial fibrosis.（2） Compared with the control group，mice in the low-dose and high-dose emodin groups showed increased collagen fiber deposition around glomeruli and tubules，a significantly elevated collagen volume fraction （P＜0.01），more pronounced thickening of the glomerular basement membrane，mesangial hyperplasia，vacuolar changes in renal tubules，and inflammatory infiltration in the interstitium. The area of red-stained type I collagen fibers was significantly increased in kidney tissue. The mRNA and protein expression levels of α-SMA in the high-dose emodin group were significantly elevated （P＜0.05，P＜0.001）. （3） Compared with the control group，the MDA content in kidney tissue was significantly increased （P＜0.01） and the GSH level was significantly decreased （P＜0.01） in the high-dose emodin group. The expression levels of 4-HNE in kidney tissue were significantly increased in both the low-dose and high-dose emodin groups （P＜0.05，P＜0.001）. （4） After 24 and 48 hours of intervention，the viability of TCMK-1 cells in the 10-100 µmol·L-1 emodin groups was significantly reduced compared with the control group （0 µmol·L-1 ）（P＜0.01， P＜0.001）. After 3 hours of intervention， the green or red fluorescent signals in TCMK-1 cells from the 20 and 40 µmol·L-1 emodin groups were significantly enhanced，indicating a significant increase in intracellular ROS accumulation.（5） Compared with the control group，the protein expression of TRIB3 in kidney tissue was significantly upregulated in both the low-dose and high-dose emodin groups （P＜0.05，P＜0.01），and the mRNA expression of TRIB3 was significantly upregulated in the high-dose emodin group （P＜0.05）. The protein and mRNA expression of TRIB3 in TCMK-1 cells from the 20 and 40 µmol·L-1 emodin groups were significantly upregulated （P＜0.01，P＜0.001）. Conclusion Long-term，high-dose administration of emodin can cause structural and functional damage to mouse kidneys. The underlying toxic mechanism may be related to inducing abnormal expression of TRIB3，leading to oxidative stress injury in renal tubular epithelial cells，which subsequently contributes to renal fibrosis.

R285.5

国家自然科学基金项目（82405508）；江苏省药品监督管理局药品监管科学科研计划项目（202406）；江苏省药品监督管理局科研计划 项目（202309）。