Department of Traditional Chinese Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, China; School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515, China.
Qingdao City Central Hospital, Qingdao, Shandong, 266042, China.
Phytomedicine. 2024 Dec;135:156142. doi: 10.1016/j.phymed.2024.156142. Epub 2024 Oct 9.
Diabetic kidney disease (DKD) is one of the most common microvascular complications in patients with diabetes mellitus. In this condition, renal tubular epithelial mesenchymal transition (EMT) is an important factor accelerating the progression of DKD and a major cause of renal fibrosis and end-stage renal disease. However, the therapeutic effect is unsatisfactory because of the lack of effective drugs. Jia Wei Qingxin Lotus Seed Drink (QISD) is a traditional Chinese medicine compound formula that has shown to be effective in the clinical treatment of DKD. However, the potential of QISD in DKD-EMT treatment has yet to be fully explored.
This study aimed to investigate the role of QISD in ameliorating DKD-EMT injury and its mechanism.
The active ingredients of QISD were identified via ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS). A DKD mouse model was constructed by high-fat diet feeding and intraperitoneal injection of STZ (60 mg/kg), and QISD (14.46, 28.92, and 57.84 g/kg/day) was administered by gavage for 12 consecutive weeks. Dapagliflozin (1 mg/kg/d) was used as a positive control. Renal pathological damage was observed by HE, PAS, and Masson staining. The expression levels of EMT-related proteins and pathway proteins were detected via immunohistochemistry, RT-qPCR, and western blot. In in vitro experiments, EMT injury was induced in human kidney tubular epithelial cells (HK-2) by using lipopolysaccharide (LPS). A combination of CCK8 assay, wound healing assay, small-molecule inhibitor intervention, and overexpression lentiviral transfection was used to investigate the effects of QISD on cell migration ability, adhesion ability, fibrotic factor formation, and mesenchymal properties.
Animal experiments showed that QISD improved blood glucose, body weight, symptoms of excessive drinking and eating, and renal pathological injury in mice, reduced extracellular matrix deposition, delayed renal EMT injury, and inhibited the activation of the histone demethylase JMJD1C. UHPLC-MS/MS and molecular docking indicated that baicalin, wogonoside, oroxylin A-7-O-β-D-glucuronide, and glulisine A found in QISD could bind to JMJD1C. The ameliorating effect of QISD on DKD-EMT injury might be related to JMJD1C. The improvement of DKD-EMT injury by QISD was accompanied by the reduction of SP1 and ZEB1 expression. The SP1 overexpression not only reversed the therapeutic effect of JIB-04, an inhibitor of JMJD1C, on DKD-EMT but also exacerbated the expression of ZEB1 and downstream EMT-related factors. Thus, QISD might affect the expression of the epithelial marker E-cadherin by inhibiting the JMJD1C/SP1/ZEB1 signaling pathway, consequently preventing the transformation of epithelial cells to mesenchymal cells and ameliorating DKD-EMT injury.
This study was the first to demonstrate that QISD might ameliorate DKD-EMT injury by inhibiting the JMJD1C/SP1/ZEB1 signaling pathway. These findings provide strong pharmacologic evidence for the clinical use of QISD in the treatment of DKD.
糖尿病肾病（DKD）是糖尿病患者最常见的微血管并发症之一。在这种情况下，肾小管上皮细胞间充质转化（EMT）是加速 DKD 进展的重要因素，也是肾纤维化和终末期肾病的主要原因。然而，由于缺乏有效药物，治疗效果并不理想。加味清心莲子饮（QISD）是一种中药复方配方，已在 DKD 的临床治疗中显示出疗效。然而，QISD 在 DKD-EMT 治疗中的潜力尚未得到充分探索。
本研究旨在探讨 QISD 改善 DKD-EMT 损伤的作用及其机制。
采用超高效液相色谱-质谱/质谱联用技术（UHPLC-MS/MS）鉴定 QISD 的活性成分。通过高脂饮食喂养和腹腔注射 STZ（60mg/kg）构建 DKD 小鼠模型，连续灌胃 12 周给予 QISD（14.46、28.92 和 57.84g/kg/d）。达格列净（1mg/kg/d）作为阳性对照。通过 HE、PAS 和 Masson 染色观察肾脏病理损伤。通过免疫组化、RT-qPCR 和 Western blot 检测 EMT 相关蛋白和通路蛋白的表达水平。在体外实验中，用脂多糖（LPS）诱导人肾小管上皮细胞（HK-2）发生 EMT 损伤。采用 CCK8 测定、划痕愈合试验、小分子抑制剂干预和过表达慢病毒转染，研究 QISD 对细胞迁移能力、黏附能力、纤维化因子形成和间充质特性的影响。
动物实验表明，QISD 改善了小鼠的血糖、体重、多饮多食症状和肾脏病理损伤，减少了细胞外基质的沉积，延缓了肾脏 EMT 损伤，并抑制了组蛋白去甲基化酶 JMJD1C 的激活。UHPLC-MS/MS 和分子对接表明，黄芩苷、黄芩素、氧化槐定碱-7-O-β-D-葡萄糖醛酸和谷氨酰胺 A 是 QISD 中的化合物，可与 JMJD1C 结合。QISD 改善 DKD-EMT 损伤的作用可能与 JMJD1C 有关。QISD 改善 DKD-EMT 损伤的同时还伴随着 SP1 和 ZEB1 表达的降低。SP1 的过表达不仅逆转了 JMJD1C 抑制剂 JIB-04 对 DKD-EMT 的治疗作用，还加剧了 ZEB1 和下游 EMT 相关因子的表达。因此，QISD 可能通过抑制 JMJD1C/SP1/ZEB1 信号通路影响上皮标志物 E-钙黏蛋白的表达，从而阻止上皮细胞向间充质细胞的转化，改善 DKD-EMT 损伤。
本研究首次证明，QISD 可能通过抑制 JMJD1C/SP1/ZEB1 信号通路来改善 DKD-EMT 损伤。这些发现为 QISD 在治疗 DKD 中的临床应用提供了有力的药理学证据。
