Wang Xiangjing, Zhang Lina, Huang Ke, Lou Chengli, Xia Yuanying, Zhou Yijing
Department of Nephrology, Jiaxing Hospital of Traditional Chinese Medicine, Jiaxing, China.
Department of General Practice, Jiabei Street Community Health Service Center, Jiaxing, China.
J Diabetes Res. 2025 Jul 17;2025:1159325. doi: 10.1155/jdr/1159325. eCollection 2025.
Diabetic nephropathy (DN), a prevalent microvascular complication of diabetes, is characterized by chronic inflammation, oxidative stress, and renal thrombosis. This study is aimed at assessing the therapeutic effects of costunolide (COS) on DN and investigating its mechanism of action in reducing inflammation and platelet activation-mediated thrombosis by inhibiting the formation of neutrophil extracellular traps (NETs). A DN mouse model was established using a high-sugar, high-fat diet combined with streptozotocin (STZ) administration, followed by treatment with varying doses of COS. The efficacy of COS was assessed through renal function indicators, including 24-h urinary protein levels, serum creatinine, and blood urea nitrogen, alongside renal histopathological analyses using hematoxylin-eosin, Masson's trichrome, and periodic acid-Schiff staining. Transcriptomic analysis was performed to identify gene expression changes in renal tissues after COS treatment. Based on transcriptomic findings, the impact of COS on inflammatory and platelet activation-related markers (IL-1, IL-6, TNF-, CCL2, and CD41) was further evaluated. Additionally, the expression of NET formation-related factors (MPO, CitH3, IGTAM, PAD4, C3, and fibrinogen) was analyzed using immunofluorescence, western blot, and ELISA. To validate the in vivo findings, isolated neutrophils were treated with COS in vitro to assess its inhibitory effects on NET formation, including markers such as SYTOX Green, CitH3, ROS, and PAD4. COS treatment significantly improved renal function and mitigated histopathological damage in DN mice. Transcriptomic analysis indicated that COS modulated pathways associated with inflammation and platelet activation, including the complement and coagulation cascades, biosynthesis of cofactors, cytokine-cytokine receptor interactions, NET formation, and NOD-like receptor signaling. COS markedly reduced the expression of inflammatory markers (IL-1, IL-6, TNF-, and CCL2) and the platelet activation marker CD41 in renal tissues. Moreover, COS decreased the expression of NET-related proteins, including MPO, CitH3, PAD4, IGTAM, C3, and fibrinogen, while lowering the CitH3/H3 ratio. In vitro, COS significantly inhibited PMA-induced NET formation in neutrophils, as evidenced by reduced SYTOX Green + CitH3 expression and decreased levels of PAD4 and ROS. COS alleviates inflammation and platelet activation-mediated thrombosis in DN mice, potentially by inhibiting excessive NET formation. These findings highlight the therapeutic potential of COS in managing DN.
糖尿病肾病（DN）是糖尿病常见的微血管并发症，其特征为慢性炎症、氧化应激和肾血栓形成。本研究旨在评估木香烯内酯（COS）对DN的治疗效果，并通过抑制中性粒细胞胞外诱捕网（NETs）的形成来研究其减轻炎症和血小板活化介导的血栓形成的作用机制。使用高糖高脂饮食联合链脲佐菌素（STZ）给药建立DN小鼠模型，随后用不同剂量的COS进行治疗。通过肾功能指标评估COS的疗效，包括24小时尿蛋白水平、血清肌酐和血尿素氮，同时使用苏木精-伊红染色、Masson三色染色和过碘酸-希夫染色进行肾脏组织病理学分析。进行转录组分析以确定COS治疗后肾组织中的基因表达变化。基于转录组学结果，进一步评估COS对炎症和血小板活化相关标志物（IL-1、IL-6、TNF-、CCL2和CD41）的影响。此外，使用免疫荧光、蛋白质印迹和酶联免疫吸附测定法分析NET形成相关因子（MPO、CitH3、IGTAM、PAD4、C3和纤维蛋白原）的表达。为了验证体内研究结果，在体外使用COS处理分离的中性粒细胞，以评估其对NET形成的抑制作用，包括SYTOX Green、CitH3、活性氧（ROS）和PAD4等标志物。COS治疗显著改善了DN小鼠的肾功能并减轻了组织病理学损伤。转录组分析表明，COS调节了与炎症和血小板活化相关的信号通路，包括补体和凝血级联反应、辅因子的生物合成、细胞因子-细胞因子受体相互作用、NET形成和NOD样受体信号传导。COS显著降低了肾组织中炎症标志物（IL-1、IL-6、TNF-和CCL2）和血小板活化标志物CD41的表达。此外，COS降低了NET相关蛋白的表达，包括MPO、CitH3、PAD4、IGTAM、C3和纤维蛋白原，同时降低了CitH3/H3比率。在体外，COS显著抑制了佛波酯（PMA）诱导的中性粒细胞NET形成，SYTOX Green + CitH3表达降低以及PAD4和ROS水平降低证明了这一点。COS可能通过抑制过度的NET形成来减轻DN小鼠的炎症和血小板活化介导的血栓形成。这些发现突出了COS在管理DN方面的治疗潜力。
