目的:从杨桃根中分离的2-十二烷基-6-甲氧基环己-2,5-二烯-1,4-二酮(DMDD),已被证明对糖尿病肾病(DKD)具有治疗作用。本研究旨在评估DMDD对DKD的影响,并探讨其潜在机制。建立DMDD作为治疗DKD的新型药物。
方法:用高糖(HG)诱导人肾小管上皮细胞(HK-2)模拟DKD,然后进行DMDD处理。使用细胞计数试剂盒-8(CCK-8)测定评估DMDD的细胞毒性。通过transwell和划痕试验评估HK-2细胞的迁移能力。探讨Smad7和miR-21-5p的作用,在HK-2细胞中采用慢病毒转染。此外,QRT-PCR检测上皮间质转化(EMT)和TGFβ1/Smad2/3通路相关蛋白的表达,蛋白质印迹,和免疫荧光技术。
结果:这项研究表明DMDD显著抑制细胞迁移和波形蛋白的表达,α-SMA,HG条件下HK-2细胞中的TGFβ1和p-Smad2/3。同时,DMDD增强E-cadherin和Smad7的蛋白质表达。有趣的是,Smad7沉默后,DMDD的治疗效果被取消.进一步的研究表明,DMDD有效抑制miR-21-5p表达,由HG上调。miR-21-5p的下调抑制TGFβ1/Smad2/3通路的激活和HG诱导的EMT。相比之下,miR-21-5p的过表达否定DMDD的治疗益处。
结论:DMDD通过调节miR-21-5p/Smad7途径减轻HG诱导的HK-2细胞中的EMT,从而抑制DKD中的肾纤维化。这些发现表明DMDD有望成为DKD的潜在治疗剂。
OBJECTIVE: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD\'s effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment.
METHODS: The human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of
Smad7 and miR-21-5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFβ1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques.
RESULTS: This study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFβ1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and
Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon
Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21-5p expression, which is upregulated by HG. Downregulation of miR-21-5p inhibits the activation of the TGFβ1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21-5p negates DMDD\'s therapeutic benefits.
CONCLUSIONS: DMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21-5p/
Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.