从杨桃根部分离得到的 DMDD 通过抑制 TLR4-BAMBI-Smad2/3 信号通路对高糖诱导的 HK-2 细胞 EMT 发挥保护作用。
Protective effect of DMDD, isolated from the root of Averrhoa carambola L., on high glucose induced EMT in HK-2 cells by inhibiting the TLR4-BAMBI-Smad2/3 signaling pathway.
机构信息
Pharmaceutical College, Guangxi Medical University, Nanning, 530021, Guangxi, China; Pharmacy Department, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China.
Pharmaceutical College, Guangxi Medical University, Nanning, 530021, Guangxi, China.
出版信息
Biomed Pharmacother. 2019 May;113:108705. doi: 10.1016/j.biopha.2019.108705. Epub 2019 Mar 13.
BACKGROUND
Hyperglycemia stimulated epithelial-mesenchymal transition (EMT) plays a critical role in initiating and progressing renal fibrosis in diabetic kidney disease (DKD). It is crucial to explore novel renal protective drugs for the treatment of DKD.
OBJECTIVE
The present study is to confirm our hypothesis and to accumulate the information for the application of DMDD (2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione) as a novel therapeutic agent to potentially inhibit renal fibrogenesis and EMT in the DKD.
METHODS
High glucose induced renal proximal tubular epithelial cell line (HK-2 cells) was cultured and treated with DMDD. The cell viability and DMDD cytotoxicity were assessed by CCK8. Immunofluorescence was used for detection of TLR4 and downstream protein in normal and high glucose induced HK-2 cells. HK-2 cells were transfected with lentivirus codifying for BAMBI (BMP and activin membrane bound inhibitor) and interfering RNA for determination of the effect of BAMBI over-expression and silencing, respectively. TLR4-BAMBI-Smad2/3 pathway was analyzed by means of RT-PCR and western blot.
RESULTS
A high concentration (60mM) of glucose induced significant EMT process and TLR4 expression was increased obviously in this circumstance. DMDD inhibited high expressions of TLR4 and Smad2/3 in HG induced cells and decreased the expression of BAMBI. In addition, the effects of decreased BAMBI expression and increased Smad2/3 expression in HG cultured cells were reversed in the cells of TAK-242 (TLR4 signaling inhibitor) intervention. BAMBI gene silencing dramatically increased EMT process and the over-expression of BAMBI was opposite in HK-2 cells with HG condition. These observations of EMT were ameliorated when the HK-2 cells were pre-treated with DMDD.
CONCLUSIONS
Our study demonstrates that DMDD treatment improves EMT in the HG induced HK-2 cells. In addition, DMDD significantly inhibits EMT by TLR4-BAMBI-Smad2/3 pathway, which hints that DMDD may be an alternative approach in diabetic renal injury.
背景
高血糖刺激上皮-间充质转化（EMT）在糖尿病肾病（DKD）中起始和进展肾纤维化中起着关键作用。探索新型肾保护药物治疗 DKD 至关重要。
目的
本研究旨在验证我们的假设，并为 DMDD（2-十二烷基-6-甲氧基环己-2,5-二烯-1,4-二酮）作为一种潜在的抑制 DKD 肾纤维化和 EMT 的新型治疗剂的应用积累信息。
方法
培养高糖诱导的人近端肾小管上皮细胞系（HK-2 细胞）并给予 DMDD 处理。通过 CCK8 评估细胞活力和 DMDD 细胞毒性。免疫荧光用于检测正常和高糖诱导的 HK-2 细胞中的 TLR4 和下游蛋白。用编码 BAMBI（BMP 和激活素膜结合抑制剂）的慢病毒转染 HK-2 细胞，并转染干扰 RNA，以分别确定 BAMBI 过表达和沉默的影响。通过 RT-PCR 和 Western blot 分析 TLR4-BAMBI-Smad2/3 通路。
结果
高浓度（60mM）葡萄糖诱导明显的 EMT 过程，TLR4 表达明显增加。DMDD 抑制 HG 诱导细胞中 TLR4 和 Smad2/3 的高表达，并降低 BAMBI 的表达。此外，在 TLR4 信号抑制剂 TAK-242 干预的细胞中，HG 培养细胞中 BAMBI 表达降低和 Smad2/3 表达增加的作用被逆转。在 HG 条件下，HK-2 细胞中 BAMBI 基因沉默显著增加 EMT 过程，而过表达 BAMBI 则相反。在 DMDD 预处理的 HK-2 细胞中，这些 EMT 观察结果得到改善。
结论
我们的研究表明，DMDD 治疗可改善 HG 诱导的 HK-2 细胞中的 EMT。此外，DMDD 通过 TLR4-BAMBI-Smad2/3 通路显著抑制 EMT，提示 DMDD 可能是糖尿病肾损伤的一种替代治疗方法。
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