Department of Nephrology, Jinshan Hospital, Fudan University, Shanghai, China.
Folia Histochem Cytobiol. 2021;59(4):259-270. doi: 10.5603/FHC.a2021.0025. Epub 2021 Dec 1.
The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism.
The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis.
The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed.
TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.
糖尿病肾病（DKD）的进展与肾小管上皮细胞-间充质转化（EMT）和肾小管间质纤维化密切相关。丹参酮 IIA（TSIIA）是从丹参等传统中药中提取的，已被证明具有抗纤维化作用。本研究旨在探讨 TSIIA 对高糖诱导的人近端肾小管细胞（HK-2 细胞）EMT 的影响及其可能的机制。
通过光学显微镜和 CCK-8 试验测量暴露于不同浓度葡萄糖的细胞增殖。用 30mM 葡萄糖和不同浓度的 TSIIA（5μM 或 10μM）刺激细胞 48 小时。用维生素 D 受体（VDR）-siRNA 转染细胞，并用高糖和 TSIIA 处理进一步处理细胞。通过 qPCR 检测α平滑肌肌动蛋白（a-SMA）mRNA 的表达，以确保 EMT 的成功诱导，并通过 qPCR 检测 VDR-siRNA 的成功转染。用 Western blot 分析检测 a-SMA、E-钙黏蛋白、VDR、β-连环蛋白和糖原合成酶激酶 3b（GSK-3b）的蛋白表达。
结果表明，高葡萄糖浓度抑制 HK-2 细胞增殖并促进 EMT。TSIIA 可通过增加 VDR 蛋白水平并抑制β-连环蛋白和 GSK-3b 蛋白水平来逆转高糖诱导的 EMT，提示 VDR 与 Wnt/β-连环蛋白通路呈负相关。用 VDR-siRNA 转染并在高葡萄糖浓度下孵育细胞后，Wnt 通路中 VDR 对β-连环蛋白和 GSK-3b 表达的抑制作用被抑制，β-连环蛋白通路被激活。当 VDR 水平通过 TSIIA 恢复时，VDR 对该通路的抑制作用也得到恢复，通路的激活受到抑制。
TSIIA 通过上调 VDR 水平减轻高糖诱导的 HK-2 细胞 EMT，这可能与 VDR 对 Wnt 通路的抑制作用有关。
