Department of Nephrology, Key Laboratory of Kidney Disease and Blood Purification in Hunan, The Second Xiangya Hospital, Central South University, Hunan, China.
Jiangsu Province Hospital, Nanjing Medical University First Affiliated Hospital Nanjing, Jiangsu, China.
Chem Biol Drug Des. 2021 Feb;97(2):293-304. doi: 10.1111/cbdd.13781. Epub 2020 Sep 7.
Norcantharidin (NCTD), the demethylated analog of cantharidin isolated from Mylabris, is known to inhibit renal fibrosis. However, the underlying mechanism is largely unknown. The present study investigates whether NCTD exerts this effect through regulation of the protein phosphatase 2A catalytic subunit (PP2Ac)-Smad3 pathway. HK-2 human renal proximal tubule cells exposed to transforming growth factor (TGF)-β1 were used as an in vitro model of renal fibrosis. The levels of total Smad3, C-terminal-phosphorylated Smad3 (p-Smad3), PP2Ac, and fibronectin (Fn) were evaluated by Western blotting. A PP2Ac overexpression plasmid and the PP2Ac inhibitor okadaic acid (OA) were used for functional analyses. The subcellular localization of Smad3 was visualized by immunofluorescence labeling. The results showed that PP2Ac overexpression increased Smad3 phosphorylation and nuclear translocation in HK-2 cells, while pharmacologic inhibition of PP2Ac with OA had the opposite effect. NCTD suppressed Fn and p-Smad3 expression and TGF-β1-induced nuclear entry of Smad3, but these effects were abrogated by inhibition of PP2Ac. Thus, the anti-renal interstitial fibrosis effect of NCTD is exerted through inhibition of PP2Ac-mediated C-terminal phosphorylation of Smad3. These findings highlight the therapeutic potential of NCTD for the treatment of renal interstitial fibrosis.
去甲基斑蝥素（NCTD）是从斑蝥中分离得到的斑蝥素的去甲基类似物，已知其能抑制肾纤维化。然而，其作用机制在很大程度上尚不清楚。本研究探讨了 NCTD 是否通过调节蛋白磷酸酶 2A 催化亚基（PP2Ac）-Smad3 途径发挥这种作用。用转化生长因子（TGF）-β1 处理 HK-2 人肾近端小管细胞作为肾纤维化的体外模型。通过 Western blot 法检测总 Smad3、C 末端磷酸化 Smad3（p-Smad3）、PP2Ac 和纤连蛋白（Fn）的水平。使用 PP2Ac 过表达质粒和 PP2Ac 抑制剂 okadaic acid（OA）进行功能分析。通过免疫荧光标记观察 Smad3 的亚细胞定位。结果表明，PP2Ac 过表达增加了 HK-2 细胞中 Smad3 的磷酸化和核转位，而用 OA 抑制 PP2Ac 则产生相反的效果。NCTD 抑制 Fn 和 p-Smad3 的表达，并抑制 TGF-β1 诱导的 Smad3 核内进入，但这些作用被抑制 PP2Ac 所阻断。因此，NCTD 的抗肾间质纤维化作用是通过抑制 PP2Ac 介导的 Smad3 C 末端磷酸化发挥的。这些发现强调了 NCTD 治疗肾间质纤维化的治疗潜力。
