Department of Nephropathy, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, PR China.
Department of Infective Disease, the First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, PR China.
Phytomedicine. 2022 Nov;106:154400. doi: 10.1016/j.phymed.2022.154400. Epub 2022 Aug 29.
Paeoniflorin (PF) was found to exhibit renal protection from diabetic kidney disease (DKD) in previous trials, but its specific mechanism remains to be elucidated.
This study furtherly explored the specific mechanism of PF in protect podocyte injury in DKD.
We observed the effects of PF on renal tissue and podocytes in DKD by constructing the vitro and vivo models after measuring the pharmacokinetic characteristics of PF. Target proteins of PF were found through target prediction, and verified by molecular docking, CESTA, and SPR, and then furtherly explored the downstream regulation mechanism related to podocyte autophagy and apoptosis by network prediction and co-immunoprecipitation. Finally, by using the target protein inhibitor in vivo and knocking down the target protein gene in vitro, it was verified that PF played a role in regulating autophagy and apoptosis through the target protein in diabetic nephropathy.
This study found that in STZ-induced mice model, PF could improve the renal biochemical and pathological damage and podocyte injure (p < 0.05), upregulate autophagy activity (p < 0.05), but inhibit apoptosis (p < 0.01). Vascular endothelial growth factor receptor 2 (VEGFR2), predicted as the target of PF, directly bind with PF reflected by molecular docking and surface plasmon resonance detection. Animal studies demonstrated that VEGFR2 inhibitors have a protective effect similar to that of PF on DKD. Network prediction and co-immunoprecipitation further confirmed that VEGFR2 was able to bind PIK3CA to regulate PI3K-AKT signaling pathway. Furthermore, PF downregulated the phosphorylation of PI3K and AKT (p < 0.05). In vitro, similarly to autophagy inhibitors, PF was also found to improve podocyte markers (p < 0.05) and autophagy activity (p < 0.05), decrease caspase 3 protein (p < 0.05) and further inhibited VEGFR2-PI3K-AKT activity (p < 0.05). Finally, the results of VEGFR2 knockdown were similar to the effect of PF in HG-stimulated podocytes.
In conclusion, PF restores autophagy and inhibits apoptosis by targeting the VEGFR2-mediated PI3K-AKT pathway to improve renal injury in DKD, that provided a theoretical basis for PF treatment in DKD.
先前的试验表明，芍药苷（PF）可减轻糖尿病肾病（DKD）导致的肾脏损伤，但具体机制尚不清楚。
本研究进一步探讨 PF 保护 DKD 中足细胞损伤的具体机制。
通过构建体外和体内模型，测量 PF 的药代动力学特征，观察 PF 对 DKD 肾组织和足细胞的影响。通过靶标预测找到 PF 的靶标蛋白，并通过分子对接、CESTA 和 SPR 进行验证，然后通过网络预测和共免疫沉淀进一步探讨与足细胞自噬和凋亡相关的下游调节机制。最后，通过体内使用靶蛋白抑制剂和体外敲低靶蛋白基因，验证 PF 通过靶蛋白在糖尿病肾病中发挥调节自噬和凋亡的作用。
本研究发现，在 STZ 诱导的小鼠模型中，PF 可改善肾脏生化和病理损伤及足细胞损伤（p < 0.05），上调自噬活性（p < 0.05），但抑制凋亡（p < 0.01）。血管内皮生长因子受体 2（VEGFR2）被预测为 PF 的靶标，通过分子对接和表面等离子体共振检测直接与 PF 结合。动物研究表明，VEGFR2 抑制剂对 DKD 具有与 PF 相似的保护作用。网络预测和共免疫沉淀进一步证实，VEGFR2 能够与 PIK3CA 结合，调节 PI3K-AKT 信号通路。此外，PF 下调 PI3K 和 AKT 的磷酸化（p < 0.05）。在体外，与自噬抑制剂相似，PF 还改善了足细胞标志物（p < 0.05）和自噬活性（p < 0.05），降低了 caspase 3 蛋白（p < 0.05），进一步抑制了 VEGFR2-PI3K-AKT 活性（p < 0.05）。最后，VEGFR2 敲低的结果与 PF 在高糖刺激的足细胞中的作用相似。
总之，PF 通过靶向 VEGFR2 介导的 PI3K-AKT 通路恢复自噬并抑制凋亡，改善 DKD 中的肾脏损伤，为 PF 治疗 DKD 提供了理论依据。
