| 方芦炜,马红珍,丁越越,夏虹,王琳.黄芪提取物介导VEGF/PI3K/AKT信号通路减缓高糖足细胞损伤的机制研究[J].浙江中西医结合杂志,2025,35(6): |
| 黄芪提取物介导VEGF/PI3K/AKT信号通路减缓高糖足细胞损伤的机制研究 |
| Study on the Mechanism of Huangqi extract Mediating the VEGF/PI3K/AKT Signaling Pathway to Alleviate High Glucose-induced Podocyte Damage |
| 投稿时间：2024-11-18 修订日期：2025-05-06 |
| DOI： |
| 中文关键词: 黄芪，高糖，足细胞，VEGF/PI3K/AKT信号通路 |
| 英文关键词:Astragalus, High glucose, Podocyte, VEGF/PI3K/AKT signaling pathway |
| 基金项目:浙江省医药卫生科技项目(2022KY229)；马红珍名老中医专家传承工作室项目(GZS2017006) |
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| 中文摘要: |
| 目的：探讨黄芪提取物通过VEGF/PI3K/AKT（血管内皮生长因子/磷脂酰肌醇-3-羟激酶/丝/苏氨酸蛋白激酶）信号通路调控AKT、VEGFA（血管内皮生长因子A）等蛋白表达，缓解高糖足细胞损伤的作用及机制。方法：足细胞体外培养，并在高糖环境下予以不同剂量黄芪提取物（0、25、50、75、100、125、150、175、200和250 μg/mL）干预，通过足细胞毒性实验（CCK-8法）确定黄芪提取物的最佳药物浓度，Real-time PCR检测PI3K、AKT、VEGFA、VEGFR2（血管内皮生长因子受体2）、Nephrin（肾病蛋白）mRNA表达，免疫印迹法检测PI3K、AKT、VEGFA、VEGFR2、Nephrin等蛋白的表达。结果：（1）CCK-8法示150 μg/mL黄芪提取物为干预足细胞活力的最佳浓度。（2）与空白对照组相比，高糖组足细胞PI3K[(3.36±0.20)比(1.00±0.05)，P<0.001]、AKT[(2.01±0.05)比(1.00±0.04)，P<0.01]、VEGFA[(1.75±0.08)比(1.02±0.30)，P<0.01]、VEGFR2[(2.53±0.67)比(1.00±0.18)，P<0.001] mRNA表达水平升高，Nephrin[(0.42±0.02)比(1.00±0.03)，P<0.01] mRNA表达水平降低。使用100、150、200 μg/mL黄芪提取物干预后，足细胞PI3K[分别(1.88±0.43)比(3.36±0.20)，P<0.05、(2.11±0.21)比(3.36±0.20)，P<0.05；(1.95±0.67)比(3.36±0.20)，P<0.05；]、AKT[分别(1.66±0.27)比(2.01±0.05)，P<0.05；(1.24±0.27)比(2.01±0.05)，P<0.05；(1.28±0.02)比(2.01±0.05)，P<0.01；]、VEGFA[分别(1.01±0.21)比(1.75±0.08)，P<0.05；(1.00±0.21)比(1.75±0.08)，P<0.05；(1.16±0.13)比(1.75±0.08)，P<0.05；]、VEGFR2[分别(1.47±0.06)比(2.53±0.67)，P<0.01；(1.21±0.15)比(2.53±0.67)，P<0.01；(1.48±0.14)比(2.53±0.67)，P<0.01；] mRNA表达水平逐渐降低，Nephrin[分别(0.74±0.01)比(0.42±0.02)，P<0.05；(0.85±0.16)比(0.42±0.02)，P<0.01；(0.71±0.09)比(0.42±0.02)，P<0.05；] mRNA表达水平逐渐升高，呈现剂量依赖性关系。（3）与空白对照组相比，高糖组足细胞VEGFA[(3.14±0.25)比(1.04±0.11)，P<0.001]及VEGFR2[(2.33±0.34)比(1.03±0.17)，P<0.001]、磷酸化PI3K（P-PI3K）[(3.87±0.33)比(1.07±0.14)，P<0.001]、磷酸化AKT（P-AKT）[(1.70±0.05)比(1.00±0.03)，P<0.01]蛋白表达升高，Nephrin[(0.37±0.12)比(1.02±0.05)，P<0.05]蛋白表达水平降低。使用100、150、200 μg/mL黄芪提取物干预后，足细胞VEGFA[分别(2.13±0.35)比(3.14±0.25)，P<0.01；(1.64±0.25)比(3.14±0.25)，P<0.001；(1.12±0.36)比(3.14±0.25)，P<0.001；]及VEGFR2[分别(1.53±0.24)比(2.33±0.34)，P<0.01；(1.24±0.12)比(2.33±0.34)，P<0.01；(0.95±0.24)比(2.33±0.34)，P<0.001；]、P-PI3K[分别(2.88±0.46)比(3.87±0.33)，P<0.05；(1.83±0.26)比(3.87±0.33)，P<0.01；(1.15±0.31)比(3.87±0.33)，P<0.001；]、P-AKT[分别(1.11±0.03)比(1.70±0.05)，P<0.01；(0.82±0.03)比(1.70±0.05)，P<0.001；(0.60±0.03)比(1.70±0.05)，P<0.001；]蛋白表达逐渐降低，而Nephrin[分别(0.72±0.15)比(0.37±0.12)，P<0.05；(0.87±0.26)比(0.37±0.12)，P<0.01；(1.06±0.11)比(0.37±0.12)，P<0.001；]蛋白表达逐渐升高。结论：黄芪提取物能有效减轻高糖诱导的足细胞损伤，其作用机制可能与抑制VEGF/PI3K/AKT等信号通路、调控AKT、PI3K、VEGFA、VEGFR2等关键靶点有关。 |
| 英文摘要: |
| Objective: To investigate the effect and mechanism of Astragalus extract in alleviating high glucose-induced podocyte injury by regulating the expression of proteins such as AKT and VEGFA through the VEGF/PI3K/AKT (vascular endothelial growth factor/phosphatidylinositol-3-hydroxykinase/serine/threonine protein kinase) signaling pathway.
Methods: Podocytes were cultured in vitro and treated with different doses of Astragalus extract (0, 25, 50, 75, 100, 125, 150, 175, 200, and 250 μg/mL) under high glucose conditions. The optimal concentration of Astragalus extract was determined using the CCK-8 cytotoxicity assay. Real-time PCR was used to detect mRNA expression levels of PI3K, AKT, VEGFA, VEGFR2 (vascular endothelial growth factor receptor 2), and Nephrin. Western blotting was employed to measure protein expression levels of PI3K, AKT, VEGFA, VEGFR2, and Nephrin.
Results: (1) The CCK-8 assay indicated that 150 μg/mL Astragalus extract was the optimal concentration for enhancing podocyte viability.
(2) Compared with the blank control group, the high glucose group exhibited increased mRNA expression levels of PI3K [(3.36±0.20) vs. (1.00±0.05),P<0.001], AKT [(2.01±0.05) vs. (1.00±0.04),P<0.01], VEGFA [(1.75±0.08) vs. (1.02±0.30),P<0.01], and VEGFR2 [(2.53±0.67) vs. (1.00±0.18),P<0.001], along with reduced mRNA expression of Nephrin [(0.42±0.02) vs. (1.00±0.03),P<0.01]. After intervention with 100, 150, and 200 μg/mL Astragalus extract, mRNA levels of PI3K [1.88±0.43 (P<0.05), 2.11±0.21 (P<0.05), and 1.95±0.67 (P<0.05) vs. 3.36±0.20], AKT [1.66±0.27 (P<0.05), 1.24±0.27 (P<0.05), and 1.28±0.02 (P<0.01) vs. 2.01±0.05], VEGFA [1.01±0.21 (P<0.05), 1.00±0.21 (P<0.05), and 1.16±0.13 (P<0.05) vs. 1.75±0.08], and VEGFR2 [1.47±0.06 (P<0.01), 1.21±0.15 (P<0.01), and 1.48±0.14 (P<0.01) vs. 2.53±0.67] gradually decreased, while Nephrin mRNA levels [0.74±0.01 (P<0.05), 0.85±0.16 (P<0.01), and 0.71±0.09 (P<0.05) vs. 0.42±0.02] increased, showing a dose-dependent relationship.
(3) Compared with the blank control group, the high glucose group showed increased protein expression of podocyte VEGFA [(3.14±0.25) vs. (1.04±0.11), P<0.001], VEGFR2 [(2.33±0.34) vs. (1.03±0.17), P<0.001], phosphorylated PI3K (P-PI3K)[(3.87±0.33) vs. (1.07±0.14), P<0.001], and phosphorylated AKT (P-AKT) [(1.70±0.05) vs. (1.00±0.03), P<0.01], along with reduced Nephrin protein expression [(0.37±0.12) vs. (1.02±0.05), P<0.05]. After treatment with 100, 150 and 200 μg/mL Astragalus extract, protein levels of VEGFA [(2.13±0.35) vs. (3.14±0.25), P<0.01; (1.64±0.25) vs. (3.14±0.25), P<0.001; (1.12±0.36) vs. (3.14±0.25), P<0.001], VEGFR2 [(1.53±0.24) vs. (2.33±0.34), P<0.01; (1.24±0.12) vs. (2.33±0.34), P<0.01; (0.95±0.24) vs. (2.33±0.34), P<0.001], P-PI3K [(2.88±0.46) vs. (3.87±0.33), P<0.05; (1.83±0.26) vs. (3.87±0.33), P<0.01; (1.15±0.31) vs. (3.87±0.33), P<0.001], and P-AKT [(1.11±0.03) vs. (1.70±0.05), P<0.01; (0.82±0.03) vs. (1.70±0.05), P<0.001; (0.60±0.03) vs. (1.70±0.05), P<0.001] gradually decreased, whereas the protein expression of Nephrin [(0.72±0.15) vs. (0.37±0.12), P<0.05; (0.87±0.26) vs. (0.37±0.12), P<0.01; (1.06±0.11) vs. (0.37±0.12), P<0.001] gradually increased.
Conclusion: Astragalus extract effectively alleviates high glucose-induced podocyte injury, potentially by inhibiting the VEGF/PI3K/AKT signaling pathway and regulating key targets such as AKT, PI3K, VEGFA, and VEGFR2. |
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