二甲双胍通过 AMPK 依赖性激活 PI3K/AKT 通路来减轻主动脉瓣间质细胞的钙化。

BACKGROUND: Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide. However, no effective treatment could delay or prevent the progression of the disease due to the poor understanding of its pathological mechanism. Many studies showed that metformin exerted beneficial effects on multiple cardiovascular diseases by mediating multiple proteins such as AMPK, NF-κB, and AKT. This study aims to verify whether metformin can inhibit aortic calcification through the PI3K/AKT signaling pathway. METHODS: We first analyzed four microarray datasets to screen differentially expressed genes (DEGs) and signaling pathways related to CAVD. Then aortic valve samples were used to verify selected genes and pathways through immunohistochemistry (IHC) and western blot (WB) assays. Aortic valve interstitial cells (AVICs) were isolated from non-calcific aortic valves and then cultured with phosphate medium (PM) with or without metformin to verify whether metformin can inhibit the osteogenic differentiation and calcification of AVICs. Finally, we used inhibitors and siRNA targeting AMPK, NF-κB, and AKT to study the mechanism of metformin. RESULTS: We screened 227 DEGs; NF-κB and PI3K/AKT signaling pathways were implicated in the pathological mechanism of CAVD. IHC and WB experiments showed decreased AMPK and AKT and increased Bax in calcific aortic valves. PM treatment significantly reduced AMPK and PI3K/AKT signaling pathways, promoted Bax/Bcl2 ratio, and induced AVICs calcification. Metformin treatment ameliorated AVICs calcification and apoptosis by activating the PI3K/AKT signaling pathway. AMPK activation and NF-κB inhibition could inhibit AVICs calcification induced by PM treatment; however, AMPK and AKT inhibition reversed the protective effect of metformin. CONCLUSIONS: This study, for the first time, demonstrates that metformin can inhibit AVICs in vitro calcification by activating the PI3K/AKT signaling pathway; this suggests that metformin may provide a potential target for the treatment of CAVD. And the PI3K/AKT signaling pathway emerges as an important regulatory axis in the pathological mechanism of CAVD.

背景：钙化性主动脉瓣疾病（CAVD）是全球最常见的瓣膜疾病。然而，由于对其病理机制的了解不足，没有有效的治疗方法可以延缓或阻止疾病的进展。许多研究表明，二甲双胍通过调节 AMPK、NF-κB 和 AKT 等多种蛋白对多种心血管疾病发挥有益作用。本研究旨在通过 PI3K/AKT 信号通路验证二甲双胍是否可以抑制主动脉钙化。

方法：我们首先分析了四个微阵列数据集，以筛选与 CAVD 相关的差异表达基因（DEGs）和信号通路。然后，使用免疫组织化学（IHC）和蛋白质印迹（WB）检测主动脉瓣样本来验证选定的基因和通路。从非钙化主动脉瓣中分离出主动脉瓣膜间质细胞（AVICs），然后用含磷酸盐的培养基（PM）或含二甲双胍的 PM 培养，以验证二甲双胍是否可以抑制 AVICs 的成骨分化和钙化。最后，我们使用 AMPK、NF-κB 和 AKT 的抑制剂和 siRNA 研究二甲双胍的作用机制。

结果：我们筛选出 227 个 DEGs；NF-κB 和 PI3K/AKT 信号通路与 CAVD 的病理机制有关。IHC 和 WB 实验显示，钙化主动脉瓣中的 AMPK 和 AKT 减少，Bax 增加。PM 处理显著降低了 AMPK 和 PI3K/AKT 信号通路，促进了 Bax/Bcl2 比值，并诱导了 AVICs 钙化。二甲双胍通过激活 PI3K/AKT 信号通路改善了 AVICs 的钙化和凋亡。AMPK 激活和 NF-κB 抑制可以抑制 PM 处理诱导的 AVICs 钙化；然而，AMPK 和 AKT 抑制逆转了二甲双胍的保护作用。

结论：本研究首次证明，二甲双胍可以通过激活 PI3K/AKT 信号通路抑制 AVICs 的体外钙化；这表明二甲双胍可能为 CAVD 的治疗提供了一个潜在的靶点。PI3K/AKT 信号通路成为 CAVD 病理机制中的一个重要调节轴。