Astragaloside IV Ameliorates Diabetic Nephropathy by Regulating NFAT5/Notch/Snail Pathway

Abstract

Background: Diabetic nephropathy (DN) is a common complication of diabetes with intricate mechanisms. Our objective was to investigate the potential improvement effects of astragaloside IV (ASV) in a DN cell model and its underlying molecular mechanisms. Methods: High glucose (HG) was employed to induce DN cell model in HK-2 cells, a human renal tubular epithelial cell line. In the initial in vitro experiment, aimed at assessing the impact of ASV on the DN cell model, HK-2 cells were subjected to ASV treatment following HG stimulation. The experimental groups were comprised of Control group, HG group, and HG + ASV treatment group. In the second in vitro experiment, five groups were studied, including Control group, HG group, HG + ASV treatment group, HG + ASV + pcDNA group, and HG + ASV + nuclear factor of activated T cells 5 (NFAT5) group, to elucidate the mechanisms underlying ASVs influence on DN progression. To evaluate the levels of NFAT5, reverse transcription-quantitative PCR (RT-qPCR) was employed. Cell proliferation and apoptosis were measured using flow cytometry, Cell Counting Kit-8 (CCK-8), and 5-Ethynyl-2-deoxyuridine (EdU) assays. Western blot analysis was performed to assess the expression of Collagen Type I (Col. I), α-Smooth Muscle Actin (α-SMA), Fibronectin (FN), Notch1, E-cadherin, Transforming Growth Factor-beta 1 (TGF-β1), NFAT5, Vimentin, Hes1, Jagged1, and Snail. Results: ASV treatment effectively alleviated HG-induced cell damage by enhancing the proliferative capacity, inhibiting cell apoptosis, and suppressing epithelial-mesenchymal transition (EMT) and fibrosis in HK-2 cells (p < 0.05 or p < 0.01). Following HG stimulation, NFAT5 level was increased, and the Notch/Snail pathway was activated (p < 0.01). These effects were significantly ameliorated by ASV treatment (p < 0.01). ASV also demonstrated a direct ability to reduce NFAT5 expression (p < 0.01). Moreover, the positive impact of ASV on cell proliferation and its inhibitory effects on cell apoptosis, EMT, and fibrosis were compromised by NFAT5 overexpression in HK-2 cells after HG stimulation (p < 0.01). Additionally, ASV treatment restrained HG-induced activation of the Notch/Snail pathway (p < 0.01), which was counteracted by NFAT5 overexpression (p < 0.01). Conclusion: ASV demonstrated the protective effects against cell damage induced by HG in HK-2 cells through downregulating NFAT5 and inactivating the Notch/Snail pathway.