Ethnopharmacological relevance: Formononetin (FMN), one of the main isoflavones isolated from Astragalus membranaceus (Fisch.) Bunge, has multiple pharmacological and renal-protective effects. Our previous study suggested FMN as a candidate compound for the treatment of chronic renal failure (CRF). However, the mechanism underlying the repressive effect of FMN on the development of CRF is still unknown.
Aims of the study: To investigate the protective effect of FMN on CRF using in vivo and in vitro models and elucidate the potential underlying mechanism.
Materials and methods: An in vivo model of adenine-induced CRF and an in vitro model of human proximal tubule epithelial cells (HK-2) stimulated with transforming growth factor (TGF)-β1 were used. Serum levels of renal function parameters and inflammatory cytokines were evaluated. Histological analysis was performed to determine the extent of renal injury and fibrosis. Network pharmacology and mRNA sequencing were used to explore the potential mechanism. PPI analysis and molecular docking were used to identify key targets. Polymerase chain reaction and western blotting were used to determine the mechanism underlying the effect of FMN on CRF.
Results: FMN decreased the levels of renal function biochemical markers, including serum creatinine, blood urea nitrogen, and 24 h urine protein content. Treatment with FMN improved renal tubule injury and extracellular matrix (ECM) components, including collagens I and III. In addition, FMN significantly inhibited epithelial-mesenchymal transition (EMT); decreased the expression of fibronectin, N-cadherin, vimentin, α-SMA, and TGF-β1; and restored the expression of E-cadherin. The effect of FMN on renal interstitial fibrosis contributed to decreasing the expression of PI3K, p-Akt, and interleukin (IL) 4, restoring the expression of nitric oxide synthase 3 (NOS3), and reducing the release of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-alpha), both in vivo and in vitro. FMN treatment improved renal function and deposition of ECM components, reduced protein levels of EMT markers in rat kidneys and HK-2 cells, decreased the release of inflammatory cytokines, and inhibited the PI3K/Akt signaling pathway.
Conclusions: FMN treatment significantly reduced the release of inflammatory cytokines and inhibited the effects of the PI3K/Akt signaling pathway on the key targets IL-4 and NOS3. Our results suggest FMN therapy as a novel therapeutic strategy for treating CRF.
Keywords: Anti-Fibrosis; Anti-inflammatory; Chronic renal failure; Formononetin; PI3K/Akt signaling pathway.
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