Gypenosides suppress fibrosis of the renal NRK-49F cells by targeting miR-378a-5p through the PI3K/AKT signaling pathway

The massive synthesis and secretion of extracellular-matrix (ECM) components by activated fibroblasts in the kidneys causes irreversible renal interstitial fibrosis. Gypenosides (GPs) have been shown to decelerate this process, in which micro RNAs (miRNAs) play an important regulatory role. This study aimed to evaluate the mechanism underlying the suppressive effect of GPs on renal fibrosis. Materials and

GPs inhibit the PI3K/AKT signaling by up-regulating miR-378a-5p in TGF-β1-stimulated NRK-49F cells and thereby reduce their massive secretion of ECM components. Given that this in-vitro model of renal interstitial fibrosis closely mimics the in-vivo pathogenesis, our results most likely apply to the in-vivo conditions.

This study used TGF-β1-stimulated NRK-49F renal cells as an in-vitro model of renal interstitial fibrosis. First, the concentration range of GPs that significantly affects the cytoactive was determined. Then, the anti-fibrotic effects of various concentrations of GPs in the in-vitro model were assessed via immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Non-coding-RNA sequencing combined with bioinformatics was used to predict the mechanistic basis of the anti-fibrotic effect of GPs, and qRT-PCR was used to verify the sequencing

TGF-β1 stimulation up-regulated α-SMA, COL1, and COL3 in NRK-49F cells, and this effect was suppressed by GPs. Additionally, TGF-β1 stimulation significantly changed the expression levels of 151 miRNAs, and GPs significantly suppressed the effect of TGF-β1 on the levels of 18 of these miRNAs. Among them, miR-3588 and miR-378a-5p were down-regulated, and miR-135b-5p and miR-3068-5p were up-regulated upon TGF-β1 induction. Of these miRNAs, miR-378a-5p was predicted to target the mRNAs of numerous proteins mainly enriched in the PI3K/AKT signaling pathway. The miRNA transfection experiments with the miR-NC mimic and PI3K inhibitor as controls showed that miR-378a-5p overexpression could suppress the TGF-β1-induced up-regulation of α-SMA, COL1, PI3K, and AKT, including the phosphorylated form (p-AKT).