Ganoderic acid A ameliorates renal fibrosis by suppressing the expression of NPC1L1

Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosis and to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Methods: Transforming growth factor beta1 (TGF-(31) was used to treat Human Kidney-2 (HK-2) cells to establish a renal fibrosis model. The differentially expressed genes in the control (CTRL) group, TGF-(31 group, and TGF-(31 + GAA group were screened via transcriptome sequencing technology and verified by qPCR and Western blot experiments. The NPC1L1 gene overexpression plasmid was constructed. The expression levels of N-cad, E-cad, and Slug-related proteins in CTRL, TGF-(31, TGF-(31+GAA (25 mu g/mL), and TGF-(31+GAA (25 mu g/mL) + NPC1L1 Overexpression (OE) groups were detected by qPCR and Western blot analysis. Western blot analysis was used to identify the extracellular matrixassociated proteins Tenascin-C, a-SMA, and fibrosis-related protein Collagen I. Fibrosis marker protein Fibronectin was detected and quantified by immunofluorescence. Results: Transcriptomic sequencing revealed that TGF-(31 stimulation led to 267 differentially regulated genes, with 118 up-regulated and 149 down-regulated, while further modulation of 213 genes, comprising 112 up-regulated and 101 down-regulated genes, was observed in the GAA intervention group. The target gene in these processes was found to be NPC1L1 by investigations using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). qPCR and Western blot results confirmed that TGF-(31 increased NPC1L1 expression, which was attenuated by GAA. Additionally, TGF-(31 upregulated N-cad and Slug. However, GAA reversed this effect and NPC1L1 overexpression partially rescued the GAA effect. TGF-(31 also decreased E-cad expression, reversed by GAA, and NPC1L1 overexpression antagonized this reversal. Furthermore, TGF-(31 promoted Collagen I, a-SMA, and Tenascin-C expression, and GAA reduced these levels, effects that were reversed by NPC1L1 overexpression. Immunofluorescence results showed that TGF-(31 increased fibronectin expression, which was decreased by GAA, and increased by NPC1L1 overexpression. Conclusion: GAA ameliorates renal fibrosis by antagonizing NPC1L1 gene expression inhibiting epithelialmesenchymal transition and reducing extracellular matrix formation.