Abstract
OBJECTIVE: To investigate the potential therapeutic effects of trifolin on hypertension-induced renal injury, as well as the key targets and pathways involved. METHODS: The mRNA transcriptional profiles of peripheral blood clinical samples from hypertensive patients were analyzed using Gene Expression Omnibus (GEO), a high-throughput gene expression database. The network pharmacology method was employed to screen key targets of trifolin in treating hypertension-induced renal injury. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. NRK-52E cells, a rat renal proximal tubular cell line, were used to construct an angiotensin Ⅱ (Ang Ⅱ)-stimulated cell model. Flow cytometry was performed to assess cell apoptosis rates and Western blotting was performed to determine the expression levels of apoptosis-related proteins, including Bax, Bcl-2, cleaved caspase-3, and caspase-3, and the phosphorylation and total protein levels of the key MAPK pathway proteins, including ERK, p38 MAPK, and JNK. RESULTS: Analysis of the dataset GSE75360 revealed that, compared with healthy controls, 3331 genes were upregulated and 3197 genes were downregulated in peripheral blood mononuclear cells of hypertensive patients. According to network pharmacology analysis, 472 potential targets of trifolin were identified, including CASP3 and MAPK1. Protein-protein interaction network analysis showed that these targets were closely associated with apoptosis regulatory signaling pathways. GO and KEGG pathway enrichment analyses indicated that trifolin was significantly enriched in pathways associated with negative regulation of apoptosis, apoptotic signaling pathways, and the MAPK signaling pathway. The in vitro experiments confirmed that, compared with the Ang Ⅱ group, trifolin intervention inhibited apoptosis in Ang Ⅱ-stimulated NRK-52E cells, suppressed the expression of Bax and cleaved caspase-3, promoted Bcl-2 expression, and inhibited the phosphorylation of p38 MAPK, ERK, and JNK (P < 0.05). CONCLUSION: Trifolin may exert its protective effect against hypertension-induced renal injury by inhibiting Ang Ⅱ-induced NRK-52E cell apoptosis and regulating the MAPK signaling pathway, representing an important mechanism underlying its therapeutic action.