OBJECTIVE To explore the mechanism of Yishen Qingli Huoxue Formula (YQHF) improving renal fibrosis by inhibiting HIF1-α using data mining, molecular docking, and in vivo and in vitro experiments.

METHODS The expression changes of HIF1-α in renal biopsy tissues of patients with chronic kidney disease (CKD) in the GEO database were analyzed. Molecular docking was used to clarify the interaction mode between YQHF effective monomers and HIF1-α. Thirty SD rats were randomized to sham, model, low-dose YQHF, high-dose YQHF, and losartan potassium groups (n=6 per group). Unilateral ureteral obstruction (UUO) was used to induce renal fibrosis. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured, and kidney sections were stained with HE and Masson to assess pathology and fibrosis. Renal HIF1-α protein expression was quantified by Western blot. A renal fibrosis cell model was established by inducing NRK-52E cells with TGF-β1, and the cells were divided into control, model, YQHF, HIF1-α inhibitor, HIF1-α inhibitor+YQHF, HIF1-α agonist, and HIF1-α agonist+YQHF groups. Western blot analysis was used to detect the protein expression levels of HIF1-α, COL-1, and α-SMA, and to observe the mechanism of YQHF-containing serum in protecting renal tubular epithelial cells.

RESULTS Data mining showed HIF1-α expression in the CKD group was significantly higher than in the control group (P < 0.01). Molecular docking indicated YQHF core components had good binding affinity to HIF1-α. In vivo, compared with the sham group, HE staining revealed tubular atrophy and inflammatory-cell infiltration, and Masson staining showed increased collagen deposition in UUO model rats (P < 0.01). Serum creatinine and blood urea nitrogen were also elevated in the model group (P < 0.05), together with up-regulated renal expression of COL-1, α-SMA and HIF-1α (P < 0.01). After intervention with either high-dose or low-dose YQHF or losartan potassium, these pathological changes were attenuated: collagen deposition decreased (P < 0.01), creatinine and BUN fell to varying degrees (P < 0.05), and renal COL-1, α-SMA and HIF-1α levels were down-regulated (P < 0.01); immunohistochemistry confirmed reduced HIF-1α in UUO kidneys (P < 0.01). In NRK-52E cells, TGF-β1 stimulation markedly increased COL-1, α-SMA and HIF-1α protein levels (P < 0.01). Both YQHF and chloramphenicol alone down-regulated these proteins (P < 0.05, P < 0.01), and their combination produced stronger inhibition of HIF-1α than YQHF alone (P < 0.05). Conversely, the HIF-1α agonist fenbendazole-d3 reversed YQHF's anti-fibrotic effect, re-elevating COL-1, α-SMA and HIF-1α (P < 0.01), with no significant difference versus agonist alone.

CONCLUSION YQHF may inhibit extracellular matrix deposition and delay renal fibrosis progression by suppressing HIF1-α accumulation, providing new theoretical evidence for traditional Chinese medicine in treating renal fibrosis.