Effects and mechanisms of Supplemented Shenqi Dihuang Decoction Formula improving renal aging and tubulointerstitial fibrosis in diabetic kidney disease by inhibiting ferroptosis

This study aimed to explore the effects and mechanisms of Supplemented Shenqi Dihuang Decoction Formula(SSDDF) in ameliorating renal aging(RA) and tubulointerstitial fibrosis(TIF) in diabetic kidney disease(DKD) by inhibiting ferroptosis, in comparison with empagliflozin(EMPA), a novel hypoglycemic agent. For the in vivo experiment, a type 2 DKD rat model with histopathological characteristics of RA and TIF was established. After successful modeling, the model rats were divided into the low-dose SSDDF(SSDDF-L) group, high-dose SSDDF(SSDDF-H) group, EMPA group and normal saline control group, and were administrated by gavage for six consecutive weeks for intervention. For the in vitro experiment, a senescent model of renal tubular epithelial cells was constructed by treating NRK-52E cells with high glucose(HG), which was then intervened with SSDDF-L containing serum, SSDDF-H containing serum, EMPA, as well as ferroptosis and iron metabolism inhibitors. In both in vivo and in vitro studies, aging phenotypes and related physiological indicators including body weight(BW) and blood glucose(BG), renal injury indicators including serum creatinine(Scr), blood urea nitrogen(BUN) and urinary N-acetyl-β-D-glucosaminidase(UNAG), aging characteristics of renal tissues and cells including senescence-associated β-galactosidase(SA-β-gal) staining and protein expression of klotho and p16, TIF pathological features and cellular fibrotic injury, senescence-associated secretory phenotype(SASP), and ferroptosis characteristics(lipid peroxidation-dependent ferroptosis detected by Ferro Orange fluorescent probe and mitochondrial transmission electron microscopy) were examined and compared. Meanwhile, an "SSDDF-component-target" network was constructed via network pharmacology, and Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were combined to screen the core signaling pathways. Non-targeted metabolomics was adopted to analyze the characteristics of renal tissues, screen potential biomarkers and enrich related metabolic pathways.The results indicated that the effect of SSDDF in ameliorating RA and TIF in DKD rats might be associated with the regulation of ferroptosis signaling pathways. In the in vivo experiment, SSDDF ameliorated the aging phenotypes, BW, BG, UNAG levels, as well as RA and TIF in DKD model rats, with SSDDF-H exerting a superior effect to SSDDF-L; notably, SSDDF-H showed a better effect than EMPA in improving Scr and BUN levels. SSDDF also ameliorated renal lipid peroxidation-dependent ferroptosis in DKD rats, where SSDDF-H had a better effect than SSDDF-L and an equivalent effect to EMPA. In the in vitro experiment, SSDDF-H containing serum multi-targetedly improved HG-induced senescence, fibrotic injury, SASP expression, ferroptosis marker levels and mitochondrial ultrastructural damage in renal tubular epithelial cells, with its effect comparable to that of ferroptosis or iron metabolism inhibitors. In conclusion, both SSDDF and EMPA can multi-targetedly ameliorate RA and TIF in DKD model rats with similar anti-aging and anti-fibrotic effects, and their core mechanism is the inhibition of renal lipid peroxidation-dependent ferroptosis. Based on this, the clinical establishment of a combined therapy regimen of SSDDF plus EMPA featured by "coordination of traditional Chinese and Western medicine" for elderly patients with DKD is expected to exert a dual synergistic effect in anti-aging and anti-fibrosis.