Salvianolic Acid B Attenuates Ferroptosis in Acute Kidney Injury by Targeting PRDX5

Abstract

Acute kidney injury (AKI) is a common side effect of the chemotherapy agent cisplatin, and ferroptosis serves as the primary mechanism underlying cell death in renal tubular epithelium in such cases. Salvianolic acid B (SAB), a compound derived from Salvia miltiorrhiza, has demonstrated promising anti-inflammatory and antioxidant properties. However, its impact on ferroptosis in the context of AKI remains to be fully explored. In this study, we utilized cisplatin-induced and folic acid-induced AKI models to investigate the protective mechanisms of SAB on renal tissue and tubular epithelial cell injury. The impact of SAB on renal cell ferroptosis was thoroughly examined and confirmed in both AKI models. To predict the potential mechanism through which SAB regulates ferroptosis, we employed an online target prediction database and subsequently verified the specific target proteins involved. Furthermore, we used drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking techniques to assess the binding capacity of SAB to the target protein. Our results reveal that SAB alleviated cisplatin- and folic acid-induced renal dysfunction in vivo and improved cisplatin-induced HK-2 cell injury. Mechanistically, SAB targeted and bound to PRDX5, enhancing its redox activity, which in turn potentiated the inhibitory effect of SLC7A11 and GPX4 on cisplatin-induced ferroptosis. Silencing PRDX5 in HK-2 cells could partially abrogate the protective effect of SAB. These results provide strong evidence for the potential of SAB in the treatment of AKI.

Keywords: PRDX5; acute kidney injury; ferroptosis; lipid peroxidation; salvianolic acid B.

FIGURE 1

SAB treatment mitigates cisplatin‐induced damage in HK‐2 cells. (A) Cell viability assay of SAB in HK‐2 cells. (B) SAB restored cell viability after stimulation with cisplatin. (C‐D) qRT–PCR analysis of KIM‐1 and NGAL. (E) Immunofluorescence analysis of KIM‐1. (F‐H) Western blot analysis of KIM‐1 and NGAL. The results are presented as mean ± SEM, and all experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001. SAB, Salvianolic acid; Cis, Cisplatin.

FIGURE 2

SAB treatment alleviates cisplatin‐induced ferroptosis and lipid peroxidation in HK‐2 cells. (A‐C) qRT‐PCR analysis of ACSL4, COX2, and GPX4. (D‐H) Western blot analysis of ACSL4, GPX4, SLC7A11, and FSP1. (I‐J) Lipid ROS staining and corresponding statistical analysis of fluorescence intensity (scale bar = 100 μm). (K) Detection of MDA levels. (L) Detection of GSH levels. The results are presented as mean ± SEM, and all experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001, ns, not significant.

FIGURE 3

SAB protects against renal dysfunction and pathological damage in cisplatin‐induced AKI mice. (A) The schematic diagram of experimental design. (B) Representative gross‐morphological images of kidney cross section. (C‐E) The creatinine, BUN, and ALT/AST levels (n = 6). (F‐G) HE staining of kidney sections and tubular injury score. (H‐I) Masson staining of kidney sections and corresponding statistical analysis. (J‐K) WB analysis of KIM‐1 and corresponding statistical analysis. (L‐M) Immunohistochemical analysis of KIM‐1 and corresponding statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

SAB treatment alleviates cisplatin‐induced renal inflammatory response in mice. (A‐C) qRT‐PCR analysis of TNF‐α, IL‐1β and MCP‐1. (D‐E) Immunohistochemical analysis of TNF‐α and corresponding statistical analysis. (F‐G) Immunohistochemical analysis of IL‐1β and corresponding statistical analysis. (H‐J) WB analysis of TNF‐α and IL‐1β and corresponding statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001, ns, not significant. n = 6.

FIGURE 5

SAB treatment inhibits ferroptosis in cisplatin‐induced AKI mice. (A‐D) qRT‐PCR analysis of GPX4, ACSL4, SLC7A11, and COX‐2. (E‐F) Immunohistochemical analysis of 4‐HNE and corresponding statistical analysis. (G‐I) WB analysis of GPX4 and SLC7A11 and corresponding statistical analysis. (J) Detection of MDA levels. (K) Detection of GSH levels. *p < 0.05; **p < 0.01; ***p < 0.001, ns, not significant. n = 6.

FIGURE 6

PRDX5 plays a critical role in SAB‐mediated inhibition of ferroptosis. (A‐B) Western blot analysis of PRDX5 in HK‐2 cells and corresponding statistical analysis. (C‐D) Western blot analysis of PRDX5 in AKI mice and corresponding statistical analysis. (E) CETSA‐Western blot analysis showed the protection of PRDX5 by SAB at different temperature gradients. (F) DARTS‐Western blot analysis showed the resistance of PRDX5 to pronase digestion under the treatment of SAB. (G) Results of molecular docking. The results are presented as mean ± SEM, and all experiments were repeated three times. ***p < 0.001.

FIGURE 7

SAB inhibits cisplatin‐induced ferroptosis in HK‐2 cells through a PRDX5‐dependent mechanism. (A‐B) Validation of the efficiency of PRDX5 silencing. (C) Immunofluorescence analysis of KIM‐1. (D‐H) Western blot analysis of GPX4, SLC7A11, FSP1, and KIM‐1. (I) Detection of the MDA content. (J) Detection of the GSH content. (K) Detection of the lipid ROS levels. The results are presented as mean ± SEM, and all experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001, ns, not significant.

FIGURE 8

Mechanistic diagram of SAB‐mediated alleviation of AKI through the regulation of PRDX5‐mediated ferroptosis in renal tubular epithelial cells.