SKP alleviates the ferroptosis in diabetic kidney disease through suppression of HIF-1α/HO-1 pathway based on network pharmacology analysis and experimental validation

Abstract

Background: Diabetic kidney disease (DKD) represents a microvascular complication of diabetes mellitus. Shenkang Pills (SKP), a traditional Chinese medicine formula, has been widely used in the treatment of DKD and has obvious antioxidant effect. Ferroptosis, a novel mode of cell death due to iron overload, has been shown to be associated with DKD. Nevertheless, the precise effects and underlying mechanisms of SKP on ferroptosis in diabetic kidney disease remain unclear.

Methods: The active components of SKP were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Protein-protein interaction (PPI) network and Herb-ingredient-targets gene network were constructed using Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted utilizing the Metascape system database. Additionally, an in vivo model of DKD induced by Streptozotocin (STZ) was established to further investigate and validate the possible mechanisms underlying the effectiveness of SKP.

Results: We retrieved 56 compounds and identified 223 targets of SKP through the TCMSP database. Key targets were ascertained using PPI network analysis. By constructing a Herb-Ingredient-Targets gene network, we isolated the primary active components in SKP that potentially counteract ferroptosis in diabetic kidney disease. KEGG pathway enrichment analysis suggested that SKP has the potential to alleviate ferroptosis through HIF signaling pathway, thereby mitigating renal injury in DKD. In animal experiments, fasting blood glucose, 24 h urine protein, urea nitrogen and serum creatine were measured. The results showed that SKP could improve DKD. Results from animal experiments were also confirmed the efficacy of SKP in alleviating renal fibrosis, oxidative stress and ferroptosis in DKD mice. These effects were accompanied by the significant reductions in renal tissue expression of HIF-1α and HO-1 proteins. The mRNA and immunohistochemistry results were the same as above.

Conclusions: SKP potentially mitigating renal injury in DKD by subduing ferroptosis through the intricacies of the HIF-1α/HO-1 signaling pathway.

Keywords: Diabetic kidney disease (DKD); Ferroptosis; HIF-1α/HO-1 signaling pathway; Network pharmacology; Shenkang pills (SKP).

Fig. 1

SKP alleviates kidney injury in DKD mice. A After 1 week of adaptive feeding, the mice were fed with HFD for 4 weeks and then intraperitoneally injected with STZ 40 mg/kg/ day for 5 consecutive days. After the model was successfully established, the drug groups were given oral feeding, and the model group (n = 6) was given the same volume of normal saline. The treatment lasted for 7 weeks. B–G SKP and Val alleviated kidney damage and fasting blood glucose (FBG) in DKD mice. B Changes in body weight of mice in each group during drug administration. C FBG level. D 24-h urine protein. E BUN levels. F Serum creatinine level. G H&E staining and PAS staining of kidney tissues of mice (400 × magnification, bar = 100 µm). *P < 0.05, **P < 0.01, and ***P < 0.001vs. control group. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. DKD group, ns no significance

Fig. 2

SKP alleviated renal fibrosis in DKD mice. A Western blot was performed to determine the expression of α-SMA Fibronectin and Vimentin in kidney tissues of each group. B–D The quantitative analysis for Western blotting results of α-SMA, Fibronectin and Vimentin. E, F Quantification of renal tissue fibrosis in mice. G Masson and Sirius red staining of kidney tissues of mice (400 × magnification, bar = 100 µm). *P < 0.05, **P < 0.01, and ***P < 0.001vs. control group. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. DKD group, ns no significance

Fig. 3

SKP reduced ferroptosis of kidney tissues in DKD mice. A, B The levels of MDA (Malonaldehyde) and GSH(Glutathione) in kidney tissues of mice. C The mRNA levels of GPX4 in the kidneys of each group. D The quantitative analysis for immunohistochemical staining assays of GPX4. E Immunohistochemical staining assays of GPX4 in kidney tissues. (400 × magnification, bar = 100 µm). F, G Western blot for GPX4 in kidney tissues of each group and the quantitative analysis for Western blotting results of GPX4. H The levels of Iron Content in kidney tissues. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. DKD group, ns no significance

Fig. 4

Network pharmacology analysis. A The Herb-ingredient-targets gene network. Green circle nodes represented herbs in SKP, rectangles represented compounds in SKP, and blue hexagonal nodes represented the intersectant targets. A1 to H1 represented shared components of two or more herbs in SKP, the details were listed in Additional file 1: Table S1. B protein–protein interaction (PPI) network of the putative targets. The red circle nodes in the middle represent the eight targets with the highest degree values. C Venn diagram of the targets of SKP, DKD and ferroptosis

Fig. 5

Diagrams of GO and KEGG enrichment analyses. A–D The color of the dot represented the − log10 (Pvalue) value, the size represented the gene count

Fig. 6

Heatmap of binding between main components and targets. The darker the color, the better the binding

Fig. 7

SKP inhibited the HIF-1α/HO-1 Signaling Pathway. A–D Western blot for HIF-1α and HO-1 in kidney tissues of each group and the quantitative analysis for Western blotting results of HIF-1α and HO-1. E, F The mRNA levels of HIF-1α and HO-1 in the renal tissues of each group. G Representative pictures of ROS changes in mice kidneys (bar = 50 µm). H Fluorescence intensity of ROS by image J. I–K Immunohistochemical staining assays of HIF-1α and HO-1 in kidneys and the quantifications of the expressions. (400 × magnification, bar = 100 µm). L Prussian blue staining of kidney tissues of mice. (800× magnification, bar = 50 µm). M Ferrous ion content in mice kidney tissues. *P < 0.05, **P < 0.01, and ***P < 0.001vs. control group. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. DKD group, ns no significance

Fig. 8

Research progress of SKP anti-ferroptosis ameliorating DKD