Vitexin enhances mitophagy and improves renal ischemia-reperfusion injury by regulating the p38/MAPK pathway

Abstract

Vitexin (VI) is a naturally occurring flavonoid derived from the leaves and seeds of Vitex, recognized for its strong antioxidant properties. This study aims to explore its effects on renal ischemia-reperfusion injury (IRI) and investigate the underlying mechanisms. We utilized hypoxia-reoxygenation (H/R) models with HK-2 cell lines and renal ischemia-reperfusion (I/R) models in mice, applying vitexin preconditioning to assess its influence on renal IRI. Our findings reveal that vitexin mitigated oxidative stress, decreased cell apoptosis, and reduced the expression of renal damage indicators such as kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), along with an overall improvement in renal function. To further investigate the mechanism, we used network pharmacology and molecular docking techniques to predict potential vitexin targets in renal IRI. Results from Western blotting and immunofluorescence assays indicate that vitexin may promote mitophagy by suppressing the phosphorylation of the pivotal p38 protein in the p38/MAPK signaling pathway, offering protection against renal IRI. The findings indicate that vitexin could potentially be used as a therapeutic agent to alleviate renal IRI.

Keywords: Renal ischemia-reperfusion injury; mitophagy; network pharmacology; vitexin.

Figure 1.

Vitexin preconditioning reduces H/R injury in HK-2 cells. (a) Cell viability after 24 h of hypoxia followed by 2, 6, and 12 h of reoxygenation. #p < .05, ##p < .01 compared to the H₂₄R₂ group. (b) Cell viability following treatment with various concentrations of vitexin (0, 5, 10, 20, 40, 60 μM). ****p < .0001 compared to the 0 μM group. (c) Cell viability following H₂₄R₂ treatment after preconditioning with vitexin at safe concentration gradients (5, 10, 20, 40 μM). ####p < .0001 compared to the 0 μM group. (d–f) Representative Western blot images and expression levels of intracellular acute kidney injury markers (KIM-1 and NGAL). *p < .05, ***p < .001, ****p < .0001 compared to the control group. ^#p < .05, ^##p < .01 compared to the H/R + DMSO group.

Figure 2.

Vitexin preconditioning mitigates oxidative stress in vivo and in vitro. (a and b) Representative fluorescent images (×100) and summarized data illustrating intracellular ROS levels. (c and d) Representative fluorescent images (×200) and summarized data illustrating mitochondrial ROS levels in cells. (e) MDA levels in mouse renal tissue. (f) SOD levels in mouse tissue. Scale bar = 100 μm in (a and c). **p < .01, ***p < .001, ****p < .0001 compared to the control group; ^#p < .05, ^##p < .01, ^###p < .001 compared to the H/R + DMSO or I/R + DMSO groups.

Figure 3.

Vitexin alleviates renal IRI in mice. (a) Representative Western blot images of acute kidney injury markers in mouse renal tissue. (b and c) Show the expression levels of acute kidney injury markers. (d and e) Represent plasma Cr and BUN levels in mice, respectively. (f and g) Representative HE-stained images of mouse renal tissue (×400) and corresponding tubular injury scores. Scale bar = 50 μm in (f). **p < .01, ***p < .001, **p < .0001 compared to the control group; ^#p < .05, ^###p < .001 compared to the I/R + DMSO group.

Figure 4.

Vitexin alleviates cell apoptosis induced by renal IRI. (a–d) Representative Western blot images and expression levels of apoptosis-related proteins in mouse renal tissue. (e and f) Representative fluorescent images (×200) and summarized data illustrating the level of apoptosis in mouse renal tissue. Scale bar = 100 μm in (e). *p < .05, **p < .01, ***p < .001, ****p < .0001 compared to the control group; ^#p < .05, ^##p < .01, ^####p < .0001 compared to the I/R + DMSO group.

Figure 5.

Vitexin regulates mitophagy in renal IRI via the p38/MAPK pathway. (a) Venn diagram illustrating the intersection analysis of vitexin’s potential drug targets, mitophagy-related genes, and genes associated with RIR injury. (b) The visualization of the 18 intersecting genes. (c and d) GO and KEGG functional enrichment analyses display the potential biological functions and mechanisms in which the 18 overlapping genes may be involved. (e) Molecular docking images from 2D and 3D perspectives illustrate the binding sites of vitexin with SRC, KRAS, and MAPK14. (f) a table presenting the binding energies of vitexin with SRC, KRAS, and MAPK14 is provided.

Figure 6.

Vitexin enhances mitophagy by downregulating the p38/MAPK pathway. (a–e) Representative Western blot images and expression levels of the key protein p38 in the p38/MAPK pathway and mitophagy-related proteins in mouse renal tissue. (f and g) Representative fluorescent images (×200) and summarized data illustrating the percentage of renal tubules with mitophagosome formation. Scale bar = 100 μm in (f). *p < .05, **p < .01, ***p < .001, ****p < .0001 compared to the control group; ^#p < .05, ^##p < .01, ^###p < .001 compared to the I/R + DMSO group.