Betanin attenuates renal ischemia reperfusion injury in rats: Insights into NLRP3, molecular docking, and molecular dynamics simulation

Abstract

Acute kidney injury (AKI) is a common and clinically consequential complication of renal ischemia-reperfusion injury (IRI), arising across diverse settings that include kidney transplantation, cardiac bypass surgery, sepsis, and hypotensive episodes. Activation of the NLRP3/caspase-1/Gasdermin D (GSDMD) pyroptotic axis has been implicated as a key driver of IRI-associated renal inflammation and represents a candidate target for nephroprotective intervention. Betanin, the principal betalain pigment of Beta vulgaris L. (red beetroot) roots, possesses recognised antioxidant and anti-inflammatory properties, but its effect on renal IRI has not previously been examined. Betanin was isolated from B. vulgaris L. roots and structurally characterised by chromatographic and spectroscopic analysis. Adult male Wistar rats were allocated to sham, betanin-only, IRI, and IRI+betanin groups (n = 6/group) and pre-treated with betanin (100 mg/kg/day, oral gavage) for five consecutive days before bilateral renal pedicle occlusion (45 minutes of ischemia followed by 24 hours of reperfusion). Renal function, oxidative and antioxidative markers, renal mRNA levels of NLRP3, IL-18, and IL-1β (normalised to GAPDH), and renal histopathology were evaluated. In parallel, network pharmacology analyses (protein-protein interaction, Gene Ontology, and KEGG pathway enrichment) together with molecular docking and a 100-nanosecond molecular dynamics simulation were used to characterise betanin’s predicted binding affinity and conformational stability within the GSDMD active site. Renal IRI significantly increased serum creatinine and BUN, renal MDA, and the renal mRNA levels of NLRP3, IL-18, and IL-1β, while reducing renal SOD activity, changes accompanied by tubular necrosis, brush-border loss, and inflammatory infiltration on histopathology. Betanin pre-treatment markedly attenuated each of these IRI-induced biochemical, molecular, and histopathological derangements. Network pharmacology analyses identified caspase-1 and GSDMD as central hub proteins within the betanin-associated interactome, with Gene Ontology and KEGG enrichment converging on pyroptosis and NOD-like receptor signalling. Molecular docking showed a strongly negative binding energy (−7.43 kcal/mol) for the betanin-GSDMD complex, and the 100-ns molecular dynamics simulation confirmed a stable, energetically favourable interaction throughout the simulation period. Collectively, these findings indicate that betanin attenuates renal IRI through concurrent antioxidant, anti-inflammatory, and anti-pyroptotic actions converging on the NLRP3/caspase-1/GSDMD axis. Given the prophylactic dosing paradigm employed, these results are most directly applicable to clinically predictable ischemic settings, such as scheduled kidney transplantation or cardiac surgery; extension to unpredictable ischemic events (e.g., sepsis, hypotensive episodes) would require validation using a post-injury, therapeutic dosing regimen. These findings support betanin’s further evaluation as a candidate small-molecule modulator of the pyroptotic cascade in ischemic renal injury.