Isobavachin Attenuates Renal and Cardiac Injury in Chronic Hyperuricemia via Modulation of NF‐κB and NLRP3 Signaling Pathways

Figures

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  Figure 1. The potential targets of isobavachin in hyperuricemia‐related cardiac and renal injury. (A) The chemical structure of isobavachin. (B) Venn diagram showing the overlap between isobavachin, hyperuricemia, heart injury and kidney injury. (C) The PPI network of the shared targets. (D, E) Topological GO and KEGG enrichment analyses of the shared targets. (F) Molecular docking of isobavachin and NF‐κB. (G–I) Isobavachin inhibited NF‐κB in LPS and ATP‐induced inflammation activation cell model. Data are presented as means ± SD (n = 3). ### p < 0.001 versus the control group; *p < 0.05, ***p < 0.001 versus the LPS + ATP group.

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  Figure 2. Effects of isobavachin on high‐UA‐induced damage and UA induced NF‐κB/NLRP3 pathway in mRTEC and H9C2 cells. Cell viability assays of isobavachin and its protective effects against UA‐induced injury in mRTEC (A) and H9C2 (B) cells. (C, D) Representative micrographs showing cell morphology and viability of mRTEC (A) and H9C2 (B) cells staining with Calcein‐AM (green, live cells) and PI (red, dead cells). Representative immunoblots and quantification of relative protein levels of p‐NF‐κB/NF‐κB, p‐IκB‐α/IκB‐α, NLRP3, ASC, caspase 1, and IL‐1β in mRTEC (E) and H9C2 (F) cells. mRTEC and H9C2 cells were stimulated with UA (20 mg/dL) in the absence or presence of isobavachin (5, 10, and 20 μM) for 24 h. Data are presented as means ± SD (n = 3). ### p < 0.001 versus the control group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the UA 20 group.

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  Figure 3. Effect of isobavachin on physiological and biochemical parameters in chronic HUA mice. (A) Schematic diagram of the experimental protocol. (B) Body weight monitoring. (C) Plasma UA concentration monitoring. (D) Urine UA concentration in the last week. NC: normal control group, HUA: hyperuricemia group, ISO: isobavachin (5 mg/kg/day), AP: allopurinol (5 mg/kg/day). Data are presented as means ± SD (n = 8). ### p < 0.001 versus the NC group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the HUA group.

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  Figure 4. Effect of isobavachin on the renal pathological changes in chronic HUA mice. (A) H&E staining. (B) The quantitative scoring of the tubular injury in kidney tissues. (C) Serum CRE levels. (D) Serum BUN levels. (E–G) Relative mRNA levels of renal Kim‐1, NGAL and Klotho. (H) Representative immunoblots and relative protein levels of renal Klotho. Data are presented as means ± SD (n = 6–8). ## p < 0.01, ### p < 0.001 versus the NC group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the HUA group; ns, no significance.

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  Figure 5. Effect of isobavachin on the the renal oxidative stress, inflammation and fibrosis in chronic HUA mice. (A–C) The renal oxidative stress markers MDA content, GSH content and SOD activity. (D) Relative mRNA levels of renal inflammation factors IL‐1β, IL‐6 and TNF‐α. (E, F) Masson staining with the analysis of the collagen deposition area in kidney tissues. (G) Relative mRNA levels of renal fibrosis biomarkers Collagen I, Fibronectin, TGF‐β1, α‐SMA and E‐cadherin. (H, I) Representative immunoblots and relative protein levels of renal Fibronectin, E‐cadherin, α‐SMA, Caludin‐1, pro‐TGF‐β1 and mature TGF‐β1. The data are represented as means ± SD (n = 3–6). # p < 0.05, ## p < 0.01, ### p < 0.001 versus the NC group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the HUA group; ns, no significance.

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  Figure 6. Echocardiographic assessment of cardiac structures in chronic HUA mice. (A) Representative echocardiographic images. (B) Left ventricular anterior wall thickness at diastole (LVAWd). (C) Left ventricular anterior wall thickness at systole (LVAWs). (D) Left ventricular internal dimension at diastole (LVIDd). (E) Left ventricular internal diameter at systole (LVIDs). The data are represented as means ± SD (n = 4). # p < 0.05, ## p < 0.01 versus the NC group; *p < 0.05 versus the HUA group; ns, no significance.

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  Figure 7. Effect of isobavachin on cardiac injury and fibrosis in chronic HUA mice. (A) Representative images of H&E and Masson's trichrome staining. (B) Serum CK levels. (C) Serum AST levels. (D) Cardiac LDH activity. (E) MDA content. (F) GSH content. (G) SOD activity. (H–J) Relative mRNA levels of IL‐1β, IL‐6 and TNF‐α. The data are represented as means ± SD (n = 6). # p < 0.05, ## p < 0.01, ### p < 0.001 versus the NC group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the HUA group; ns, no significance.

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  Figure 8. Effects of isobavachin on the NF‐κB/NLRP3 pathway in the kidney and heart of chronic HUA mice. (A) Representative immunoblots of renal NF‐κB/NLRP3 pathway proteins. (B) Quantification of relative protein levels of renal p‐NF‐κB/NF‐κB, p‐IκB‐α/IκB‐α, NLRP3, ASC, caspase 1, and IL‐1β. (C) Representative immunoblots of cardiac NF‐κB/NLRP3 pathway proteins. (D) Quantification of relative protein levels of cardiac p‐NF‐κB/NF‐κB, p‐IκB‐α/IκB‐α, NLRP3, ASC, caspase 1, and IL‐1β. The data are represented as means ± SD (n = 3). # p < 0.05, ### p < 0.001 versus the NC group; *p < 0.05, **p < 0.01, ***p < 0.001 versus the HUA group; ns, no significance.

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  Figure 9. Schematic diagram illustrating the protective effects of isobavachin on chronic HUA‐inducted kidney and heart injury. Isobavachin alleviates HUA‐induced oxidative stress, inflammation, and fibrosis in the kidney and heart, potentially through its dual role in promoting UA excretion and inhibiting NF‐κB signaling and NLRP3 inflammasome activation.