Bruceine A protects nuclear receptor 4A1 from ubiquitin-degradation to alleviate mesangial proliferative glomerulonephritis

Abstract

The nuclear receptor 4A1(NR4A1) plays a crucial role in maintaining cellular homeostasis and is involved in various disease processes; however, its functional role and pharmacological potential in mesangial proliferative glomerulonephritis (MsPGN) remain unexplored. In this study, we found that downregulation of NR4A1 promotes the pathogenesis of MsPGN by regulating inflammatory and proliferative responses in mesangial cells (MCs), whereas overexpression of NR4A1 reverses these processes. Bruceine A (BA) binds to NR4A1 at residues D481/Q568 and exhibits NR4A1-dependent anti-inflammatory and anti-proliferative effects both in vitro and in vivo. Notably, adeno-associated virus serotype 9 (AAV9)-mediated overexpression of NR4A1 alleviates glomerular injury and inflammatory cascades, while knockout of NR4A1 impairs the renoprotective effects of BA. BA binds to the ligand-binding domain (LBD) of NR4A1 and further sterically blocks K48-linked polyubiquitination at K558, thereby stabilizing NR4A1 protein levels. This stabilization enables NR4A1 to auto-activate its own promoter, amplifying the transcriptional repression of nuclear factor kappa-B (NF-κB) signaling phosphorylation, which ultimately attenuates inflammatory cascades and mesangial proliferation to confer renal protection. This study provides a promising therapeutic avenue for the development of next-generation therapies against MsPGN.

Fig. 1

NR4A1 downregulation promotes IgAN progression via modulating inflammatory and proliferative responses in MCs. a Single-cell RNA sequencing revealed significant downregulation of NR4A1 in MCs from IgAN patients compared to controls. b, c Representative immunohistochemistry images showing NR4A1 expression in renal tissues from patients with IgAN and healthy controls. d, e siRNA knocked down the gene and protein expression of NR4A1. f Western blot was performed to quantify NR4A1 and NF-κB/p-NF-κB protein levels. g Effect of NR4A1 knockdown on CCL2 protein expression. h, i Edu cell proliferation experiment. j Effect of NR4A1 knockdown on inflammation and proliferation-related genes. Data were expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001 vs model group

Fig. 2

NR4A1 overexpression reverses the inflammatory response and proliferation of MCs. a, b NR4A1 overexpression plasmid increased the expression of NR4A1 gene and protein in MCs. c Western blot analysis of NR4A1 and NF-κB/p-NF-κB protein expression. d Effect of NR4A1 on inflammation-related genes. Data were expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001 vs model group. e, f Effect of NR4A1 overexpression on UACR and 24 h urine protein quantification. g Western blot analysis of the changes in NR4A1 and NF-κB/p-NF-κB proteins. h Renal immunofluorescence showed changes in NR4A1 protein. Bars =100 µm. Magnification: ×200. i Representative images of glomeruli from AAV9-NR4A1-treated rats stained with PAS and HE staining, Bars = 50 µm. Magnification: ×400. Data were shown as mean ± SEM (n = 6). Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001 vs model group

Fig. 3

BA binds to NR4A1 with high affinity and forms close interactions. a Molecular docking diagram of the NR4A1 agonist and BA. b BA and NR4A1 protein interaction using CETSA assay. c BA and NR4A1 protein interaction using DARTS assay. d Binding proteins identification of BA using a human proteome microarray. e Affinity between BA and the NR4A1/LBD WT protein measured by MST. f Affinity between BA and the NR4A1/LBD WT protein measured by SPR. g MST assay detected the affinity between BA and NR4A1/LBD mutant protein. h SPR assay detected the affinity between BA and NR4A1/LBD mutant protein

Fig. 4

Effects of BA on proteinuria and pathological changes. a The chemical structure of BA. b Experimental flow diagram. c, d Effects of BA on 24 h urinary protein excretion and UACR on days 3 and 7 in anti-Thy1 nephritis rats. e–h Representative images of glomeruli from BA or OM treated animals with anti-Thy1 nephritis stained with PAS and HE staining, Bars = 50 µm. Magnification: ×400. i Effects of BA on IL-6, CCL2, NF-κB, TNF-α and NR4A1. Data were presented as the mean ± SEM (n = 6). Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the model group

Fig. 5

BA inhibits MCs proliferation and inflammation in vitro. a, b Effect of BA on inhibiting the proliferation of MCs by Edu incorporation assay. c, d Flow cytometry analysis of the effects of BA on the cell cycle distribution of MCs. e Effects of BA on IL-6, CCL2, NF-κB, α-SMA, TGF-β1 and Cyclin E. f Western blot analysis of NR4A1 and NF-κB/p-NF-κB protein expression. Data were presented as the mean ± SEM (n = 3). Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the model group

Fig. 6

BA binds to NR4A1 in a competitive manner with CsnB in vitro and in vivo (a, b) Effects of BA on 24 h urinary protein excretion and UACR. c Representative images of glomeruli from BA or OM treated animals with anti-Thy1 nephritis stained with PAS and HE staining, Bars = 50 µm. Magnification: ×400. d–f Semi-quantitative analysis of renal pathological changes. g Western blot analysis of NR4A1 and NF-κB/p-NF-κB protein expression in each group. Data were presented as the mean ± SEM (n = 6). Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the model group. h, i Colony formation assay. j BA and CsnB on the genes NR4A1, IL-6, CCL2, NF-κB, α-SMA and Cyclin E in vitro. k Western blot analysis of NR4A1 and NF-κB/p-NF-κB protein expression in each group. Data were presented as the mean ± SD (n = 3). Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the model group

Fig. 7

BA alleviates anti-Thy1 nephritis dependent on NR4A1. a, b Effects of NR4A1 deficiency on 24-hour urinary protein excretion and UACR. c Representative glomerular images from BA-treated or NR4A1-deficient rats with anti-Thy1 nephritis, stained with PAS and HE (scale bar = 50 µm; magnification ×400). d Impact of NR4A1 deficiency on the expression of NR4A1, IL-6, CCL2, NF-κB, α-SMA and Cyclin B. e Western blot analysis of NR4A1, NF-κB/p-NF-κB protein levels in each group. Data are expressed as mean ± SEM (n = 6). Statistical significance was determined as follows: *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the model group

Fig. 8

BA stabilizes NR4A1 by inhibiting K48-linked polyubiquitination at K558 to confer renal protection. a MCs were treated with CHX (100 μg/mL) for different times, western blot examined the protein expression of NR4A1. b MCs were treated with Chloroquine (20 μM) for different times, western blot examined the protein expression of NR4A1. c MCs were treated with MG132 (20 μM) for different times, western blot examined the protein expression of NR4A1. d Inhibition of NR4A1 ubiquitination expression by BA. e BA inhibits K48 ubiquitin chain modification. f BA fails to modulate NR4A1 ubiquitination upon mutation of residues D481 and Q568. g K558 serves as a critical ubiquitination site in NR4A1. h Dual-luciferase assays demonstrated NR4A1 binding to its autoregulatory promoter. i Dual-luciferase assays demonstrated NR4A1 inhibited the transcriptional activity of NF-κB

Fig. 9

BA stabilizes NR4A1 by inhibiting K48-linked polyubiquitination at K558 to ameliorate MsPGN. (Left) Under pathological conditions, K48-linked polyubiquitination mediates NR4A1 degradation, thereby impairing its autoregulatory transcriptional activity. Subsequent NF-κB activation promotes inflammatory cytokine release and MCs proliferation, exacerbating kidney injury. (Right) BA inhibits K48-linked polyubiquitination at K558, stabilizing NR4A1. This restores NR4A1-mediated transcriptional auto-amplification and NF-κB suppression, attenuating inflammation and mesangial hyperplasia to confer renal protection