Piceatannol attenuates thoracic aortic aneurysm progression by targeting STK33

Figures

• 

  Fig. 1. Integrated analytical framework for identifying and validating therapeutic targets and potential drugs for TAA

• 

  Fig. 2. 31 significant druggable gene targets were identified through Mendelian randomization analysis. The forest plot shows odds ratio (OR) with 95% confidence interval (CI) for each drug target-TAA association

• 

  Fig. 3. Enrichment analysis and PPI network of 31 significant drug targets. (A)-(B): GO and KEGG enrichment analysis for MR significant results, respectively; C-D PPI network and node rank of each drug targets

• 

  Fig. 4. Molecular docking of candidate drugs and its potential target proteins. (A) STK33 docking piceatannol; (B) GLS docking piceatannol; (C) ARSA docking piceatannol; (D) CTSB docking beta-Solamarine; (E) FAS docking beta-Solamarine; (F) GLS docking 3,5-dinitrocatechol; (G) ARSA docking 3,5-dinitrocatechol; (H) SARDH docking periodate-oxidized adenosine; (I) SARDH docking periodate-oxidized adenosine; (J) SARDH docking periodate-oxidized adenosine; (K) SARDH docking periodate-oxidized adenosine

• 

  Fig. 5. Piceatannol attenuates TAA incidence, mortality, and aortic dilation while restoring contractile markers in BAPN-induced mice. Three-week-old C57BL/6J mice were administered 0.5% BAPN in their drinking water for 28 days, supplemented with daily oral gavage of piceatannol at 20 mg/kg. Piceatannol treatment significantly reduced the total incidence (A) and mortality (B) of TAA compared to the BAPN-only group, with kaplan-meier curves (C) demonstrating a marked improvement in overall survival (n = 21). Representative ex vivo images (D) and echocardiographic measurements at 21 days (E) revealed that piceatannol effectively limited the maximum aortic diameter in the ascending aorta (asc) and aortic arch (arch) segments (n = 8). Furthermore, Western blot analysis (F) and subsequent quantitative densitometry (G-I) showed that piceatannol treatment partially restored the protein expression levels of the VSMC contractile markers MYH11, α-SMA, and CNN1, which were significantly downregulated by BAPN (n = 4). Data are expressed as mean ± SEM, with survival differences analyzed by the log-rank (mantel-cox) test and aortic measurements evaluated via two-way ANOVA followed by Sidak’s multiple…

• 

  Fig. 6. Piceatannol attenuated elastin degradation in BAPN-induced TAA mice. Three-week-old C57BL/6J mice were treated with 0.5% BAPN in drinking water for 21 days. (A) Representative images of HE staining and EVG staining (B) and elastin degradation score. Scale bar was shown in the image, n = 8. Data are presented as median (interquartile range). Statistical comparisons among multiple groups were performed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. p < 0.05 was considered statistically significant

• 

  Fig. 7. The mRNA expression of STK33 is downregulated in TAA aortic tissues of both humans and mice, as well as in AngII-stimulated aortic smooth muscle cells. (A-C) relative expression level of STK33, GLS and ARSA in AngII induced mouse TAA tissue from GSE290902 datasets; n of control group = 11; n of AngII group = 8; (D-F) relative expression level of STK33, GLS and ARSA in human TAA tissue from GSE202267 datasets; n of control group = 11; n of TAA group = 25; (G-H) after stimulating rat primary aortic smooth muscle cells with AngII for 48 hours, the mRNA expression levels of STK33, GLS, and ARSA were detected by RT-qPCR; n = 6; (J-L) three-week-old C57BL/6J mice were treated with 0.5% BAPN in drinking water for 21 days, the aorta mRNA expression levels of STK33, GLS, and ARSA were detected by RT-qPCR; n = 6. For publicly available GEO datasets, differential expression was analyzed using unpaired two-tailed Student’s t-tests. For RT-qPCR experiments, comparisons between two groups were performed using unpaired two-tailed Student’s t-tests after confirmation of normal distribution. Data are presented as mean ± SEM. p < 0.05 was considered statistically significant

• 

  Fig. 8. The protein expression of STK33 is downregulated in human TAA aortic tissues. (A) Representative H&E staining and immunohistochemical staining of STK33 of aortic sections from control and TAA patients. (B) relative quantitative analysis of STK33 protein expression. (C) Representative Western blot images showing STK33 protein expression in control and TAA aortic tissues. (D) quantitative analysis of STK33 protein expression normalized to GAPDH. Data are presented as mean ± SEM. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. p < 0.05 was considered statistically significant

• 

  Fig. 9. Analysis of the piceatannol-STK33 complex by molecular dynamics simulation. (A) root mean square deviation (RMSD) trajectory of the complex. (B) root mean square fluctuation (RMSF) per residue of the protein. (C) radius of gyration (Rg) of the protein throughout the simulation. (D) number of hydrogen bonds between piceatannol and STK33 over time. (E, F) three-dimensional (3D) and two-dimensional (2D) free energy landscapes of the complex

• 

  Fig. 10. Piceatannol inhibits AngII-induced vascular smooth muscle cells phenotypic transformation and TAA via targeting STK33. (A)-(B) three-week-old C57BL/6J mice were treated with 0.5% BAPN in drinking water for 21 days, the aorta protein expression levels of STK33, were detected by Immunohistochemistry; n = 4. (C) rat primary aortic smooth muscle cells were treated with 1 μmol/L AngII, with or without 100 μmol/Lpiceatannol, for 48 hours. The mRNA expression levels of SM22α, α-SMA, CNN1, and STK33 were then measured by RT-qPCR. (D) rat primary aortic smooth muscle cells were stimulated with xx mol AngII and concurrently treated with xx mol piceatannol. To suppress STK33 expression, the cells were also transfected with STK33-specific siRNA. After 48 hours of treatment, the mRNA expression levels of SM22α, α-SMA, CNN1, and STK33 were analyzed by RT-qPCR. Data are presented as mean ± SEM. For comparisons involving more than two groups, one-way ANOVA followed by Tukey’s multiple comparisons test was performed. For experiments with two independent variables (e.g., AngII and siRNA treatment), two-way ANOVA followed by Sidak’s multiple comparisons test was used. Normality was assessed prior…