Citronellal improves endothelial dysfunction by affecting the stability of the GCH1 protein

Abstract

Endothelial dysfunction (ED) serves as the pathological basis for various cardiovascular diseases. Guanosine triphosphate cyclopyrrolone 1 (GCH1) emerges as a pivotal protein in sustaining nitric oxide (NO) production within endothelial cells, yet it undergoes degradation under oxidative stress, contributing to endothelial cell dysfunction. Citronellal (CT), a monoterpenoid, has been shown to ameliorate endothelial dysfunction induced by in atherosclerosis rats. However, whether CT can inhibit the degradation of GCH1 protein is not clear. It has been reported that ubiquitination may play a crucial role in regulating GCH1 protein levels and activities. However, the specific E3 ligase for GCH1 and the molecular mechanism of GCH1 ubiquitination remain unclear. Using data-base exploration analysis, we find that the levels of the E3 ligase Smad-ubiquitination regulatory factor 2 (Smurf2) negatively correlate with those of GCH1 in vascular tissues and HUVECs. We observe that Smurf2 interacts with GCH1 and promotes its degradation via the proteasome pathway. Interestingly, ectopic Smurf2 expression not only decreases GCH1 levels but also reduces cell proliferation and reactive oxygen species (ROS) levels, mostly because of increased GCH1 accumulation. Furthermore, we identify BH ₄/eNOS as downstream of GCH1. Taken together, our results indicate that CT can obviously improve vascular endothelial injury in Type 1 diabetes mellitus (T1DM) rats and reverse the expressions of GCH1 and Smurf2 proteins in aorta of T1DM rats. Smurf2 promotes ubiquitination and degradation of GCH1 through proteasome pathway in HUVECs. We conclude that the Smurf2-GCH1 interaction might represent a potential target for improving endothelial injury.

Keywords: Smurf2; citronellal; degradation; endothelial dysfunction.

Figure 1

Protective effect of CT on H ₂O ₂-induced endothelial cell injury and the aorta in T1DM rats (A,B) Effect of CT on blood lipid levels in T1DM rats ( n=6). (C,D) Effect of CT on endothelium-dependent relaxation induced by acetylcholine (Ach) or sodium nitroprusside (SNP) in organ lumen ( n=6). (E) Effect of CT on cell proliferation of HUVECs ( n=3). (F) Effect of H 2O 2 on cell viability of HUVECs ( n=3). (G) Survival rate after treatment of H 2O 2-induced endothelial cell injury with different concentrations of CT ( n=3). (H) The effect of CT on FBG level of rats in each group ( n=6). (I) H&E staining of carotid arteries ( n=6). Scale bar: 100 μm. (J) Masson staining of carotid arteries ( n=6). Scale bar: 100 μm. (K) The ultrastructural changes of endothelial injury induced by CT in T1DM rats were observed by TEM ( n=6). Scale bar: 10 μm. Data were analyzed by one-way ANOVA by Tukey post hoc tests or Bonferroni post hoc tests. All data are expressed as the mean ±SEM. * P<0.05 vs Control group, # P<0.05 vs T1DM group or H 2O 2 group.

Figure 2

CT suppresses the protein expressions of Smurf2 and GCH1 in carotid arteries of T1DM rats (A) The immunofluorescence staining of Smurf2 and GCH1 in carotid arteries (Smurf2, green; GCH1, red) ( n=6). Scale bar: 50 μm. (B) The immunohistochemistry analysis of GCH1 and Smurf2 in carotid arteries ( n=6). Scale bar: 100 μm. (C,D) Quantitative analysis of immunofluorescence of GCH1 (Red) and Smurf2 (Green) in carotid arteries ( n=6). (E,F) Quantitative analysis of positive intensity of GCH1 and Smurf2 ( n=6). (G) The expression levels of Smurf2 and GCH1 proteins in carotid arteries were detected by western blot analysis ( n=6). (H,I) Quantitative analysis of (G). Data were analyzed by one-way ANOVA by Tukey post hoc tests or Bonferroni post hoc tests. All data are expressed as the mean±SEM. * P<0.05, ** P<0.01 vs Control group, # P<0.05 vs T1DM group.

Figure 3

CT inhibits apoptosis of endothelial cells in vivo and in vitro (A,B) TUNEL staining in carotid arteries ( n=6) and HUVECs ( n=3). Scale bar: 50 μm. (C) Quantitative analysis of positive cell apoptosis rate in carotid arteries ( n=6). (D) Quantitative analysis of positive cell apoptosis rate in HUVECs ( n=3). Data were analyzed by one-way ANOVA by Tukey post hoc tests or Bonferroni post hoc tests. All data are expressed as the mean±SEM. * P<0.05, *** P<0.001 vs Control group, # P<0.05, ### P<0.001 vs T1DM group or H 2O 2 group.

Figure 4

The interaction of Smurf2 with the GCH1 protein was determined by co-immunoprecipitation experiments (A) CT suppressed the protein expressions of Smurf2 and GCH1 in HUVECs ( n=3). (B,C) Quantitative analysis of (A) ( n=3). (D) The interaction of Smurf2 with GCH1 was determined by reciprocal coIP assay in HUVECs. Input groups were used as an internal protein loading control ( n=3). (E) Supplementation with the proteasome inhibitor MG132 can prevents Smurf2-induced degradation of GCH1 ( n=3). (F) Quantitative analysis of (E) ( n=3). (G) Immunofluorescence staining of eNOS in carotid arteries ( n=6). Scale bar: 50 μm. Data were analyzed by one-way ANOVA by Tukey post hoc tests or Bonferroni post hoc tests. All data are expressed as the mean±SEM. * P<0.05 vs Control group, # P<0.05, ## P<0.01 vs T1DM group or H 2O 2 group.