Icariside II induces rapid phosphorylation of endothelial nitric oxide synthase via multiple signaling pathways

Icariside II, as a favonoid compound derived from epimedium, has been proved to involed in a variety of biological and pharmacological effects such as anti-inflammatory, anti-osteoporosis, anti-oxidation, anti-aging, and anti-cancer but its mechanism is unclear, especially in terms of its effect on post-transcriptional modification of endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS plays an important role in the synthesis of nitric oxide in endothelial cells, which is closely related to erectile dysfunction, atherosclerosis, Alzheimer's disease, and other diseases. Our study aims to investigate the effect and mechanism of Icariside II on the rapid phosphorylation of eNOS. In this study, human umbilical vein endothelial cells (HUVECs) were stimulated with Icariside II in the presence or absence of multiple inhibitors (1 µM), including LY294002 (PI3K-inhibitor), MK-2206 (AKT-inhibitor), Bisindolylmaleimide X (AMPK-inhibitor), H-89 (CaMKII-inhibitor), KN-62 (PKA-inhibitor), Dorsomorphin (PKC-inhibitor). The proliferation of HUVECs was assessed using cell counting kit-8 (CCK-8). The release of nitric oxide (NO) within HUVECs was detected via fluorescence probe (DAF-FM). Western blot was used to examine the effect of Icariside II on the expression of eNOS, phosphorylation of eNOS, and common signaling pathways proteins. In this study, Icariside II was found to promote the cell proliferation and rapid NO release in HUVECs. The phosphorylation of eNOS-Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 min (p < 0.05). Meanwhile, the phosphorylation of eNOS-Thr495 was significantly decreased after 45 min of stimulation (p < 0.05). Following the intervention with multiple inhibitors, it was found that MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) could significantly inhibit the phosphorylation of eNOS-Ser1177 caused by Icariside II (p < 0.05), while MK-2206, LY294002, and Bisindolylmaleimide X reversed the alleviated phosphorylation of eNOS-Thr495. We concluded that Icariside can regulate rapid phosphorylation of eNOS- Ser1177 and eNOS-Thr495 via multiple signaling pathways, resulting in the up-regulation of eNOS and the increased release of NO.

 

Comments:

It appears that you have provided a summary of a research study involving Icariside II and its effects on endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs). This study aimed to investigate the mechanism by which Icariside II affects the phosphorylation of eNOS and its subsequent impact on nitric oxide (NO) production in endothelial cells. Here's a breakdown of the key findings and implications of the study:

1. **Icariside II Promotes Cell Proliferation and NO Release in HUVECs**:
   - Icariside II was found to promote the proliferation of HUVECs, which suggests its potential role in endothelial cell growth and maintenance.
   - It also led to the rapid release of nitric oxide (NO) within HUVECs. NO is an important molecule that plays a crucial role in regulating blood vessel function and overall cardiovascular health.

2. **Phosphorylation of eNOS-Ser1177 and eNOS-Thr495**:
   - Phosphorylation of eNOS at specific sites is a critical regulatory mechanism for its activity. In this study, Icariside II had different effects on the phosphorylation of eNOS at two distinct sites:
     - Phosphorylation of eNOS at Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 minutes.
     - Phosphorylation of eNOS at Thr495 was significantly decreased after 45 minutes of stimulation.

3. **Inhibitor Experiments**:
   - The study involved the use of various inhibitors to investigate the signaling pathways involved in Icariside II's effects on eNOS phosphorylation.
   - MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) were among the inhibitors tested.
   - These inhibitors were found to have significant effects on the phosphorylation of eNOS at Ser1177, indicating that multiple signaling pathways are involved in this process.
   - MK-2206, LY294002, and Bisindolylmaleimide X also reversed the decreased phosphorylation of eNOS at Thr495, suggesting a complex interplay between these pathways.

4. **Conclusion**:
   - The study concludes that Icariside II can regulate the rapid phosphorylation of eNOS at Ser1177 and Thr495 via multiple signaling pathways.
   - This regulation results in an up-regulation of eNOS and an increased release of NO.
   - The findings suggest that Icariside II may have potential therapeutic implications in conditions related to endothelial dysfunction, such as erectile dysfunction, atherosclerosis, and other cardiovascular diseases.

In summary, this research provides valuable insights into the molecular mechanisms by which Icariside II influences endothelial nitric oxide synthase activity and NO production in HUVECs. Further studies may be needed to explore the clinical relevance and potential therapeutic applications of these findings in various diseases and conditions.