Advances in nano research

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Effects of exploration and molecular mechanism of CsV on eNOS and vascular endothelial functions

  • Zuo, Deyu (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Jiang, Heng (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Yi, Shixiong (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Fu, Yang (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Xie, Lei (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Peng, Qifeng (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Liu, Pei (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Zhou, Jie (Department of Rehabilitation Medicine, Chongqing Traditional Chinese Medicine Hospital) ;
  • Li, Xunjia (Department of Nephrology, Chongqing Traditional Chinese Medicine Hospital)
  • Received : 2022.01.27
  • Accepted : 2022.03.06
  • Published : 2022.05.25
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Abstract

This study aimed to investigate the effects and potential mechanisms of Chikusetsusaponin V (CsV) on endothelial nitric oxide synthase (eNOS) and vascular endothelial cell functions. Different concentrations of CsV were added to animal models, bovine aorta endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) cultured in vitro. qPCR, Western blotting (WB), and B ultrasound were performed to explore the effects of CsV on mouse endothelial cell functions, vascular stiffness and cellular eNOS mRNA, protein expression and NO release. Bioinformatics analysis, network pharmacology, molecular docking and protein mass spectrometry analysis were conducted to jointly predict the upstream transcription factors of eNOS. Furthermore, pulldown and ChIP and dual luciferase assays were employed for subsequent verification. At the presence or absence of CsV stimulation, either overexpression or knockdown of purine rich element binding protein A (PURA) was conducted, and PCR assay was employed to detect PURA and eNOS mRNA expressions, Western blot was used to detect PURA and eNOS protein expressions, cell NO release and serum NO levels. Tube formation experiment was conducted to detect the tube forming capability of HUVECs cells. The animal vasodilation function test detected the vasodilation functions. Ultrasonic detection was performed to determine the mouse aortic arch pulse wave velocity to identify aortic stiffness. CsV stimulus on bovine aortic cells revealed that CsV could upregulate eNOS protein levels in vascular endothelial cells in a concentration and time dependent manner. The expression levels of eNOS mRNA and phosphorylation sites Ser1177, Ser633 and Thr495 increased significantly after CsV stimulation. Meanwhile, CsV could also enhance the tube forming capability of HUVECs cells. Following the mice were gavaged using CsV, the eNOS protein level of mouse aortic endothelial cells was upregulated in a concentration- and time-dependent manner, and serum NO release and vasodilation ability were simultaneously elevated whereas arterial stiffness was alleviated. The pulldown, ChIP and dual luciferase assays demonstrated that PURA could bind to the eNOS promoter and facilitate the transcription of eNOS. Under the conditions of presence or absence of CsV stimulation, overexpression or knockdown of PURA indicated that the effect of CsV on vascular endothelial function and eNOS was weakened following PURA gene silence, whereas overexpression of PURA gene could enhance the effect of CsV upregulating eNOS expression. CsV could promote NO release from endothelial cells by upregulating the expression of PURA/eNOS pathway, improve endothelial cell functions, enhance vasodilation capability, and alleviate vessel stiffness. The present study plays a role in offering a theoretical basis for the development and application of CsV in vascular function improvement, and it also provides a more comprehensive understanding of the pharmacodynamics of CsV.

Keywords

Acknowledgement

This work was supported by Chongqing Postdoctoral Innovative Talent Support Program (CQBX2021001) and the Special Project for Performance Incentive and Guidance of Scientific Research Institutions in Chongqing (cstc2021 jxjl130020, cstc2021jxjl130002).

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