Sains Malaysiana 53(3)(2024): 591-604

http://doi.org/10.17576/jsm-2024-5303-09

Thymoquinone Reverses Homocysteine-Induced Endothelial Dysfunction via Inhibition of Endoplasmic Reticulum-Stress Induced Oxidative Stress Pathway

(Timoquinon Membalikkan Disfungsi Endotelium Aruhan Homosistein ​​melalui Perencatan Laluan Tekanan Oksidatif Aruhan Retikulum Endoplasma)

SITI SARAH M. SOFIULLAH¹, DHARMANI DEVI MURUGAN², SUHAILA ABD MUID^3,4, WU YUAN SENG^5,6, NOR HISAM ZAMAKSHSHARI ⁷, QUAN FU GAN⁸, MELONNEY PATRICK ^3,4, NORASIKIN AB AZIS⁹, SRINIVASA RAO SIRASANAGANDLA¹⁰ & CHOY KER WOON^1,*

¹Department of Anatomy, Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Jalan Hospital, 47000 Sungai Buloh, Petaling Jaya, Selangor, Malaysia
²Department of Pharmacology, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia
³Institute of Pathology, Laboratory and Forensic Medicine (I-PperForM), Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Jalan Hospital, 47000 Sungai Buloh, Petaling Jaya, Selangor, Malaysia
⁴Department of Biochemistry, Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Jalan Hospital, 47000 Sungai Buloh, Petaling Jaya, Selangor, Malaysia
⁵Centre for Virus and Vaccine Research, School of Medical and Life Sciences, Sunway University, 47500 Subang Jaya, Selangor, Malaysia
⁶Department of Biological Sciences, School of Medical and Life Sciences, Sunway University, 47500 Subang Jaya, Selangor, Malaysia
⁷Department of Chemistry, Faculty of Resources Science and Technology, University Malaysia Sarawak, Kota Samarahan, 94300, Kuching, Sarawak, Malaysia
⁸Pre-clinical Department, Faculty of Medicine and Health Science, UTAR Sg Long Campus, 43000 Kajang, Selangor, Malaysia
⁹Department of Pharmacology, Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, 47000, Selangor, Malaysia
¹⁰Department of Human & Clinical Anatomy, College of Medicine & Health Sciences, Sultan Qaboos University, PO Box 35, PC 123, Al-Khoud, Muscat, Oman

Received: 31 July 2023/Accepted: 9 February 2024

Abstract

Hyperhomocysteinemia causes endoplasmic reticulum (ER) stress, which elevates reactive oxygen species (ROS) and induces endothelial dysfunction, the hallmark of cardiovascular diseases. Nigella sativa seeds contain thymoquinone (TQ), a cardioprotective bioactive component. Nevertheless, research on investigating the effectiveness of TQ in preventing endothelial dysfunction caused by homocysteine (Hcy) is scarce. Therefore, the purpose of this work was to examine the role of TQ in restoring Hcy-induced endothelial dysfunction as well as the mechanisms behind this role. Male Sprague-Dawley (SD) rat aortas were isolated and then co-treated in an organ bath with Hcy and TQ, tauroursodeoxycholic acid (TUDCA), apocynin, or Tempol to examine vascular function. Furthermore, human umbilical vein endothelial cells (HUVECs) were treated with Hcy and TQ, Tempol, apocynin, TUDCA or H₂O₂ to determine the cell viability via a phase contrast microscope and dye exclusion test. ER stress pathway involvement, ROS and NO bioavailability were investigated using immunoassays and fluorescence staining, respectively. The binding affinity of TQ to GRP78 has been identified using molecular docking. According to our findings, Hcy hindered endothelium-dependent relaxation in an isolated aorta and caused apoptosis in HUVECs. TQ, TUDCA, Tempol, and apocynin were able to counteract these negative effects. In HUVECs, treatment with TQ decreased ROS levels, increased NO bioavailability, and decreased GRP78 and NOX4 protein. According to the molecular docking study outcomes, TQ could attach to GRP78 effectively via a hydrogen bond and a hydrophobic connection to the amino acid at GRP78 ATP binding pocket. Taken together, the findings show that TQ protected endothelial function caused by Hcy via inhibiting ER stress-mediated ROS and eNOS uncoupling.

Keywords: Endoplasmic reticulum stress; endothelial dysfunction; homocysteine; oxidative stress; thymoquinone

Abstrak

Hiperhomosisteinemia meninggikan tekanan retikulum endoplasma (RE) yang boleh meningkatkan spesies oksigen reaktif (SOR), yang membawa kepada disfungsi endotelium sel dan penyakit kardiovaskular. BijiNigella sativa mengandungi timoquinon (TQ), komponen bioaktif berkardio-protektif. Walau bagaimanapun, tiada kajian yang menilai kesan TQ terhadap disfungsi endotelial yang disebabkan oleh homosistein (Hcy). Oleh itu, penyelidikan ini bertujuan untuk mengkaji kesan dan mekanisme TQ dalam menormalkan disfungsi endotelial yang disebabkan oleh Hcy. Aorta diasingkan daripada tikus Sprague-Dawley (SD) jantan yang diinkubasi dengan Hcy dan dirawat bersama dengan atau tanpa TQ, TUDCA, apocynin atau Tempol dalam mandian organ untuk mengkaji fungsi vaskular. Di samping itu, sel endotelial vena umbilik manusia (HUVECs) diinkubasi dengan Hcy dan TQ, Tempol, apocynin, TUDCA atau H₂O₂ untuk menilai keviabelan sel dengan menggunakan mikroskop kontras fasa dan ujian pengecualian pewarna. Penglibatan laluan tekanan ER, ROS dan NO bioketersediaan diakses masing-masing melalui immunoasai dan pewarnaan pendarfluor. Dok molekul telah dilakukan untuk menilai pertalian mengikat TQ kepada GRP78. Keputusan kami mendedahkan bahawa Hcy merosakkan disfungsi endotelial dalam aorta dan apoptosis dalam HUVECs. Kesan ini telah dinormalkan oleh TQ, TUDCA, Tempol dan apocynin. Rawatan dengan TQ mengurangkan tahap ROS, meningkatkan bioketersediaan NO serta mengurangkan protein GRP78 dan NOX4 dalam HUVECs. Hasil kajian dok molekul menunjukkan bahawa TQ boleh mengikat dengan baik kepada GRP78 melalui ikatan hidrogen dan interaksi hidrofobik dengan asid amino pada poket pengikat ATP GRP78. Kesimpulannya, TQ membaikipulih fungsi endotelial yang dirosakkan oleh Hcy melalui perencatan ROS pengantara tekanan ER dan meningkatkan bioketersediaan NO.

Kata kunci: Disfungsi endotelium; homosistein; tekanan oksidatif; tekanan retikulum endoplasma; timoquinon

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*Corresponding author; email: choykerwoon@uitm.edu.my