Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Diabetes. Jun 15, 2024; 15(6): 1317-1339
Published online Jun 15, 2024. doi: 10.4239/wjd.v15.i6.1317
Heyingwuzi formulation alleviates diabetic retinopathy by promoting mitophagy via the HIF-1α/BNIP3/NIX axis
Jia-Jun Wu, Shu-Yan Zhang, Lin Mu, Zhi-Guo Dong, Yin-Jian Zhang
Jia-Jun Wu, Shu-Yan Zhang, Zhi-Guo Dong, Yin-Jian Zhang, Department of Ophthalmology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
Lin Mu, Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China
Co-first authors: Jia-jun Wu and Shu-yan Zhang.
Author contributions: Jia-jun Wu and Shu-yan Zhang contributed equally to this study, they are co-first authors of this manuscript. Wu JJ, Zhang SY, and Zhang YJ conceived and designed the experiments, and performed the experiments; Wu JJ, Mu L, and Dong ZG analyzed the data; Zhang SY and Mu L contributed to the reagents and materials; Wu JJ wrote the manuscript; and all authors have read and approved the final manuscript.
Supported by the National Key Research and Development Project of China, No. 2019YFC1711605; National Natural Science Foundation of China, No. 81904257; and Medical Innovation Research Project of Science and Technology Commission of Shanghai Municipality, No. 21Y11923100.
Institutional animal care and use committee statement: All experimental procedures were by the Institutional Animal Care and Use Committee and approved by the Ethics Committee of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine approved the study (Approval No. p2022045).
Conflict-of-interest statement: All authors certify that there is no conflict of interest.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at zhangyinj@126.com.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Yin-Jian Zhang, MMed, Chief Physician, Department of Ophthalmology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, No. 725 Wanping South Road, Xuhui District, Shanghai 200032, China. zhangyinj@126.com
Received: December 26, 2023
Revised: February 22, 2024
Accepted: April 1, 2024
Published online: June 15, 2024
Processing time: 168 Days and 11.6 Hours
Abstract
BACKGROUND

Diabetic retinopathy (DR) is the primary cause of visual problems in patients with diabetes. The Heyingwuzi formulation (HYWZF) is effective against DR.

AIM

To determine the HYWZF prevention mechanisms, especially those underlying mitophagy.

METHODS

Human retinal capillary endothelial cells (HRCECs) were treated with high glucose (hg), HYWZF serum, PX-478, or Mdivi-1 in vitro. Then, cell counting kit-8, transwell, and tube formation assays were used to evaluate HRCEC proliferation, invasion, and tube formation, respectively. Transmission electron microscopy was used to assess mitochondrial morphology, and Western blotting was used to determine the protein levels. Flow cytometry was used to assess cell apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. Moreover, C57BL/6 mice were established in vivo using streptozotocin and treated with HYWZF for four weeks. Blood glucose levels and body weight were monitored continuously. Changes in retinal characteristics were evaluated using hematoxylin and eosin, tar violet, and periodic acid-Schiff staining. Protein levels in retinal tissues were determined via Western blotting, immunohistochemistry, and immunostaining.

RESULTS

HYWZF inhibited excessive ROS production, apoptosis, tube formation, and invasion in hg-induced HRCECs via mitochondrial autophagy in vitro. It increased the mRNA expression levels of BCL2-interacting protein 3 (BNIP3), FUN14 domain-containing 1, BNIP3-like (BNIP3L, also known as NIX), PARKIN, PTEN-induced kinase 1, and hypoxia-inducible factor (HIF)-1α. Moreover, it downregulated the protein levels of vascular endothelial cell growth factor and increased the light chain 3-II/I ratio. However, PX-478 and Mdivi-1 reversed these effects. Additionally, PX-478 and Mdivi-1 rescued the effects of HYWZF by decreasing oxidative stress and apoptosis and increasing mitophagy. HYWZF intervention improved the symptoms of diabetes, tissue damage, number of acellular capillaries, and oxidative stress in vivo. Furthermore, in vivo experiments confirmed the results of in vitro experiments.

CONCLUSION

HYWZF alleviated DR and associated damage by promoting mitophagy via the HIF-1α/BNIP3/NIX axis.

Keywords: Diabetic retinopathy; HIF-1α/BNIP3/NIX axis; Mitophagy; Heyingwuzi formulation; Traditional Chinese medicine

Core Tip: Our study demonstrates the protective effects and action mechanisms of the Heyingwuzi formulation against diabetic retinopathy (DR) in vivo and in vitro. Here, we developed a new treatment formulation for DR based on traditional Chinese medicine and revealed its action mechanism involving the promotion of mitophagy via regulation of the hypoxia-inducible factor-1α/BCL2-interacting protein 3 (BNIP3)/BNIP3-like (BNIP3L, also known as NIX) axis.