The Neuroprotective Role of Ginsenoside Rg1 Against Cerebral Ischemia-Reperfusion Damage Through Inhibition of Mitophagy via Blocking Mitophagosome-Lysosome Fusion

Sep 30, 2026

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Ginsenoside Rg1 shows promise in reducing cerebral ischemia–reperfusion injury, yet its molecular mechanism is not fully defined. The study established an in vitro CIRI model using SH-SY5Y and SK-N-AS cells under oxygen–glucose deprivation followed by reoxygenation (OGD/R), and an in vivo MCAO/R mouse model. Rg1 improved cell survival, mitochondrial function, and reduced injury by preserving membrane potential and lowering mitochondrial ROS. Mitophagy occurred after OGD but was inhibited during reperfusion by blocking mitophagosome–lysosome fusion, increasing accumulation of mitochondrial and mitophagy-related proteins without worsening dysfunction.

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Abstract

Introduction

Cell Proliferation ORIGINAL ARTICLE OPEN ACCESS The Neuroprotective Role of Ginsenoside Rg1 Against Cerebral Ischemia–Reperfusion Damage Through Inhibition of Mitophagy via Blocking Mitophagosome-Lysosome Fusion Lin Ai1 | Hangui Ren2,3,4 | Yuan Wang1 | Mengfan Liu2,3 | Yufei Qiu1 | Jiling Feng5 | Rongchen Dai2,3 | Wang Fu1 | Yongpeng Wang1 | Zhichao Xi2,3 | Hongxi Xu2,3 | Feng Wang1 1Department of Neurology, Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine, Shanghai, China | 2School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China | 3Engineering Research Center of Shanghai Colleges for TCM New Drug Discovery, Shanghai, China | 4Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China | 5Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Correspondence: Zhichao Xi ([xizhichao@shutcm.edu.cn](xizhichao@shutcm.edu.cn)) | Hongxi Xu ([hxxu@shutcm.edu.cn](hxxu@shutcm.edu.cn)) | Feng Wang ([dq2381@shutcm.edu.cn](dq2381@shutcm.edu.cn)) Received: 3 February 2025 | Revised: 15 May 2025 | Accepted: 19 May 2025 Funding: This work was supported by the Postdoctoral Fellowship Program and China Postdoctoral Science Foundation, GZC20241082; The Shanghai University of Traditional Chinese Medicine Budget Project, 23KFL041; Shanghai Science and Technology Commission: Scientific and Technological Innovation Action Plan Medical Innovation Research Special Project, 23Y11906400; Natural Science Foundation of Shanghai Municipality, 23ZR1448700; Shanghai Super Postdoctoral Program, 2024524; Discipline Construction of Pudong Health Bureau of Shanghai Discipline group of cerebrovascular system diseases, PWZxq2022-01; CAI Dingfang Shanghai famous traditional Chinese Medicine inheritance studio, YC-2023-0121; Shanghai “Science and Technology Innovation Action Plan” Rising Star Program, 202424YF2746900 . Keywords: cerebral ischemia–reperfusion injury | ginsenoside Rg1 | ischemic stroke | mitophagosome-lysosome fusion | mitophagy ABSTRACT Ginsenoside Rg1 has shown promise in ameliorating cerebral ischemia–reperfusion injury (CIRI). However, its precise molecular mechanisms remain unclear. In this study, an in vitro CIRI model was established using SH-SY5Y and SK-N-AS neuronal cell lines subjected to oxygen–glucose deprivation followed by reoxygenation (OGD/R) . For the in vivo model, C57BL/6J mice underwent middle cerebral artery occlusion and subsequent reperfusion (MCAO/R). The protective effects of Rg1 against OGD/R injury were analysed using the CCK-8 assay and the PI exclusion method. The in vivo neuroprotective effects of Rg1 against CIRI were evaluated using various assessments, including brain blood flow, neurological deficits, behavioural tests, TTC, H&E, Nissl and TUNEL staining. Mitophagy was assessed by detecting mitophagy-initiating proteins via Western blotting, transmission electron microscopy, immunohistochemistry and immunofluorescence staining. Additionally, mitochondrial function was assessed by ATP measurement, the JC-1 assay and MitoSOX-based flow cytometry. Our results show that Rg1 significantly mitigated cell death caused by OGD/R and substantially enhanced cell viability in vitro. Moreover, Rg1 alleviated OGD/R-induced mitochondrial dysfunction, as indicated by preserved mitochondrial membrane potential and decreased mitochondrial ROS levels. Mitophagy was induced after OGD treatment, which was subsequently inhibited by Rg1 during reperfusion. Mechanistically, Rg1 disrupted the fusion of mitophagosomes with lysosomes rather than inhibiting mitophagy initiation, leading to an accumulation of mitochondrial proteins and mitophagy-initiating proteins. Notably, prolonged inhibition of mitophagy by Rg1 did not induce cytotoxicity or exacerbate mitochondrial dysfunction. Furthermore, administration of Rg1 in MCAO/R mice significa

FAQs

What models were used to study Rg1 against cerebral ischemia–reperfusion damage?

An in vitro OGD/R model was created using SH-SY5Y and SK-N-AS neuronal cell lines. An in vivo MCAO/R model was performed in C57BL/6J mice.

How did Rg1 affect cell survival and mitochondrial injury in vitro?

Rg1 significantly reduced OGD/R-induced cell death and improved cell viability. It also alleviated mitochondrial dysfunction, shown by preserved mitochondrial membrane potential and decreased mitochondrial ROS.

What specific step of mitophagy is inhibited by Rg1?

Rg1 disrupted mitophagosome–lysosome fusion rather than blocking mitophagy initiation, leading to accumulation of mitochondrial and mitophagy-initiating proteins.

Sep 30, 2026

Uploaded by Patrick