Isorhamnetin Ameliorates 6-OHDA-Induced SH-SY5Y Cell Damage by Targeting FosL1 to Activate the Akt/mTOR Pathway

Academic Background

Parkinson’s Disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra Pars Compacta (SNpc). This neuronal loss is closely associated with mitochondrial dysfunction and oxidative stress. 6-Hydroxydopamine (6-OHDA), a metabolite of dopamine, can induce the accumulation of Reactive Oxygen Species (ROS), leading to damage in dopaminergic neurons. Therefore, studying how to mitigate 6-OHDA-induced neuronal damage by inhibiting oxidative stress, cellular senescence, and apoptosis is of great significance for the treatment of Parkinson’s disease.

Isorhamnetin (ISO), a flavonoid with antioxidant and anti-apoptotic properties, has been shown to have neuroprotective effects in various disease models. However, the mechanism of ISO in Parkinson’s disease remains unclear. This study aims to explore whether ISO alleviates 6-OHDA-induced SH-SY5Y cell damage by targeting FosL1 to activate the Akt/mTOR signaling pathway.

Source of the Paper

This paper was co-authored by Shaochen Qin, Xiaobo Wan, Shanshan Kong, Kunmei Xu, Jungong Jin, Shiming He, and Mingsheng Chen. The authors are affiliated with the Department of Neurology and Acupuncture at the Affiliated Hospital of Shanxi University of Chinese Medicine, as well as the Department of Neurosurgery at Xi’an International Medical Center Hospital. The paper was first published on November 19, 2024, in the Journal of Neurophysiology, with the DOI 10.1152/jn.00351.2024.

Research Process

1. Cell Culture and Treatment

The study used human SH-SY5Y cells (purchased from the American Type Culture Collection, CRL-2266), cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When cell density reached 80%, the cells were treated with different concentrations of 6-OHDA (25, 50, 100 μM) and/or ISO (12.5, 25, 50 μM) for 12 hours. Finally, 100 μM 6-OHDA and 50 μM ISO were selected for subsequent experiments.

2. Cell Viability Assay

Cell viability was detected using the CCK-8 kit. The results showed that 100 μM 6-OHDA significantly inhibited the viability of SH-SY5Y cells, while ISO significantly improved the 6-OHDA-induced decrease in cell viability.

3. Detection of Cellular Senescence and Apoptosis

Cellular senescence and apoptosis-related protein expression were detected through SA-β-gal staining, Western blot (WB), and immunofluorescence. The results showed that 6-OHDA significantly increased the number of SA-β-gal-positive cells and the expression of senescence-related proteins p21 and p16, while ISO significantly reduced these indicators. Additionally, ISO significantly decreased the number of apoptotic cells and the expression levels of cleaved caspase-3 and Bax induced by 6-OHDA.

4. Oxidative Stress and Mitochondrial Function Detection

Intracellular ROS generation was detected using DCFH-DA staining, mitochondrial membrane potential (MMP) was assessed using JC-1 staining, and oxidative stress levels were evaluated by measuring MDA and SOD content. The results showed that 6-OHDA significantly increased ROS generation and MDA content while decreasing SOD levels and MMP, whereas ISO significantly reversed these changes.

5. FosL1 Overexpression and Akt/mTOR Signaling Pathway Detection

Molecular docking simulations suggested that ISO might target FosL1. WB results showed that 6-OHDA significantly increased FosL1 expression and decreased p-Akt and p-mTOR expression, while ISO significantly reversed these changes. FosL1 overexpression experiments further confirmed that ISO exerts neuroprotective effects by inhibiting FosL1 to activate the Akt/mTOR signaling pathway.

Key Findings

1. 6-OHDA-Induced SH-SY5Y Cell Damage: 6-OHDA significantly reduced cell viability, increased cellular senescence and apoptosis, and induced oxidative stress and mitochondrial dysfunction.
2. Neuroprotective Effects of ISO: ISO significantly improved 6-OHDA-induced decreases in cell viability, cellular senescence, and apoptosis, while alleviating oxidative stress and mitochondrial dysfunction.
3. ISO Activates the Akt/mTOR Pathway by Targeting FosL1: ISO activates the Akt/mTOR signaling pathway by inhibiting FosL1 expression, thereby exerting neuroprotective effects. FosL1 overexpression experiments further confirmed this mechanism.

Conclusion

This study reveals that ISO alleviates 6-OHDA-induced SH-SY5Y cell damage by targeting FosL1 to activate the Akt/mTOR signaling pathway. This finding provides new insights and potential therapeutic targets for the treatment of Parkinson’s disease.

Research Highlights

1. Key Discovery: ISO significantly improves 6-OHDA-induced neuronal damage by inhibiting FosL1 to activate the Akt/mTOR signaling pathway.
2. Innovative Methodology: This study is the first to use molecular docking simulations to identify FosL1 as a potential target of ISO and to experimentally validate this mechanism.
3. Application Value: As a natural compound, ISO has potential neuroprotective effects and may serve as a new strategy for treating Parkinson’s disease.

Additional Valuable Information

The experimental data from this study support the mechanism by which ISO exerts neuroprotective effects by inhibiting FosL1 to activate the Akt/mTOR signaling pathway. This discovery not only provides new insights for the treatment of Parkinson’s disease but also offers a reference for research into other neurodegenerative diseases.

---

Through this study, we have gained a deeper understanding of the neuroprotective mechanisms of ISO in Parkinson’s disease and provided important theoretical foundations for future drug development and clinical treatments.