Saffron과 Passiflora incarnata L.의 항산화 작용에 의한 Fetuin-A 발현 증가 및 파킨슨병 모델에서의 소뇌 Purkinje 세포 보호 효과 = Antioxidant effects of Saffron and Passiflora incarnata L. enhance Fetuin-A expression and protect cerebellar Purkinje cells in a Parkinson’s disease mouse model

다국어 초록 (Multilingual Abstract)

Aging is recognized as a major risk factor for neurodegenerative diseases, among which Parkinson’s disease (PD) is characterized by motor impairments and the progressive loss of dopaminergic neurons in the substantia nigra. While earlier research has primarily focused on alterations within the nigrostriatal pathway, growing evidence suggests that structural and functional abnormalities of the cerebellum also contribute to the clinical manifestations of PD. In particular, the vulnerability and degeneration of Purkinje cells have emerged as critical pathological features associated with PD-related motor dysfunction.
Fetuin-A, a multifunctional glycoprotein predominantly synthesized in the liver but also expressed in the central nervous system, has been implicated as a regulatory factor reflecting neuronal injury and prognosis. Previous findings demonstrated that reduced expression of Fetuin-A in cerebellar Purkinje cells is closely associated with morphological abnormalities and increased cellular vulnerability in PD models.
Resveratrol (RES), Saffron-derived antioxidant (SDA), and Passiflora incarnata L.(PI) are natural extracts with potent antioxidant activities, known to enhance neuronal survival by scavenging reactive oxygen species (ROS), preserving mitochondrial function, and attenuating inflammatory signaling. Considering that oxidative stress contributes to both the reduction of Fetuin-A and increased susceptibility of Purkinje cells in PD, these extracts may exert protective effects through the modulation of Fetuin-A expression.
In the present study, we evaluated the effects of these three natural extracts on Fetuin-A expression and Purkinje cell preservation in an MPTP-induced PD mouse model. Additionally, primary cerebellar neurons derived from E18 embryos were used to assess the antioxidant and neuroprotective effects of each extract under MPP⁺-induced PD-like conditions in vitro.
Pretreatment with natural extracts significantly attenuated the MPTP-induced downregulation of Fetuin-A in cerebellar Purkinje cells and reduced the number of TUNEL-positive and cleaved caspase-3 (CC3)-positive cells, indicating suppression of apoptotic activation. The proportion of Fluoro-Jade C–positive Purkinje cells was also markedly reduced, demonstrating a strong inhibitory effect on Purkinje cell degeneration. These protective effects were accompanied by partial preservation of motor function. Consistent results were observed in vitro, where extract pretreatment preserved Fetuin-A expression and reduced ROS accumulation in MPP⁺-treated neurons.
Collectively, these findings suggest that natural extracts with antioxidant properties mitigate the MPTP-induced decrease in Fetuin-A expression and contribute to the preservation of Purkinje cell integrity. This study provides evidence supporting the potential of these natural compounds as preventive therapeutic strategies targeting cerebellar pathology in Parkinson’s disease.

목차 (Table of Contents)

• List of tables iii
• List of figures iiii
• List of Abbreviations vi
• Abstract viii
• I. Introduction 1
• II. Materials and Methods 3
• 1. Preparation and oral administration of natural extracts 3
• 2. Experimental animals and administration schedule of MPTP and natural extracts 3
• 3. Behavioral test for motor function assessment 6
• 4. Tissue Preparation for histological and protein analysis 6
• 5. Nissl staining for morphological evaluation of cerebellar
• Purkinje cells and substantia nigra neurons 7
• 6. Immunohistochemistry for assessing neuronal activity and apoptosis in cerebellar Purkinje cells 8
• 7. Antibodies 9
• 8. Immunofluorescence staining for detection of Fetuin-A expression in cerebellar Purkinje cells 11
• 9. TUNEL assay for detection of DNA fragmentation in cerebellar Purkinje cells 11
• 10. Fluoro-Jade C staining for detecting degenerating Purkinje cells 12
• 11. Western blot analysis for detection of Fetuin-A expression in cerebellar tissue 12
• 12. Primary culture of cerebellar Purkinje cells 13
• 13. Tretment of primary cultured Purkinje cells with natural extracts and MPP+ 15
• 14. Assessment of cell viability following natural extracts and MPP+ treatment using the MTT assay 15
• 15. Immunocytochemistry for Fetuin-A and Calbindin in primary cerebellar neurons 16
• 16. Measurement of intracellular reactive oxygen species using the DCFDA assay 17
• 17. Statistical analysis 18
• III. Results 19
• 1. Changes in body weight, food intake, and water intake during MPTP and natural extracts treatment 19
• 2. MPTP-induced loss of dopaminergic neurons in the substantia nigra 19
• 3. MPTP-induced neuronal death in the substantia nigra was shown using Nissl staining 20
• 4. Natural extracts alleviated the MPTP-treated mice’s motor function 20
• 5. Natural extracts mitigated the diminution in Purkinje cell density per unit length 21
• 6. Natural extracts mitigated the MPTP-induced decrement of Fetuin-A expression in cerebellar Purkinje cells 21
• 7. MPTP-induced reduction of extract-mediated enhancement in Fetuin-A levels were documented, albeit without achieving statistical significance 22
• 8. Cleaved caspase-3 expression was elevated in the MPTP group and attenuated by natural extract treatment 22
• 9. TUNEL-positive Purkinje cells were increased in the MPTP group and reduced by all extract treatments 23
• 10. Natural extracts reduced MPTP-induced Fluoro-Jade C-positive Purkinje cell degeneration 23
• 11. Effect of natural extract pretreatment on MPP+-induced cytotoxicity 24
• 12. Effects of natural extract pretreatment on MPP+-induced reduction of Fetuin-A expression in primary cerebellar Purkinje cells 26
• 13. Pretreatment with natural extracts attenuates MPP+-induced ROS accumulation in primary cerebellar neurons 26
• IV. Discussion 48
• V. References 53
• VI. Supplement 57
• 국문요약 62
• 감사의 글 64