Role And Mechanism Of Cytarabine In Promoting Mitophagy Via AMPK/PINK1/Parkin Pathway In A Model Of Parkinson’s Disease

Background:Parkinson’s disease(PD)has become the second most prevalent neurodegenerative disease after Alzheimer’s disease.Current treatment strategies for PD aim to alleviate the symptomatic features of the disease rather than addressing the complex pathology at its root.Therefore,the search for therapeutic agents with a multi-pronged effect on PD progression is envisaged as an effective tactic.Cytarabine has a long history of being demonstrated to prevent dopaminergic neuronal death at on-neurotoxic concentrations and shows promise as a potential PD therapy.We hypothesized that cytarabine may have potential therapeutic value in PD.Objective:To establish an in vitro model of PD using rotenone(ROT)-induced SH-SY5Y cell mimicry,to observe whether cytarabine has a protective effect on ROT-induced SH-SY5Y cell injury,and to explore the effects of cytarabine on PD and its potential mechanisms.Methods:(1)SH-SY5Y cells were treated with different concentrations of cytarabine,and its active effect on neuronal cells was detected by the resazurin assay.(2)SH-SY5Y cells were treated with different concentrations of ROT for 24 h to establish an in vitro model of PD,and the optimal concentration of ROT was determined by the resazurin assay.(3)A control group,a ROT group,and low,medium,and high dose groups of cytarabine were established,and cell activity in each group was assessed using the resazurin assay,and cell morphology was observed under the microscope.(4)Correlation assay of the oxidative stress effect of cytarabine on ROT-induced SH-SY5Y cells:the total antioxidant capacity,SOD content,GSH content,MDA content,and NAD~+/NADH ratio of different groups of cells were measured by the kit.(5)Assays for cytarabine’s effect on ROT-induced mitochondrial dysfunction in SH-SY5Y cells:Mito-Tracker Red CMXRos for mitochondrial viability and morphology;JC-1 for mitochondrial membrane potential(MMP);Seahorse XF Cell Mito Stress Test Kit for mitochondrial function.(6)Correlation assay of the effect of cytarabine on ROT-induced mitochondrial autophagic pathway in SH-SY5Y cells:Western blot was used to detect expression levels of AMPK,p-AMPK,PINK1,Parkin,VADC1,and DJ-1 in different groups of cells.Results:(1)The results of the resazurin assay demonstrated no toxic effects on SH-SY5Y cells at concentrations ranging from 2.5μM to 80μM(P<0.05).(2)Based on the edelweiss assay findings,ROT at 20μM was chosen as the best concentration for the PD model(P<0.001).(3)The resazurin assay findings revealed that cytarabine reduced the ROT-induced decrease in cell activity,number,and morphological alterations(P<0.001);the YO-PRO-1/PI kit results demonstrated that cytarabine reversed ROT-induced apoptosis and necrosis.(4)According to the oxidative stress-related kit,cytarabine significantly improved the total antioxidant capacity,SOD content,GSH content,decreased NAD~+/NADH content and increased MDA content in ROT-induced SH-SY5Y cells(P<0.001),indicating that cytarabine attenuated ROT-induced SH-SY5Y cell damage by resisting oxidative stress.(5)The results of JC-1,Mito-Tracker Red CMXRos fluorescence staining,and Seahorse XF Cell Mito Stress Test Kit showed that cytarabine significantly ameliorated ROT-induced MMP depletion,reduced mitochondrial viability(P<0.001),altered mitochondrial morphology(P<0.05),and decreased mitochondrial function(P<0.05),indicating that cytarabine attenuated ROT-induced damage in SH-SY5Y cells through anti-mitochondrial dysfunction.(6)Western blot analysis revealed that cytarabine significantly increased the protein content of p-AMPK,PINK1,Parkin,DJ-1,and VDAC1in ROT-induced SH-SY5Y cells(P<0.05),whereas the AMPK inhibitor Compound C(CC)inhibited this trend.At the same time,cytarabine’s residual protective properties were diminished.Conclusion:Our findings support cytarabine’s possible neuroprotective impact on Parkinson’s disease.It promotes mitophagy by activating the AMPK/PINK1/Parkin signaling pathway,counteracting ROT-induced oxidative stress and mitochondrial dysfunction,and realizing its neuroprotective effects.Cytarabine,as a beneficial alternative treatment,has the potential to be included in the clinical practice of Parkinson’s disease soon.