Melatonin Inhibited The Mpp⁺ Induced Apoptosis Of Mn9d Cells Via Upregulating Uqcrc1

Background: Parkinson’s disease（PD）is the second largest neurodegenerative disease,causing motor symptoms such asstatic tremor,bradykinesia,postural disturbance,and stiffness,besides nonmotor symptoms such as cognitive dysfunction and depression.The perioperative period is an important cause of the morbidity of PD patients,and preoperative sleep disturbance and circadian rhythm disorders will increase the risk of anesthesia.With the aggravation of aging in China,more and more PD patients need to receive anesthesia.Improving the nonmotor symptoms of PD patients in the perioperative period will help reduce postoperative cognitive dysfunction and accelerate recovery.Melatonin is an endogenous indoleamine hormone,mainly secreted by the pineal gland.It is clinically used to regulate biological rhythms and improve cognitive dysfunction.At the same time,melatonin has many biological functions,including anti-tumor,anti-inflammatory,anti-oxidant.What melatonin protect neurons makes it to be a potential treatment strategy for many neurological disorder diseases.However,the molecular mechanism of melatonin in the treatment of Parkinson’s disease is still unclear.Through bio-informatics analysis,it was found that the change of ubiquinol-cytochrome c reductase core protein 1（UQCRC1）in Parkinson’s disease play an important role in the disease.Located in the inner mitochondrial membrane,UQCRC1 is a key subunit of the mitochondrial respiratory chain complex III,involved in electron transport and ATP generation.Melatonin has the property of being able to pass through the mitochondrial membrane,so whether melatonin plays a neuroprotective role by regulating UQCRC1 deserves further investigation.Objective: Our research’s aim is to explore the impact and mechanism of melatonin on MPP+-induced the neuronal damage of MN9 D cells.Methods: The mouse dopaminergic neuron MN9 D cells were selected as the research object,and the MN9 D cells were treated with MPP+ to construct a PD cell model.The cytotoxicity of MPP+ and the neuroprotective effect of melatonin were detected by CCK-8 method,and the appropriate drug concentration was screened for subsequent experiments.According to the previous results,200 μM MPP+ was used to construct a PD cell model.After MPP+ pretreated for 12 h,the final concentration of200 μM melatonin was added for treatment,and the co-culture was continued for 24 h.Cell viability was detected by CCK-8;the cell morphology in each group was observed and compared under light microscope;Bax,Bcl-2,Cyt C and Cleaved-Caspase-3protein expressions were detected by Western-blot;apoptosis level was detected by Hoechst33258/PI staining.Mitochondrial reactive oxygen species detection kit and mitochondrial membrane potential detection kit were used to assess cellular mitochondrial function;western-blot and immunofluorescence were used to detect the expression of UQCRC1.To further explore the relationship between melatonin’s inhibition of MPP+-induced apoptosis and UQCRC1,si RNA was used to downregulate the expression of UQCRC1,and the apoptosis level was detected.Results:（1）The cell viability of MN9 D cells was significantly decreased after MPP+treatment in a dose-dependent manner when compared to the control group（P<0.05）,MT treatment could significantly inhibit the MPP+-induced cell viability decrease in a dose-dependent manner（P<0.05）.The morphology of the cells was observed after treatment with MPP+,including that the cells became round,the protrusions were decreased,and the number of cell was decreased.Compared with the MPP+ group,the cell state of the treatment group was improved.（2）Compared with the control group,the number of apoptotic cells in the MPP+group increased.Western-blot results showed that MPP+ up-regulated Bax,Cyt C and Cleaved-Caspase-3 protein expressions,and down-regulated Bcl-2.In addition,the results of immunofluorescence showed that MPP+ increased the co-localization of Cyt C with mitochondria.Melatonin treatment group could inhibit MPP+-induced apoptosis.（3）Compared with the control group,MPP+ increased the generation of reactive oxygen species in MN9 D cells and decreased mitochondrial membrane potential,thereby inducing mitochondrial dysfunction,while melatonin treatment ameliorated the damage of mitochondrial function in MN9 D cells by MPP+.（4）Compared with the control group,the expression of UQCRC1 in the MPP+group was decreased,and its expression was up-regulated after melatonin treatment.After suppressing UQCRC1 by si RNA,the expression of UQCRC1 in the melatonin group and the melatonin-treated group decreased,while the expressions of Bax,Cyt C and Cleaved-Caspase-3 were increased,and the expression of Bcl-2 was decreased.Conclusion: Melatonin attenuates MPP+-induced MN9 D cell damage via inhibiting the decline of cell viability,protecting neuronal morphology,reducing apoptosis,and mitigating mitochondrial damage.Its potential mechanism of such functions is achieved by up-regulating the expression of UQCRC1.