| Parkinson's disease?PD?is a neurodegenerative disorder that occurs frequently in the elderly.It is characterized by motor dyskinesia,stiff muscle,resting tremor and postural reflex.The pathological feature of PD is selective dopamine?DA?neuron loss in the substantia nigra pars compacta?SNpc?.Currently,the pathogenesis of PD is not clear.Genetic factors,oxidative stress,mitochondrial dysfunction,apoptosis,environmental factors and iron accumulation may participate in the pathogenesis of PD.Studies have shown that the accumulation of iron in the substantia nigra?SN?is the leading cause of neuronal damage in PD.Maintaining iron homeostasis is particularly important for controlling the occurrence and development of the disease.Hepcidin,a hormone-like substance that has been found in recent years to regulate the concentration of iron in cells,plays an important role in maintaining iron homeostasis.When the body is in an iron deficient state,activated hepcidin promotes iron uptake by the small intestine and increases the cellular iron content.Conversely,when the body iron content is increased,hepcidin decreases and the excessive iron-induced damage is attenuated.The physiological function of hepcidin is that it binds to the ferroportin 1?Fpn1?,an iron transporter on the cell membrane,causing Fpn1 to undergo internalization and degrade Fpn1 thereby inhibiting iron efflux.Increased expression of hepcidin may lead to iron accumulation in cells,thereby stimulating the occurrence of oxidative stress by Fenton reaction and thus produce irreversible damage to the cell.To date,there is no effective drugs to prevent or reverse the cell damage and death during the course of PD.Therefore,it is a new research hotspot to search for effective,low toxic and less side effects drugs to treat PD in clinics.Myricetin is a natural flavonoid extracted from the leaves and barks of the Chinese bayberry.Nowadays,the anti-oxidant,anti-inflammatory and anti-cancer effects of flavonoids have aroused widespread concern.In previous studies,myricetin has been shown to have neuroprotective effects,and its mechanism may be related to anti-oxidation and anti-apoptotic effects.Myricetin may also improve chronic stress-induced depression,learning and memory loss in chronic stressed animal models.However,the exact neuroprotective mechanism of myricetin is still not yet clear.Studies have shown that myricetin can inhibir the expression of hepcidin,and the secretion of hepcidin can lead to the accumulation of iron,we hypothesized that myricetin may inhibit the production ofhepcidin,thereby triggering neuroprotective effects.However,the intrinsic mechanisms of myricetin-mediated neuroprotection in the pathogenesis of PD and the possible intracellular signal pathways involved in it is still uncertain.In this study,the neurotoxic drug rotenone was used to treat dopaminergic MES23.5cells to prepare the PD cell model.Rotenone can act on the mitochondrial respiratory chain complex I to induce the mitochondrial damage of the cells,leading to neuronal death.By using methyl thiazolyl tetrazolium?MTT?assay,flow cytometry?FCM?,real-time PCR,laser scanning confocal microscopy,western blot analysis and other research methods,in the present study,we explored the protective mechanisms of myricetin on rotenone-induced cell damage in MES23.5 cells.The results are as follows:1.After treated with different concentrations of rotenone for 24 h,the cell viability by MTT assay reached 66.1%of control in 400 n M rotenone treatment group,which was significantly different from that of the control?P?0.001?.After treated with different concentrations of myricetin(10-11 M-10-4 M),compared with 400 nM rotenone,10-6 M myricetin had the most significant protective effect?P?0.001?,and 10-10 M and 10-5 M myricetin treatment also showed a protective effect?P?0.01?,however,weaker than 10-6M myricetin treatment.So rotenone in 400 nM and myricetin in 10-6 M were used in the following experiments.2.After treated with 400 nM rotenone for 24 h,the fluorescent dye Rh 123 was used to detect the mitochondrial membrane potential.The results showed that compared with the control group,rotenone treatment significantly decreased mitochondrial membrane potential?P<0.001?,while 10-6 M myricetin treatment may alleviate this effect,compared with rotenone treatment group,the mitochondrial membrane potential increased by 9.4%in myricetin treatment group,the difference was significant?P?0.01?.3.After treated with 400 nM rotenone for 24 h,the level of reactive oxygen species?ROS?in MES23.5 cells was 2.5 times higher than that of the control group?P<0.001?.This effect was partly inhibited by 10-66 M myricetin treatment?P?0.05,compared with rotenone treatment group?.4.The expression of hepcidin mRNA in MES23.5 cells was detected by real-time fluorescence quantitative PCR.After treated with 400 nM rotenone for 24 h,the mRNA expression of hepcidin was up-regulated by 1.5-fold compared with control?P<0.001?.Myricetin in 10-6 M partly reversed this result,and showed a 39.6%reduction in mRNA expression compared with that of rotenone treatment group.The result is significantly different?P<0.05?.5.Calcein was used as an fluorescence indicator to detect the intracellular iron levels.Fluorescence quenching may reflex the iron transport ability of the cells.After 400 nM rotenone treatment for 24 h,we could not detect a significant increase in the fluorescence intensity compared with the control group.10-6 M myricetin treatment can partly reverse this effect,the fluorescence intensity was significantly increased compared with the rotenone treatment group.6.After treated with 400 nM rotenone for 24 h,the expression of Fpn1 mRNA in MES23.5 cells was decreased by 38.3%compared with the control?P?0.001?.10-6 M myricetin treatment may inhibit rotenone-induced Fpn1 mRNA down-regulation?increased by 35.9%in myricetin treatment group compared with rotenone treatment group?,the difference was significant?P<0.05?.7.After treated with 400 nM rotenone for 24 h,the protein expression of Fpn1 was determined by western blot.The results showed that compared with the control,expression of Fpn1 in rotenone treatment group decreased by 34.7%,the difference was significant?P<0.01?.Myricetin in 10-6 M treatment partly reserved this decrease by48.3%compared with rotenone treatment group.The result is significantly different?P<0.05?.8.After treated with 400 nM rotenone for 24 h,the expression of phosphorylated STAT3was determined by western blot.The results showed that the expression of p-STAT3 in rotenone treatment group increased by 47.9%compared with the control.The difference was significant?P<0.001?.10-66 M myricetin treatment inhibits rotenone-induced up-regulation of p-STAT3 expression by 28.2%.The results were significantly different?P<0.001,compared with rotenone treatment group?.9.After treated with 400 nM rotenone for 24 h,the expression of phosphorylated SMAD was determined by western blot.The results showed that the expression of p-SMAD1/5/9in rotenone treatment group increased by 42.5%compared with the control.The difference was significant?P<0.01?.10-6 M myricetin treatment inhibits rotenone-induced up-regulation of p-SMAD1/5/9 expression by 23.2%.The results were significantly different?P<0.05,compared with rotenone treatment group?.In summary,the results in the present study showed that myricetin may protect against rotenone-induced cell damage in MES23.5 cells.The mechanisms underlying myricetin-mediated neuroprotection may be attributed to myricetin-induced activation of JAK-STAT and BMP-SMAD signaling pathways,thereby inhibiting hepcidin expression,and then facilitating iron outflow.In addition,myricetin may directly inhibit rotenone-induced ROS production and protect mitochondria against rotenone-induced neurotoxicity.This study not only provides a further experimental basis for the neuroprotective mechanism of myricetin,but also provides the basis for the new use of flavonoids and the further development of new drugs for clinical prevention of PD. |
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