The Neuroprotective Effect And Mechanism Of Curcumin In Parkinson’s Disease Model By Regulating Ferroptosis Pathway

Objective: Parkinson’s disease(PD)is the second most common neurodegenerative disease in the world and the fastest growing neurodegenerative disease.The loss of dopaminergic neurons and the formation of lewy bodies in the substantia nigra are major pathological changes of Parkinson’s disease.Parkinson’s disease is characterized by motor symptoms such as tremor,rigidity,slow movement,postural gait disorders.The prevalence of PD is about 1% in people over 60 years old and more than 4% in people over 85 years old.With the aging population and longer life expectancy,the number of people with PD is increasing year by year.The disease not only seriously affects the health and quality of life of the elderly patients,but also brings a heavy economic burden to family and society.The etiology of PD is complex and the pathogenesis is not clear.Because of lack of fundamental treatment PD patients mainly rely on medication to relieve their symptoms.Therefore,it is urgent to explore new drugs and therapies for PD according to the pathogenesis of it.Recent studies suggest that ferroptosis may be involved in the development of Parkinson’s disease.Ferroptosis is a new type of programmed cell death.In terms of morphology,ferroptosis is characterized by shrinkage of mitochondria,increased membrane density and reduction or vanishing of mitochondrial cristae,which is different from apoptosis,necrosis and autophagy or the other types of cell death.Large amounts of lipid metabolites produced by iron-dependent lipid peroxidation combined with imbalanced redox reaction would promote cell death,and which are essential features of ferroptosis.Ferroptosis is mainly regulated by iron metabolism,lipid metabolism and glutathione metabolism.The accumulation of iron and lipid metabolites or the reduced glutathione(GSH)consumption all can promote ferroptosis.Recently,researchers have found that iron overload,lipid peroxide accumulation,incresed reactive oxygen species(ROS)and the reduction of GSH and glutathione peroxidase 4(GPX4)are present in Parkinson’s disease.They suggest that ferroptosis may be involved in the occurrence and development of Parkinson’s disease,and inhibiting ferroptosis is expected to be a new therapeutic direction for Parkinson’s disease.Curcumin is a kind of polyphenol compound which is extracted from the rhizome of curcuma.It is the main chemical component of curcuma and is considered as the most pharmacologically active monomer compound.Some earlier studies have confirmed that curcumin has strong antioxidant activity and lipophilic characteristics and it could penetrate the blood-brain barrier and has a protective effect against stroke and some other central nervous system diseases.In recent years,its application in preventing or delaying neurodegenerative diseases has rapidly received attention.However,whether curcumin could protect dopaminergic neuron from ferroptosis in Parkinson’s disease remains unclear.In this study,an animal and cell model of Parkinson’s disease will be established by 6-hydroxydopamine(6-OHDA),we will explore the condition of ferroptosis and investigate the effect and mechanism of curcumin on ferroptosis in Parkinson’s disease.Methods: In the first part,an animal model of Parkinson’s disease was established by brain stereotactic injection of 6-OHDA.After it was successfully established,behavioral test,HE staining,immunohistochemistry and western blot were used to verify the effectiveness of curcumin in 6-OHDA-induced Parkinson’s disease model and screened out the optimal therapeutic dose for subsequent experiments.Then,perls staining,western blots,transmission electron microscopy,malondialdehyde(MDA)and GSH kits were used to detect the content of iron,ferroptosis related proteins,MDA,GSH and the morphology of mitochondrial.They could help to explore the condition of ferroptosis and investigate the effect and mechanism of curcumin on ferroptosis in Parkinson’s disease.In the second part,firstly,erastin was used to induce ferroptosis in MN9 D cells,and trypan blue staining was used to determine whether curcumin had an inhibitory effect on ferroptosis.Then,6-OHDA was used to induce MN9 D cells to establish a Parkinson’s disease model.Western blot,transmission electron microscopy,flow cytometry,MDA and GSH kits were employed to further clarify the regulatory effect of curcumin on ferroptosis in Parkinson’s disease.In the third part,animal model of Parkinson’s disease was established,and the protein expressions of Nrf2,ATF3 and SLC7A11 were detected by western blot.Nrf2 was knocked down with small interfering RNA in6-OHDA-induced MN9 D cells,and the m RNA and protein expression of Nrf2 and SLC7A11 were detected by RT-q PCR and western blot.To explore the mechanism of curcumin in Parkinson’s disease model via regulating ferroptosis,the levels of ferroptosis related proteins,ROS and the morphology of mitochondrial in cells were detected by western blot,flow cytometry and transmission electron microscopy.Cell activity was detected by CCK8 kit.Results: 1.After modeling,the number of rotation to the healthy side in 30 min of model rats was more than 210 r,and which was more than control group.Neurons in substantia nigra on the injured side were loose and the expression of TH-positive neurons on the injured side were significantly reduced compared with the healthy side.All the above results indicated that the PD model was established successfully.2.Compared with the control group,the number of rotation to the healthy side in 30 min of the medium-dose and high-dose curcumin treatment rats was significantly reduced.The medium-dose and high-dose of curcumin treatment could improve the pathological injury of neurons in rats with Parkinson’s disease,and significantly increased the expression of TH protein in the striatum and substantia nigra of 6-OHDA-induced rats.3.Western blot results showed that the expression of GPX4,FTH1 and SLC7A11 in striatum and substantia nigra in the model group were significantly decreased compared with the control group,while the expression of PTGS2 was significantly increased.Curcumin treatment could reverse the expression of ferroptosis related proteins and which was similar to the effect of ferroptosis inhibitor Liproxstatin-1(Lip-1).4.Perls staining results showed that lots of iron deposited in the striatum and substantia nigra of 6-OHDA induced rats,transmission electron microscopy showed that mitochondria in the substantia nigra of 6-OHDA induced rats was shrinked and with increased membrane density and reduction or vanishing of mitochondrial cristae.Curcumin treatment could reduce iron deposition and improve mitochondrial morphology in neurons.5.The results of MDA and GSH detection indicated that curcumin could significantly reduce MDA and increase GSH level in substantia nigra of 6-OHDA-induced rats.6.Trypan blue staining result showed that curcumin could reduce erastin induced MN9 D cell death and it suggested that curcumin had an inhibitory effect on ferroptosis.7.Western blot results showed that curcumin could significantly increase the expression of GPX4,FTH1,SLC7A11 and decrease the expression of PTGS2 in 6-OHDA-induced MN9 D cells by a concentration-dependent manner.And compared with 100 nm and 1 μM curcumin,10μM curcumin could not only significantly decrease the level of MDA but increase GSH in 6-OHDA-induced MN9 D cells.8.Consistent with the results of inverted fluorescence microscope,flow cytometry showed that ROS in 6-OHDA-induced MN9 D cells was higher than control group,1 μM and 10 μM curcumin treatment could significantly reduce it.9.Western blot results showed that the expressions of Nrf2 and SLC7A11 in total proteins and nuclear proteins in the substantia nigra of PD rats were lower than control rats and curcumin treatment could significantly increase it.The expression of ATF3 in the nuclear protein of 6-OHDA-induced rats was higher than control rats,but curcumin treatment could not reduce or change the expression of ATF3 in the total protein or nuclear protein of 6-OHDA-induced rats.The above results suggested that ATF3 was not the main factor of curcumin regulating ferroptosis in 6-OHDA-induced rats.10.Nrf2 was knocked down by small interfering RNA transfection and the m RNA and protein expression of Nrf2 in 6-OHDA-induced MN9 D cells were decreased.Western blot results showed that knocking down Nrf2 could decrease the expression of SLC7A11,GPX4 and FTH1,and increase the expression of PTGS2.And knocking down Nrf2 could reverse the regulatory effect of curcumin on the expression of ferroptosis related proteins in 6-OHDA-induced MN9 D cells.11.Inverted fluorescence microscopy and flow cytometry results showed that Nrf2 knockdown could increase intracellular ROS levels,revers the down-regulation effect of curcumin on ROS in 6-OHDA-induced MN9 D cells.12.Electron microscopy showed that mitochondria in the 6-OHDA-induced cells was shrinked and with reduction or vanishing of mitochondrial cristae,curcumin treatment could improve the morphology of mitochondrial.But Nrf2 knockdown could reverse the protective effect of curcumin on mitochondria.13.The results of CCK8 detection suggested that Nrf2 knockdown could aggravate 6-OHDA-induced MN9 D cells damage,reduce cell viability and inhibit the protective effect of curcumin on MN9 D cells.Conclusion: 1.Ferroptosis was observed in 6-OHDA-induced rat models of Parkinson’s disease,and the neuroprotective effect of curcumin was closely related to its inhibition of ferroptosis.2.Curcumin can reduce intracellular ROS levels,improve the expression of ferroptosis related proteins and have an inhibitory effect on ferroptosis in6-OHDA-induced cell models.3.Curcumin treatment could not reduce or change the expression of ATF3 in total protein and nuclear protein of 6-OHDA model rats.Curcumin attenuates ferroptosis in 6-OHDA-induced model of Parkinson’s disease via activating Nrf2/SLC7A11 signaling pathway.