The Effect Of Apoferritin On The Protection Against MPTP-induced PD Mouse Model

Parkinson’s disease(PD)is the second most common neurodegenerative disease after Alzheimer’s disease,and is common in middle-aged and elderly people.The pathological feature of PD is that dopamine neurons in the substantia nigra pars compacta(SNpc)are damaged,leading to the gradual deficits of motor functions.However,the etiology and pathogenesis are not yet fully understood.More and more evidence confirms that the iron content in the substantia nigra(SN)of PD patients is significantly increased.Elevated unstable iron can increase the production of reactive oxygen species(ROS)through the Fenton reaction and then aggravate oxidative stress of dopamine neurons.Iron is also an important participant in ferroptosis.Ferroptosis is an iron-dependent programmed cell death discovered and named by Professor Brent R.Stockwell as early as 2012,and ferroptosis has been found in PD patients.It has been reported that ferroptosis is closely related to the onset of PD.Therefore,to prevent iron deposit and iron-dependent ferroptosis in PD is an important therapeutic target.Ferritin is an important iron storage protein within the cells,that can maintain cellular iron homeostasis by chelating iron.Ferritin is composed of two types: H-ferritin and L-ferritin,H-ferritin has ferrous oxidase activity and can oxidize ferrous iron into ferric iron.L-ferritin has a nucleation site to promote the formation of iron core and complete iron storage.When the body’s iron is excessive,the excess iron can be stored in ferritin;when the body is lack of iron,ferritin can maintain the body’s iron balance by releasing the stored iron.The recent findings show that the extracellular ferritin interacts with the cell through the specific binding of H-ferritin to transferrin receptor 1(Tf R1).Apoferritin is an iron-free form of ferritin,derived from horse spleen serum.It is composed of 24 subunits and has the ability to store iron,indicating that it might participate in the regulation of iron deposition in PD.However,the role of apoferritin in the PD mouse model induced by1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)and possible mechanism are unclear.Therefore,in this study,Western Blot,immunofluorescence,Perls’ iron staining and behavioral techniques are used to investigate the role and possible mechanisms of apoferritin in MPTP-induced PD mouse model.The results are shown below:1.The results of body weight changes showed that the weight of PD mice treated with MPTP was significantly lower than that of the control group,and the difference was statistically significant(P<0.05).Pretreatment with 10 mg/kg or 15 mg/kg apoferritin can effectively antagonize the weight loss of mice caused by MPTP,and the difference is statistically significant(P<0.05).There was no statistically significant difference between the apoferritin treatment group and the control group.2.The pole-climbing test results showed that compared with the control group,MPTP treated mice had significantly longer climb time and longer time to turn their heads.10mg/kg apoferritin or 15 mg/kg apoferritin pretreatment could significantly improve the pole-climbing ability(P<0.05).There was no significant difference between the 10 mg/kg apoferritin pretreatment group and the 15 mg/kg apoferritin pretreatment group.Therefore,10 mg/kg apoferritin pretreatment was used for subsequent experiments.There was no statistically significant difference between the apoferritin treatment group and the control group.3.The results of the open-field test showed that the total moving distance of the mice in the MPTP treatment group was significantly lower than that of the control group,and the difference was statistically significant(P<0.01).The total moving distance of mice in the10 mg/kg apoferritin pretreatment group was significantly increased compared with the MPTP group,and the difference was statistically significant(P<0.05).4.The results showed that apoferritin given by gastric gavage or intravenous injection(10mg/kg)can significantly improve the motor dysfunction of MPTP-induced PD mice.And there was no significant difference in the time of climb and turn their heads between gastric gavage group and intravenous injection group.Therefore,intragastric administration was chosen for subsequent experiments.5.After MPTP treatment,compared with the control group,the number of TH-positive cells and the expression level of TH protein in the SN of mice were significantly reduced,and the difference was statistically significant(P<0.001).10 mg/kg apoferritin pretreatment can effectively antagonize the loss of TH-positive neurons and the decrease of TH protein expression in the SN of mice induced by MPTP,and the difference is statistically significant(P<0.05).6.After MPTP treatment,compared with the control group,the number of Iba1-positive cells in the SN of mice increased significantly,and the difference was statistically significant(P<0.01).Compared with the MPTP group,the number of Iba1-positive cells in the SN of mice was significantly reduced after apoferritin pretreatment,and the difference was statistically significant(P<0.05).7.After MPTP treatment,compared with the control group,the number of iron-positive cells in the SN of mice increased significantly,and the difference was statistically significant(P<0.001).Apoferritin pretreatment can effectively inhibit the increase in the number of iron-positive cells in the SN of mice,and the difference is statistically significant(P<0.05).8.After MPTP treatment,compared with the control group,the protein expression of the iron transport protein divalent metal ion transporter(DMT1)was significantly increased,and the protein expression of transferrin receptor(Tf R1)decreased in the SN of mice,and the difference was statistically significant(P<0.01).Apoferritin pretreatment can effectively inhibit MPTP-induced up-regulation of DMT1 and down-regulation of Tf R1 in the SN of mice,and the difference was statistically significant(P<0.05).9.After MPTP treatment,compared with the control group,the expression of the ferroptosis related protein long-chain acyl-Co A synthetases 4(ACSL4)increased significantly in the SN of mice,while the protein level of ferroptosis suppressor protein1(FSP1)decreased significantly.The difference was statistically significant(P<0.01).Pretreatment with apoferritin can effectively inhibit the increase of ACSL4 protein and the down-regulation of FSP1 induced by MPTP,the difference was statistically significant(P<0.05).10.After MPTP treatment,compared with the control group,the protein expression of the ferroptosis related protein glutathione peroxidase 4(GPX4)in the SN of mice was reduced,while apoferritin pretreatment did not change the protein expression of GPX4.After MPTP treatment,the level of glutathione/oxidized glutathione(GSH/GSSG)in the SN of mice did not change significantly,compared to the control.11.After intracerebroventricular injection of 3 μg apoferritin for 30 minutes,the levels of L-Ferritin in the cerebrospinal fluid and serum increased significantly compared with the sham operation group(P<0.05).In addition,after injection of 3 μg apoferritin into the lateral ventricle for 1 h,3 h,6 h,12 h,24 h,the level of L-Ferritin in the cerebrospinal fluid of mice with apoferritin injection for 1 h and 3 h was significantly increased compared with the sham operation group(P<0.001).The level of L-Ferritin in the SN of mice was significantly increased after apoferritin injection for 6 h,compare to the sham operation group(P<0.01).12.Compared with the control group,the level of L-Ferritin protein in primary cultured VM neurons and in the SN of mice treated with apoferritin increased significantly,and the difference was statistically significant(P<0.05).There was no significant difference in H-ferritin protein levels of the VM neurons and the SN of mice treated with apoferritin,compared to the control.In summary,apoferritin can significantly improve MPTP-induced motor dysfunction by restoring the expression of TH protein and TH-positive neurons in the SN.In addition,apoferritin pretreatment can inhibit MPTP-induced iron accumulation by down-regulating iron import protein DMT1.At the same time,apoferritin can effectively prevent MPTP-induced ferroptosis by inhibiting the up-regulation of ACSL4 and down-regulation of FSP1 in the SN of mice.These results indicate that apoferritin exerts a neuroprotective effect by inhibiting iron accumulation and regulating ferroptosis.The above results provide new targets and ideas for the treatment of PD.