| Iron is the most abundant transition metal in the human brain.The distribution of iron in the brain of healthy adults is uneven,with iron being highest in the basal ganglia and low in the gray matter,white matter,midbrain and cerebellum.As people age,iron is deposited in brain regions such as substantia nigra(SN)and globus pallidus.In various neurodegenerative diseases,such as Alzheimer’s disease(AD)and Parkinson’s Disease(PD),iron is deposited in specific brain regions and iron homeostasis is disturbed.Lysosome,as a major regulator of iron metabolism,plays a key role in cellular iron transport and homeostasis.Studies have reported that iron levels in the liver and spleen of mice were increased after intraperitoneal administration of iron.However,the effects of iron overload in peripheral tissues(such as liver and spleen)on lysosomal function after intraperitoneal administration of iron,and whether peripheral iron can enter the brain region to affect lysosomal function and motor function need to be further explored.C57BL/6 mice were intraperitoneally injected with iron dextran(FeDx,200 mg/kg),while the control group was given the same amount of normal saline every other day for 1,2,3 or 4 weeks,respectively.Western blot method was applied to detect the liver,spleen,hippocampal(HC),striatum(STR),substantia nigra and olfactory bulb(OB)of L ferritin(L-ferritin),Cyl-Co A synthetase long-chain family member 4(ACSL4)and glutathione peroxidase 4(GPX4),while detecting the expression of lysosome associated proteins,such as cathepsin B(CTSB),cathepsin D(CTSD),glucocerebrosidase(GCase),lysosome integrated membrane protein 2(LIMP-2)and lysosomal associated membrane proteins-1(LAMP-1).Iron content in the serum and cerebrospinal fluid(CSF)was measured using an iron content kit.Immunofluorescence and immunohistochemical staining were used to detect the number of dopaminergic neurons and fiber density.Gait analysis was used to evaluate the motor function of mice.The results are as follows:1.After 1,2,3 or 4 weeks of intraperitoneal injection of FeDx,L-ferritin levels in liver and spleen were significantly higher than those in the control group,with statistically significant differences.ACSL4 and GPX4 protein levels in liver and spleen were not significantly different at 1-4 weeks.The levels of CTSB,CTSD,GCase and LIMP-2protein in liver increased at 1-4 weeks,and the difference was statistically significant.The protein levels of CTSD,GCase and LIMP-2 in spleen increased at 1-4 weeks,while the protein levels of CTSB in liver increased at 2-4 weeks,and the difference was statistically significant.After 1 week of intraperitoneal injection of FeDx,there was no significant change in LAMP-1 protein level in liver,and protein expression decreased at2-4 weeks.The results suggested that intraperitoneal injection of FeDx induced iron overload and lysosome dysfunction in liver and spleen of mice.2.Intraperitoneal injection of FeDx caused a slight increase in serum iron concentration after 1 week,and the serum iron level increased over time after 2-4 weeks,the difference was statistically significant.Iron concentrations in CSF remained near the limit of quantitation(LOQ)at 1-4 weeks.The results suggested that intraperitoneal injection of FeDx resulted in a time-dependent increase in serum iron concentration,but no significant increase in CSF iron concentration.3.Intraperitoneal injection FeDx 1-2 weeks,OB in L-ferritin protein levels,indicates that OB are early iron deposit,and not observed changes in HC and STR.After 3 to 4 weeks of intrabitoneal injection of FeDx,L-ferritin protein levels in OB decreased to normal levels,while L-ferritin protein levels in HC and STR increased.Iron staining positive cells of SN and STR increased 3 or 4 weeks after intraperitoneal injection of FeDx.The results suggested that iron deposition of OB,SN,HC and STR was induced by intrapitoneal injection of FeDx.4.After 1-4 weeks of intraperitoneal injection of FeDx,CTSB and CTSD protein levels in SN,HC and STR remained unchanged.The protein levels of CTSB and CTSD remained unchanged in OB after 1 to 3 weeks of intrabitoneal injection,but were down-regulated in OB after 4 weeks,and LAMP-1 in HC after 4 weeks.The levels of GCase and LIMP-2 proteins in SN,HC,STR and OB showed no significant changes at 1-4 weeks.The results suggested that no significant lysosomal dysfunction was observed in OB,SN,HC and STR after intrapitoneal injection of FeDx.5.After two intranasal administration of Ferric ammonium citrate(FAC),L-ferritin,CTSB,GCase and LIMP-2 protein levels increased in OB,while there was no significant difference between CTSD and LAMP-1 protein levels.In primary ventral midbrain neurons/astrocytes treated with 100 μM FAC,there were no significant differences in the protein levels of CTSB,CTSD,GCase,LIMP-2 and LAMP-1.In primary ventral midbrain neurons,LIMP-2 protein levels increased after 1 m M FAC treatment,but there were no significant differences in CTSB,CTSD,GCase and LAMP-1 protein levels.After 1 m M FAC treatment,the protein levels of LIMP-2 and LAMP-1 in primary ventral midbrain astrocytes were increased,while GCase protein levels were not significantly different and CTSB protein levels were increased.The results suggested that FAC induced upregulation of CTSB expression and impaired lysosome function in OB and primary ventral midbrain astrocytes.6.After 4 weeks intraparitoneal injection of FeDx,the fiber density and protein level of tyrosine hydroxylase(TH)in STR was reduced,but the number of TH-positive neurons in SN was not significantly different.However,although iron deposits first appeared in OB brain tissue,TH protein levels in OB were not significantly different at 1-4 weeks.The results suggested that intraperitoneal injection of FeDx caused minor damage to the nigrostriatal system.7.After intraperitoneal injection of FeDx,static gait indexes of mice were changed,and indexes such as maximum contact area,maximum contact mean intensity,print length,print width and print area decreased on different PAWS.However,no significant changes in dynamic gait indicators were observed.The results suggest that intraperitoneal injection of FeDx does not cause significant gait disturbance.Mice were intrabitoneally injected with FeDx to induce iron overload in liver and spleen,and iron deposition in OB,SN,HC and STR.Serum Feconcentration increased with time dependence,but CSF Feconcentration did not increase significantly.CTSB,CTSD,GCase and LIMP-2 proteins were significantly upregulated in liver and spleen,but not in brain.CTSB expression was up-regulated in acute iron overload OB and primary ventral midbrain astrocytes.The number of dopaminergic neurons in SN remained unchanged,and the mice did not cause significant gait disturbance.The results suggested that intraperitoneal injection of FeDx induced iron overload and lysosomal dysfunction in liver and spleen,and iron deposition in brain regions(OB,SN,HC and STR),but not enough to cause significant lysosomal dysfunction and movement disorders. |
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