The Effect Of Iron On The Secretion Of Ferritin In Cultured Astrocytes And The Underlying Mechanisms

Parkinson disease?PD?is one of the most common neurodegenerative diseases in the elderly.Its clinical features include resting tremor,muscle stiffness,and bradykinesia,which have seriously affected people's quality of life.Studies have shown that environmental factors,genetic factors,aging,oxidative stress might be involved in the pathogenesis of PD.An autopsy of PD patients revealed a large amount of iron deposits in the substantia nigra?SN?area of the brain.The abnormal deposition of SN iron can cause iron metabolism disorders in the brain,which is one of the key causes of PD.In the brain,iron is mainly bound to ferritin.Ferritin is composed of heavy chain?H-Ferritin?and light chain?L-Ferritin?,which can store up to 4,500 iron atoms.It is a key protein that could chelate and store iron.Studies have shown that the level of ferritin in the SN of PD patients decreased,and the load of ferritin is higher than normal.The increase in iron content and the absence of upregulation of ferritin make DA neurons more vulnerable to oxidative stress damage in the SN.In recent years,the discovery of extracellular ferritin indicates that ferritin might not only be an intracellular iron storer,but might also be an important factor involved in the regulation of tissue and systemic iron.Studies have shown that astrocytes may be an important source of secreted ferritin in the brain.However,the role and the underlying regulating mechanisms of iron in the secretion of ferritin from astrocyte are unclear.Therefore,in this experiment,we intend to study the role of high iron levels in secretion of ferritin and clarify the possible regulating mechanisms in the primary cultured astrocytes.In this experiment,ferric ammonium citrate?FAC?was used to prepare high-iron models of cells.Western blots,ELISA,small interference lentivirus and other techniques are used to study the effect of high iron status on ferritin protein expression and secretion in the primary cultured astrocytes,and to explore its possible mechanisms.The results are as follows:1.After treatment with 100?mol/L FAC for 24 hrs,L-Ferritin secreted by primary cultured astrocytes increased significantly compared with the normal control group?P<0.01?,while the levels of L-Ferritin secreted by primary cultured astrocytes did not change significantly after treatment with neurotoxin?200?mol/L MPP⁺?.In addition,H-Ferritin secreted by the primary cultured astrocytes decreased after FAC or MPP⁺treatment,and the difference is statistically significant compared with the normal control group?P<0.01?;2.FAC can induce an increase in the protein levels of L-Ferritin and H-Ferritin?P<0.001?and a decrease in the protein levels of IRP₁ in the primary cultured astrocytes;The difference is statistically significant compared with the control?P<0.05?.3.Pretreatment with 100?mol/L CQ for 30 min can significantly inhibit FAC-induced increase of L-Ferritin secretion?P<0.001?;CQ had no effect on the FAC-induced decrease in H-Ferritin secretion.4.After FAC treatment for 24 hrs,the level of L-Ferritin in the cell supernatant was significantly increased compared with the control group?P<0.001?;the level of L-Ferritin in the cell supernatant of the FAC/Rab27a-Sh RNA group was not significantly changed compared with the FAC group.The level of H-Ferritin secreted by the cells in the FAC-treated group was significantly lower than that of the control group?P<0.001?;and Rab27a-ShRNA could inhibit FAC-induced decrease in astrocyte H-Ferritin content in the supernatant,and the difference was statistically significant?P<0.05?.5.After FAC treatment for 24 hrs,the expression of L-Ferritin protein in the primary cultured astrocytes was significantly increased compared with the control group;while the expression of L-Ferritin protein in the FAC/Rab27a-ShRNA group was lower than that of the FAC group?P<0.01?.H-Ferritin protein expression was significantly increased in the FAC treatment group compared with the control group;and H-Ferritin protein expression was increased in the FAC/Rab27a-ShRNA group compared with the FAC group,and the difference was statistically significant?P<0.01?.6.After FAC treatment for 24 hrs,the protein expression of Rab27a was significantly lower than that of the control group?P<0.05?.7.After FAC treatment for 24 hrs,the expressions of sec22b and TRIM16 protein were significantly decreased compared with the control group?P<0.01?;there was no statistically significant change of sec22b and TRIM16 protein expressions in the FAC/CQ group compared to the FAC group,CQ treatment alone has no effect on the protein expressions of sec22b and TRIM16;8.After FAC treatment for 24 hrs,the expressions of sec22b and TRIM16 protein were significantly decreased compared with the control group?P<0.01?;and the expressions of sec22b and TRIM16 protein were decreased in the FAC/Rab27a-ShRNA group compared with the FAC group?P<0.05?;Rab27a-ShRNA alone could also reduce the expression of sec22b and TRIM16 protein?P<0.01?.In summary,the high iron levels can increase the secretion of L-Ferritin and decrease the secretion of H-Ferritin in the primary cultured astrocytes,suggesting that FAC might affect the secretion of two subunits of ferritin in the primary cultured astrocytes through different mechanisms.In addition,the decrease of H-Ferritin secretion induced by FAC might be related to Rab27a and decreased secretory autophagy which is related to sec22b and TRIM16.And iron-induced secretion of L-Ferritin can be blocked by the chloroquine,suggesting that it may be related to fusion of lysomes and autophagy vesicles.This experiment provides new evidence for the mechanisms underlying the expression and secretion of ferritin from primary cultured astrocytes in high-iron environments,and provides new targets for the treatment of PD.