Neuroprotection Of Astaxanthin In Neural Cell Damage Model And The Underlying Mechanism Of Autophagy Involving In The Protection Process

Astaxanthin?AX?is a carotenoid that widely found in marine plants and animals.It has many physiological functions such as anti-tumor,enhancing immune function,improving cardiovascular disease,and so on.AX can protect the nervous system by improving the intracellular antioxidant capacity and affecting autophagy,however,the mechanism has not been completely explained.Autophagy is a process in which damaged organelles and cytoplasmic proteins could be degradated through autophagosomes binding to lysosomes or other enzymatic vesicles,thus it could maintain stable intracellular metabolism and material regeneration.Autophagy plays a role in a variety of physiopathological responses,including neurodegenerative diseases.Mitochondria is the main energy-providing organelles,and homeostasis of mitochondria depends on an impaired mitochondrial clearance mechanism,mitophagy.Autophagy plays an important role in neurodegenerative diseases.In this experiment,rat pheochromocytoma cell PC12 and human brain astroglioma cell U87MG were used to establish the neural cell damage model induced by Amyloid-?fragment A?25-35 to examine the protective effect of AX in the neural cell damage model and the role of autophagy in this process.Objective:1.To study the protective effect and mechanism of AX in neural cell damage models;2.To explore the role of autophagy and mitophagy in the protection of AX.Method:1.Establishment of neural cell damage modelEstablish a neural cell damage model using rat pheochromocytoma cell PC12 and human brain astrocytoma cell U87MG,and A?_25-35 was used as inducers.2.The protective effect of astaxanthin?1?MTT method to determine the concentration of AX medication;?2?Hochest staining to detect apoptosis;?3?Western blotting to detect the expression of nerve cell marker proteins GAP43 and TUBB3;?4?Detection of SOD,GSH-Px activity and MDA content of peroxidase-related enzyme system in cells;?5?Detecting the content of reactive oxygen species ROS in cells;?6?Western blotting to detect the expression of oxidation-related proteins p62 and NRF2;?7?Western blotting to detect the expression of mitochondrial-related proteins AMPK,TOMM20 and Mitofusin 2;?8?Detection of mitochondria by immunofluorescence.3.The role of autophagy in the protection of astaxanthin?1?Western blotting to detect the expression levels of autophagy-related proteinsLC3Av1,LC3B,p62 and Parkin;?2?Detect cell viability after using 3-MA autophagy inhibitor;?3?Detection of mitochondrial autophagy by immunofluorescence;?4?Electron microscopy to detect autophagic vesicles and mitochondria;?5?Co-IP to detect LC3B and Parkin protein interaction.Results:1.Establishment of neural cell damage modelMTT results showed that with the increase of A?concentration,cell viability gradually decreased.2.The protective effect of AX?1?MTT results showed that compared with the A?model group,cell viability gradually increased with the increase of AX concentration.The AX concentration was determined as 0.1?M,1?M,10?M;?2?Hoechst results showed that AX inhibits apoptosis induced by A?;?3?Western blotting showed that the expression levels of neurons marker proteins GAP43 and TUBB3 were increased compared with the A?model group,indicating that AX has protective effect on cells;?4?Enzymatic test results showed that compared with the control group,the model group caused a significant decrease in SOD and GSH-Px enzyme activity and increased MDA content;AX pretreated cells inhibited the effect of A?;?5?ROS results in cells showed that ROS generation increased and a large number of aggregates were found in the A?model group;ROS content decreased in the AX group;?6?Western blotting showed that after treated with A?for 24 hours,the expressionof p62 was significantly reduced while the expression of phosphorylated p62 andnuclear NRF2 were increased compared with the control group.Simultaneously,p62,phosphorylated p62 and nuclear NRF2 expression were increased in AX group;?7?Western blotting showed that compared with the control group,the expressionof TOMM20,Mitofusin 2 and phosphorylated AMPK decreased significantly aftertreated with A?for 24 hours.Compared with model group,expression of TOMM20,Mitofusin 2 and phosphorylated AMPK were increased in AX group;?8?Immunofluorescence showed that the expression of TOMM20 was low in theA?model group,indicating a reduced number of normal mitochondria.In the AXgroup,the expression of TOMM20 was increased,indicating that the number ofnormal mitochondria was large.3.The role of autophagy in the protection of AX?1?Western blotting results showed that compared with the control group,theexpression levels of LC3Av1-?,LC3B-?and Parkin were significantly increasedwhile p62 expression was decreased treated with A?for 24 hours.Compared withthe model group,the expression levels of LC3Av1-?and LC3B-?decreased,andthe expression of p62 increased in AX group.?2?After the cells were treated with the autophagy inhibitor 3-MA,MTT resultsshowed that compared with the control group,3-MA had no significant effect oncell viability.In A?model group,whether or not used of 3-MA,cell viability waslower than that of the control group.Furthermore,without 3-MA,cell viability ofAX group gradually increased while cells using 3-MA cannot increase cell viabilityeven with AX;?3?Detection of mitophagy by immunofluorescence,the results showed that theexpression level of LC3B in the model group increased significantly,and theexpression of Parkin increased which showed an increase in aggregation and adiffuse distribution in cytoplasm.LC3B aggregation decreased and Parkinaggregation increased,indicating a reduced LC3B expression and increased Parkinexpression;?4?Electron microscopy results showed that in the A?model group,the number ofautophagic vesicles increased and the number of damaged mitochondria increased.In the AX group,the number of autophagic vesicles decreased and the number ofmitochondria of normal structure increased;?5?Co-IP results show that LC3B and Parkin do not interact with each other.Conclusions:In the A?_25-35 induced nerve cell damage model,p62 phosphorylation and NRF2 nuclear location promoted by AX enhanced nerve cell resistance to A?,which lead to enhanced activity of antioxidant enzyme system,increased ROS removal,and maintained mitochondrial stability.AX reduced the total autophagy level of cells by enhancing their ability to resist A?.And at low autophagy levels,AX strengthened mitophagy,thus promoting the removal of damaged mitochondria,maintaining the normal state of nerve cells and reducing the degree of damage to nerve cells.