The Study Of Selenium Protective Role In The Injury Of Neuronal Cells And Its Relationship With The Mitochondrial Pathway

Objective:It is investigated that selenium and selenoprotein H (SelH) and protect nerve cells frominjury caused by excess glutamate. And the relationships between such protective effects andthe mitochondrial pathway are discussed. Our results would provide new insight to protectingnerve cells.Methods：Hippocampal neurons HT-22, SelH transfection hippocampal neurons SelH andcorresponding non-transfection hippocampal neurons HT+V are chosen for experiments. Inthe logarithmic growth phase, HT-22cells are divided into the following groups in order toinvestigate the protective effects of selenium.①The control group;②The injury group:Cultivated by glutamate with final concentration6mM,③The protective group: Jointlycultivated by glutamate with final concentration6mM and sodium selenite with finalconcentration100nM. Similarly, SelH and HT+V cells are divided into the following groupsto investigate the protective effects of selenoprotein H.①The SelH control group,②TheSelH injury group: Cultivated by glutamate with final concentration4mM,③The HT+Vcontrol group,④The HT+V injury group: Cultivated by glutamate with final concentration6mM. MTT method is used to detect the rates of apoptosis after6,10,24hours cultivation.Inverted microscope and CFM are used to observe the changes of cell morphology andmicro-structure. The generation of ROS is detected after DHE staining. Mitochondria andlysosome are marked by Mito Tracker Green and Lyso Tracker Red respectively, and the formatting process of autophagy is observed through CFM. The expressions of Caspase-3,9and Apaf-1associated with the mitochondrial pathway of apoptosis are measured byimmunofluorescence and western-blotting.Results:Part1:①The inhibition with rate of8.71%±0.009are observed after HT-22cells havebeen cultivated by6mM glutamate for6hours. With the time evolution, the inhibition ratesare increase. The inhibition rates is decrease obviously with jointly cultivated by glutamateand100nM sodium selenite.②The control group cells shows normal morphology,completely cell membrane and nucleus plentiful organelles; Double nucleus is obviously andintact in cells. Mitochondrial crest is clear. Cytoplasm has rough endoplasmic reticulum withnormal morphology, granules are uniform distributed. The glutamate injury groups show anincrease in apoptotic cells. Apoptotic cell nuclei is incomplete or fragmentation evendisappear, cell organelles significantly reduced; Double nucleus is ambiguous, mitochondrialstructural is damaged, and the outline does not distinguish or even disappear. Cytoplasmappears a lot of lysosomes. The rough endoplasmic reticulum are extended significantly, andgranules are incomplete. Large number of cell nuclei disappear and show crumbled nucleolus,scarce organelles, the hyperinflation endoplasmic reticulum, and vacuoles are formed in thecell structure. The group jointly treated by glutamate and100nM sodium selenite showsignificantly reduction injuries compared with injury group.③After ROS-specific labeledprobe DHE marking, we find a few production of ROS in the control group. The generation ofROS is obviously increased in injury group. The projective group show significantlyreduction of ROS production and few increase comparing with injury group and control grouprespectively (P<0.05).④Mitochondria and lysosome are marked by Mito Tracker Greenand Lyso Tracker Red respectively. Mitochondria are colored clear outline clear, almost nocoloration lysosomes in control group. However mitochondrial staining was significantlyreduced in injury group, and lysosomal staining was significantly enhanced (P<0.05). These mean autophagy bodies also increase in injury group, and significantly improved in jointlytreated group.⑤The results from Immunofluorescence and western-blotting show that theexpression of caspase-3,9and Apaf-1was significantly decreased in protective groupscomparing with injury group.Part2:①The results from MTT show that HT+V and SelH cells are inhibited after6hours under4mM glutamate cultivating, and the inhibition rates will increase with timeevolution (10H, and24H). Comparing with the same time glutamate cultivating, SelH groupsshow low inhibition rates contrasting to HT+V groups(P<0.05).②In control group, cellsshows normal morphology, completely cell membrane and nucleus plentiful organelles.Double nucleus is obviously and intact in cells. Mitochondrial crest is clear. Cytoplasm hasrough endoplasmic reticulum with normal morphology, granules are uniform distributed. Allglutamate injury groups show an increase in apoptotic cells. Apoptotic cell nuclei isincomplete or fragmentation even disappear, cell organelles significantly reduced. Doublenucleus is ambiguous, mitochondrial structural is damaged, and the outline does notdistinguish or even disappear. Cytoplasm appears a lot of lysosomes. The rough endoplasmicreticulum are extended significantly, and granules are incomplete. Large number of cell nucleidisappear and show crumbled nucleolus, scarce organelles, the hyperinflation endoplasmicreticulum, and vacuoles are formed in the cell structure.③After DHE, a ROS-specificlabeled probe, marking, we find ROS hardly produced in the control group. But the generationof ROS is obviously increase in injury group. The injury group of SelH groups showsignificantly reductive production of ROS comparing with HT+V groups and few increasecomparing with control group (P<0.05).④Double marking of Mitochondria and lysosomeshow that the mitochondria are colored clear and outline clear, almost no colorationlysosomes in control group. However mitochondrial staining was significantly reduced ininjury group, and lysosomal staining was significantly enhanced (P <0.05), while autophagybodies also increases. In injury group of SelH sells, autophagy significantly improved in contrast to the HT+V cells.⑤The results from immunofluorescence and western-blottingshow that the expressions of caspase-3,9and Apaf-1of HT+V cells in glutamic injury groupsare significantly increase comparing with their control groups(P<0.05), while theexpressions of SelH cells in glutamic injury groups are less increase than HT+V groups andsome of them are statistically insignificant.Conculusions:(1) Selenium (sodium selenite) can significantly alleviate the hippocampal neuronaldamage.(2) SelH can significantly alleviate the hippocampal neuronal damage.(3) Selenium and selH are functionalized by reducing mitochondrial autophagy, ROSgeneration and stable mitochondrial pathway, and result in protective role for nerve cells.