Effect Of Mitochondrial CB1 Receptor On Mitochondrial Fission In A Rat Hippocampal Neuron Model Of Hypoxia/ Re-oxygenation Injury

ObjectiveTo evaluate the effect of mitochondrial CB1 receptor?mtCB1?on mitochondrial fission in a rat hippocampal neuron model of hypoxia/re-oxygenation?H/R?injury.MethodsPrimarily cultured hippocampal neurons obtained from Wistar rats were divided into6 groups using a random number table.?1?normal group?N group?:hippocampal neurons were cultured in normal medium,without any administration;?2?I/R group:the hippocampal neurons were subjected to oxygen-glucose deprivation?OGD?for 6h followed by re-oxygenation for 20h;?3?ACEA+I/R group:ACEA were added into the medium with final concentration 1?mol/L during the 20h re-oxygenation;?4?ACEA+AM251+I/R group:ACEA and AM251 were add with respectively final concentration1?mol/L,10?mol/Lduringthe20hre-oxygenation;?5?ACEA+Hemopressin+I/R group:ACEA and Hemopressin were add into culture medium with respectively final concentration 1?mol/L,10?mol/L during the re-oxygenation for 20h;?6?Vehicle group?V+I/R group?:DMSO was add into culture medium with final concentration<0.1%during the 20h re-oxygenation.The CCK-8 assay was utilized to detect the cell viability in three subgroups,in order to identify the period of drug action;The invert microscope was adapted to observe the Morphology of hippocampal neurons of every group;Laser scanning confocal microscope was used to measure Ca²⁺concentration and ROS content in cytoplasm;The transmission electron microscope was utilized to observe the mitochondrial ultrastructure;The apoptosis rate was tested by flow cytometry;Western blot was adopted to examine the expression of apoptosis-inducing factor?AIF?,dynamin-related protein 1?Drp1?,fission 1 protein?Fis1?,apoptosis–related protein cytochrome c?Cytc?,and Rho-associated coiled-coil containing protein kinase?ROCK1?.Results1.The cells viability of reperfusion subgroup obviously exceed to the other two subgroups in ACAE+I/R group and ACEA+Hemopressin+I/R group?P<0.05?,and there were no significant differences were observed in ACEA+AM251+I/R group and Vehicle group?P>0.05?;therefore,we chose the reperfusion period for 20 h as our target research period in the next series of experiments;2.Compared to the N group,the apoptosis rate,Ca²⁺concentration,ROS content,the expression of AIF,Drp1,Fis1,Cytc and ROCK1 were significantly increased in the other five groups?P<0.05?;3.Compared to the I/R group,those detection indexes mentioned above were significantly decreased in ACEA +I/R group and ACEA+Hemopressin+I/R group?P<0.05?,and there were no significant differences were observed in ACEA+AM251+I/R group and Vehicle group?P>0.05?;4.Compared to the ACEA+Hemopressin+I/R group,those detection indexes mentioned above were significantly increased in ACEA+AM251+I/R group?P<0.05?;5.the mitochondrial ultrastructure of N group possessed the integrity structure and explicit mitochondrial outer membrane.On the contrary,in the I/R group,Vehicle group,and ACEA+AM251+I/R group mitochondrion had lose the mitochondrial out membrane and cristae,without the typical structure,some mitochondria appear vacuolated.Meanwhile,the mitochondrial substructure of the ACEA+I/R group and ACEA+ Hemopressin +I/R group were less typically obliterated and the mitochondrial cristae can be seen under the transmission electron microscope.ConclusionThe reduction of ROS induced by mt CB1 can alleviate the expression of ROCK1 and Ca²⁺ concentration in cytoplasm,and inhibit the mitochondrial fission,and eventually attenuate the H/R injury of hippocampal neurons.