| Objective:To observe the change of mitochondrial dynamics orchestrates mitophagy in hippocampal neurons of rats with spleen-qi deficiency,investigate the occurrence mechanism of spleen-qi deficiency from the mitochondrial perspective.Materials and methods:1 Grouping Adult male Sprague Dawley rats(n=16)were habituated for two weeks,temperature(22±2°C),humidity(55±5%)and a light/dark cycle(light on at 8:00 and off at20:00).Rats were divided into two BW-matched groups,control group and model group,8 in each group.Rats in the control group were provided with standard feeding method,whereas rats in the model group were treated with the following protocol.2 The modeling and evaluation method2.1 Modeling:It consisted of 15 days of intermittent fasting(IF)and forced swimming(FS).IF was comprised of two days of fasting followed by one day of satiation.FS was conducted by placing rats into a cylinder(60 cm in height and 25 cm in diameter)which containing water at 28°C-30°C,and rats were considered to be exhaustion when failing to raise their heads above the water.2.1 Model evaluation:a.Emaciation:the body mass decreased obviously to the control group.b.Lack of food:food and water intake measured in the metabolic cage for 24h were significantly reduced to the control group;C.Mental exhaustion:the movement distance and standing times in the open-field experiment within 5min were detected,and the movement distance and standing times decreased significantly to the control group;D.Weakness:the grasping power of the limbs reduced significantly to the control group.E.Withered hair:the conclusions were judged by three persons respectively.3 Function and morphology3.1 Behavior:The open-field experiment was utilized to detect the distance of movement and the number of standing times of rats.3.2 Harvest:Rats were sacrificed by decapitation after anesthetized with 10%chloral hydrate(0.35ml/100g)and then hippocampal tissues were immediately taken out and cut into pieces of 1mm~3at 4?,and finally fixed with 2.5%glutaraldehyde in phosphate buffer and the rest tissues cryopreserved at-80?.3.3 Morphology:After dehydration in ethanol with graded concentrations,specimens were treated with propyleneoxide and embedded in Epon.Ultrathin sections(60 nm)stained with uranyl acetate and lead citrate was examined in a transmission electron microscope(JEM-1200EX;Jeol,Tokyo,Japan)at 120 kV.(1)Mitochondria:number,volume,mitochondrial cristae morphology.(2)Mitophagic flux:number of phagocytic vesicles,autophagosomes and autophagosomes.3.4 Mitochondrial function:Total protein/mitochondrial deposit were extracted from frozen tissue,the ATP level and hydrogen peroxide level were detected with colorimetric method then MMP was detected with JC-1 method.3.5 Mitochondrial dynamics Total protein was extracted from frozen tissue and the expressions of Drp1,Mff,Fis1,Mfn1,Mfn2 and Opa1 were detected with Western blot method.3.6 Mitophagy Protein was extracted from total protein/mitochondrial deposit and the expressions of PINK1?Parkin/LC3B-II and p62 were detected with Western blot method.3.7 Statistical Analysis Differences between groups were examined by pair-sample t-test(IBM SPSS,version 20.0;SPSS,Chicago,Illinois)and values were reported as mean±SEM.A P value of<0.05 denotes a statistically significant difference.4 Results4.1 Model evaluation Rats were robust,responsive,with dense and shiny coat in the control group and emaciated,slowly reaction in the model group.Body weight,food or water intake,exercise distance,standing times and grasping power of the forelimbs of the model group all decreased to the control group.4.2 Mitochondrial morphology There were depleted cristae and inner mitochondrial membranes(IMM)but fewer autophagic-like structures in the model group than that in the control group.4.3 Mitochondrial function The ATP contents of hippocampal neurons in the control group and the model group were 0.014±0.003 versus 0.011±0.002 mmol/g,respectively(n=8,P<0.01);Mitochondrial membrane potential of hippocampal neurons in two groups were0.698±0.042 versus 0.580±0.007,respectively(n=8,P<0.01);The hydrogen peroxide contents of hippocampal neurons mitochondrion in two groups were 0.774±0.119 versus1.589±0.193 mM,respectively(n=8,P<0.01).4.4 Expressions of proteins related to mitochondrial fission Drp1,Mff and Fis1 protein expressions in two groups were 1.60±0.060 versus 1.450±0.090(n=4,P<0.01),0.842±0.095versus 1.151±0.023(n=4,P<0.01),0.740±0.037 versus 0.859±0.054(n=4,P>0.05),respectively.4.5 Expressions of proteins related to mitochondrial fusion Mfn1,Mfn2 and Opa1 protein expressions in two groups were 0.618±0.035 versus 0.667±0.013(n=4,P>0.05);0.691±0.023 versus 0.924±0.046(n=4,P<0.01),0.804±0.049 versus 0.562±0.068(n=4,P<0.05),respectively.4.6 Expressions of proteins related to mitophagy PINK1,Parkin,LC3B-II and p62 protein expressions in two groups were 1.193±0.145 versus 0.663±0.115,1.257±0.152 versus 1.031±0.180,0.616±0.072 versus 0.450±0.073;0.693±0.288 versus 1.195±0.111(n=4,P<0.05),respectively.5 Conclusions5.1 The spleen qi deficiency is closely related to mitochondrial fission strengthen and fusion stagnation in hippocampal neurons.5.2 The spleen qi deficiency is closely related to mitophagy suppression in hippocampal neurons. |
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