| Objective:To explore the possible mechanism of uneasiness caused by spleen qi deficiency by detecting rats’behavior,hippocampal neuronal mitochondrial morphology,basic function nad its autophagy level in spleen qi deficiency.Materials and methods:1.Animal groups 16 SPF-level adult rats were divided into the normal group and model group accodring to the random data table,8 in each group.2.Establishment and evaluation of spleen qi deficiency model Establishing method:in 15days,rats in the model group were treated by 5 circles of free access to feed and water ad libitum for one day and fasted for 2 days,as well as forced swimming to fatigue every day.Evaluation standard:emaciation,poor appetite,mental and physical fatigue,and hair colorlessness.The protocol was approved by the Animal Ethics Committee of Liaoning University of Traditional Chinese Medicine,China(2015205).3.Behavior test The Etho Vision XT Version 9 software and TM-vision behavior testing system were used.After fed 2h later,rats were placed individually into the center sectors of the arena and their behaviors were recorded in 5 min,calculating central and peripheral time,motion distance,mean motion velocity and rearing times.4.Specimen collection After rats were anesthetized with 10%chloral hydrate by intraperitoneal injection(0.35mL/100g),their bilateral hippocampal tissues were taken out,some of them were stored at-80℃;some were cut into pieces of 1mm~3at 4℃and fixed with2.5%glutaraldehyde in phosphate buffer;some added with mitochondrial isolation buffer were homogenized and centrifuged(1,000 g,5min,4℃)to draw the supernatants which were centrifuged again in the same condition,and then the supernatants were draw and centrifuged(1,200g,10min,4℃)to acquire the pellets which were saved in 100μL PBS(p H7.4)to form mitochondrial suspension.5.Mitochondrial morphology of hippocampal neurons The 2.5%glutaraldehyde solution was extracted and used to fix the experimental tissue,washed with PBS method,and then 1%osmic acid was used to fix the tissue.After dehydration,the tissue was sealed in epoxy resin with ethanol and acetone solution.After slicing,3%uranyl acetate lead citrate was taken out and stained on both sides.Mitochondrial morphology was examined in a transmission electron microscope.6.Evaluation of the mitochondrial function in hippocampal neurons Mitochondrial suspension was used to determine mitochondrial membrane potetials(MMP)by the JC-1,and adenosine triphosphatase(ATP)and reactive oxygen spieces(ROS)levels by ELISA,according to their protocols of the manufacturers.7.Expression of mitochondrial associated proteins in hippocampal neurons Western blotting was used to check related protein expressions.Take out the frozen tissue and thaw it at room temperature.After thawing,add the prepared mitochondrial protein lysate,put it into the sterile box for 30 minutes,take it out and grind it on ice,centrifuge it with centrifuge, centrifuge it at 4℃for 10 minutes,and then take the supernatant after observation Methods the concentration of the experimental protein was detected carefully,and then the buffer solution was added.The buffer solution was 2×SDS-PAGE protein buffer prepared in the experiment.It was boiled in 100℃water,denatured and inactivated,and then electrophoresed on the membrane.The membrane was transferred and cut.It was cooled in 37℃ water,and then dehydrated.The experiment was completed with 5%skimmed milk powder/test for 2h,then 4mL of PTEN-induced putative kinase 1(PINK1)(1∶1000),parkin(1∶1000),Light chain 3(LC3-I)(1∶5000)and LC3-II(1∶2000)were added respectively,and the temperature was kept at 4℃and placed on the shaking table overnight;then the membrane was washed,and the experiment was completed by tbst method,and the Goat anti rabbit antibody(1:5000)prepared before the experiment was put into it,and the antibody was labeled with HRP.Put 4mL of the above antibody into GAPDH(1∶1000)box,incubate it in room temperature for 1.5h,and then wash the film.In order to achieve the purpose of development and exposure,add ECL luminous solution,and apply alphaview was used to complete the experiment and obtain the protein expression data.7.Statistical analysis The data were expressed as mean±standard deviation(?±S)and treated by t test using SPSS 18.0 software.A value of P<0.05 or less was considered statistical significance.Results:1.Behaviors Motion distances in the open field were 6524.32±1435.32cm and5772.60±1621.93cm(n=8,P>0.05),central times were 21.19±10.18s and 41.76±22.86s(n=8,P<0.05),perpherary times were 278.85±10.11s and 258.27±22.86s(n=8,P<0.05),mean velocities were 4.57±0.78cm/s and 3.31±1.00cm/s(n=8,P<0.05),rearing times were15.4±3.2 times/5min and 8.5±2.9 times/5min(n=8,P<0.01),repectively,in the normal group and model group.2.Hippocampal neuronal mitochondria morphology Compared with those in the normal group,both mitochondrial cristae and autophagy-like structures were less.3.Hippocampal mitochondrial function ATP levels were 0.217±0.088 pmo L/g and0.090±0.043 pmo L/g(n=8,P<0.01),MMPs were 0.728±0.170 and 0.591±0.012(n=8,P<0.05),ROS levels were 13.55±5.44 UI/mL and 11.76±3.59UI/mL(n=8,P>0.05),repectively,in the normal group and model group.4.Hippocampal mitochondrial PINK1/Parkin pathway PINK1 expressions were0.804±0.074 and 0.416±0.124(n=4,P<0.05)and Parkin expressions were 0.831±0.081 and0.606±0.041(n=4,P<0.05),repectively,in the normal group and model group.5.Hippocampal mitochondrial flux LC3-Ⅱ expressions were 106.06±12.79 and 57.60±10.05(n=4,P<0.05),LC3-I expressions were 166.86±6.05 and 172.58±11.13(n=4,P>0.05),LC3-II/I ratios were 0.647±0.088和0.355±0.074(n=4,P<0.05),repectively,in the normal group and model group.Conclusion:1.Uneasiness caused by spleen qi deficiency might be caused by hippocampal neuronal energy shortage due to mitochondrial damage and mitophagy inhibition. |
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