| ObjectiveIn recent years,studies have found that early life stress such as parental neglect,abuse,and mental stress can increase the risk of depression.But the underlying molecular mechanism of depression remains unclear.Sinisan(SNS),as the basic prescription of Traditional Chinese Medicine for regulating the liver,has obvious antidepressant effects in clinical and experimental study.Therefore,maternal separation(MS)model was used to simulate early life stress in this experiment,and to study the mechanism of depression induced by early life stress and intervention effects of SNS on depression.It will provided experimental evidences for the clinical application in preventing and treating depression induced by early life stress.Methods1.Effects of MS rats on depression-like behavior and synaptic plasticityThe 60 pups of SD rats on the birth day(postnatal day 0,PND0)were randomly divided into male maternal separation group(M-MS),male non-maternal separation group(M-NMS),female maternal separation group(F-MS)and female non-maternal separation group(F-NMS),15 pups per group.MS modeling was performed from PND1 to PND21.From PND56 to PND63,sucrose preference test and forced swimming test were used to evaluate depression-like behavior in rats,and open field test was used to evaluate anxiety-like behavior in rats.After the behavioral experiments,Nissl staining was used to detect the pathological status and number of neurons in the hippocampus and prefrontal cortex.Immunohistochemistry was used to detect the expression of Synaptophysin(SYN)protein in the CA1,CA2,CA3,DG ares of hippocampal and the prefrontal cortex.Western blotting(WB)was used to detect the the expression levels of SYN,Growth-associated binding protein 43(GAP-43)and Postsynaptic density 95(PSD-95)in hippocampus and cortical tissue.2.Metabonomics analysis of brain tissue in MS ratsChromatography-mass spectrometry technology was used to analyze brain tissue samples,and data processing was performed on the obtained metabolite map.The differential metabolites between groups were screened by multivariate statistics,and the MetPA database was used to enrich pathways of differential metabolites.3.The mechanism of SNS regulates mitochondrial function to mediated synaptic plasticity of hippocampus in MS rat modelThe 72 male pups of SD rats on PND0 were randomly divided into control group,MS group(Model),fluoxetine group(Positive),low-dose of SNS group(Low),medium-dose of SNS group(Medium)and high-dose of SNS group(High),12 pups per group.From PND1 to PND21,pups of Model group,Positive group and SNS groups were separated from their dams.From PND60 to PND90,rats were given intragastric administration with pure water,fluoxetine(5.0 mg/kg),and SNS(2.5 g/kg,5.0 g/kg and 10 g/kg).Sucrose preference test,open field test and forced swimming test were performed from PND83 to PND90.Subsequently,transmission electron microscopy(TEM)was used to observe the structure of synapses and mitochondria in the hippocampus.Immunohistochemistry was used to detect the expression of PSD-95 and SYN proteins in the CA1,CA2,CA3,DG regions of hippocampal.The ATP assay kit was used to detect the content of ATP in the hippocampus.WB was used to detect the expression levels of SYN,PSD-95,Mitofusin 2(Mfn2),Dynamin-related protein 1(Drp1)and Fission 1(Fis1)in hippocampus.The correlation between depression-like behavior and synaptic plasticity protein expression levels,ATP content,mitochondrial fusion/fission protein expression levels were performed with by Pearson’s correlation test.Results1.Effects of MS rats on depression-like behavior and synaptic plasticity(1)The results of body weight and behavioral tests showed that compared with the control group,MS reduced the weight gain,sucrose preference(%),central region time,and central region distance of male and female rats,and increased the immobility time of male rats.(2)The results of Nissl staining showed that the neurons in the hippocampus and prefrontal cortex of male and female MS rats were loosely arranged.Compared with the control group,the number of neurons in the hippocampus CA1,CA2,CA3 and prefrontal cortex of male MS rats were significantly reduced and the number of neurons in the hippocampus CA1,CA2,CA3,DG areas and prefrontal cortex of female MS rats were also significantly reduced.(3)The results of immunohistochemistry and WB showed that compared with the control group,MS reduced the protein expression levels of SYN,GAP-43 and PSD-95 in the hippocampus and cortex of male and female rats.2.Metabonomics analysis of brain tissue in MS rats(1)Metabolomics results showed that the sex hormone metabolites(estradiol,testosterone,androstenedione,estrone,estriol,5β-dihydrotestosterone)of the MS groups in male and female did not significantly difference compared with the control group,(2)The pathway enrichment results showed that pantothenate and CoA biosynthesis,arginine and proline metabolism,glutathione metabolism,and phenylalanine,tyrosine,and tryptophan biosynthesis were significantly altered.3.Effects of SNS on depression-like behavior and synaptic plasticity in MS rat model(1)The results of body weight and behavioral results showed that the weight gain,the sucrose preference(%)and central region distance of the model group were significantly reduced,and the immobility time was significantly increased compared with the control group.Compared with the model group,the weight gain,the sucrose preference(%)and central region distance of the positive group and SNS groups were significantly increased,and the immobility time was significantly decreased.(2)The TEM results of the synaptic structure showed that the asymmetric synaptic PSD thickness and asymmetric synaptic cleft length in the hippocampus of model group were significantly reduced compared with the control group.Compared with the model group,they were increased significantly of the positive group and SNS groups.However,there was no statistical difference in the symmetric synaptic cleft length between the groups.(3)The results of immunohistochemistry and WB showed that the expression levels of PSD-95 and SYN protein in the hippocampus of the model group were significantly reduced compared with the control group.Compared with the model group,they were increased significantly in the positive group and SNS groups.4.Effects of SNS on the structure and function of mitochondria in the hippocampus of MS rat model(1)The TEM results of the mitochondrial structure showed that the mitochondria in the hippocampus of the control group were mostly rod-shaped,the mitochondrial crista was clear,and the outer membrane structure was completed.The mitochondria in the hippocampus of the model group were obviously swelled and vacuolated,the mitochondrial crista were arranged disorderly,and partly dissolved and disappeared.Compared with the model group,the mitochondrial pathological changes of the positive group and SNS groups have been significantly improved.(2)The ATP assay results showed that ATP levels were significantly reduced in the model group compared with the control group.Compared with the model group,the levels of the positive group,the low and high dose of SNS groups were increased significantly,but there was no significant difference in middle dose of SNS group.(3)The results of WB showed that compared with the control group,the protein expression levels of Mfn2 in the hippocampus of the model group was significantly reduced.and the expression levels of Drp1 and Fis1 were significantly increased.Compared with the model group,the Mfn2 protein expression levels of the positive group and the SNS groups were significantly increased,and the Drpl and Fisl protein expression levels were significantly reduced.(4)The results of correlation analysis showed that the sucrose preference(%)was positively correlated with the ATP levels and the expression levels of PSD-95,SYN and Mfn2,and negatively correlated with the expression levels of Drpl and Fis1.In contrast,the immobility time was negatively correlated with ATP levels and the expression levels of PSD-95,SYN and Mfn2,and positively correlated with the expression levels of Drpl and Fis1.Conclusion1.Maternal separation induces anxiety-like and depression-like behavior in rats and reduces the expression levels of synaptic plasticity protein in hippocampus and cortex to impairs hippocampal synaptic remodeling.2.Maternal separation significantly alter the brain metabolic pathways of pantothenate and CoA biosynthesis,arginine and proline metabolism,glutathione metabolism,and phenylalanine,tyrosine,and tryptophan biosynthesis.3.SNS may regulate hippocampal synaptic remodeling by alleviating mitochondrial structural damage and energy metabolism disorders,thereby improving depression damage caused by early stress... |
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