| Objective:To observe the influence of Xingpi Jieyu formula on the synaptic plasticity of hippocampal neurons based on AMPK/SIRT1/PGC-1α pathway associated with mitochondrial biosynthesis in rats with depression through chronic restraint stress.Methods:The experimental study is divided into four parts.Part 1:140 SD male rats were divided randomly into control group(CON),model group(MOD),Xingpi Jieyu formula group(XPJYF),Escitalopram Oxalate Tablets group(EOT),Xingpi Jieyu formula combined with Escitalopram Oxalate Tablets group(XPJYF+EOT).Each group was divided into 3 weeks and 6 weeks groups according to the stress time.In addition to CON,the other groups were subjected to chronic restraint stress(CRS)for 21 days.After the modeling started,the drug was given simultaneously by intragastric administration.The behavioral observations were made before modeling and at 3 or 6 weeks of modeling.After 3 weeks of modeling,the degree of spleen deficiency was evaluated,and the success of the model was evaluated by behavioral test.After sampling for each group,the HE staining and Nissl staining were used to observe the pathological changes of neurons in hippocampal CA1 region and the intervention effect of XPJYFPart 2:The grouping and intervention of rats were the same as study 1.The changes of synapses and mitochondria ultrastructure in hippocampal CA1 region were observed;mitochondrial and neuronal synaptosomal indexes were analyzed;The changes of neuronal axons and dendritic branches were observed by Golgi staining.Extract the hippocampal synaptosomes,the expression of synaptic plasticity proteins were detected by immunofluorescence staining,and the contents of mitochondrial synthetic proteins were detected by western blot.The activities of mitochondrial respiratory chain enzyme complex I-IV and mitochondrial enzyme in hippocampus were detected.Part 3:Drug-containing sera were prepared for XPJYF’s L,M and H dose groups.The primary hippocampal neurons were isolated and cultured and identified in vitro,and the XPJYF cytotoxicity test was carried out by CCK8 method.A model of hippocampal neuron injury induced by corticosterone(CORT)was established and divided into CON,CORT,CORT+X(H),CORT+X(M),CORT+X(L),CORT+E,CORT+E+X(H),CORT+E+X(M),CORT+E+X(L).The SYN and PSD-95 expression were detected by cellular immunofluorescence.The imaging of hippocampal neurons and mitochondria was observed.Mitochondrial biosynthesis and synaptic remodeling protein expression were detected by Western blot.The contents of 5-HT and DA and their receptors were detected by ELISA and RT-PCR.Part 4:The grouping and intervention of rats were the same as study 1.The expression of NeuN/CD31/GFAP in hippocampal CA1 region was observed by immunofluorescence staining.The expression of AMPK/SIRT1/PGC-1α signaling pathway gene in hippocampus was detected by RT-PCR.The protein expressions of NeuN,GFAP,VEGF and Collagen Ⅳwere detected by Western blot.Result:1.Part 11.1 Macro characterization:Before modeling,rats in each group were in good mental state,responsive and able to move freely.After 3 weeks of modeling,the stress response decreased,and the expression was tired,the activity decreased,the hair was dry or yellow,and the stool gradually thinned,which was more obvious at 6 weeks.Compared with the model group,the treatment group showed an obviously improvement.1.2 Change of body mass:Compared with CON group,the body mass of rats in MOD group decreased(P<0.01).Compared with MOD group,the body mass of XPJYF group,EOT group and combined groups increased(P<0.01,P<0.05).Compared with XPJYF group,the body mass in 3 weeks of EOT group and in each combined groups increased(P<0.01,P<0.05),but decreased in the 6 weeks of EOT group(P<0.05).Compared with the EOT group,the body mass in the combined group increased(P<0.01,P<0.05)1.3 Behavioral comparison:Compared with CON group,the sugar water preference rate(SPT)in MOD group was decreased(P<0.01),but the forced swimming immobility time(FST)was prolonged(P<0.01).Compared with MOD group,the SPT in 3 weeks of EOT and combined group was increased(P<0.01,P<0.05),and also the same as in each 6 weeks of treatment group.But the FST was decreased in these group(P<0.01,P<0.05).Compared with each XPJYF and EOT groups,the SPT in combined group was increased(P<0.01,P<0.05),but the FST was decreased(P<0.01,P<0.05).Compared with the baseline time,the SPT in each treatment group was decreased(P<0.01),the FST in each combined group and in 6 weeks of XPJYF and EOT group was prolonged(P<0.01,P<0.05).1.4 The serum amylase and Urine D-xylose excretion rate:Both serum amylase and Urine D-xylose excretion rate were decreased after 3 weeks modeling(P<0.01),and even decreased in 6 weeks compared with 3 weeks modeling(P<0.01).1.5 Pathological changes of hippocampal CA1 region:HE staining:The number,distribution and morphology of neurons in hippocampal CA1 region in CON group were normal,and the round nuclei were clear and centered.In 3 weeks of MOD group,the number of neurons decreased,the arrangement was irregular,with different cell size,and some cells were triangular,with deeply stained nucleus,which nucleoli were not clear.The neuron damage in 6 weeks of MOD group was further aggravated than 3 weeks group.The quantity and shape of each drug group were improved in different degrees,and the improvement effect was obvious in 6 weeks group and the combined group.Nissl staining:In CON group,the neurons in hippocampal CA1 region were normally distributed.and abundant Nissl bodies could be seen in the cytoplasm.The Nissl bodies near the nucleus were "tabby like",and the distal nuclei were fine granules like,and the nucleoli were clear.In 3 weeks of MOD group,the number of cell layers decreased,the arrangement was disordered,the morphology was irregular,the membrane was not complete,and the number of cytoplasmic Enni bodies decreased.At 6 weeks of MOD group,the cell damage was aggravated,the morphology of the cells was obviously wrinkled,the cytoplasm was deeply stained,the nucleus was not clear,some cells were reduced in Nissolith,and the phenomenon of central chromatolysis was observed.The cell damage in each treatment group gradually recovered,and the improvement was obvious in 3 weeks of EOT group and 6 weeks of XPJYF group.2.Part 22.1 There were more neuronal processes,normal mitochondria,complete synaptic structure and more synaptic vesicles in CON group.After 3 weeks of modeling,the mitochondria in the synapse were swollen,the cristae were broken,and the number of synaptic vesicles decreased.At 6 weeks of modeling,the damage was more obvious,including unclear synaptic space,reduction of synaptic vesicles,aggregation and distribution of synaptic vesicles,mitochondrial membrane destruction,cristal rupture and matrix vacuolation,and above injury was alleviated to different extent in each treatment group.2.2 Mitochondrial stereology:Compared with CON group,the volume density(Vvm)of MOD groups was increased(P<0.01),while the surface number density(Nm),specific surface density(δ)and specific membrane surface density(δm)were decreased(P<0.01).Compared with MOD group,the Vvm in each treatment group decreased(P<0.01,P<0.05),and the Nm in each 6 weeks groups increased(P<0.01).The 8 and δm in XPJYF,combined groups and in 6 weeks of EOT group were increased(P<0.01,P<0.05).Compared with XPJYF group,the δ in 6 weeks of EOT group was decreased(P<0.01),the δm in each combined groups was increased(P<0.01,P<0.05),and the Vvm in 6 weeks of combined group decreased(P<0.01).2.3 Synaptosomology:Compared with CON group,the number density(Nv),surface density(Sv)and the number density(Ns)of synaptic vesicles in each MOD groups were decreased(P<0.01).Compared with MOD group,the Nv,Sv and Ns in 6 weeks of XPJYF and combined group were increased(P<0.01,P<0.05).2.4 Golgi staining:Compared with CON group,the number of Sholl intersection points and the density of dendritic spines in each MOD groups was reduced(P<0.01).Compared with MOD group,the number of Sholl intersection points in each XPJYF and combined groups were increased(P<0.01,P<0.05).The density of dendritic spines in each 6 weeks groups of XPJYF and EOT were increased(P<0.01,P<0.05),and the same as each combined groups.Also,the density of dendritic spines in 6 weeks of combined group was higher than that in XPJYF and EOT group(P<0.01)2.5 Synaptic plasticity proteins in hippocampal synaptosomes:Compared with CON group,the level of GAP-43 and PSD-95 in 3 weeks of MOD group were increased(P<0.01),while those were decreased in 6 weeks groups(P<0.05),and the SYN and Syntaxin 1 in each MOD groups were decreased(P<0.01).Compared with MOD group,the level of GAP-43 and PSD-95 in each XPJYF and combined groups were increased(P<0.01,P<0.05),and the same as in 3 weeks of EOT group.The SYN in each XPJYF and combined groups was increased(P<0.01),and Syntaxin 1 in each 6 weeks of treatment groups was increased(P<0.01,P<0.05).Compared with the XPJYF group,the SYN in 6 weeks of EOT group was decreased(P<0.05),and the GAP-43 in 6 weeks of combined group was increased(P<0.01).2.6 Mitochondrial biosynthetic proteins in hippocampal:Compared with MOD group,the SIRT1,PGC-1α,AMPK-al,TFAM and NRF1 in MOD group were decreased(P<0.01).Compared with MOD group,the SIRT1 and AMPK-α1 in 3 weeks of EOT group and the SIRT1,PGC-1α and TFAM in each combined groups were increased(P<0.01,P<0.05).The PGC-1α in 6 weeks group and AMPK-al in 3 weeks group of combined was increased than XPJYF group(P<0.01).3.Part 33.1 Cytotoxicity test:The result of immunofluorescence identification showed that the cultured hippocampal neurons were consistent with the characteristics of neuron cells.By CCK8 test,the serum concentrations of Xingpi Jieyu formula in each group were determined to be 10%in the high-dose group,10%in the medium-dose group and 20%in the low-dose group,and the action time was determined to be 24h.3.2 Expression of mitochondrial biosynthetic proteins:Compared with CON group,the level of SIRT1,PGC-1α,NRF1 and Tfam were decreased in CORT and CORT+E groups(P<0.01).Compared with CORT group,the level of SIRT1,NRF1 and Tfam in CORT+X(H)and CORT+X(M)groups were increased(P<0.01),the level of SIRT1 in CORT+E group was decreased(P<0.05),and the level of PGC-1α in CORT+X(H)group was increased(P<0.01).Compared with CORT+E group,the level of PGC-1α and Tfam in CORT+E+X(H)group were increased(P<0.05),and the level of NRF1 and Tfam in CORT+E+X(M)group were increased(P<0.01).3.3 Expression of Synaptic plasticity proteins:Compared with CON group,the level of GAP-43,SYN and PSD-95 in CORT group and CORT+E group were decreased(P<0.01).Compared with CORT group,the level of GAP-43 and SYN in CORT+X(H)and CORT+X(M)groups were increased(P<0.01),the same as PSD-95 in CORT+X(H)group.Compared with CORT+E group,the GAP-43,SYN and PSD-95 in each of the CORT+E+XPJYF group were increased(P<0.01,P<0.05).3.4 Contents of 5-HT and DA and the gene expression of their receptors:Compared with CON group,the level of 5-HT,DA,5-HT1AR mRNA and DRD1 mRNA in CORT group and CORT+E group were decreased(P<0.01).Compared with CORT group,the level of 5-HT and DA in CORT+X(H)group were increased(P<0.05),the level of 5-HT in CORT+E group was decreased(P<0.05),and the levels of 5-HT1AR mRNA and DRD1 mRNA in CORT+X(H)and CORT+X(M)group were increased(P<0.01).Compared with CORT+E group,the 5-HT in CORT+E+X(H)group increased(P<0.05),and the 5-HT1AR mRNA and DRD1 mRNA levels in each CORT+E+XPJYF groups were increased(P<0.01,P<0.05).4.Part 44.1 Expression of mitochondrial synthetic genes:Compared with CON group,the expression of SIRT1 mRNA and PGC-1α mRNA in MOD group were decreased(P<0.01).Compared with MOD group,the levels of SIRT1 mRNA,PGC-1α mRNA and AMPK-α1 mRNA in each treatment groups were increased(P<0.01).The level of NRF1 mRNA were increased in 6 weeks of XPJYF group,and also increased in each EOT and combined groups(P<0.01).The expression of Tfam mRNA in each XPJYF and combined groups and in 3 weeks of EOT group were increased(P<0.01,P<0.05).Compared with XPJYF group,the levels of SIRT1 mRNA,PGC-1α mRNA and NRF1 mRNA in combined group were increased(P<0.01),the level of SIRT1 mRNA was increased in 3 weeks of EOT group(P<0.01).4.2 Expression of synaptic microenvironment proteins:Compared with CON group,the level of NeuN,GFAP and VEGF in MOD groups were decreased(P<0.01).Compared with MOD group,the level of NeuN,GFAP and VEGF in each XPJYF and combined groups were increased(P<0.01,P<0.05),the GFAP in each EOT groups increased(P<0.01),the VEGF in 3 weeks of EOT group was increased(P<0.01).Compared with XPJYF group,the GFAP in each EOT groups were decreased(P<0.05),the GFAP in each combined groups were increased(P<0.01),the VEGF in 3 weeks of combined group increased(P<0.05).Conclusion:1.After 3 weeks of chronic restraint stress,the animal model met the criteria of depression with syndrome of stagnation of liver qi and spleen deficiency.Neurons in hippocampal CA1 region of brain were damaged,and the degree of damage increased with the prolongation of stress time.Both the Xingpi Jieyu formula and escitalopram oxalate can reduce the neurons damage and improve depression-like behavior in varying degrees,and the long-term protective advantage of Xingpi Jieyu formula is more obvious than that of escitalopram oxalate,and the combined application of these two drugs is more significant2.The synaptic ultrastructure destruction,mitochondrial damage and synaptic plasticity in hippocampal CA1 region of depressive model rats are directly related to mitochondrial biosynthesis,which is mediated by AMPK/SIRT1/PGC-1α signaling pathway.Both Xingpi Jieyu formula and escitalopram oxalate can dynamically regulate this signaling pathway and promote the synaptic plasticity of hippocampal neurons,but their advantages are different.The escitalopram oxalate has more advantages in regulating neurotransmitters,while Xingpi Jieyu formula is more obvious in improving mitochondrial function,and the long-term effect of Xingpi Jieyu formula is better than that of escitalopram oxalate3.The damage of astrocytes,microvascular endothelial cells and microvascular basement membrane in the synaptic microenvironment of hippocampal neurons in depression is related to synaptic plasticity.The Xingpi Jieyu formula can increase mitochondrial biosynthesis by regulating the signaling pathway of AMPK/SIRT1/PGC-1α,and protect the synaptic microenvironment and neuroremodeling in depression.Moreover,this effect is more advantageous than that of escitalopram oxalate,and the combined application of these two drugs had a significant effect. |
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