| Objective:Previous studies have found that brain-type creatine kinase(CK-BB)is the target of protopanaxadiol(PPD),and PPD is an activator of CK-BB.In this study,by observing the effects of PPD in corticosterone(CORT)-induced hippocampal CK-BB enzyme activity,neural structural plasticity and depression-like behaviors,the mechanism of PPD targeting CK-BB to exert antidepressant effect was explained.Methods:Experiment 1:Effects of long-term CORT injection on hippocampal creatine kinase(CK),neural structural plasticity and depression-like behaviors in miceSixty C57BL/6 mice were randomly divided into 6 groups,namely 1W control group(Control),1W CORT group;3W Control group,3W CORT group;5W Control,5W CORT group.The mice in CORT group were given intraperitoneal injection of CORT(30 mg/kg/d)at random time points,and the mice in control group was injected with the same dose of normal saline.After administration,behavioral experiments and hippocampal CK enzyme activity assay were performed in each group.Western-Blot was used to determine hippocampal brain-derived neurotrophic factor(BDNF),neurofilament light chain(NF-L),CK-BB and synapse vesicular protein(SYP).The dendritic spine density and dendritic length of neurons in the hippocampal CA1,CA3 and DG regions were quantitatively analyzed by the stereology Neurolucida(MBF Bioscience)method.Experiment 2:Effects of CK inhibitor on hippocampal CK,neurostructural plasticity and depression-like behaviorsTwenty male SD rats were randomly divided into 4 groups:sham group(Sham),CORT group,inhibitor group(Inhibitor),CORT+Inhibitor group.CORT is intraperitoneal injected with a dose of 20 mg/kg/d.Inhibitor lateral ventricle administration method:CK inhibitor(β-guanidinopropionic acid,β-GPA)was dissolved in cerebrospinal fluid,and then injected into the capsule micro-osmotic pressure pump(Alzet Model 2006),the capsule pump was embedded in rats subcutaneously on the back and connected to the rat brain cannula,the concentration of CK inhibitor in the capsule pump is 10 mmol/L(200 μL).After 5 weeks of administration of the inhibitor at a constant speed(0.15 μL/h)to the capsule pump,the depression-like behavior and CK enzyme activity of the rats were detected.Western-Blot was used to detect the proteins of BDNF,NF-L,CK-BB and SYP in the hippocampus of the rats.Dendritic spine density and dendritic length of neurons in hippocampal CA1.CA3 and DG regions were quantitatively analyzed by the stereological Neurolucida method.Experiment 3:PPD regulates hippocampal CK,neurostructural plasticity and depression-like behaviors in model animalsSixty C57BL/6 mice were randomly divided into 6 groups:Control group,CORT group,CORT+fluoxetine group(Fluoxetine),CORT+PPD high-dose group(50 mg/kg))(H),CORT+PPD medium-dose group(25 mg/kg)(M),CORT+PPD low-dose group(12.5 mg/kg)(L).CORT was administered by intraperitoneal injection at a dose of 30 mg/kg/d.In the control group,the CORT was replaced by normal saline,fluoxetine was intragastrically administered at a dose of 10 mg/kg/d,and PPD was administered by intragastric administration.After 5 weeks,the behavioral tests were performed and CK enzyme activities were determined.Western-Blot was used to determine the protein expressions of BDNF,NF-L,CK-BB and SYP in the rat hippocampus.Dendritic spine density and dendritic length of neurons in hippocampal CA1,CA3 and DG regions were quantitatively analyzed by the stereological Neurolucida method.ResultsExperiment 1:Long-term CORT injection induces depression-like behaviors in mice,reduces hippocampal CK enzyme activities,and impairs hippocampal neurostructural plasticityCompared with the 5W control group,the immobility time in the FST and TST experiments of the mice in the 5W CORT group was significantly increased(P<0.01),and the enzymatic activity of CK was significantly decreased(P<0.01).Western-Blot results showed that compared with the 5W control group,the expressions of BDNF(P<0.01),CK-BB(P<0.05),NF-L(P<0.05)and SYP(P<0.05)in the 5W CORT group of mice was significantly down-regulated.The results of stereology showed that compared with the 5W control group,the dendritic spine density and dendritic length of neurons in the CA1 and CA3 areas of mice in the 5W CORT group were significantly decreased(P<0.05).The dendritic spine density(P<0.01)and dendritic length(P<0.05)of neurons in DG area were significantly decreased.Experiment 2:CK inhibitor reduces the activity of CK in hippocampus of rats,and damages the neuroplasticity of hippocampus,leading to depression-like behaviorsCompared with the Sham group,the rats in CORT group(P<0.01)and the Inhibitor group(P<0.01)had significantly lower percentages of sucrose preference.In the FST,the immobility time of the rats in CORT group(P<0.05)and the Inhibitor group(P<0.05)was significantly prolonged.In TST,the immobility time of the rats in the CORT group(P<0.01)and the Inhibitor group(P<0.01)was significantly prolonged.When compared with Sham group,the activity of CK was significantly decreased in CORT group(P<0.01)and Inhibitor group(P<0.01).Western-Blot results showed that compared with the Sham group,the expressions of CK-BB(P<0.05)in the CORT group and the Inhibitor group was significantly decreased.When compared with the Sham group,the BDNF(P<0.05),NF-L P<0.05)and SYP(P<0.01)decreased significantly in CORT group;the BDNF(P<0.05),NF-L(P<0.05)and SYP(P<0.01)in Inhibitor group decreased significantly.Stereological results showed that compared with the Sham group,the dendritic spine density and dendritic length of neurons in the CA1,CA3 and DG regions were significantly reduced in the CORT group(P<0.05)and the Inhibitor group(P<0.05).Experiment 3:PPD can up-regulate hippocampal CK activity in depression model mice and antagonize CORT-induced hippocampal neuroplasticity damage and depression-like behaviorsCompared with the CORT group,the immobility time of the mice in the high-dose PPD group in FST(P<0.01)and TST(P<0.05)was significantly shortened.High-dose PPD group(P<0.05)and the middle-dose PPD group(P<0.05)mice had significantly increased CK enzyme activities.Western-Blot results showed that compared with the CORT group,the PPD high-dose group mice had higher levels of BDNF(P<0.01),CK-BB(P<0.05),NF-L(P<0.05)and SYP(P<0.01).Stereological results showed that compared with the CORT group,the spines density of neurons in the high-dose PPD group of CA1(P<0.01),the high-dose PPD group of CA3(P<0.05),the high-dose PPD(P<0.05)and the middle-dose PPD(P<0.05)group of DG was significantly increased.Compared with the CORT group,the dendrite length of neurons in the high-dose PPD group of CA1 region(P<0.01),the high-dose PPD group of CA3 region(P<0.05),and the high-dose PPD group of DG region(P<0.05)was significantly increased.Conclusions1.Long-term CORT injection can lead to the decrease of expression and activity of hippocampal creatine kinase,thereby impairing the plasticity of hippocampal neural structure and inducing depression-like behaviors.2.After PPD enters the brain tissue and combines with CK-BB,it can increase the activity of creatine kinase and antagonize the plastic damage of hippocampal neural structure caused by long-term corticosterone injection,thereby exerting antidepressant activity. |
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