| This experiment is composed of two parts,animal experiment and cell experiment.Through chronic immobilization stress and oxidative stress,the nerve protective effect of Xiaoyaosan Decoction on rats with depression can be discussed from Wnt/β-catenin signal pathway.The first part:Effect of Xiaoyaosan Decoction on hippocampal nerve regeneration in rats with chronic stress from Wnt/β-catenin signal pathway1 Objective(1)To observe the changes of Wnt/β-catenin pathway in rats with syndrome of stagnation of liver qi and spleen deficiency,so as to explore the biological of nerve regeneration.(2)To investigate the effect of Xiaoyaosan Decoction on hippocampal nerve regeneration in rats with depression from Wnt/β-catenin signal pathway,so as to provide experiment basis for treating stress-induced diseases.2 MethodsTotal 60 male rats were randomly divided into the following groups:control group,model group,Xiaoyaosan group and fluoxetine group,15 rats in each group.Rats in experimental groups suffered from immobilization stress for 21 days.The rat model was tested as the model of syndrome of stagnation of live qi and spleen deficiency by animal characterization,bodyweight,food intake,open field test and sucrose preference test.Immunofluorescence was used to detect BrdU/DCX and BrdU/NeuN positive cell number.And Western blot and RT-PCR were used to detect the change of Wnt/β-catenin signal pathway.3 Mainly results(1)Through the 21 days chronic inmmobilization stress,body weight and food intake of rats in model group decreased(P<0.01),but those of rats in Xiaoyaosan group and fluoxetine group significantly increased(P<0.05).Open field test showed that the movement capacity of rats in model group decreased(P<0.05),but those of rats in Xiaoyaosan group and fluoxetine group significantly increased(P<0.05).Sucrose preference test showed that sucrose consumption rate of the model group was obviously lower than that of the control group(P<0.05),and that of the Xiaoyaosan and fluoxetine was significantly higher than the model group(P<0.05).All the results above suggested that rat model of syndrome of stagnation of live qi and spleen deficiency was successfully established.(2)The BrdU/DCX、BrdU/NeuN double-labeled positive cells of model group decreased significantly compared with control group(P<0.05).The BrdU/DCX、BrdU/NeuN double-labeled positive cells increased compared with control group(P<0.05).(3)Compared with control group,the protein expression of Wnt1、Wnt3a、β-catenin、LEF-1 decreased and the protein expression of GSK-3β increased in model group(P<0.01or P<0.05).After treatment with Xiaoyaosan and fluoxetine,the protein expression of Wnt1、Wnt3a、β-catenin、LEF-1 decreased compared with model group(P<0.01or P<0.05).The protein expression of GSK-3β in Xiaoyaosan group decreased compared with model group(P<0.05).(4)Compared with control group,the gene expression of Wntl、Wnt3a、β-catenin、Axin、LEF-1 decreased and the protein expression of GSK-3β increased in model group(P<0.01or P<0.05).After treatment with Xiaoyaosan and fluoxetine,the protein expression of Wntl、Wnt3a、β-catenin、Axin decreased compared with model group(P<0.01or P<0.05).The gene expression of GSK-3β in Xiaoyaosan and fluoxetine group increased compared with model group(P<0.05).The gene expression of LEF-1 in Xiaoyaosan and fluoxetine group had no significant difference compared with model group(P>0.05).4.Conclusion(1)Rat model of syndrome of stagnation of live qi and spleen deficiency was successfully established.(2)Xiaoyaosan can significantly improve the behavioral manifestations of syndrome of stagnation of live qi rats.(3)Depression is closely related with nerve cell degeneration.(4)Xiaoyaosan can can activate Wnt/β-catenin signaling pathway to increase neuronal regeneration,which may be one of its antidepression mechanism.(5)Xiaoyaosan Decoction have neuroprotective effects,by increasing nerve cell regeneration and recovering hippocampal nerve function.The second part:Protective effect of Xiaoyaosan Decoction on hippocampal neural synapse under oxidative stress from Wnt/β-catenin signal pathway1 ObjectiveTo observe the effect of Xiaoyaosan Decoction on oxidative stress hippocampal neural synapse regulated by Wnt/β-catenin signal pathway2 MethodsHipppcampal neurons were isolated from rats and cultured in the serum-free culture media.The primarily cultures neurons were divided into control group,H2O2 group(100μmol/L),SSa(2.5 mg/L、5 mg/L、10.0 mg/L)+H2o2.The cell viability was determined by MTT analysis.Immunofluorescence was used to detect VGAT/Gephyrin and VGLUT/PSD95 integrated puncta number.And Western blot was used to detect Wnt/β-catenin signal pathway protein.3 Mainly results(1)Compared with control group,the cell viability of H2O2 group(100μmol/L),SSa(2.5 mg/L)+H2O2 group decreased to(67.81±9.45)%、(82.74±8.95)%(P<0.01or P<0.05);Compared with H2O2 group,the cell viability of H2O2 group,SSa(2.5 mg/L)+H2O2 group increased to(94.82±4.45)%、(92.49±5.2)%(P<0.05).(2)Compared with control group,VGAT/Gephyrin integrated puncta number of H2O2 group(100μmol/L),SSa(2.5 mg/L)+H2O2 group decreased(P<0.01or P<0.05).Compared with H2O2 group,VGAT/Gephyrin integrated puncta number of H2O2 group(100μmol/L),SSa(2.5 mg/L、5 mg/L、10.0 mg/L)+H2O2group increased(P<0.05).Compared with control group,VGLUT/PSD95 integrated puncta number of H2O2 group(100μmol/L),SSa(2.5 mg/L)+H2O2 group decreased(P<0.01or P<0.01).Compared with H2O2 group,VGLUT/PSD95 integrated puncta number of H2O2 group(100μmol/L),SSa(2.5 mg/L、5 mg/L、10.0 mg/L)+H2O2 group increased(P<0.01).(3)Compared with control group,the protein expression of Wnt7a、β-catenin、LEF-1 decreased and the protein expression of GSK-3β increased in H2O2 group(P<0.01or P<0.05).After treatment with SSa(5 mg/L、10.0 mg/L),the protein expression of Wnt7a、β-catenin、LEF-1 increased and GSK-3β decreased compared with H2O2 group(P<0.01or P<0.05).The protein expression of GSK-3β in Xiaoyaosan group decreased compared with model group(P<0.05).The protein expression of LEF1 increased and GSK-3β decreased in SSa(2.5 mg/L)+H2O2 group compared with SSa(10.0 mg/L)+H2O2group(P<0.05).The protein expression of Wnt7a、LEF1 increased and GSK-3β decreased in SSa(2.5 mg/L)+H2O2 group compared with SSa(5 mg/L)+H2O2group(P<0.05).Threr is no significant difference in the protein expression of Wnt7a、β-catenin、LEF-1、GSK-3β between SSa(10 mg/L)+H2O2group compared with SSa(5 mg/L)+H2O2group(P>0.05).4.Conclusion(1)SSa can increase the neuron synapse number and have neuroprotective effects.(2)Xiaoyaosan can can activate Wnt/β-catenin signaling pathway to play antidepression function.(3)SSa(5 mg/L)can be chosen as the best dosageThe protective effect of Xiaoyaosan and SSa on hippocampal neuron under the condition of stress may be regulated by Wnt/β-catenin signaling pathway. |
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