| Objective: The antidepressant effect of Shenhu Wendan Decoction was evaluated by mouse behavioral despair model and rat unpredictable mild stress model,and it was discussed from monoamine neurotransmitters,inflammatory cytokines,ERK signaling pathways and neurotrophic factors.Methods: 1.Pharmacodynamic studies:(1)Male ICR mice were randomly divided into 7 groups according to their body weight: blank control group,fluoxetine hydrochloride group(0.02g/kg),Chaihu Shugan powder(9.29g/kg crude drug),Shenhu Wendan Decoction Low-dose group(2.94g/kg crude drug),medium-dose group(5.88g/kg crude drug),high-dose group(11.76g/kg crude drug)and highest dose group(23.52g/kg crude drug),each group was given continuous gavage for 16 days,the open field test was conducted on the 13 th day,the tail suspension experiment was performed on the 14 th day,and the forced swimming test was performed on the 15 th day.(2)Male Wistar rats were built with CUMS combined with orphan depression model.The sucrose preference experiment was used to determine the successful modeling.Combined with the behavioral result of the open field test,the rats were randomly divided into 7 groups: blank control group,model group and positive control group(fluoxetine hydrochloride 0.01g/kg),Chinese medicine positive control group(Chaihu Shugan Powder 6.5g/kg crude drug),Shenhu Wendan Decoction low dose group(4.12g/kg crude drug),Shenhu Wendan Decoction medium dose group(8.24g/kg crude drug)and Shenhu Wendan decoction high dose group(16.48g/kg crude drug),a blank control group was also set up.The rats in each group were given intragastric administration for 15 consecutive days.The blank control group and the model group were given distilled water of the same volume.Except the blank control group,the other groups continued to receive stimulations for 15 days.The sucrose consumption experiments and the open field tests were performed on the 0,21 st and 35 th day.2. Mechanism research:(1)Monoamine neurotransmitter :The content of monoamine neurotransmitters(5-HT,DA and NE)in hippocampus,cortex and hypothalamus of each group was detected by ELISA.(2)Neuroimmunity :The levels of inflammatory cytokines IL-1β,IL-6,TNF-α,IL-4 and IL-10 in rat serum and hippocampus were determined by ELISA.(3)ERK pathway:Western blot was used to detect the protein expression of Raf,p-Raf,Rsk,MEK,p-MEK,ERK1/2,p-ERK1/2,CREB,p-CREB and BDNF in hippocampus of each group;the gene expression levels of ERK1/2,CREB,BDNF and Trk B in rat hippocampus were detected by PCR method.The expressions of ERKl/2 and Trk B in hippocampus of rats were detected by immunohistochemistry Results: 1.Pharmacodynamic studies:(1)Results of the mouse behavioral despair model: Compared with the blank control group,the suspension time of the mice in the fluoxetine group and high dose group of Shenhu Wendan decoction group was significantly shortened(P<0.01),time of suspension of mice in Chaihu Shugan San group and the middle dose group of Shenhu Wendan Decoction was shortened(P<0.05);the swimming time of mice fluoxetine group,Chaihu Shugan San group,Shenhu Wendan decoction medium dose group and the high-dose group of Shenhu Wendan decoction was significantly shortened(P<0.01),and the swimming time of the low-dose group of Shenhu Wendan Decoction was shortened(P<0.05); there was no significant difference in the total distance of movement and the number of rearings among the fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction groups(P<0.05).(2)Rat behavioral and pathological staining results:After 21 days of modeling,the body weight,sucrose preference and behavioral results in the open field test of the model group were significantly different from those of the blank control group(P<0.05).After 14 days of continuous administration,the weight of rats in the high dose group of Shenhu Wendan Decoction increased significantly compared with the model group(P<0.05);the sucrose preferences of high,medium and low dose groups were significantly higher than that of the model group(P<0.05).The total moving distances and the numbers of rearing in the high-dose group were significantly increased than those in the model group(P<0.05).In Nissl staining,the pyramidal cells in the hippocampal CA 3 region of the model group were disordered,the stratification of neurons was unclear,the Nissl body was stained or disappeared,and the nucleus pyknosis was observed.Compared with the model group,the neurons in the high and medium dose groups of Shenhu Wendan Decoction were layered clearly,arranged neatly,the membrane was almost intact,the chromatin distribution in the nucleus was more uniform,and the Nissl body was lightly stained.2.Mechanism studies:(1)Monoamine neurotransmitter measurement results: Compared with the model group,the levels of 5-HT in the hippocampus,cortex and hypothalamus of the blank control group,fluoxetine group,Chaihu Shugan San group and Shenhu Wendan group were significantly increased(P<0.01); the content of DA in the cortex of fluoxetine group increased(P<0.05),the content of DA in the hippocampus and hypothalamus of the blank control group,Chaihu Shugan San group and Shenhu Wendan group increased significantly(P<0.01);the content of NE in hippocampus of fluoxetine group increased(P<0.05)while in hippocampus of blank control group,Chaihu Shugan San group and Shenhu Wendan group it increased significantly(P<0.01).(2)Neuroimmunoassay results:The protein expression levels of Raf,p-Raf,p-MEK,ERK1/2,p-ERK1/2,Rsk,CREB,p-CREB and BDNF in the hippocampus of depressed rats in Shenhu Decoction groups were significantly higher than those in the model group(P<0.05),in which the high dose group was the most effective.At the same time,the high dose group of Shenhu Wendan Decoction could significantly up-regulate the expression of ERK1/2,CREB,BDNF and Trk B m RNA in hippocampus(P<0.05),and up-regulate the average optical density of ERK1/2 and Trk B(P<0.05).(3)ERK signal pathway test results: WB results: Compared with the blank control group,the protein expressions of Raf,p-Raf,MEK,p-MEK,ERK1/2,p-ERK1/2,Rsk,CREB and BDNF in the model group were significantly decreased(P<0.01);Compared with the model group,the expression of Raf protein in hippocampus of fluoxetine group and Shenhu Wendan decoction group increased significantly(P<0.01),and the expression of Raf protein in hippocampus of Chaihu Shugan San group increased(P<0.05).The expression of p-Raf protein in hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group increased significantly(P<0.01);the expression of MEK protein in hippocampus of fluoxetine group increased.(P<0.05), The expression of p-MEK protein in hippocampus of fluoxetine group was significantly increased(P<0.01).The expression of p-MEK protein in hippocampus of Chaihu Shugan Powder and Shenhu Wendan Decoction group increased(P<0.05).The expressions of ERK1/2 and p-ERK1/2 in hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group increased significantly(P<0.01); The protein expression of Rsk in hippocampus of fluoxetine group and Shenhu Wendan decoction group increased significantly(P<0.01).The protein expression of CREB in hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group increased(P<0.05).The protein expression of p-CREB in hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group increased significantly(P<0.01).The protein expression of BDNF in the hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group increased significantly(P<0.01). rt-PCR results: Compared with the blank control group,the expression of ERK1 m RNA,ERK2 m RNA,BDNF m RNA and Trk B m RNA in the hippocampus of the model group were significantly decreased(P<0.01),and the expression of CREB m RNA was decreased(P<0.05). Compared with the model group,the expression of ERK1/2 m RNA in the hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group was significantly increased(P<0.01). The expression of CREB m RNA in hippocampus of fluoxetine group and Chaihu Shugan powder group was significantly increased(P<0.01),and the expression of CREB m RNA in hippocampus of Shenhu Wendan Decoction group was increased(P<0.05). The expressions of BDNFm RNA and Trk B m RNA in hippocampus of fluoxetine group and Shenhu Wendan decoction group were significantly increased(P<0.01),and the expressions of BDNFm RNA and Trk B m RNA in hippocampus of Chaihu Shugan San group were increased(P<0.05).IHC results: Compared with the blank control group,the optical density values of ERK1/2 and Trk B in the hippocampus of the model group were significantly lower(P<0.01);Compared with the model group,the optical density values of ERK1/2 and Trk B in the hippocampus of fluoxetine group,Chaihu Shugan San group and Shenhu Wendan decoction group were significantly increased(P<0.01).Conclusions: 1.Shenhu Wendan Decoction has a certain anti-depression effect,which can shorten the immobility time of suspense and forced swimming in behavioral despair mouse model,and reverse the decrease of sucrose preference,weight loss,total moving distance and the number of erects in CUMS rats;2.The antidepressant effect of Shenhu Wendan Decoction is related to the up-regulation of monoamine neurotransmitters such as 5-HT,DA and NE in the brain;3.Shenhu Wendan Decoction may regulate the immune system by up-regulating the anti-inflammatory cytokines IL-4,IL-10 and down-regulating the proinflammatory cytokines IL-1β,IL-6 and TNF-α.In this way,Shen Hu Wendan Decoction restores the dynamic balance of the immune system and prevents depression;4.Shenhu Wendan Decoction may play an anti-depressant role by activating the key indicators(Raf,MEK,ERK,RSK,CREB)in the ERK signal transduction pathway and regulating the neurotrophic factor BDNF and its receptor Trk B. |
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