The Effects Of Chaihu-Shugan-San And Its Components On The Behavior And The Expression Of P38MAPK And ERK5in The Brain Of Depressed Rats

ObjectiveTo evaluate the antidepressant-like effects of Chaihu-Shugan-San (CHSGS) and its components and further to investigate the CHSGS’s effects upon signal transduction of p38mitogen-activated protein kinase (p38MAPK) and of extracellular signal-regulated kinase5(ERK5) expressions in the frontal lobe and hippocampus of rats with depression induced by chronic unpredicted mild stress, so as to explore the molecular mechanism implicated in CHSGS antidepressant effect.Methods1. Male healthy adult SD rats were randomly divided into six groups: Normal; Model; CHSGS; Component Ⅰ; Component Ⅱ; and Fluoxetine.2. The experimental rat model of chronic-stress depression was replicated by chronic unpredictable mild stress (CUMS) for28days. Every non-normal rat was randomly subjected to one of different stimuli. Each rat in each group was intragastrically administered with a corresponding isovolumic liquid4.5ml/kg/day. The dosage of medicines administered to the rats equaled to that of a70kg-adult.3. Every rat was weighed on day1(before the CUMS),14and28of the experiment. The Open-field test and Sucrose consumption test were administered on day1,14and28of the experiment.4. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) in different groups had been tested by radio immunity technique.5. The underlying antidepressant mechanism were explored by measuring the effects of CHSGS, and its components, on the expression of p38MAPK, ERK5, phosphorylation-p38(p-p38MAPK) and phosphorylation-ERK5(p-ERK5) in the frontal lobe and hippocampus of rats using immunohistochemistry and western blotting. 6. Determining the mRNA levels of p38MAPK and ERK5in the pre frontal and the hippocampus of rats using Real-time Polymerase Chain Reaction (RT-PCR).Results1. Body weight was recorded on day1,14and28. The beginning weight difference between the groups was no statistically significant (P>0.05). The end weight CUMS model group had a significant decrease compared with the normal control group (P<0.01). Open-field activity and Sucrose consumption were measured on day1,14and28. There were no significant differences in the number of either crossings or rearings in the open-field test (P>0.05) among all groups, and no significant differences in the sucrose consumption among all groups on day1(P>0.05). After CUMS was administered, the number of crossing, rearing and grooming in the CUMS model group significantly decreased, the time for staying in the central square in open-field test increased and the total liquid consumption, sucrose consumption and the percentage of sucrose preference significantly decreased when compared to the normal group on day14and28(P<0.01).2. The CHSGS group showed a significant increase in the body weight, in crossings, rearings and the sucrose consumption and the percentage of sucrose preference, while the time of staying in the central squared decreased when compared to the Model group on days14and28day (P<0.05; P<0.01). The rats in the component Ⅰ, the component Ⅱ and fluoxetine group showed no significant difference in the weight gain and behavior indices when compared to the Model on day14(P>0.05). After28days of CUMS, the CHSGS rats showed the reverse of almost all the behavior alterations observed in model group. Similar changes were observed in the fluoxetine group (P<0.01; P<0.05). Component Ⅰ and Ⅱ each had only a partial effect on the depression indicators measured when compared to the Model group (P<0.05).3. The concentration of TNF-α and IL-6in serum increased significantly in depressant model rats when compared to the normal control group (P<0.01). Compared with the Model group, the levels of TNF-α and IL-6decreased in the CHSGS and the Fluoxetine groups (P<0.01). The levels of TNF-a increased both in the component Ⅰ and Ⅱ when compared to the normal (P<0.01). Compared with the Model group, there were no difference in the serum level of TNF-α (P>0.05). Compared with the Model group, the level of IL-6in the component Ⅰ group was decreased (P<0.05).4. The p38MAPK and ERK5protein were expressed both in the prefrontal and hippocampus of the normal rats. The Model group showed a significant increase in the level of p38MAPK with decreased in the gray value in the prefrontal and hippocampus when compared to the normal group using immunohistochemistry technique (P<0.01). The p38MAPK protein levels decreased and gray values increased in the prefrontal and hippocampus both in the CHSGS and in the fluoxetine groups when compared to the Model group (P<0.01; P<0.05). The p38MAPk expression increased in the hippocampus of the component Ⅰ and Ⅱ when compared to the normal group (P<0.01; P<0.05). Compared with the Model group, both were no statistical signification (P>0.05). There were no significant differences in the prefrontal and hippocampus ERK5expressions between the groups (P>0.05). Protein levels of p38, p-p38MAPK, ERK5, p-ERK5and β-actin were measured in the prefrontal and the hippocampus of all groups using Western-blotting technique. Both the prefrontal and hippocampus p38, p-p38and p-p38/p38MAPK levels of the Model group increased compared to that of the Normal group (P<0.05; P<0.01). The CHSGS group and the Fluoxetine group showed a significant decrease in the p38,p-p38and p-p38/p38MAPK levels both in the prefrontal and the hippocampus when compared to the Model group (p<0.05). Protein levels of p38, p-p38and p-p38/p38MAPK in Component Ⅰ and Ⅱ were not significantly different from those in the Model group (p>0.05, either). There were no significant differences in the prefrontal and the hippocampus ERK5levels between the groups (P>0.05). However, there were significant frontal lobe and hippocampus p-ERK5and p-ERK5/ERK5levels differences between the groups (P<0.01, P<0.05). Prefrontal and hippocampus p-ERK5levels of the Model group decreased significantly compared to that of the Normal group (P<0.01). p-ERK5and p-ERK5/ERK5levels in the frontal lobes and the hippocampi of CHSGS group rats increased compared to the Model control group (P<0.05; P<0.01). The positive control, Fluoxetine group also had a significant increased in hippocampus p-ERK5levels and in prefrontal and hippocampus p-ERK5/ERK5levels when compared to the Model group (P<0.05). There was no significant difference in the p-ERK5and P-ERK5/ERK5levels of the CHSGS group and the Fluoxetine group (p>0.05). p-ERK5and p-ERK5/ERK5values in Component Ⅰ and Ⅱ were no significantly different from those in the Model group (p>0.05).5. p38MAPK and ERK5mRNA analyses of frontal lobe and hippocampus samples from the Normal, Model, CHSGS, Component Ⅰ and Ⅱ, and the Fluoxetine group were performed to investigate the potential involvement of p38and ERK5mRNA in the depressed rats which had been administrated CHSGS and its Components (Ⅰ and Ⅱ). RT-PCR analysis demonstrated that p38MAPK mRNA expression was upregulated and2-△△CT value increased in the prefrontal and hippocampus of the Model group when compared to the Normal group (P<0.05, P<0.01). p38MAPK mRNA2-△△CT values in the prefrontal showed no difference among the CHSGS group, Components (Ⅰ and Ⅱ) and Flouxetine group when compared to the Model group (P>0.05). The hippocampus p38MAPK mRNA2-△△CT values both in the CHSGS and Fluoxetine groups showed decrease when compared to the Model group (P<0.05). RT-PCR analysis demonstrated there were no significant differences among groups in the frontal lobe and the hippocampus ERK5mRNA (P>0.05). The frontal lobe and hippocampus ERK5mRNA2-△△CT values show no difference between the groups (P>0.05).Conclusion1. Depressed rats had decreased weight gain; decreased locomotor activity in the open-field; and, reduced sucrose consumption, all of which mimic the symptoms of depression in humans and indicated success in depressing the rats. Fluoxetine rats showed the reverse of almost all the behavior alterations obversed in Model group, which further indicated success in depressing the rats.2. The CHSGS treatment shows results similar to fluoxetine treatment and appears to have significantly prevented these abnormal changes. These support the conclusion that CHSGS has an antidepressant effect. Component Ⅰ and Ⅱ seems to have partially reduced the severity of the behavioral changes and slow weight gain. CHSGS had superior antidepressant effect than either of two of its individual component, Ⅰ or Ⅱ. The results proved that liver-soothing and stagnation-dispersing played an important role in treating depression. The results also proved the herbs with the blood-activating effect could enhance the antidepressant effect of liver-soothing and stagnation-dispersing.3. The concentration of TNF-α and IL-6in serum increased significantly in depressant model rats by CUMS. Chaihu-Shugan San can decreased the concentration of TNF-α and IL-6.4. The present study demonstrates that chronic mild stress increased p38MAPK levels, and decreased p-ERK5levels in the brain, especially in the hippocampus and induced depressive-like behaviors. CHSGS alleviated these depressive-like behaviors, downregulated p38MAPK and upregulated p-ERK5experessions, which suggests that CHSGS therapeutic effect on depression may involved the p38MAPK and ERK5signal pathway. The research results provided a reliable experiment basis for depressive treatment.