| Objective : The changes of behavior,morphology and number of microglia in hippocampus,and levels of proinflammatory factors such as interleukin-1β,interleukin-6 and tumor necrosis factor-α in depressed rats stimulated by chronic unpredictable mild stress were observed after the intervention of Kaixinshuyu Decoction,to clarify the antidepressant effect and mechanism of Kaixinshuyu Decoction.Method:After one week of adaptive feeding,60 SPF male SD rats were randomly divided into control group,model group,fluoxetine group,Kaixinshuyu Decoction high,medium and low dose group,10 rats in each group.The control group did not do any treatment,and the other 5 groups used 21 th chronic unpredictable mild stimulation to establish a depression rat model.From the 21 st day of the experiment,the treatment group was given fluoxetine hydrochloride capsules and Kaixinshuyu Decoction high,medium and low dose decoction before modeling,the control group and the model group were given normal saline for 21 days(continuous modeling during administration).Behavior evaluation was conducted on the 0 th,21 ST and 42 nd day of the experiment(observe the mental state,coat color and glossiness,reaction sensitivity and body weight changes of rats in each group;to evaluate the general condition of rats and their autonomous and exploratory behaviors in unfamiliar environments).The baseline level of behavior of rats in each group was determined on the 0 th day of the experiment,combining behavioral test to evaluate the success of depression model on the 21 st day,samples were taken after the behavioral test on the 42 nd day.After taking materials,the contents of interleukin-1β,interleukin-6 and tumor necrosis factor-α in serum and hippocampus of rats in each group were detected by Elisa method.The morphology of microglia and the number of Iba-1 positive cells in hippocampus of rats in each group were detected by immunohistochemistry.Results:1.Effect of Kaixinshuyu Decoction on behavior of depression rats:(1)General observation: The rats in the control group have pure white and shiny fur,large appetite,energetic,sensitive reaction,easily provoked and frightened,and normal stools.In the model group,rats showed yellow coat color,lack of luster,decreased food intake,poor spirit,huddled up,slow response and decreased resistance during gastric perfusion.The general condition of rats in each treatment group was improved compared with the model group.(2)Body weight measurement : On the 0th day of the experiment,there was no significant difference in body weight between the groups(P > 0.05).On the 21 st and 42 nd day of the experiment,compared with the control group,the body weight of the model group decreased significantly(P < 0.001);compared with the model group,the weight of rats in fluoxetine group,Kaixinshuyu Decoction middle and high dose groups increased(P < 0.001,P < 0.01,P < 0.05).(3)Open field test: On the 0th day of the experiment,there was no significant difference in the number of crawling grids and vertical standing times between the groups(P > 0.05).On the 21 st and 42 nd day of the experiment,compared with the control group,the number of crawling squares and the number of vertical standing in the model group decreased(P < 0.01,P < 0.001);compared with the model group,the number of crawling cells and the number of vertical standing in fluoxetine group and Kaixinshuyu decoction group increased(P < 0.01,P < P<0.05,P<0.001).2.Effects of Kaixinshuyu Decoction on serum cytokines IL-1β,IL-6 and TNF-α in depressed rats:Compared with the control group,the protein contents of IL-1β,IL-6 and TNF-α in the serum of the model group increased significantly(P < 0.001,P < 0.01).Compared with the model group,the serum levels of IL-1β,IL-6 and TNF-α in fluoxetine group and low,medium and high dose groups of Kaixinshuyu Decoction decreased significantly(P < 0.001,P < 0.01,P < 0.05).3.The effect of Kaixinshuyu decoction on the content of IL-1β,IL-6 and TNF-α in hippocampus of depressed rats :Compared with the control group,the contents of IL-1β,IL-6 and TNF-α in the hippocampus of the model group were significantly increased(P < 0.001,P < 0.01,P < 0.05).Compared with the model group,the levels of IL-1β and TNF-α protein in fluoxetine group and Kaixinshuyu Decoction medium dose group decreased significantly(P < 0.01,P < 0.05).The level of IL-6 in each treatment group decreased significantly(p < 0.001,p < 0.01,P < 0.05).4.The effect of Kaixinshuyu Decoction on the morphology of microglia and its marker Iba-1 in hippocampus of depressed rats:(1)Microglia morphology :The number of microglia in the hippocampus of the control group was less and the cell body is small,uniform in size,evenly distributed,and has many and long branches.Compared with the control,there weremore microglia in hippocampus of rats in model group,the cell body shortened,and the cell branches decreased.Compared with the model group,Fluoxetine group,Kaixinshuyu Decoction medium and high dose group can effectively reduce the number of microglia,maintain the small cell body and its branch morphology of microglia.In the low-dose Chinese medicine group,the microglia cell body was shortened,the branches were more,and the number of cells was not significantly reduced.(2)Expression of microglial marker Iba-1 protein:On the 42 nd day of the experiment,compared with control group,the level of Iba-1 protein in hippocampus of rats in model group was significantly increased(P < 0.001).Compared with the model group,the levels of Iba-1 protein in the fluoxetine group and the low,medium and high dose groups of Kaixinshuyu Decoction were significantly decreased(P < 0.001,P < 0.01).Conclusion:1.In this study,a rat model of depression was established by chronic unpredictable mild stress(CUMS)stimulation.Behavioral tests confirmed that the depression rat model was successfully prepared.2.The depression rat model established by CUMS stimulation.In the behavioral test,compared with the control group,the weight gain of the model group and the number of crawling grids and vertical standing in the open field test were significantly reduced.Kaixinshuyu Decoction can increase the weight of rats and significantly increase the number of crawling grids and vertical standing times in the open field test,and was proved to have antidepressant effect.3.The levels of pro-inflammatory factors IL-1β,IL-6 and TNF-α in hippocampus and serum of CUMS depressed rats were significantly increased,and the expression of microglia marker Iba-1 was increased,suggesting that the peripheral and brain tissues of CUMS depressed rats were in an inflammatory activation state and the microglia in hippocampus were over-activated.It is speculated that CUMS stress stimulation can cause peripheral and central immune responses,and the overactivation of microglia may be the inducing factor of neuroinflammation.Kaixinshuyu Decoction can reduce the contents of IL-1β,IL-6 and TNF-α in hippocampus and serum,inhibit the activation of microglia,reduce the inflammatory response.4.This experimental study found that one of the antidepressant mechanisms of Kaixinshuyu Decoction may be related to reducing the levels of pro-inflammatory factors in the hippocampus and serum,inhibiting the activation of microglia in the hippocampus,and then alleviate the inflammatory response related. |
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