| Objective:Rats exposed to chronic stress will produce symptoms of liver depression and spleen deficiency,and also show depression-like behavior.Xiaoyaosan(XYS)has been shown to have significant antidepressant effects and is often used to treat depression in clinical practice.Basic studies have also shown that XYS can improve depression-like behavior induced by chronic stress in rats and mice.Nerve damage is one of the most important causes of depression.Studies have shown that HemerlB/c has been reported to significantly increase in hippocampal CA1 region of chronic stress rats,and affect the downstream mTOR pathway through mGLur5,leading to decreased expression of synaptophysin related proteins PSD95 and SYP,resulting in nerve damage.Therefore,the purpose of this study was to observe whether the antidepressant effect of XYS on depression model of liver depression and spleen deficiency syndrome replicated by chronic mild unpredictable stress(CUMS)and glutamate-induced HT-22 hippocampal neuron cell injury model was related to the Homer1-mGluR5 and mTOR pathways.Methods:1.In vivo experimentsThe rats were divided into normal control group(NC)and model group.The rats model of depression,liver stagnation and spleen deficiency combined with disease and syndrome was simulated by CUMS for 6 consecutive weeks.At the 3rd week of CUMS,the model was evaluated by sugar-water experiment,and those that showed no statistical difference in sugarwater consumption between the model group and the NC group were not included in the subsequent experiment.The other rats were randomly divided into 3 groups with 12 rats in each group:model group(CUMS),fluoxetine group(FLU)and Xiaoyaosan group(XYS).XYS was intragastric at the periphery of the 4th～6th model,and fluoxetine was used as positive control drug.After 6 weeks,the general state of rats,the preference for sugar water,forced swimming,open field test and other behavioral tests were used to evaluate the performance of ’liver depression’,and the performance of ’spleen deficiency’ was evaluated by weight,food intake and other methods.At the same time,the efficacy of XYS on the model rats with depression syndrome of liver stagnation and spleen deficiency was observed by using prescription to test the syndrome.The effective components of XYS were identified by liquid mass spectrometry(UPLC-MS)to provide the basis for the effective substances of XYS.The content of glutamate(Glu)in hippocampal CA1 homogenate was determined by chemical colorimetry.Western blot and immunohistochemistry were used to detect the protein expression levels of Homer1b/c,mGLur5,p-mTOR,mTOR,p70S6K,p-p70S6K,p-4EBP1,4EBP1,PSD95 and SYP in hippocampal CA1 region.PCR was used to detect Homer1.mRNA levels of mGluR5,PSD95 and SYP.2.In vitro experiment(1)The experimental results of CCK8 cell activity showed that the drug-containing serum of XYS had no effect on the activity of HT-22 cells.The optimal administration concentration of the drug-containing serum of XYS was 10%,the optimal model concentration of glutamate was 5mM,and the concentration of the untransfected cells screened by purinomycin was 1.5ug/ml.(2)Homer 1 could be silenced by lentivirus transfected HT-22 cells,and the silencing efficiency of Homer1 gene reached 22.15%of that of the normal group,and the silencing level reached 35.94%when translated into Homer1b/c protein.There were significant differences with the normal group and follow-up studies could be conducted.(3)Glutamate induced HT22 cells.Results1.In vivo experiments(1)Six weeks of CUMS was used to replicate the rat model of depression,liver stagnation and spleen deficiency.After treatment with XYS,the hair color of CUMS rats was restored to luster,the stool of rats was restored to normal shape,and the activity was increased.It can increase the body weight and food intake of rats with depression syndrome of liver stagnation and spleen deficiency.After the treatment of XYS for 6 weeks,the depression-like behavior of the rats was obviously alleviated,the inactivity time of Forced swimming test(FST)was reduced,the total movement distance of the Open field test(OFT)was increased,the stay time of the central area was increased,and the drinking amount of sugar water was increased.All these indicate that the treatment of XYS with prescription for the detection of the syndrome shows the efficacy of depression,liver stagnation and spleen deficiency in rats.(2)The results of UPLC-MS showed that the active components of XYS were paeoniflorin,liquiritin,isoliquiritin,liquiritigenin,saikosaponin A,curcumin,and ferulic acid.(3)XYS can reduce glutamate content in hippocampal CA1 region of rats induced by CUMS.(4)XYS can reduce the relative content of Homer1 mRNA in hippocampal CA1 region of CUMS rats,and increase the relative content of mGluR5,PSD95 and SYP mRNA.(4)The expression of Homer1b/c in hippocampal CA1 region of CUMS rats was decreased,and the expression of mGluR5,pmTOR/mTOR,p-p70S6K/p70S6K,p-4EBP1/4EBP1 was promoted,thereby promoting the expression of synaptic marker proteins PSD95 and SYP.2.In vitro experiment(1)The experimental results of CCK8 cell activity showed that the drug-containing serum of XYS had no effect on the activity of HT-22 cells.The optimal administration concentration of the drug-containing serum of XYS was 10%,the optimal model concentration of glutamate was 5mM,and the concentration of the untransfected cells screened by purinomycin was 1.5 ug/ml.(2)Homer1 could be silenced by lentivirus transfected HT-22 cells,and the silencing efficiency of Homer1 gene reached 22.15%of that of the normal group,and the silencing level reached 35.94%when translated into Homer1b/c protein.There were significant differences with the normal group and follow-up studies could be conducted.(3)Glutamate induced HT22 cells produced significant toxicity,and XYS containing serum could play a protective role,which increased the activity of HT-22 cells and reduced the toxicity.(4)After the intervention of XYS with glutamate-induced HT-22 cells,the normal spindle and fibrous morphology could be restored.(5)After interfering with glutamate-induced HT-22 cells with XYS drug-containing serum,the mRNA relative content of Homer1 was decreased,and the mRNA relative content of mGluR5,PSD95 and SYP was increased.It can significantly reduce the expression of Homer1b/c and promote the expression of mGluR5,p-mTOR/mTOR,p-p70S6K/p70S6K and p-4EBP1/4EBP1,thus promoting the expression of synaptic marker proteins PSD95 and SYP,which is consistent with the results after Homer1 silencing.This suggests that Homer1 may be the target of XYS,but further experimental verification is needed.Conclusions(1)Six weeks of CUMS can replicate the rat model of depression,liver stagnation and spleen deficiency combined with disease and syndrome,and XYS has significant efficacy in the detection of syndrome by prescription.(2)XYS can improve depression-like behavior,increase food intake and weight;Decrease the immobility time in the forced swimming experiment,increase the movement distance in the open field experiment,increase the residence time in the central area,and increase the sugar water consumption;(3)XYS can reduce the content of glutamate and reduce the neurotoxicity of glutamate.The mechanism of its action may be that it can activate the downstream mTOR pathway and promote the activation of transcriptional protein-related p70S6K and 4EBP1 by reducing the expression of Homer1b/c in the hippocampal CA1 region of CUMS rats and increasing the expression of mGluR5.Thus,the expression of synaptic marker proteins PSD95 and SYP is increased,which plays a neuroprotective role.In conclusion,this study provides a preliminary experimental basis,suggesting that XYS disperses play a protective role of nerve and synapse in the treatment of depression,liver depression and spleen deficiency syndrome,and the mechanism of action may be related to the Homer1-mGluR5 and the downstream mTOR pathway. |
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