Studies On The Antidepressant Effect And Mechanism Of Chaihu Shugan San Via Targeting Tryptophan Metabolism

[OBJECTIVE]As a kind of traditional Chinese medicine prescription,Chaihu Shugan San has been proven to have good antidepressant effects in clinical.The purpose of this study was to evaluate the therapeutic effects of Chaihu Shugan San on rat model of depression induced by chronic unpredictable mild stress（CUMS）,and detect the changes of tryptophan metabolism pathway in rats under the intervention of Chaihu Shugan San,futher to explore the antidepressant mechanism of Chaihu Shugan San based on the regulation mechanism of indoleamine 2,3-dioxygenase（IDO）of tryptophan（Trp）-kynurenine（KYN）pathway and tryptophan 2,3-dioxygenase（TDO）.[METHODS]1.In this experiment,32 male Sprague-Dawley rats were random Ly divided into four groups according to their weight and the results of behavioral experiments:normal group（Control group）,model group（CUMS group）,fluoxetine group(CUMS+Flu group,20 mg·Kg^-1),Chaihu Shugan San group(CUMS+CSGS group,19.8 g·Kg^-1).Except the Control group,the other three groups of rats were subjected to CUMS procedure for 28 days according to the schedule,and then administered for 8 weeks by oral gavage.The Control group and the CUMS group were given pure water,and the groups were given corresponding doses of the drug.In the process of drug administration,except for the Control group,the other three groups were given CUMS stimulation at the same time.Before and after CUMS procedure and administration,behavioral experiments（sucrose preference experiment,open field experiment,forced swimming experiment）were used to evaluate the therapeutic effect of Chaihu Shugan San on depressed rats.2.The liquid chromatography tandem mass spectrometry technique was developed to determine the concentration of 14 neurotransmitters including Trp and its metabolites in plasma,hippocampus and urine of rats,and the method was verified according to the FDA guidelines.3.The established HPLC-MS/MS method was used to determine the levels of Trp and its metabolites in the plasma and hippocampus of four groups of rats.RT-PCR and western blotting methods were employed to determine m RNA expression and the content of IDO in hippocampus and TDO in liver.In order to further explore the upstream regulation mechanism of Chaihu Shugan San on IDO enzyme in hippocampus,ELISA method was used to determine the concentration of inflammatory factors in hippocampus of each group of rats,and Western Blot was used to determine the upstream regulatory proteins of inflammatory factors,such as TLR-4,NF-κB,P2X7R,NLRP3,caspase-1.[RESULTS]The results of behavioral experiments showed that after 28 days of CUMS procedure,compared with the Control group,the other three groups of rats showed significant reduced exercise capacity,lack of pleasure,and reduction in autonomous activity（P<0.05）.After Chaihu Shugan San administering for 8 weeks,the depressed state was significantly improved,both the total open-field exercise distance and sucrose preference index were significantly increased,and the duration time of forced swimming immobility was significantly reduced（P<0.05）.In this experiment,LC-MS/MS was successfully established to determine concentrations of 14 metabolites in rat plasma and hippocampus.The results showed that the contents of Trp and 5-HT in hippocampus and plasma of CUMS rats were significantly reduced,and the concentration of quinolinic acid（QUIN）was significantly increased,the ratio of KYN to Trp was also significantly increased（P<0.05）.After administration of Chaihu Shugan San and fluoxetine,the levels of Trp and 5-HT in the hippocampus and plasma were increased significantly,and the ratio of KYN to Trp was decreased（P<0.05）.The results of studies on rat IDO enzyme and its upstream regulation mechanism in hippocampus showed that the content and m RNA expression levels of IDO in the hippocampus of CUMS rats were significantly increased compared with the normal group,and the levels of inflammatory factors interleukin-6（IL-6）,interleukin-1β（IL-1β）were significantly increased,the protein content of TLR-4,NF-κB,P2X7R,NLRP3,caspase-1 increased significantly（P<0.05）;the administration of Chaihu Shugan San and fluoxetine could significantly reduce the protein and m RNA expression of IDO,and reduce the concentration of inflammatory factors,the protein expression of TLR-4,NF-κB,P2X7R,NLRP3,and caspase-1（P<0.05）.As to TDO in liver,compared with the Control group,CUMS procedure could significantly increased the expression of its protein and m RNA;In comparison,Chaihu Shugan San treatment could significantly reduce the expression level of TDO.[CONCLUSION]In conclusion,Chaihu Shugan San could significantly improve the depressed state of rats caused by CUMS procedure.On the one hand,the mechanism may be associsted with inhibiting TLR-4 and P2X7R signal pathways,thus to futher reduce the levels of inflammatory factors in hippocampus and inhibit the protein and m RNA expression of IDO in hippocampus,which might contribute to reducing the conversion of Trp to KYN.On the other hand,Chaihu Shugan San could significantly reduce the protein and m RNA expression of TDO.In short,Chaihu Shugan San might exert the multi-targets’antidepressant effect by inhibiting Trp metabolism both in the central and peripheral matrix.