Exploring The Antidepressant Mechanism Of Chaihu Shugan San Based On The Regulation Of Glucocorticoid Signal Pathway

[OBJECTIVE]Chaihu Shugan San(CSGS)is a classic prescription for the treatment of depression in traditional Chinese medicine based on the principle of “Relieving the Depressed Liver”.However,the antidepressant mechanisms of CSGS have not been fully elucidated and more studies on these antidepressant mechanisms are needed.In this study,a UPLC-MS/MS method was established to determine the concentration of glucocorticoid(GC)metabolites in plasma and prefrontal cortex(PFC)of rats and applied to the detection of GC metabolites in CUMS depression model rats to explore the effect of CSGS on depressive behavior and the effect of GC differential metabolisms in rats.Based on the regulation of CSGS on the level of GC in depressed rats,the role of its intervention on the upstream and downstream signals of GC in the antidepressant mechanism was further studied.[METHODS]1.UPLC-MS/MS technique was used to establish a method for the determination of GC metabolites(CORT,11-DHCORT,DCORT)in rat plasma and PFC tissue.The method was investigated.2.After one week of adaptive feeding、open field test(OFT)and weight screening,the rats were randomly divided into normal group(Control group),model group(CUMS group),fluoxetine hydrochloride group(CUMS+Flu group),and Chaihu Shugan San group(CUMS+CSGS group).The experimental groups(CUMS group,CUMS+Flu group,CUMS+CSGS group)were given CUMS modeling stimulation for 4 weeks.The CUMS+Flu group and CUMS+CSGS group were given fluoxetine hydrochloride and Chaihu Shugan San respectively for 8 weeks,and the modeling stimulation was carried out at the same time.Behavioral tests were performed at 0,4,and 12 weeks to evaluate the pharmacodynamic effects of CSGS;Plasma and PFC samples were collected at 12 weeks,and a UPLC-MS/MS method established in the first part was used to determine GC metabolites.RT-PCR and Western blot were used to detect the expression and content of key metabolic enzymes of GC in each group.3.Based on the screening results and literature investigation,RT-PCR and Western blot were used to further explore the effects of CSGS on the downstream signal proteins of GC(GR,FKBP51,HSP-70,etc.)in the liver and PFC of depressed rats,and its potential effect on inflammatory mediators(HAT,NF-κB).[RESULTS]1.UPLC-MS/MS technique and substitute substrate method were used to establish a method for the detection of GC metabolites(CORT,11-DHCORT,DCORT)in rat plasma and PFC.The methodological verification results showed that low matrix effect and relatively high extraction recovery can be obtained by solid-liquid extraction after protein precipitation.Besides,the accuracy and precision of the method meet the requirements of biological sample analysis.2.(1)After 4 weeks of CUMS modeling stimulation,each experimental group showed depression-like behavior according to the results of OFT,SPT,and FST behavioral tests compared with the Control group,indicating that CUMS modeling was successful.After 8 weeks of administration,CSGS could significantly relieve the state of depression in rats,as shown by the increase of the total distance of exercise,and the sugar water preference index(P<0.05).The antidepressant pharmacodynamic effect of CSGS was confirmed at the overall animal level.(2)The UPLC-MS/MS method established in the first part was used to detect the concentration of GC metabolites in plasma and PFC of rats in each group.The differential metabolisms of GC in depressed rats were found,while CSGS could reduce the concentration of CORT and correct the differential metabolisms.(3)Based on the discovery of differential metabolisms of GC in depressed rats,RT-PCR and Western blot were used to determine the expression of GC metabolic enzyme m RNA and protein in rat liver and PFC.The results showed that,compared with the CUMS group,CSGS could significantly reduce the content and expression of CORT synthase 11β-HSD1 in peripheral liver tissue,and increase the content and expression of metabolic enzyme11β-HSD2(P<0.05).In PFC,it can significantly increase the content and expression of 11β-HSD2(P<0.05),and decrease the content and expression of 11β-HSD1,but there was no significant difference.3.The results of detection of downstream proteins of GC signal in each group showed that compared with Control group,the expression and content of GR in liver and PFC of CUMS group decreased,and the expression and content of GR co-chaperone proteins(FKBP51 and HSP-70)were significantly abnormal(P<0.01).CSGS and fluoxetine hydrochloride could significantly increase the expression and content of GR and affect the expression and content of GR co-chaperone proteins.[CONCLUSION]In this study,the rat model of CUMS depression was established and the concentration of GC in plasma and PFC of rats was detected by UPLC-MS/MS technique.It was found that there were differential metabolisms of GC in depressed rats,and CSGS could reduce the concentration of CORT and relieve the depressive behavior of rats.The mechanisms may be as follows: 1)CSGS regulates the level of CORT in vivo by affecting the metabolic enzymes in the peripheral liver and central PFC of GC;2)by affecting the expression of GR and its co-chaperone,which are the key proteins of downstream signal transduction in GC,the negative feedback inhibition effect of GC is normal;3)there may be crosstalk between the downstream signals of GC and inflammatory reactions.CSGS may play a multi-pathway and multi-target antidepressant effect based on the regulation of these signal pathways.