Antidepressant Efficacy Of Therapeutic Administration Of Chaigui Granule And The Mechanism Of Regulating CYP19A1-E₂-ERKs Pathway

Rationales:Chaigui granules are obtained by the research group through preliminary material research on Xiaoyao San,screening antidepressant components,analyzing chemical components and combining clinical observations,tailoring the Xiaoyao San and optimizing the extraction process.New drugs have been obtained so far.Clinical trial approval（2018L03149）.In previous preclinical studies,the treatment method of preventive administration（administration while modeling）is currently recognized.However,the clinical treatment of depression is mostly diagnosed first and then drug treatment,so Although Chaigui Granules has been approved by clinical trials,in order to explore the clinical treatment effect on patients with depression,this study uses the CUMS depression model for therapeutic administration based on previous research Drug,administration while continuing to build models to further evaluate the antidepressant efficacy of Chaigui granules,providing scientific basis for clinical research.On the basis of the prophylactic administration in the early stage,when discussing the regulating effect of Chaigui Granules on the neuroendocrine system,it was found that it can significantly improve the decrease of T content in rats caused by CUMS stress.However,a large number of studies have proved that Xiaoyao powder can significantly improve the depression-like behavior caused by gonadal resection,and it is speculated that Chaigui granules have a good regulatory effect on the low levels of sex hormones caused by depression.After a large number of literature investigations,it was found that T and E₂ in the hippocampus have neuroprotective effects and can exert antidepressant effects from multiple channels.Sex hormones in the hippocampus can not only regulate the inhibition of 5-HT and NE reuptake,balance the excitability and inhibition system in the brain,but also exert antidepressant effects by activating different cascade signalssuch as extracellular regulatory protein kinase（ERKs）;There are two main sources of internal T and E₂:（1）peripheral sex hormones can be entered through the blood circulation;（2）the brain weaves itself to produce sex hormones.Based on the above analysis,it is unclear whether Chaigui Granules can exert antidepressant effects through T and E₂ levels in the brain,and how to regulate T and E₂ levels in the brain.Therefore,this study is sufficient to establish the basis for the therapeutic administration of chronic mild unpredictable stress（CUMS）depression rats,and to explain the antidepressant effect of Chaigui Granules from the perspective of Chaigui Granules regulating sex hormones and their signaling pathway Mechanism to provide scientific basis for the rational application of drugs.Objective:Objective:1、 The therapeutic administration of CUMS,combined with the effects of Chaigui Granules on behavioral indicators,HPA axis-related hormone levels,and monoamine neurotransmitters in depressed rats,in order to clarify the antidepressants administered Chaigui Granules effect.2、 Through the changes of testosterone（T）,estradiol（E₂）and cytochrome P450 aromatase（CYP19A1）levels in the peripheral and brain of depressed rats,explore the regulation of Chaigui granules on peripheral and brain hormone levels effect.3、 Using molecular biology（Real Time q PCR and Western blot）to detect changes in m RNA and protein levels of T and E₂ receptors and the rate-limiting enzyme CYP19A1,which synthesizes E₂ in CUMS depressed rats,and combined with E₂ The effect of downstream ERKs signaling pathways was measured at the gene and protein levels,and the comprehensive explanation of Chaigui granules on the regulation of CYP19A1-E₂-ERK signaling pathways.Methods:1、 Set three dose groups of Chaigui Granules according to the results of previous dose screening experiments,use CUMS model to build a therapeutic dose,observe the effect of Chaigui Granules on the behavioral indicators of depression-like rats,and determine the HPA axis related indicators（CRH,ACTH,CORT）,monoamine neurotransmitters（Trp,5-HT,5-HIAA,Tyr,DA,NE,Glu,GABA）,clarify the antidepressant effect of Chaigui granules for therapeutic administration.2、 Enzyme-linked immunosorbent assay（ELISA）was used to determine the levels of T and E₂ in the serum and brain of rats,and the level of CYP19A1 in the brain.3、 The m RNA levels of androgen receptor（AR）,estrogen receptor（ERα,ERβ）,CYP19A1,ERK1 and ERK2 in rat brain were measured using Real Time q PCR technology;AR,The protein levels of ERα,ERβ,CYP19A1,and ERK1/2 are discussed at the molecular level to explore the antidepressant mechanism of Chaigui granules in regulating the CYP19A1-E₂-ERKs pathway.Result:1、 Results of behavioral:（1）The therapeutic administration of Chaigui granules can better improve the weight loss of depressed rats.Compared with the model group,at the 6th week（that is,the3 rd week of administration）,The dose group can significantly alleviate the slow weight gain of rats caused by CUMS（P<0.05,P<0.01）;（2）The results of the sugar and water preference index showed that at the 7th week（ie,the 4th week of administration）,each of Chaigui granules The dose groups were significantly increased（P<0.05,P<0.01）,indicating that Chaigui granules can better improve the lack of pleasure in rats caused by CUMS.（3）In the open field indicators,Chaigui granules can increase depression to varying degrees.The number of crossing grids and upright times of rats reduced the central stay time（P<0.05,P<0.01）,indicating that Chaigui particles can improve the weakened ability of rats to move and the ability to explore new things caused by CUMS stress;（4）The results of forced swimming immobility time showed that each dose group of Chaigui Granules could significantly reduce the forced swimming immobility time of depressed rats（P<0.01）,and effectively alleviate the desperate behavior of depressed rats.2、 Results of monoamine neurotransmitters:（1）In the 5-HT metabolic pathway,Chaigui Granules can reduce the levels of serum and brain Trp and 5-HIAA in depressed rats to varying degrees（P<0.05,P<0.01）Increased 5-HT levels in the brain of depressed rats（P<0.05）,indicating that Chaigui Granules can exert antidepressant effects by regulating 5-HT metabolic pathways.（2）In the NE metabolic pathway,Chaigui granules can increase the levels of Tyr,DA,and NE in serum and brain of depressed rats to varying degrees（P<0.05,P<0.01）,indicating that Chaigui granules can play a role in regulating NE metabolic pathway Antidepressant effect.（3）In the Glu metabolism pathway,Glu and GABA are important excitatory and inhibitory amino acids in the central system.Chaigui granules can increase GABA levels in serum and brain of depressed rats to varying degrees（P<0.05）,and reduce Glu levels and GABA / Glu ratio（P<0.05）,which shows that Chaigui granules can exert antidepressant effect by balancing excitatory and inhibitory systems.3、 Results of HPA axis-related hormone levels: Compared with the model group,Chaigui granules can reduce CRH,ACTH and CORT levels in CUMS depression rats to varying degrees（P<0.05）,indicating that Chaigui granules can better improve stress Caused by HPA axis hyperfunction.4、 Results of T、 E₂ and CYP19A1 levels in peripheral and brain:（1）In the peripheral serum,Chaigui granules can increase the levels of T and E₂（P<0.05）,indicating that Chaigui granules can regulate peripheral serum Neutral hormone levels;（2）In the hippocampus,Chaigui granules can increase T and E₂ levels to varying degrees（P<0.05,P<0.01）,indicating that Chaigui granules can also regulate sex hormone levels in the brain through different pathways;（3）In hippocampal tissue,Chaigui granules can significantly increase the levels of CYP19A1 in depressed rats（P<0.05,P<0.01）,and combined with the analysis of T and E₂ levels in the peripheral and hippocampus,it shows that Chaigui granules can regulate peripheral blood T by And the level of CYP19A1 in the brain,and then regulate the level of E₂ in the brain,thereby regulating the CYP19A1-E₂-ERKs pathway to play an antidepressant effect.5、 Results of Real Time q PCR:（1）Compared with the model group,Chaigui Granules can increase the m RNA levels of AR,ERα and ERβ in depressed rats to varying degrees（P<0.05,P<0.01）,indicating that Chaigui Granules can control sex hormones.The m RNA level of the receptor has a certain effect;（2）The gene level of CYP19A1 was measured,and it was found that each dose group of Chaigui granules could increase the m RNA content of CYP19A1 in depressed rats to varying degrees（P<0.05）;（3）in ERK1 The m RNA levels of ERK2 and ERK2 were measured and found that each dose group of Chaigui granules could increase the m RNA levels of ERK1 and 2 to varying degrees（P<0.05,P<0.01）;changes in sex hormone receptors,CYP19A1 and ERK1/2 m RNA levels A comprehensive analysis showed that Chaigui granules could specifically affect the expression of ERK1 and ERK2 genes downstream by regulating the specific binding of E₂ to its receptor.6、 Results of western blot:（1）Compared with the model group,Chaigui granules can increase the content of ERβ protein in depressed rats to varying degrees（P<0.05,P<0.01）,and there is a trend of callback to ERα,but no significant difference is seen.This shows that Chaigui granules have a certain effect on the protein expression level of estrogen hormone receptors;（2）The protein level of CYP19A1 was measured,and it was found that Chaigui granules can increase the protein content of CYP19A1 in depressed rats to varying degrees（P<0.01）;（3）The protein levels of ERK1/2and p-ERK1/2 were measured,and it was found that each dose group of Chaigui granules could activate the ERK1/2 signaling pathway to varying degrees（P<0.05,P<0.01）;Comprehensive analysis results show that Chaigui granules can activate the downstream ERK1/2 signaling pathway to exert antidepressant effect through the combination of E₂ and ERβ.Conclusion:The therapeutic administration of Chaigui granules has a better effect on the lack of pleasure and slow weight gain in depressed rats,significantly enhances the ability to explore activities and new things,and significantly relieves“desperate behavior”,showing a good antidepressant effect;The antidepressant mechanism of Chaigui granules may be related to its regulation of T and peripheral CYP19A1 levels in peripheral blood,and then E₂ levels in the brain,affecting ERβ gene and protein levels,promoting the specific binding of E₂ and ERβ,and finally activating downstream ERK1/2 signals Pathway related.