| Rationales:Depression is a common psychiatric disorder that afflicts nearly 350 million people worldwide.In the analysis of patients with depression,it was found that women accounted for 68%,which was much higher than men.At present,the most important hypothesis that women are more susceptible to depression than men are the difference in the physiological and reproductive structure between men and women,so it is speculated that this phenomenon may be related to frequent fluctuations in estrogen levels in women.Chaigui granule is a new antidepressant traditional Chinese medicine previously developed by the research group and has been shown to have good antidepressant efficacy.In the previous study,the research group found that Chaigui granule could exert antidepressant effects by regulating estradiol levels in male CUMS rats.However,the antidepressant effect of Chaigui granule on female CUMS rats,how Chaigui granule affects and regulates E2 levels in female CUMS rats,what are the differences between behavioral and sex hormone changes in female and male CUMS rats,and what may be the intrinsic factors causing these differences,these issues are not clear.Therefore,in this study,we first verified the antidepressant efficacy of Chaigui granule on female CUMS rats by establishing a female CUMS rat model,and further analyzed the E2 production and metabolic pathways in female CUMS rats on this basis to clarify the regulatory mechanism of Chaigui granule on E2 in female CUMS rats.Secondly,through comparing the experimental data of this study with the previous experimental data of male CUMS rats of the research group,the differences in behavioral appearance between female and male CUMS rats were clarified,and the HPA axis and sex hormone-related indicators were comprehensively analyzed to preliminarily explore the possible causes of the differences in behavioral performance between female and male rats,providing theoretical support for Chaigui granule to regulate different pathways in male and female rats to exert antidepressant efficacy,and also providing new ideas for the prevention and treatment of depression by traditional Chinese medicine in clinical practice.Objective:1.To clarify the antidepressant effect of Chaigui granule on female CUMS rats.2.To analyze the regulatory mechanism of Chaigui granule on E2 levels in serum and hippocampus of female CUMS rats from two pathways: E2 production and metabolism.3.Based on the apparent differences in behavior between female and male CUMS rats,to explore the possible intrinsic mechanism of Chaigui granule in the treatment of female and male CUMS rats.Methods:1.Two dose groups of Chaigui granule were added on the basis of the previous dose screening experiment(5 in total).By establishing the female CUMS rat model,the regulation effect of Chaigui granule on the behavioral indicators and HPA axis related indicators(CRH,ACTH,CORT)of female CUMS rats was observed,and the antidepressant efficacy of Chaigui granule on female CUMS rats was clarified.Principal component analysis was used to assign the behavioral parameters of rats,which could more clearly and intuitively reflect the recovery effect of different treatment groups of Chaigui granule after integrated analysis.2.ELISA was used to determine the levels of T,E1,E2,2-OH-E2,and 4-OH-E2 in serum and hippocampus of rats,and the effect of Chaigui granule on the levels of sex hormones related to E2 in serum and hippocampus of female CUMS rats was analyzed.3.RT-qPCR and WB were used to determine the m RNA and protein expression of key regulatory enzymes in E2 synthesis pathway(CYP19A1,HSD17B1)and key regulatory enzymes in E2 decomposition pathway(CYP1A1,CYP1B1)in the hippocampus of female CUMS rats,the regulatory effect of Chaigui granule on E2 synthesis and decomposition in the hippocampus of female CUMS rats was verified at the molecular level.First,the behavior,HPA axis and sex hormone levels of female and male CUMS rats were compared and analyzed to preliminarily understand the performance differences in behavior,HPA axis and sex hormone levels between female and male CUMS rats.Principal component analysis and Pearson correlation analysis were used to correlate the emotional index with HPA axis index and sex hormone index in female and male CUMS rats,and the internal causes of the differences in extrinsic behavioral performance between female and male CUMS rats were further analyzed.Results:1.Behavioral parameters and HPA axis related hormone levels:(1)Behavioral parameters: Compared with the control group,CUMS procedure resulted in a significant decrease in the number of crossings,the number of rearing,sucrose preference rate,and body weight(P < 0.05,P < 0.01)and a significant increase in forced swimming test immobility time(P < 0.01)in female rats.Compared with the model group,the number of crossing and the number of rearing were significantly increased in the five dose groups of Chaigui granule(P < 0.05,P < 0.01),the dose group B of Chaigui granule had the best effect in the number of crossings,and the groups B and C of Chaigui granule had the best effect in the number of rearing.The sucrose preference rate and body weight of rats in Chaigui granule A,B,C and D groups were significantly increased(P < 0.05,P < 0.01),Chaigui granule B dose group had the best improvement effect in sucrose preference test,and Chaigui granule A,B and C dose groups had the best improvement effect on body weight of rats.The forced swimming immobility time was significantly decreased in the five dose groups of Chaigui granule(P < 0.05);Compared with the control group,rats in the model group had significantly lower time to explore novel objects,time occupied in the clear box,time ratio to enter the open arm,number of open arm probes,number of platform crossings,and time to reach the platform(P < 0.05,P < 0.01).Compared with the model group,the time to explore the new object,the number of open arm probes and the time taken to reach the platform were significantly increased in the five dose groups of Chaigui granule(P < 0.05,P < 0.01),with the best improvement effect in the time to explore the new object experiment in the rats of Chaigui granule A and B groups,and the best effect in the number of open arm probes experiment in the rats of Chaigui granules B group.Chaigui granule A and B dose groups of rats in the clear box accounted for a significant increase(P < 0.05).The time ratio of rats entering the open arm in Chaigui granule A,B and C dose groups was significantly increased(P < 0.05,P < 0.01),and Chaigui granule B and C dose groups had the best effect.The number of platform crossings was significantly increased in the Chaigui granule A,B,and E dose groups(P <0.05).(2)HPA axis related indicators: Compared with the control group,the levels of CRH,ACTH and CORT in the serum of rats in the model group were significantly increased(P < 0.05,P < 0.01).Compared with the model group,the CRH level in the serum of rats in the Chaigui granule A,B and C dose groups was significantly decreased(P < 0.05).ACTH levels in serum of rats in Chaigui granule groups A,C and D were significantly decreased(P < 0.05).The level of CORT in serum of rats in Chaigui granule A,B,C and D groups was significantly decreased(P < 0.05,P < 0.01),and Chaigui granule A and D groups had the best improvement effect.The results showed that Chaigui granule also had a significant antidepressant effect on female CUMS rats.Through the assignment analysis of behavioral parameters,it was found that each dose group of Chaigui granule significantly improved depression-like behavior in female CUMS rats.Through the whole analysis of different behavioral data,Chaigui granule B dose group had the best effect on depression-like behavior improvement in female CUMS rats.2.Results of E2 related sex hormone levels in serum and hippocampus:(1)Regulation effect of E2: Compared with the control group,the E2 levels in the serum and hippocampus of rats in the model group were significantly decreased(P < 0.05).Compared with the model group,the E2 levels in the serum and hippocampus of rats in Chaigui granule groups A,B,C and D were significantly increased(P < 0.05,P < 0.01);(2)The regulatory effects of T and E1: Compared with the control group,the T and E1 levels in the serum and hippocampus of rats in the model group were significantly decreased(P < 0.05,P < 0.01).Compared with the model group,the serum T level was significantly increased in the Chaigui granule A,B and C groups(P < 0.05),and the hippocampus T level was significantly increased in the Chaigui granule A and B dose groups(P < 0.01).The serum and hippocampus E1 levels were significantly increased in the Chaigui granule 5 dose groups(P < 0.05,P < 0.01).(3)Regulation effect of 2-OH-E2 and 4-OH-E2: Compared with the control group,the 2-OH-E2 levels in the serum and hippocampus of rats in the model group were significantly decreased(P < 0.05),and the4-OH-E2 levels tended to decrease,with no significant difference.Compared with the model group,the 2-OH-E2 level in the serum of rats in Chaigui granules B and D groups was significantly increased(P < 0.05),and the 2-OH-E2 level in the hippocampus of rats in Chaigui granule A,B,C and D groups was significantly increased(P < 0.05).The results showed that Chaigui granule could not only promote the synthesis of E2 by increasing the levels of T and E1 in the serum and hippocampus of female CUMS rats,but also increase the levels of 2-OH-E2 in the serum and hippocampus of female CUMS rats to promote the decomposition of E2,so that the levels of E2 in the serum and hippocampus of Chaigui granule treated rats were maintained within the normal steady-state range.3.RT-qPCR and WB results:(1)E2 synthesis pathway: Compared with the control group,the m RNA and protein expressions of CYP19A1,a key enzyme that promotes the conversion of T to E2,and HSD17B1,a key enzyme that promotes the conversion of E1 to E2,were significantly decreased in the hippocampus of rats in the model group(P < 0.01).Compared with the model group,the m RNA expression of CYP19A1 in the hippocampus of rats in Chaigui granule B,C and D dose groups was significantly increased(P < 0.05,P< 0.01),and the CYP19A1 protein expression in the hippocampus of rats in Chaigui granule B and C was significantly increased(P < 0.05,P < 0.01).The expression of HSD17B1 m RNA in the hippocampus of rats in the five dose groups of Chaigui granule was significantly increased(P < 0.05,P < 0.01),while the expression of HSD17B1 protein in the hippocampus of rats in Chaigui granule groups B and C was significantly increased(P < 0.05);(2)E2 decomposition pathway: Compared with the control group,the m RNA and protein expressions of CYP1A1,a key enzyme that promotes the conversion of E2 to 2-OH-E2,and CYP1B1,a key enzyme that promotes the conversion of E2 to 4-OH-E2,were significantly decreased in the hippocampus of rats in the model group(P < 0.01).Compared with the model group,the m RNA expression of CYP1A1 in the hippocampus of rats in groups A and B of Chaigui granule was significantly increased(P < 0.01),and the CYP1A1 protein expression in the hippocampus of rats in the five dose groups of Chaigui granule was significantly increased(P < 0.05,P < 0.01).The m RNA expression of CYP1B1 in the hippocampus of rats in the five dose groups of Chaigui granule was significantly increased(P < 0.05,P < 0.01),while the CYP1B1 protein expression in the hippocampus of rats in Chaigui granule groups B,C,D and E was significantly increased(P < 0.01).4.Comparison and correlation analysis of behavior,HPA axis and sex hormone levels between female and male CUMS rats:(1)Comparative analysis of female and male CUMS rats: Compared with male rats,CUMS stress induced more significant depression-like behaviors,such as anhedonia,quiescence and exploratory behaviors,in female rats.This is related to the difference of HPA axis and sex hormone levels between female and male CUMS rats;(2)Correlation analysis between female and male CUMS rats: The results showed that there was a significant correlation between behavioral parameters and HPA axis and sex hormone parameters in both female and male CUMS rats,while the correlation between behavioral parameters and HPA axis and sex hormone parameters in female rats was higher than that in male rats.These results suggest that female rats may be more susceptible to fluctuating changes in HPA axis and sex hormone levels than male rats.Conclusion:Chaigui granule could improve the depression-like behavior of female CUMS rats to different extents,and had a significant regulatory effect on HPA axis-related hormones,of which Chaigui granule B dose group had the best effect.By analyzing the two pathways of E2 synthesis and decomposition in the hippocampus of CUMS rats,it was found that Chaigui granule could not only increase the conversion of T to E2 and E1 to E2 by promoting the expression of m RNA and protein of CYP19A1 and HSD17B1,key enzymes of E2 synthesis pathway.At the same time,it can also promote the expression of m RNA and protein of CYP1A1 and CYP1B1,which are key enzymes of E2 decomposition pathway,and increase the decomposition of E2 to 2-OH-E2 and 4-OH-E2,so that the E2 level in the hippocampus of female CUMS rats returns to the normal steady-state range.In performing a differential analysis between female and male CUMS rats,it was found that CUMS stress induced relatively earlier and more pronounced depression-like behavior in female rats,which may be related to the fact that female rats are more susceptible to fluctuations in HPA axis and sex hormone levels than male rats. |
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