| Objective1.To analyze the clinical efficacy and safety of the TCM psychosomatic development through a randomized controlled study.2.To investigate the mechanism of action of the TCM psychosomatic development in the treatment of menopausal syndrome using gut microbiomics,metabolomics and fMRI techniques.Method1.Clinical studyAccording to the study criteria,69 patients with menopausal syndrome who visited the gynecology outpatient clinic of Guangdong Provincial Hospital of Traditional Chinese Medicine from January 2020 to April 2022 were randomly assigned to the TCM psychosomatic development(referred to as the treatment group)and the Placebo group(referred to as the control group),and the intervention period was 12 weeks,with the observation time points of the initial consultation and the end of the 4th,8th,and 12th week of treatment.The changes and differences of TCM syndrome scale score,Greene scale,SDS scale,SAS scale,HAMA scale and HAMD scale scores were compared between the two groups before and after treatment.Safety indicators such as sex hormones,DA,NE,5-HT,lipid levels and liver and kidney functions were measured before and at the end of the 12th week after treatment to objectively evaluate the improvement of their psychosomatic symptoms.2.Mechanism study(1)The Regional homogeneity(ReHo),Amplitudeoflow-Frequencyfluctuation(ALFF)and Functional Connectivity(FC)between menopausal syndrome patients and healthy controls,treatment group and control group before and after treatment were analyzed using functional magnetic resonance imaging(fMRI).(2)Amplicon sequencing of 16S rDNA was used to analyze the differences in intestinal flora diversity and microbial species composition between patients with menopausal syndrome and healthy controls,treatment group and control group before and after treatment.(3)To annotate the relevant metabolic pathways using Liquid chromatography-tandem mass spectrometry metabolomics analysis of differential metabolites in menopausal syndrome patients compared with healthy controls,treatment group and control group before and after treatment.Results1.Clinical efficacy results(1)Efficacy analysis of TCM syndrome scale score and each entry,Greene’s total score and each dimension,SDS,SAS,HAMD and HAMA total scoreIntra-group comparison:①Treatment group:When comparing before and after treatment,it was found that TCM syndrome scale score and other entry were statistically significant(P<0.05)in the treatment group,except for the entry of hypoactive sexual desire;Greene,SDS,SAS,HAMD and HAMA total scores at 4,8 and 12 weeks of treatment were significantly lower compared with those before treatment,and the difference was statistically significant(P<0.001),indicating that the TCM Psychosomatic development could improve the somatic and psychological symptoms of menopausal syndrome patients during the treatment period lasting 12 weeks.②In the control group,In the control group,there was no statistical significance(P>0.05)for the total score and each item of the TCM symptom scale in the comparison within the group;The differences in the psychological dimension,vasodilatory dimension and total score of Greene scale,HAMD scale and HAMA scale were statistically significant after 4 weeks of treatment compared with those before treatment(P<0.05);only the differences in the scores of Greene scale,SDS scale and SAS scale were statistically significant after 8 weeks of treatment compared with those before treatment(P<0.05);in the control group,the differences in the total score of Greene scale,vasodilatory dimension and total score of Greene scale,HAMD scale and HAMA scale were statistically significant after 12 weeks of treatment compared with those before treatment(P<0.05).The differences in Greene scale total score,psychological dimension score and HAMA scale score after 12 weeks of treatment were statistically significant compared with those before treatment(P<0.05);indicating that the control group was not as significant as the treatment group in improving psychosomatic symptoms,with a slower onset time and a poorer trend of sustained decline,considering that the intra-group differences in the control group were to some extent influenced by the comfort effect and the supportive psychotherapy effect.Comparison between groups:①After treatment,the total score and each point of the TCM syndrome scale in the treatment group were lower than those in the control group,and there were statistically significant differences in the scores of the TCM syndrome scale total score,irritability,breast distension,lumbar and knee weakness,nocturia,mental depression,chest tightness and sighing,baking heat and sweating,and insomnia,except for the entry of hypersexuality(P<0.05);The differences in Greene’s total score and each dimension were statistically significant in the treatment group compared with the control group at all time points,except for the sexual dimension at the 4-week treatment time point.This indicates that the treatment group was able to improve the discomfort of patients in terms of psychological and somatic discomfort and vasodilatory dysfunction,and the efficacy was better than that of the control group.②The SAS and SDS scale scores of the treatment group were lower than those of the control group at 4,8 and 12 weeks of treatment,and the differences were all statistically significant when compared with the control group(P<0.01),indicating that the treatment group was better than the control group in improving patients’ anxiety and depressive symptoms.③The HAMD scale and HAMA scale scores of the treatment group were lower than those of the control group at all time points,and the differences between the HAMD scale and HAMA scale scores at 4 weeks,8 weeks and 12 weeks of treatment were statistically significant when compared with the control group.It showed that the treatment group could improve the psychosomatic symptoms of depression and anxiety of patients,and the efficacy was better than that of the control group.Difference comparison:The comparison of the differences in Greene,SDS and SAS scale scores between the treatment and control groups at 4,8 and 12 weeks of treatment and before treatment was statistically significant(P<0.05).The comparison of the difference between the treatment and control groups at 8 and 12 weeks of treatment and the pre-treatment HAMD and HAMA scale scores was statistically significant(P<0.05).This indicates that the improvement of Greene,SDS and SAS scale scores in the treatment group was significantly better than that in the control group after 4,8 and 12 weeks of treatment;the improvement of HAMD and HAMA scale scores in the treatment group was significantly better than that in the control group after 8 and 12 weeks of treatment.Repeated measures:Multiple time-point repeated measures ANOVA for Greene,SDS,SAS,HAMD and HAMA total scores showed that time,group,time and group interaction had an effect on the above scale scores,indicating that the efficacy of the treatment group differed from that of the control group and that the efficacy varied with time.(2)Analysis of observed indicatorsIntra-group comparison:①The differences in serum DA,NE and 5-HT levels before and after treatment in the treatment group were statistically significant(P<0.001);the differences in sex hormone and lipid profiles before and after treatment in the treatment group were not statistically significant(P>0.05).②The differences in serum DA,NE and 5-HT levels before and after treatment in the control group were not statistically significant(P>0.05);the differences in sex hormones and blood lipids before and after treatment in the control group were not statistically significant(P>0.05).This indicates that the TCM mind-body treatment program can significantly increase the serum levels of central neurotransmitters,and no statistically significant differences were found between the treatment and control groups in sex hormone and lipid profiles before and after treatment(P>0.05).Comparison between groups:①Serum DA.NE and 5-HT levels were significantly higher in the treatment group compared with the control group,and the difference was statistically significant(P<0.05),indicating that the treatment group could significantly increase serum DA,NE and 5-HT levels after treatment.②The difference between serum FSH,LH,E2,TG,TC,HDL and LDL levels in the treatment group compared with the control group was not statistically significant(P>0.05).Difference comparison:The comparison of the difference between the treatment and control groups before and after treatment showed that the difference in DA difference was statistically significant(P<0.01),while the difference in NE and 5-HT was not statistically significant(P>0.05),indicating that the TCM mind-body treatment program had a greater degree of effect on increasing dopamine serum levels;the results showed that the difference in HDL and TG difference was statistically significant(P<0.05),but the magnitude of the difference was not significant(P>0.05).However,the magnitude of the difference change was not significant,and the clinical reference significance was small considering the large individual sample variation in clinical samples resulted.In conclusion,the results of this part of the study showed that serum DA,NE and 5-HT levels increased in the treatment group,suggesting that the TCM Psychosomatic development may play a role in the treatment of menopausal syndrome by increasing the levels of monoamine neuro transmitters.(3)Safety evaluation resultsThere was no significant difference between the safety indexes of blood,urine routine,liver function,kidney function,electrocardiogram and gynecological ultrasound of patients in the treatment group and the control group before and after treatment,suggesting that the safety in laboratory examination was better in both groups.2.Mechanism study results(1)Analysis of fMRI results①Comparative analysis of patients with menopausal syndrome and healthy controls:compared with healthy controls,patients with menopausal syndrome were found to have increased synchronization of neuronal activity in the right supplementary motor area;decreased spontaneous neuronal activity in the right superior temporal gyrus,right postcentral gyrus,right superior limbic gyrus,right supplementary motor area,bilateral lingual gyrus,and divided cerebellar regions;right supplementary motor area,left superior parietal gyrus,right middle frontal gyrus,left postcentral gyrus and left superior temporal gyrus.②Comparative intra-group analysis of the treatment group and the control group:comparison of the treatment group before and after treatment revealed that the synchronization of neuronal activity in the left angular gyrus of the patients was reduced after treatment;spontaneous neuronal activity in the right medial superior frontal gyrus,left postcentral gyrus,left superior parietal gyrus,right supplementary motor area,right middle temporal gyrus,left superior parietal gyrus was increased,and spontaneous neuronal activity in some cerebellar regions was reduced;spontaneous neuronal activity in the right superior temporal gyrus,right marginal superior gyrus,the The right medial superior frontal gyrus and the left angular gyrus had altered functional connectivity.No difference changes were found in the control group before and after treatment.③Intergroup comparative analysis between treatment and control groups:after treatment,the treatment group was found to have increased spontaneous neuronal activity in the left superior temporal gyrus compared to the control group;and enhanced functional connectivity in the right superior temporal gyrus-left middle frontal gyrus.(2)Analysis of microbiomic results①Comparative analysis between patients with menopausal syndrome and healthy controls:Beneficial bacteria such as Bifidobacterium,Prevotella and Bifidobacterium played a significant role in healthy controls,while patients with menopausal syndrome had reduced flora diversity,decreased species abundance,and opportunistic pathogenic bacteria such as Actinomyces and Escherichiae played a significant role.②Comparative intra-group analysis between the treatment group and the control group:compared before and after treatment in the treatment group,the diversity of flora and the abundance of species increased in the treatment group,and beneficial bacteria such as butyric acid cocci and ruminal cocci played a significant role.In the comparison of the control group before and after treatment,there was no significant difference in the diversity and abundance of flora in the control group after treatment,and the inflammation,infection-related Microsomonas and opportunistic pathogenic bacteria Clostridium played a major dominant role.③Comparative analysis between groups of treatment group and control group:After treatment,the comparison between treatment group and control group revealed that the species abundance and biodiversity of intestinal flora in treatment group had an increasing trend compared with control group,and showed a tendency to approach to healthy control group.The abundance of intestinal SCFA-producing microbiota(Bifidobacterium,E.faecalis,Prevotella,and monomorphic Macrobacterium)was increased in the treated group compared with the control group,and showed a trend toward the healthy control group.(3)Analysis of metabolomic results①Comparative analysis of patients with menopausal syndrome and healthy controls:OPLS-DA analysis revealed that there were significant metabolic profile differences between patients with menopausal syndrome and healthy controls and the model was validated.Fatty acid differential metabolites were found to exhibit relative down-regulation in both serum and urine differential metabolites of patients compared to healthy controls.②Within-group comparative analysis of the treatment and control groups:OPLS-DA analysis revealed significant metabolic profile differences between the treatment and control groups before and after treatment,and the model was validated.The majority of fatty acid differential metabolites(serum differential metabolites:o-ACETYLCARNITINE,suberic acid,2-Deoxyglucose)were found in both serum and urine differential metabolites of the treatment group before and after treatment;urine differential metabolites:3-Hydroxy-3-methylglutaric acid(3-Hydroxy-3-methylglutaric acid),sorbic acid(Sorbic acid),and dethiobiotin(Dethiobiotin))were relatively upregulated.In the control group before and after treatment,the vast majority of fatty acid differential metabolites were still down-regulated after treatment in the control group.③Comparative group analysis between the treatment and control groups:OPLS-DA analysis revealed that there were significant metabolic profile differences between the treatment and control groups after treatment,and the model was validated.The blood and urine differential metabolite enrichment analysis after treatment in both groups suggested that the common metabolic pathway was the fatty acid anabolic pathway.The combined analysis of the differential metabolic pathways in serum and urine before and after treatment in the treatment group and in the menopausal syndrome group and the healthy control group revealed that their common differential metabolic pathway was the sphingolipid metabolic pathway.Conclusion1.Clinical efficacy section①The TCM psychosomatic development can improve the TCM syndrome scale,Greene scale dimensions and total scores,SDS,SAS,HAMD and HAMA scale scores of menopausal syndrome patients,and significantly improve the physical and psychological symptoms of menopausal syndrome patients,and its efficacy is significantly and persistently better than that of the control group.②The TCM psychosomatic development can improve serum DA,NE and 5-HT levels in patients with menopausal syndrome,suggesting that the mind-body treatment program may play a role in treating menopausal syndrome by increasing the levels of monoamine neurotransmitters.2.Mechanism study part①fMRI:The TCM psychosomatic development can restore the neuronal activity and change the functional connectivity of the emotional brain areas such as anxiety,depression,panic and functional brain areas such as cognitive memory,which may be the mechanism of its action to improve the mind-body symptoms of menopausal syndrome.②Microbiomics:The TCM psychosomatic development can increase the diversity of intestinal flora and the abundance of beneficial bacteria,especially the intestinal SCFA-producing microbiota(Bifidobacterium,E.faecalis,Prevotella,and Macrobacterium monomorphicum),which may play a role in improving the psychosomatic symptoms of menopausal syndrome through brain-gut interaction..③Metabolomics:The TCM psychosomatic development can upregulate fatty acid metabolites,and fatty acid synthesis and sphingolipid metabolic pathways may be the metabolic pathways for the treatment of menopausal syndrome. |
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