| Rationales:Chaigui granules is a new type of antidepressant Chinese medicine,which was obtained by the research group after the prescription of Xiaoyao san was disassembled and reconstituted and the process was optimized.Through five kinds of animal models of depression,we have proved the efficacy,and also found that it can improve thegastrointestinal dysfunction associated with depression,but its mechanism is not clear.Gastrointestinal function is regulated by the intestinal flora,which,as an important component of intestinal function,inhibits the growth of pathogenic bacteria and produces beneficial metabolites to maintain body health.In our previous study,we found that Chaigui granules(Compound chaigui prescription)can regulate the level of TMAO produced by intestinal flora metabolism.It is suggested that Chaigui granules may play an antidepressant role by regulating the metabolites of intestinal flora.Metabonomics study showed that the levels of glutamate,the precursor of arginine,and its metabolites citrulline and spermidine in different tissues of patients with depression and depressed animals were changed,suggesting that arginine pathway was involved in the occurrence of depression.In the use of ASV technology to assess the phenotype of depression,it was found that the prevalence of GABA producing bacteria in depression was slightly higher.GABA was mainly produced through arginine putrescine GABA metabolic pathway,arginine and ornithine metabolic pathway,indicating that the intestinal flora participated in the arginine metabolic pathway in patients with depression.Literature research found that arginine and its metabolites change in depression.Exogenous administration of arginine metabolites,such as agmatine,putrescine,spermidine and spermine,all have antidepressant effects,indicating that arginine metabolism pathway is involved in the occurrence of depression.Therefore,it is speculated that how Chaigui granules exerts antidepressant effects by regulating arginine metabolic pathways involved in the gut flora.Therefore,in this study,16 S rRNA high-throughput sequencing technology was used to analyze the effect of Chaigui granules on the improvement of intestinal microflora composition and the regulation of metabolites in arginine metabolism pathway in CUMS rats.From the perspective of intestinal flora,the mechanism of Chaigui granules’ antidepressant effect through arginine metabolism pathway mediated by intestinal flora was studied by molecular biology technology.Objective:1.Objective to clarify the role of intestinal flora in the antidepressant mechanism of Chaigui granules.2.Study on antidepressant mechanism of Chaigui granules in improving arginine metabolism pathway mediated by intestinal flora Methods:1.CUMS rats were given Chaigui granules by therapeutic administration,and sequencing studies of the flora in different parts of the gut of CUMS rats were performed using 16 S rRNA high-throughput sequencing.Observe the changes in the structure and diversity of the intestinal flora of CUMS rats,to explore the regulatory effect of Chaigui granules on the changed intestinal flora in CUMS rats and how the differential flora is involved in the arginine metabolic pathway to exert antidepressant mechanism study.2.The levels of arginine and its metabolites in hippocampus and peripheral serum of CUMS rats were determined by UPLC-MS/MS technology to explore whether the arginine metabolic pathway of depression rats is disordered and whether Chaigui granules can regulate the levels of metabolites.3.The mRNA levels of Odc,Agmat,Sat1,Spds and Spms in the hippocampus of CUMS rats were determined by Real Time qPCR,and the protein levels of Odc,Agmat,and Sat1 in the hippocampus of CUMS rats were determined by Western blot.The regulation of Chaigui granules on arginine metabolism pathway was further verified at molecular level.Result:1.The results of intestinal microflora showed that: there were differences in the structure of intestinal flora in different parts of the intestine,and Chaigui granules had different regulatory effects on it at different classification levels.The species of jejunum flora were the most abundant,and the species of cecum flora were the most concentrated.Compared with caecum,jejunum and ileum flora had certain similarity.The results of flora diversity showed that Chaigui granules had the most significant effect on jejunum flora,with significant difference(P<0.05).Chaigui granules had no effect on ileum,but had significant effect on caecum Chao1 index,indicating that Chaigui granules had stronger effect on jejunum flora.The results showed that Thermus and Ochrobactrum were involved in the arginine biosynthesis pathway,while Leptothrix,Acinetobacter and Pseudomonas were involved in the arginine metabolism pathway.It plays an antidepressant role by regulating the differential flora involved in arginine pathway.2.Results of determination of each metabolite of the arginine pathway: Compared with the control group,the levels of Arg and Cit in the serum of CUMS rats were significantly increased(P<0.05,P<0.01,and the levels of Glu,Orn,Spd,Spm,Put and GABA were significantly decreased(P<0.05,P<0.01)by the administration of Chaigui granules,and each metabolite was addback significantly(P<0.05,P<0.01),with Orn regulating the most significant(P<0.05,P<0.01),It indicated that Chaigui granules improved arginine and its metabolite levels to some extent.The Chaigui extract group had no significant regulation on Arg and Spd,but there was a tendency of callback to Spd,which indicated that Chaigui granules had a strong regulatory effect on Arg.The levels of Glu,Arg,Orn and Cit in the hippocampus of CUMS rats can be significantly reduced by Chaigui granules(P<0.05,P<0.01),and the regulation of Arg is the most significant.Polyamines Spm,Spd,Put and GABA levels were significantly increased by Chaigui granules(P<0.05,P<0.01),while Chaigui extract group had no significant adjustment to Glu and Spd.Although there is no significant difference between the two administration groups,the degree of regulation is different.Combined with the serum results,it is found that Chaigui granules has a more significant regulatory effect on Arg.Comprehensive comparison shows that Chaigui granules have a significant callback effect on substances in the arginine metabolism pathway.This shows that Chaigui granules may play an antidepressant effect by regulating the metabolic pathway of arginine.3.Real Time qPCR results showed that: Odc,as the key rate limiting enzyme of polyamine synthesis,significantly increased the mRNA level in the hippocampus of CUMS rats(P<0.05,P<0.01),while Agmat,Spds and Spms significantly increased the mRNA level in the hippocampus of CUMS rats(P<0.05,P<0.01)The mRNA level was also significantly increased(P<0.05,P<0.01),indicating that Odc catalyzed Orn to Put level was inhibited,and the gene expression levels were significantly decreased after Chaigui granules intervention(P<0.05,P<0.01).As the rate limiting enzyme of polyamine metabolism,Sat1 mRNA level in hippocampus of CUMS rats decreased significantly(P<0.05),polyamines could not be metabolized continuously,resulting in the accumulation of metabolic enzymes Spms and Spds.Administration of Chaigui granules could increase Sat1 mRNA level of CUMS rats(P<0.05,P<0.01).The results showed that Chaigui granules could regulate the rate limiting enzyme genes Odc and Sat1 in arginine metabolism pathway mRNA expression,and then affect the changes of downstream polyamine metabolites to play the role of anti depression.4.Western blot results showed that: compared with the control group,the protein level of SAT1 in the hippocampus of CUMS rats was significantly decreased(P<0.01),and the protein level of ODC was significantly increased(P<0.05,P<0.01).After intervention with Chaigui granules,the protein level was significantly reversed,which was consistent with the above results,indicating that Chaigui granules can regulate the expression of rate limiting enzyme in arginine metabolism pathway plays a regulatory role.The protein level of AGMAT in the hippocampus of CUMS rats was significantly increased(P<0.05,P<0.01),indicating that the conversion of agmatine to putrescine was inhibited,resulting in the decrease of putrescine content.Chaigui granules could significantly regulate the expression level of AGMAT protein(P<0.05,P<0.01).The results show that Chaigui granules can play an antidepressant effect by regulating the expression of the rate-limiting enzyme genes ODC and SAT1 in the arginine metabolic pathway,and affecting the changes in the downstream polyamine metabolites of arginine.Conclusion: The results showed that Chaigui granules could significantly improve the imbalance of jejunum flora induced by depression.Functional prediction analysis showed that the differential flora was involved in arginine pathway.The mechanism of action may be to restore the balance of intestinal flora and normalize the arginine metabolism pathway.By increasing the expression level of SAT1,it can promote the transformation of Spm to Spd and Spd to Put,so as to accelerate the metabolism of arginine downstream metabolites polyamines,and then accelerate the transformation of Orn and agmatine to Put,and Arg to Orn,Cit and agmatine,so as to avoid substance accumulation and accelerate the whole process arginine metabolism pathway,so that the body metabolism returned to normal,and then restore health. |
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