| Purposes:EGCG（Epigallocatechin gallate）is one of the primary bioactive compounds in green tea,categorized as tea polyphenol.EGCG plays crucial regulatory roles in human health maintenance via multiple strategies represented by antioxidative mechanisms,which is long established as the key substance in green tea to achieve nutritional merits.As the second major neurodegenerative disease in the world,Parkinson’s disease（PD）seriously affects human health.Studies have shown that EGCG can relieve PD symptoms to some extent,but the mechanism of action of EGCG is still unclear.As it has been reported that the intestinal microbial structure of PD patients is changed compared with that of healthy people,and the consumption of EGCG can have modulatory effect on intestinal microbiota.Therefore,the purpose of this study was to investigate whether the effect of EGCG on the rescue of Parkinson’s disease was achieved by reshaping the intestinal microbial structure,as well as to figure out the gut-brain signaling mechanisms involved.Methods:In this study,we used Drosophila melanogaster to establish Parkinson’s genetic(PINK1B9 or PINK1 knockdown)and environmental（rotenone exposure）models,and subjected to EGCG intervention.First,the fruit flies were allowed to grow on the food containing EGCG with varying concentrations for 20 days,and the repair effect of EGCG was determined by detection of drosophila’s behavioral and morphological manifestations.After determining the repair effect,16S r RNA was adopted to measure the gut microbial structure in response to EGCG intervention,and the ensuing antibiotic interference demonstrated that gut microbiota was implicated in mediating the improved effect of EGCG against PD.Subsequently,strain-specific q PCR was used to identify the bacterial strains playing essential roles in EGCG intervention.In order to further dissect the key genes involved in the studied gut-brain pathway,transcriptome screening was performed.Finally,recombination of the screened genes with the PINK1B9 flies were completed to identify key genes through behavioral and morphological analysis.Results:1)feeding flies with 0.5 m M EGCG for 20 days could effectively improve various phenotypes,（including climbing index from 15.33±0.60 to 8.55±0.27;flight/jumping index increased from 4.20±0.49 to 8.35±0.76;the number of neurons in the PPL1 area increased from 8.00±0.26 to 9.33±0.42;the proportion of neurons containing abnormal mitochondrial morphology decreased from 27.22%±3.91 to15.97%±2.18,etc.）in Drosophila models of Parkinson’s disease caused by genetic factors and/or environmental factors,indicating that EGCG could improve PD symptoms in the studied models,and the rescue effect of EGCG is time-and dose-dependent.2)antibiotic interference can eliminate the restoration effect of EGCG on drosophila of PD model,indicating that the improving effect of EGCG is realized by affecting the intestinal microbial structure of drosophila,specifically by reducing the relative abundance of Lactobacillus plantarum KJ01 in drosophila;3)four different strains were isolated and identified from fly gut,and through relative abundance analysis and single strain intervention,LP KJ01 was found to be a key bacterium affecting the EGCG effects.This mechanism not only exists in flies of the genetic Parkinson’s disease model,but also applies to model flies caused by environmental factors;4)transcriptomic analysis identified 1615 genes deregulated by the mutation of PINK1,whereas the expression levels of 291 genes were additionally altered following the addition of EGCG.The enriched pathways regulated by EGCG are most commonly related to amino acid and carbohydrate metabolisms;5)by means of pathway analysis and q PCR verification,we found that six genes,namely TotM,Miro,mt:ATPase 6,P32,CG4908 and m Rp L55,varied in their expressions according to the disease model establishment and EGCG intervention,enabling them optimal candidate molecules moderating the studied gut-brain pathway;6)on the basis of PINK1B9 model,a double-knockdown fly was obtained by further knocking-down the corresponding genes.As evidenced by the results,specific knockdown of TotM expression can weaken the repair effect of EGCG,and meanwhile either EGCG or KJ01 lost the ability to interfere with the PD manifestations,indicating that TotM is a key gene involved in the EGCG-intestinal microbiota-PD improvement pathway.Conclusion:EGCG can improve PD symptoms by reducing the relative abundance of LP KJ01 in the intestinal tract of drosophila,and the intestinal microbiota-TotM pathway plays an essential role in EGCG-mediated neuroprotection.The present study first investigated the improving mechanisms of EGCG towards Parkinson’s disease in the perspective of gut microbiota,shedding light on the concrete health-promoting pathways of EGCG,as well as providing useful molecular and microbial targets for nutritional intervention of psychiatric disorders. |
