| Metabolic syndrome(MS)is a cluster of harmful conditions characterized by obesity,insulin resistance and other risk factors for cardiovascular disease.Pu-erh tea and its functionally active ingredient theabrownin have certain functions such as lowering blood lipids and blood glucose,but few studies have focused on their effects on MS.This study aims to explore the anti-MS mechanism of Pu-erh tea and theabrownin in MS mice,using transcriptomics,metabonomics and16 S r DNA sequencing.It was found that Pu-erh tea and theabrownin can significantly improve liver,jejunum and brain tissue lesions,down-regulate the serum levels of inflammatory factors of IL-1,IL-6 and TNF-α,and increase the levels of antioxidants such as MDA,SOD,GSH and INS in the serum of MS mice.Pu-erh tea and theabrownin intervention significantly affected the metabolism of glycerophospholipid,linoleic acid and purine in the serum and brain tissue,regulated the circadian rhythm pathway in the liver,increased the relative abundance of Clostridiales＿bacterium and Firmicutes＿bacterium＿ASF500,and decreased the relative abundance of Brevundimonas＿vesicularis.Pu-erh tea and theabrownin intervention may improve the symptoms of MS through the microbiota-gut-liver-brain axis by regulating circadian rhythm genes in the liver,and short-chain fatty acid transport and receptor genes.1.Pu-erh tea and theabrownin can improve MS by regulating anti-obesity,anti-inflammatory and anti-aging functions in MS mice.Animal experiments showed that Pu-erh tea and theabrownin could alleviate MS-induced symptoms.The results of hepatic inflammatory cytokines(IL-1,IL-6 and TNF-6),antioxidant index(SOD and GSH),oxidant index(MDA),insulin and blood glucose showed that metformin hydrochloride and theabrownin considerably decreased the abnormal levels of MS-induced inflammatory factors(P<0.05).SOD and GSH levels were significantly decreased,while that of MDA was elevated.In addition,antioxidant capacities were enhanced in MS mice by metformin hydrochloride and theabrownin(P< 0.05).Histopathological results indicated that Pu-erh tea was superior to theabrownin in reducing weight gain and improving lipid metabolism.Pu-erh tea and theabrownin can effectively reduce the damage and inflammation of liver and jejunum tissues.Taken together,theabrownin was superior to Pu-erh tea in reducing the MS-related inflammatory factors,oxidants and insulin resistance,and improving antioxidant capacity in MS mice.2.Effects of Pu-erh tea and theabrownin on lipid metabolism in the serum and brain tissue of MS mice.LC-MS/MS untargeted metabolomics was performed to detect changes in the metabolic profiles of serum and brain tissue samples of all groups.A total of 259 and 149 differential metabolites were identified from the serum and brain,respectively.Metabolomics Pathway Analysis(Met PA)showed that Pu-erh tea and theabrownin may significantly regulate glycerophospholipid metabolism and linoleic acid metabolism in the serum and brain of normal mice(P<0.05).In MS mice,Pu-erh tea significantly altered glycerophospholipid metabolism and linoleic acid metabolism in the serum,while theabrownin greatly affected the metabolism of glycerophospholipid and linoleic acid in the brain tissue(P<0.05).In addition,Pu-erh tea,theabrownin and metformin hydrochloride significantly affected purine metabolism in the brain tissue of MS mice.3.Pu-erh tea and theabrownin affected the expression of circadian rhythm genes in the liver of MS mice.A total of 5,252 differentially expressed genes were detected by liver transcriptomic analysis.Signaling-pathway impact analysis(SPIA)of these genes showed significant changes in the circadian rhythm gene-related pathways in the liver of MS mice treated with Pu-erh tea or theabrownin(P<0.05).Circadian rhythm genes such as Npas2,Clock,Arntl and Cry1 were altered remarkably in MS mice treated with Pu-erh tea or theabrownin(P<0.05).Furthermore,theabrownin affected more genes in the circadian rhythm pathway(such as Npas2,Clock,Arntl,Cry1,Per3,Per2 and Bhlhe40)than Pu-erh tea in MS mice.4.Pu-erh tea and theabrownin changed the gut microbiota composition in MS mice.The results of gut microbiota analysis at different time points showed a decrease in α diversity but an increase in β diversity after normal chow diet replaced by high-fat diet(P<0.05).PCo A analysis showed that high-fat diet significantly changed the composition of gut microbiota in mice.In addition,the abundance of Firmicutes/Bacteroides,Proteobacteria and Verrucomicrobia in the intervention groups at different time points of normal chow diet were significantly higher than those in groups replaced by high-fat diet.A gut microbiota analysis conducted at the end of the experiment showed that Firmicutes had a lower abundance while lipopolysaccharide and Proteobacteria had higher abundances in the MS groups compared with the K groups.The abundance of Bacteroides in the MS-Y group was the highest.The α diversity of the K-PE group and K-TB group was significantly increased compared with that of the K group(P<0.05),while the MS intervention groups had no significant change compared with that of the MS group.PCo A and NMDS analysis showed that the MS intervention groups had narrower gaps with health group(K)than the MS group,suggesting that the intervention of Pu-erh tea,theabrownin and guanidine diformate may improve the gut microbiota of MS mice.To further explore the key bacteria related to Pu-erh tea and theabrownin intervention,time series analysis,Meta Stats and Wilcoxon were used.It was found that compared with the K group,three bacteria were significantly decreased(Brevundimonas vesicularis,Veillon micrococcus,and Cutibacterium＿acnes),while another three were increased(Blautia＿coccoides,Desulfovibrio sp UNSW3 caefat S,Firmicutes bacterium ASF500)in the K intervention groups.In addition,compared with the MS group,in the MS intervention groups,the butyric acid-producing bacteria Clostridiales＿bacterium 42＿27 and the anti-inflammatory and anti-weight gaining Firmicutes bacterium ASF500 increased significantly(P<0.05).PICRUSt functional analysis showed that Pu-erh tea and theabrownin intervention affected remarkably the microbial metabolic function(amino acid and lipid metabolism,for example)in the K groups,but no significant change was found in the abundance of gut microbiota of MS mice.These data suggested that Pu-erh tea and theabrownin may alleviate MS by regulating the gut microbiota,adjusting the microbiota composition,reducing inflammation,and improving the gut physiology and immune system.5.Pu-erh tea and theabrownin may improve MS through the microbiota-gut-liver-brain axis.Partial least square-discriminant analysis combining repeated double cross-validation was performed on differential gut bacteria,differentially expressed liver genes,and differential serum and brain tissue metabolites.Nineteen intestinal bacteria,81 liver genes,71 serum and 32 brain metabolites were screened with |Pearson correlation| > 0.6.Principal component analysis(PCA)demonstrated effective discrimination among the groups.Correlation analysis with the seven key bacteria found in the above gut microbiota results identified six key bacteria in MS mice treated with Pu-erh tea and theabrownin: Clostridiales＿bacterium 42＿27,Firmicutes＿bacterium＿ASF500,Blautia＿coccoides,Brevundimonas vesicularis,Cutibacterium＿acnes,and Desulfovibrio＿sp＿UNSW3caefat S.Moreover,these bacteria are strongly related to liver circadian rhythm genes(such as Npas2,Nr1d1,Cry2,Per3,Arntl,etc.),which are associated with lipid,amino acid and purine metabolism(e.g.N,N-dimethylglycine,deoxyguanosine,deoxycholic acid(+),beta-guanidinopropionic acid,12-hydroxydodecanoic acid,etc.)(P<0.05).In order to study the interactions among the above six key bacteria,gene and metabolite indexes,as well as inflammation and aging indexes,a Spearman’s correlation analysis was conducted.The results showed that in MS and MS-PE groups,Cutibacterium＿acnes was significantly negatively correlated with decanoic acid,but positively correlated with Clock,Npas2,MDA and IL-1.Clostridiales＿bacterium 42＿27 was significantly positively correlated with brain decanoic acid and negatively correlated with Clock(P < 0.05).In MS and MS-TB groups,Firmicutes＿bacterium＿ASF500 was significantly negatively correlated with serum3-dehydrocarnitine and brain decanoic acid,Slc16a3,MDA and IL-6,and positively associated with serum metabolites(e.g.2-arachidonyl glycerol ether,2-deoxyycastasterone,succinic acid semialdehyde),lyso PI(18:0/0:0),Cry1,Cry2,Per2,Per3,Slc16a7 and INS(P < 0.05).Blautia＿coccoides was negatively correlated with Slc5a8,IL-6 and TNF-α,but positively correlated with serum metabolites DG(18:3(9Z,12 Z,15Z)/15:0/0:0),brain metabolites Lyso PI(18:0/0:0),Per3,Olfr78 and INS(P<0.05).There was a significant positive correlation between Clostridiales＿bacterium 42＿27 and serum metabolites linoelaidic acid,Clock,Npas2,IL-1,serum lipid PC(P-18:1(9Z)/16:1(9Z),2-arachidonyl glycerol ether,deoxycholic acid(-)and deoxycholic acid(+)(P<0.05).Therefore,the intervention of Pu-erh tea and theabrownin in MS mice may improve MS by changing the composition of gut microbiota,thus affecting the interaction among liver circadian rhythm genes,short-chain fatty acid transporter and receptor genes.Therefore,we speculate that short-chain fatty acid metabolism may be the potential target via which Pu-erh tea or theabrownin affect gut microbiota.To further verify the hypothesis,short-chain fatty acids were detected in mouse fecal samples.The results showed that obesity-related short chain fatty acids(Acetic acid and Propionic acid)and potentially harmful short-chain fatty acids(Isobutyric acid and Isovaleric acid)were significantly reduced in MS mice treated with Pu-erh tea(P<0.05).q PCR results showed that the expression of 5 bacteria,7 rhythm genes,1glycerophospholipid metabolism gene,7 short-chain fatty acid transfer-glycerophospholipid transformation genes and their receptors were consistent with those of RNA-Seq.Correlation analysis showed that Per3 and Cry2 were significantly increased in MS,while their expressions were significantly decreased in Pu-erh tea(P < 0.05).At the same time,Per3 and Cry2 were significantly associated with short-chain fatty acids(acetic acid),inflammation(IL-1,IL-6)and antioxidant indexes(SOD)(P < 0.05).In summary,in this study,a high fat diet-induced mouse model of MS was established to explore the potential mechanisms of Pu-erh tea and theabrownin in alleviating MS,using multi-omics techniques(16S r DNA sequencing,transcriptome,LC-MS/MS metabolome and GC-MS target metabolome,etc.).It was found that Pu-erh tea and theabrownin may improve MS through the microbiota-gut-liver-brain axis(gut microbiota-fecal short-chain fatty acid metabolism-lipid metabolism in serum and brain tissue-liver inflammation and aging factors as well as circadian rhythm genes).At the same time,it was found that Pu-erh tea and theabrownin may act on different targets in improving MS with varied efficacies..This study provides a theoretical basis for the development of probiotic products of Pu-erh tea,and theoretical support for the development of Pu-erh tea industry. |
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