Evaluating The Inhibitory Effect Of Lactic Acid Bacteria On Dipeptidyl Peptidase-4 Activity In Caco-2 Cells

Type 2 diabetes is a disease developed from metabolic syndrome.It has become a very important global health problem.China has the largest number of diabetic patients.Dipeptidyl peptidase-4（DPP-4）is a key target for the treatment of type 2 diabetes.However,synthetic DPP-4 inhibitors have some undesirable effects.Therefore,lactic acid bacteria（LAB）have become a new source of DPP-4 inhibitors due to their natural and safety characteristics.At present,studies have shown that LAB have DPP-4 inhibitory activity,but the main components and mechanism of LAB against DPP-4 activity were not clear.Therefore,in the current study,12 LAB strains from Chinese traditional fermented food stored in the laboratory were selected as the research objects,and LAB with DPP-4 inhibitory activity were screened through the constructed Caco-2 cell model.Then,the strain with the strongest inhibitory activity were identified by 16S r DNA sequencing technology and API 50CH biochemical characteristics.Furthermore,ultrafiltration technology and ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS/MS）were used to analyze the active components with DPP-4 inhibitory effect.Finally,the mechanism of the LAB strain which was exhibited the highest activity against DPP-4 was explored by ribonucleicacid sequencing（RNA-seq）technology and real-time quantitative PCR（q RT-PCR）technology.The main contents and results of the research are as follows:（1）LAB with significant inhibition of DPP-4 activity were screened based on the Caco-2 cell modelThis chapter first established the Caco-2 cell model.Based on this model,the inhibitory effects of cell-free excretory supernatants（CFS）and cell-free extracts（CFE）from LAB on DPP-4 of Caco-2 cells were analyzed.The results showed that the activity of DPP-4 in Caco-2 cells reached the peak when Caco-2 cells inoculated with high density（7×10⁵cells/m L）and cultured for 7 days,while that of Caco-2 cells reached the peak when Caco-2cells inoculated with low density（1×10⁵cells/m L）and cultured for 14 days.It was further found that the half inhibitory concentration(IC₅₀)of sitagliptin（a known DPP-4 inhibitor）did not change with the increase of culture time after Caco-2 cells inoculated with high density cultured for more than 4 days.Hence,the Caco-2 cells that inoculated with high density and cultured for 4 days were used to screen LAB strains with DPP-4 inhibitory activity.It was observed that the inhibitory activity of CFE collected from each LAB strain on DPP-4 was higher than that of CFS.The inhibition rates of CFS and CFE were 0 to 5.68%and 3.09%to24.33%respectively.The data obtained in this experiments indicated that the Caco-2 cells inoculated with high density（7×10⁵ cells/m L）after cultured for 4 days could be used as a cell model for screening DPP-4 inhibitors.The CFE of 2018SWU.B05.08004 had significant DPP-4 inhibitory activity,and the strain named YE4.The CFE from YE4 strain was abbreviated as YE4-CFE.（2）Identification of YE4 and components analysis with DPP-4 inhibitory activityIn order to clarify the species of YE4 and active components with DPP-4 inhibitory effect on Caco-2 cells,this study identified its species according to genetic and biochemical characteristics,and explored active components from YE4-CFE by ultrafiltration technology and UHPLC-MS/MS.The results revealed that Lactobacillus plantarum YE4 was identified by 16S r DNA sequencing technology,phylogenetic tree analysis and API 50CH biochemical kit.The components with larger molecular weight in YE4-CFE had lower inhibition rate on DPP-4 of Caco-2 cells,and the components less than 3 KDa had the strongest inhibitory activity on DPP-4.There were 477 compounds less than 3 KDa in YE4-CFE,and the relative proportions of 19 compounds were more than 1%,including adenine（10.573%）,acetylcholine（7.479%）,L-phenylalanine（7.177%）,guanine（6.418%）,2-hydroxycinnamic acid（5.975%）,L-tyrosine（5.683%）.The 19 compounds were classified as imidazopyrimidines,carboxylic acids and derivatives,cinnamic acids and derivatives,purine nucleosides,fatty acyls,diazines,organonitrogen compounds,indoles and derivatives and others.The results of these experiments demonstrated that the active components of YE4-CFE with DPP-4 inhibitory effect were mainly small molecules less than 3 KDa,including 19compounds such as adenine,acetylcholine,L-phenylalanine,guanine,2-hydroxycinnamic acid,L-tyrosine and so on.（3）Mechanism analysis of Lactobacillus plantarum YE4-CFE inhibiting DPP-4 activityTo explore the mechanism of YE4-CFE inhibiting DPP-4 activity,we predicted the gene effect of YE4-CFE on Caco-2 cells based on RNA-seq technology,and focused on TNF,MAPK pathway and DPP-4 gene expression by q RT-PCR technology.Datas exhibited that2296 genes were up-regulated and 1718 genes were down-regulated in Caco-2 cells after treated with YE4-CFE.These differentially expressed genes were not only related to the components of plasma membrane,cation channel activity,muscle contraction,biological adhesion and cell adhesion in GO enrichment analysis,but also related to lipid metabolism,carbohydrate metabolism,endocrine system,endocrine and metabolic diseases in KEGG database.In addition,YE4-CFE inhibited the m RNA expression of DPP-4 gene in Caco-2cells.At the same time,six genes related to TNF pathway were altered,including the m RNA expression of CFLAR was markedly alleviated,and the m RNA expression of ATF4,CREB3L3,NFκBIA,JUNB and Tnfaip3 was notably elevated by YE4-CFE treatment.The ten genes related to MAPK pathway also changed,including the m RNA expression of IRAK1,FLNA and MYC were remarkably relieved,and the m RNA expression of DDIT3,HSPA1A,ATF4,HSPA1B,STMN1,DUSP1 and NR4A1 were raised dramatically after incubated with YE4-CFE.Altogether,these findings revealed that the inhibitory effect of YE4-CFE on DPP-4was related to TNF and MAPK pathway.In summary,the CFE collected from Lactobacillus plantarum YE4（YE4-CFE）had prominent DPP-4 inhibitory activity.The inhibitory effects of YE4-CFE on DPP-4 were partly attributed to TNF and MAPK pathways,and nineteen compounds less than 3 KDa,particularly the high percentage of adenine,acetylcholine and L-phenylalanine.