The Effect And Mechanism Of Lactate On Intestinal Epithelial Barrier Restoration In Piglets With DSS-Induced Colitis

Inflammatory bowel disease(IBD),including Crohn’s disease(CD)and ulcerative colitis(UC),is a common and complicated intestinal inflammatory disease.The main manifestations are impaired intestinal barrier function,intestinal mucosa congestion,edema,and ulcer.In pig production,colitis can lead to diarrhea,delayed growth and development of piglets,resulting in a decrease in breeding income.Lactate,as a product of sugar metabolism,plays an important role in the process of colitis.In this study,both in vivo and in vitro models were used to investigate the protective effect of lactate on intestinal barrier of piglets with colitis and its possible mechanism.Firstly,under the model of acute intestinal inflammation in piglets induced by dextran sulfate sodium sulfate(DSS),the alleviating effect of dietary supplementation with lactate on the intestinal barrier of colitis piglets was explored.Then the mechanism of lactate in alleviating intestinal barrier injury was further explored through cell experiments.Experiment 1 Mitigative effect of dietary lactate supplementation on the intestinal barrier restoration in piglets with DSS-induced colitisA total of 30 DLY weaned gilts at 24 days with similar body weight were randomly divided into 3 groups after 3 days’ preparation period: control group,DSS group and lactate group.Each group had 10 replicates and 1 pig per replicate.Piglets in the control group and DSS group were fed with basic diets,while the lactate group was fed with basic diet containing 2% liquid lactate.After 7 days of feeding,on the 8th day of the experiments,piglets in the DSS group and the lactate group were intragastric administered 5% DSS solution for 4 consecutive days,and the control group was administered the same volume of saline.Piglets were slaughtered in the morning of the 12 th day of the experiment,and intestinal tissue and chyme samples were collected.The experiment lasted for 11 days.The results were as follows:(1)Compared with the control group,the jejunal villi of piglets induced by DSS were shrunk,partially broken,structurally destroyed,and the goblet cells became larger and more numerous,and gathered at the damaged villi.The villus height and the ratio of villus height to crypt depth in the jejunum was significantly decreased(P < 0.01),and the number of goblet cells was significantly increased(P < 0.01).Dietary supplementation with lactate significantly alleviated the villous atrophy and the ratio of villus height to crypt depth was markedly improved in the jejunum of piglets(P < 0.01).(2)Compared with the control group,DSS significantly reduced the m RNA expression of Occludin and ZO-1 in the colon(P < 0.05).However,compared with the DSS group,dietary supplementation of lactate significantly increased the protein level of Claudin-1 in jejunum(P < 0.01)and the m RNA expression of ZO-1 and ZO-2 in the colon(P < 0.05).(3)Compared with the control group,DSS-induced colitis significantly increased the level of IL-17 A in the colonic mucosa(P < 0.001),and significantly reduced the levels of Foxp3、TGF-β1 and IL-35 in the colonic mucosa of piglets(P < 0.001).Compared with the DSS group,dietary supplementation of lactate significantly reduced the level of IL-17A(P< 0.001)and significantly increased the levels of Foxp3、TGF-β1and IL-35 in the colonic mucosa(P < 0.05).(4)Compared with the control group,DSS-induced colitis significantly decreased the protein level of IκBα(P < 0.05),and significantly increased the ratio of Clv.casp9/Casp9 and p-STAT3/STAT3 protein in the colon(P < 0.01).Compared with the DSS group,the ratio of Clv.casp9/Casp9 and p-STAT3/STAT3 protein ratio was significantly decreased in the colon of piglets(P < 0.05).(5)Compared with the control group,DSS significantly increased the m RNA expression of HO-1 and Keap-1(P < 0.05),and significantly decreased the m RNA expression of NRF2 in the colonic mucosa of piglets(P < 0.01).Compared with the DSS group,dietary supplementation of lactate significantly increased the expression of antioxidant-related genes NRF2 and NQO1 in the colon of piglets(P < 0.01),and significantly decreased the m RNA expression of HO-1(P < 0.05).(6)Compared with the control group,there was no significant difference in the rate of Ki67-positive cells in the colonic crypts of piglets in the DSS group(P > 0.1).However,the dietary supplementation of lactate significantly increased the rate of Ki67-positive cells compared with the DSS group(P < 0.01).(7)Compared with the control group,the protein level of CD24 and Cyclin D1 were significantly decreased in the jejunum(P < 0.05),and the protein abundance of colonic PCNA、c-Myc and Cyclin D1 were significantly reduced in the DSS group(P < 0.01).Compared with the DSS group,the protein abundance of Cyclin D1、CD24 in the jejunum and protein level of PCNA、c-Myc and Cyclin D1 in colon were significantly increased(P< 0.05).(8)Compared with the control group,the protein levels of CK-1 and DVL2 in the jejunum and the protein levels of β-catenin,CK-1,DVL2、Frizzled 6 and β-Trcp in the colon were significantly reduced in the DSS group(P < 0.05).Compared with the DSS group,the protein levels of CK-1 and DVL2 in the jejunum and the protein abundance of β-catenin、CK-1、GSK-3β、Axin2、DVL2、LEF and β-Trcp protein expression in the colon were significantly increased in the lactate group(P < 0.05).(9)Compared with the control group,DSS significantly reduced the relative abundance of Bacteroidota,Spirochaetota,Muribaculaceae,UCG-002,Prevotella,Clostridia＿UCG-014,Treponema,Rikenellaceae＿RC9 and Christensenellaceae＿R-7 in the colon of piglets(P < 0.05).Compared with DSS group,dietary supplementation of lactate significantly increased the relative abundance of Euryarchaeota and Bacteroides in the colon of piglets(P< 0.05).The above results indicated that in the DSS-induced colitis in piglets,the intestinal barrier of piglets was impaired,the protein expression of tight junction were significantly reduced with the imbalance of gut microbiota,the proliferation and renewal of the intestinal epithelial cells were blocked with unbalanced the redox state.However,dietary supplementation of lactate alleviated DSS-induced intestinal barrier impairment,improved intestinal antioxidant and immune regulation functions.Moreover,we found that lactate can activate the colonic Wnt/β-catenin signaling pathway to promoted the proliferation of intestinal epithelial cells and acted on specific gut flora,thus promoting the repair of intestinal epithelial barrier injury in piglets.Experiment 2: The alleviating effect of lactate on inflammatory injury in IPECJ2 cells and its mechanismIn this study,IPEC-J2 cells were induced by different concentrations of DSS for 24 h,and the appropriate concentration of DSS was screened to establish a cell inflammatory injury model to explore the effect of DSS on cell proliferation and apoptosis.Secondly,to explore the alleviating effect of lactate / sodium lactate on inflammatory injury of IPEC-J2 cells,cells were treated with different concentrations of lactate for 24 h before the induction of DSS.The results showed that:(1)Compared with the control group,when the concentration of DSS reached 0.04 %,the cell viability and Ki67 mean fluorescence intensity were significantly reduced(P < 0.01).0.01 %,0.04 % DSS significantly increased the early,late and total apoptosis rates of cells(P < 0.05),0.08 %,0.1% and 0.5%DSS significantly increased the late apoptosis rates of cells(P < 0.01),0.08 % and 0.1% DSS significantly increased the m RNA expression of inflammatory cytokines IL-1β,IL-6 and IL-18(P < 0.01).(2)Compared with the control group,2 m M and 4 m M lactate significantly increased cell viability(P < 0.05).At the same time,16 m M and 32 m M sodium lactate significantly increased cell viability(P < 0.05).(3)Compared with DSS group,lactate had no significant effect on the cell viability,while 16 m M and 32 m M sodium lactate significantly increased cell viability(P < 0.05).Sodium lactate at 8 m M,16 m M and 32 m M significantly increased the average fluorescence intensity of Ki67(P < 0.01),and 8 m M sodium lactate had the best effect.(4)Compared with DSS group,sodium lactate at 2 m M,4 m M,8 m M,16 m M and32 m M significantly reduced the late apoptosis and total apoptosis rate(P < 0.05),and 8m M sodium lactate had the best effect.(5)Compared with DSS group,sodium lactate significantly reduced the m RNA expression of IL-1β、IL-6、IL-18 and Caspase-1(P < 0.05),and 8 m M sodium lactate had the best effect.8 m M sodium lactate was selected for subsequent experiments.(6)Compared with the control group,the ROS-positive rate of cells were significantly increased in the DSS group(P < 0.001).However,the ROS-positive rate of cells was significantly decreased in the sodium lactate group after DSS induction(P < 0.01).(7)Compared with the control group,DSS significantly decreased the proportion of cells in S phase(P < 0.01).Compared with the DSS group,sodium lactate at 8 m M significantly increased the proportion of cells in the S phase after DSS induction(P < 0.01).(8)Compared the control group,DSS significantly reduced the Lgr5-positive rate of cells(P < 0.001).Compared with DSS group,8 m M sodium lactate significantly increased the Lgr5-positive rate of cells after DSS induction(P < 0.01).The above results showed that sodium lactate can alleviate DSS-induced inflammatory injury in IPEC-2 cells,reduce the production of ROS,alleviate cell apoptosis,and significantly increase the number of Lgr5 stem cells after DSS induction,promote the proliferation of IPEC-J2 cells,alleviate DSS-induced intestinal epithelial cells injury.In summary: The results of the present study showed that dietary supplementation of lactate alleviated intestinal inflammation induced by DSS,reduced intestinal structural injury,promoted intestinal cell proliferation and can acted on specific gut microbiota,improved intestinal barrier function in piglets.Further study showed that dietary supplementation of lactate activated intestinal Wnt/β-catenin signaling pathway and improved the antioxidant and immunomodulatory functions of piglets.Cell experiments showed that sodium lactate significantly inhibited the production of ROS in intestinal epithelial cells under the induction of DSS,alleviated the apoptosis of intestinal epithelial cells,and increased the number of intestinal Lgr5 stem cells,thereby promoting the repair of intestinal barrier under inflammatory injury.