Regulation Effects Of Terpine-4-ol On Intestinal Epithelial Cell Homeostasis In Piglets Under Immune Stress

Weaning is the most challenging time in pig breeding.The gastrointestinal system and immune system of piglets are not fully developed and are vulnerable to the invasion of external pathogenic microorganisms,resulting in diarrhea and intestinal inflammation of piglets,causing intestinal structure and function disorders,disrupting the normal renewal process of intestinal stem cells,and then causing the homeostasis imbalance of intestinal epithelial cells,resulting in decreased production performance and even death,which seriously affects the economic benefits of pig breeding.The homeostasis of intestinal epithelial cells may be related to the disturbance of the regeneration process,and the regeneration of intestinal epithelial cells is derived from intestinal stem cells.How to relieve intestinal inflammatory damage of piglets from the perspective of nutritional regulation has become a research hotspot.With the implementation of the policy of total forage prohibition,the search for suitable antibiotic substitutes has been concerned.Our previous study found that tea tree oil and its main component Terpine-4-ol(TER)can relieve intestinal damage and enhance intestinal barrier function of piglets after stress.However,the effects of Terpinen-4-ol on intestinal stem cells of piglets were less studied.Therefore,based on previous studies,we hypothesize that Terpinen-4-ol May regulate intestinal epithelial cell homeostasis in piglets,but its mechanism of action remains unclear.Elucidate its potential mechanism is of great significance for the development of novel feed additives and their application in pig production.Experiment 1:Effects of terpine-4-ol on growth performance and intestinal morphology of immune-stressed pigletsThis experiment was conducted to investigate the effects of Terpinen-4-ol supplementation on growth performance and intestinal morphology of immune-stressed piglets.Forty 28-day-old(15-18 kg)healthy piglets weaned at 21 days of age(Duroc ×Landrace × large white pigs)were randomly divided into 5 treatment groups:Control group(CON group,fed a basal diet),lipopolysaccharide stimulation group(LPS group,fed a basal diet),low,medium and high dose Terpinen-4-ol groups(LT+P group,MT+P group and HT+P group,fed a basal diet supplemented with 30 mg/kg,60 mg/kg and 90 mg/kg TER),respectively.There were 8 piglets in each group.The pre-trial period was 3 days and the trial period was 21 days.On day 21(6 h before sampling),piglets in LPS,LT+P,MT+P and HT+P groups were intraperitoneally injected with LPS of 100 μg/kg body weight,and piglets in CON group were intraperitoneally injected with 0.9%sterile normal saline.Growth performance,diarrhea rate and intestinal morphology of piglets were measured.The test results show that;(1)Compared with CON group,average daily gain(ADG)and average daily feed intake(ADFI)of piglets in MT+P and HT+P groups were significantly increased(P<0.05);Compared with CON group,the ratio of feed to gain in LT+P group was significantly decreased(P<0.05),and diarrhea rate of piglets in TER group was significantly decreased(P<0.05).(2)Compared with CON group,the villi of jejunum and ileum of piglets in LPS group were different in length and thickness,and their structures were damaged and disordered.The morphology of intestinal villi in TER group was more complete than that in LPS group,and the transverse section of villi was wider.Compared with CON group,the villus height of jejunum and the chorio crypt ratio of empty and ileum of piglets in LPS group were significantly decreased(P<0.05),and the crypt depth of jejunum and ileum of piglets in LPS group was increased compared with CON group(P>0.05).The villus height and pile crypt ratio of jejunum in TER group and HT+P group were significantly increased compared with LPS group(P<0.05),and the crypt depth of jejunum in MT+P group and ileum crypt depth in LT+P and MT+P groups were significantly decreased compared with LPS group(P<0.05).In conclusion,Terpinen-4-ol can improve the growth performance and reduce the diarrhea rate of piglets.It is recommended that the supplemental dose of Terpinen-4-ol should be 60 mg/kg in piglets’ diet.At the same time,Terpinen-4-ol can increase the villus height and the ratio of pile to crypt,reduce the crypt depth,and damage the intestinal structure of piglets,which may cause the homeostasis imbalance of intestinal epithelial cells.Experiment 2:Regulation of intestinal epithelial cell homeostasis in piglets under immune stress by terpinen-4-olThis experiment was conducted to investigate the effects of dietary supplementation of Terpinen-4-ol on intestinal epithelial cell homeostasis in piglets under immune stress.The design of the experiment is the same as that of experiment 1.The proliferation of empty and ileal epithelial cells,the number of stem cells,the number of intestinal stem cell differentiation cells(intestinal absorption cells,goblet cells,endocrine cells,Pan’s cells)were measured.The results showed as follows:(1)Compared with CON group,the number of Ki67 positive cells in empty and ileum crypt and the expression of Ki67 gene related to intestinal epithelial cell proliferation were significantly decreased in LPS group(P<0.05),and the number of Ki67 positive cells in empty and ileum of weaned piglets was significantly increased by adding medium and high dose TER(P<0.05).Meanwhile,TER could increase Ki67 gene expression in empty and ileum of piglets(P<0.05).(2)Compared with CON group,Lgr5(Leucine-rich repeat containing G protein-coupled receptor 5)gene and Lgr5 protein expression in empty and empty ileum of piglets in LPS group were significantly decreased(P<0.05).TER can mitigate this decline;Compared with LPS group,Lgr5 and Bmi1(Bmilpolycomb ring finger oncogene 1)gene expressions in jejunum of LT+P group and ileum of MT+P and HT+P groups were significantly increased(P<0.05).The expressions of intestinal stem cell related genes Lgr5 and Bmi1 in HT+P group were significantly decreased compared with those in MT+P and LT+P groups(P<0.05).The relative expression of Lgr5 protein in jejunum and ileum of MT+P groups in TER group was significantly higher than that in LPS group(P<0.05),while the relative expression of Lgr5 protein in ileum of HT+P group was significantly lower than that in MT+P group(P<0.05).(3)Compared with CON group,the number of empty and ileum goblet cells and the number of Muc2(Mucin 2)positive cells in LPS group were significantly decreased(P<0.05),while the number of empty and ileum goblet cells in MT+P group was significantly increased compared with LPS group(P<0.05).Compared with LPS group,the number of Muc2 positive cells in jejunum and ileum of piglets in TER group was significantly increased(P<0.05).(4)Compared with CON group,the expression of Muc2,ChgA(Chromogranin A)in jejunum and Villin,Lyz(Lysozyme)in ileum of piglets in LPS group was significantly decreased(P<0.05).The relative expressions of Lyz gene and protein in jejunum of piglets in LPS group were significantly increased compared with CON group(P<0.05).Compared with LPS group,the Villin and ChgA gene expressions in jejunum of piglets in MT+P group and TER group were significantly increased(P<0.05),and the Muc2 gene expression in jejunum of piglets in LT+P group was significantly increased(P<0.05).The expressions of ChgA,Lyz and Muc2,Lyz genes in jejunum and ileum of piglets in LT+P and MT+P groups were significantly increased(P<0.05);The expression of Villin,Muc2,ChgA and Lyz genes in jejunum and ileum stem cells in HT+P group was significantly decreased compared with that in MT+P group.LT+P and MT+P groups could up-regulate the expression of Lyz protein in jejunum(P<0.05).The relative expression of intestinal Lyz protein in HT+P group was significantly decreased compared with that in LT+P and MT+P groups(P<0.05).In conclusion,LPS can reduce the proliferation of intestinal epithelial cells and the number of intestinal stem cells,and reduce the number of intestinal absorption cells,goblet cells,endocrine cells and Pan’s cells,resulting in the homeostasis of intestinal epithelial cells.Moreover,dietary supplementation of Terpinen-4-ol in can regulate the homeostasis of intestinal epithelial cells induced by LPS,improve intestinal health,and alleviate intestinal damage caused by immune stress in piglets.Experiment 3:Potential mechanism of terpine-4-ol in regulating intestinal epithelial cell homeostasis in piglets under immune stressThis experiment was conducted to investigate the potential mechanism of dietary supplementation of Terpinen-4-ol in regulating intestinal epithelial cell homeostasis in piglets under immune stress.The design of the experiment is the same as that of experiment 1.The expression of Wnt/β-catenin signaling pathway related genes and proteins in jejunum and ileum of piglets were determined.The results show that:(1)The gene expressions of Wntl,TCF4 and Cyclin D1 in jejunum and Wntl,β-catenin and TCF4 in ileum of piglets in LPS group were significantly lower than those in CON group(P<0.05),while the gene expressions of c-Myc in jejunum of piglets in LPS group were significantly higher than those in CON group(P<0.05).The gene expressions of Wntl,β-catenin and Cyclin D1 in jejunum and Wntl,β-catenin,TCF4 and c-Myc in ileum of piglets in TER group were significantly increased,compared with LPS group(P<0.05).The expressions of TCF4,cMyc and Cyclin D1 genes in jejunum of piglets in LT+P and MT+P groups were significantly higher than those in LPS group(P<0.05).The gene expressions of Wntl,βcatenin,TCF4,Cyclin D1 and c-Myc in empty and ileum of piglets in HT+P group were significantly decreased compared with those in MT+P group.(2)Compared with CON group,the protein relative expressions of β-catenin,Cyclin D1 and TCF4 in jejunum ileum,Wntl and c-Myc in ileum of piglets in LPS group were significantly decreased(P<0.05);The relative expression of Cyclin D1 protein in jejunum Wntl in MT+P group,TCF4 and LT+P groups in HT+P group was significantly increased compared with LPS group(P<0.05).The relative expression of β-catenin protein in jejunum in TER group was significantly increased compared with LPS group(P<0.05).Compared with LPS group,the relative expressions of Wntl,β-catenin,TCF4 and Cyclin D1 in ileum of piglets in LT+P and MT+P groups were significantly increased(P<0.05);The relative expressions of Wntl,β-catenin,Cyclin D1 in jejunum and Wntl,β-catenin,TCF4 and Cyclin D1 in ileum of piglets in HT+P group were significantly decreased compared with those in LT+P and MT+P groups(P<0.05).(3)Compared with CON group,the relative expression of β-catenin protein in empty and ileal nucleus of piglets in LPS group was significantly decreased(P<0.05);Compared with LPS group,the relative expression of nuclear β-catenin protein in jejunum of piglets in MT+P,HT+P,ileum LT+P and MT+P groups was significantly increased(P<0.05).The relative expression of β-catenin protein in ileum nucleus of piglets in HT+P group was significantly decreased compared with that in LT+P and MT+P groups(P<0.05).In conclusion,LPS can down-regulate the expression of Wnt/β-catenin signaling pathway genes and proteins,while Terpinen-4-ol May regulate intestinal epithelial cell homeostasis and relieve intestinal damage of immune-stressed piglets by up-regulating Wnt/β-catenin signaling pathway.May regulate intestinal epithelial cell homeostasis and relieve intestinal damage of immune-stressed piglets through Wnt/β-catenin signaling pathway.In conclusion,dietary supplementation of Terpinen-4-ol can improve intestinal villus crypt structure,improve growth performance and reduce diarrhea rate of weaned piglets.The recommended dosage is 60 mg/kg.LPS can reduce the proliferation of intestinal epithelial cells and the number of intestinal stem cells,and reduce the number of intestinal absorption cells,goblet cells,endocrine cells and Pan’s cells,resulting in the homeostasis of intestinal epithelial cells.However,Terpinen-4-ol may regulate intestinal epithelial cell homeostasis of piglets under immune stress through Wnt/β-catenin signaling pathway.These results can provide reference for the application of Terpinen-4-ol in piglets and provide a new nutritional target for regulating intestinal injury in piglets.