The Study On Dihydroartemisinin Alleviates Intestinal Injury Of Weaned Piglets With Intrauterine Growth Retardation And Its Underlying Mechanism

Intrauterine growth retardation(IUGR)is a serious problem in animal husbandry production,which refers to the growth and development damage of mammalian embryo/fetus or its organs during pregnancy.As a multiple animal,pig has a high natural incidence of IUGR,which is as high as 15-20%in pig production.IUGR not only increases the mortality and morbidity of newborn piglets,but also may have a permanent impact on their growth,metabolism and development after birth,which will eventually leads to slow growth and development of pigs,a decline in feed utilization,and causes serious impact on the economic benefits to the pig industry.A large number of studies have shown that IUGR reduces the growth performance,damages the intestinal morphology,reduces the intestinal immune function,and causes inflammation in the intestine of weaned piglets.Therefore,it is of great significance to regulate the growth and development and intestinal function of IUGR piglets.Dihydroartemisinin(DHA)is an important derivative of artemisinin,which is an active component of Artemisia annua.It is reported that DHA has anti-inflammatory,antibacterial and immunomodulatory functions.However,there is no rearch about the effect of DHA on IUGR piglets,and the mechanism of DHA in repairing the intestinal injury of IUGR weaned piglets is limited.Therefore,the effects of DHA on growth performance,nutrient utilization and blood index of weaned piglets were studied in this experiment.The optimal dose of DHA in weaned piglets was chosen,which provides a reference for the optimal dose of DHA in IUGR weaned piglets.In addition,we investigated the effects of DHA on growth performance and intestinal injury of IUGR weaned piglets and explore the feasibility of DHA as a feed additive to improve the intestinal health of IUGR piglets1 Effect of diet supplemented with different levels of dihydroartemisinin on growth performance,nutrient utilization and blood parameters of weaned pigletsIn Experiment 1,a total 360 21-day-old piglets were randomly divided into 6 groups with 6 replicates and 10 piglets in each replicate.The control group was fed with basal diet,and the five treatment groups were fed with basal diet supplemented with 20,40,80,160 and320 mg/kg DHA,respectively.The experimental period was 28 days.At the age of 49 days,two piglets with similar body weight from each replicate were selected and blood samples were collected by jugular venipuncture.Growth performance,nutrient utilization,serum antioxidant enzyme activity and cytokine concentration were measured.The results showed that:(1)Dietary supplementation of 80 mg/kg DHA significantly increased ADFI and ADG,and decreased F/G(P<0.05).(2)Diet supplemented with different levels of DHA could effectively reduce the diarrhea incidence and improve the digestibility of DM,OM,CP and EE,and 80 mg/kg DHA group had the best effect(P<0.05).(3)Dietary supplementation with 80 mg/kg DHA significantly increased serum TP concentration and decreased serum TG concentration(P<0.05).(4)Dietary supplementation with 80,160 and 320 mg/kg DHA increased serum T-AOC level and CAT activity of piglets(P<0.05).(5)Dietary supplementation with 80 and 160 mg/kg DHA decreased IL-1βconcentration,increased the concentrations of IL-4 and IL-10,Ig A and Ig G in the serum of piglets(P<0.05).(6)Dietary supplementation with 160 and 320 mg/kg DHA increased serum T-Bil concentration and AKP activity of piglets,and 320 mg/kg DHA increased serum AST activity of piglets(P<0.05).These results indicated that dietary DHA supplementation could improve the growth performance,nutrient utilization,serum antioxidant and immune function of weaned piglets,but excessive DHA may cause damage to the liver of piglets.In this study,the optimal dose of DHA in the diet of weaned piglets was 80 mg/kg.2 Effect of diet supplemented with dihydroartemisinin on growth performance,intestinal development,intestinal digestion and absorption of IUGR weaned pigletsIn Experiment 2,12 normal birth weight(NBW)piglets and 24 IUGR piglets were selected and weaned at 21 days of age.NBW piglets were divided into NBW-CON(NC)group(basal diet),and IUGR piglets were divided into IUGR-CON(IC)group(basal diet)and IUGR-DHA(ID)group(basal diet+80 mg/kg DHA).The experimental period was 28days.At the age of 49 days,8 piglets with similar body weight were slaughtered and jejunum and ileum samples were collected.Growth performance,intestinal development,activities of digestive enzymes and absorption related enzymes,concentrations and expression levels of intestinal nutrient transporters were determined.The results showed that:(1)IUGR significantly reduced the body weight and ADG of piglets for 0-7 d,7-14 d and 0-21 d(P<0.05).IUGR decreased the body weight,ADG and ADFI of piglets for 49 d(P<0.05).Dietary DHA supplementation significantly increased ADG and ADFI of IUGR piglets(P<0.05).(2)Compared with the NBW-CON group,the BW before slaughter,the weight of small intestine and jejunal length in the IUGR-CON group were decreased,and the relative weight of jejunum and ileum were increased(P<0.05).Diet supplemented with DHA significantly impoved the BW before slaughter,and decreased the relative weight of jejunum and relative length of ileum of IUGR piglets.(P<0.05).(3)IUGR significantly decreased intestinal VH,VCR and VSA,and increased CD of piglets(P<0.05).Dietary DHA supplementation significantly increased intestinal VH,VCR and VSA,and decreased CD of IUGR piglets(P<0.05).(4)Compared with NBW-CON group,the activities of jejunal lactase,sucrase,maltase and AKP and ileal amylase,trypsin,lactase,sucrase and Na~+-K~+-ATPase were significantly decreased in IUGR-CON group(P<0.05).Diet supplemented with DHA significantly increased the activities of jejunal amylase,lactase,maltase and AKP,and the activities of ileal amylase,trypsin and Na~+-K~+-ATPase in IUGR piglets(P<0.05).(5)IUGR significantly decreased the concentrations of jejunal GLUT2 and ileal SGLT1 of piglets,and dietary DHA supplementation increased the concentrations of jejunal GLUT2 and ileal SGLT1 in IUGR piglets(P<0.05).(6)IUGR significantly reduced the m RNA expressions of jejunal Glut2,Snat2,Fabp and Fasn,and ileal Glut2,Sglt1,Snat2,Pept1,Fabp and Fasn of piglets(P<0.05).DHA supplementation significantly increased the m RNA expressions of these genes in IUGR piglets(P<0.05).Therefore,DHA could improve the growth performance and improve the digestive and absorptive function of IUGR piglets by improving intestinal development and intestinal morphology,digestive enzyme activity,the concentrations and related gene expressions of nutrient transporters of IUGR weaned piglets.3 Effect of diet supplemented with dihydroartemisinin on intestinal mucosal epithelial barrier function in IUGR weaned pigletsThe experimental design was the same as Experiment 2.The results showed that:(1)IUGR increased the serum DAO concentration,and dietary DHA supplementation reduced the serum DAO concentration in IUGR piglets(P<0.05).(2)The numbers of GCs and IELs in jejunum and ileum of IUGR-CON group were significantly lower than those of NBW-CON group(P<0.05).The numbers of GCs and IELs in jejunum and ileum of IUGR piglets were significantly increased after DHA supplementation(P<0.05).(3)IUGR significantly reduced the concentrations of jejunal MUC2 and ileal TFF3 of piglets,and diet supplemented with DHA increased the concentrations of jejunal MUC2 and ileal TFF3 in IUGR piglets(P<0.05).(4)Compared with NBW CON group,the concentrations of s Ig A and Ig G in the ileum was significantly reduced in the IUGR-CON group(P<0.05).DHA supplementation significantly increased the concentrations of s Ig A and Ig G in jejunum and ileum of IUGR piglets(P<0.05).(5)IUGR significantly down-regulated the m RNA expressions of jejunal CD8 and MHC-I,and ileal Ig G,Fc Rn,CD4,CD8 and MHC-I m RNA,and up-regulated the m RNA expressions of IL-6 in the jejunum and ileum of piglets(P<0.05).Diet supplemented with DHA significantly increased the m RNA expressions of jejunal Ig G,CD8,MHC-I and ileal Ig G,Fc Rn,CD4,CD8 and MHC-I,and decreased the m RNA expression of jejunal IL-6 and ileal IL-6 and TNF-αof IUGR piglets(P<0.05).(6)IUGR significantly reduced the m RNA amd protein expressions of ileal claudin-1 and jejunal occludin of piglets(P<0.05).Dietary DHA supplementation significantly increased the m RNA and protein expressions of ileal claudin-1 and jejunal occludin of IUGR piglets(P<0.05).In conclusion,DHA could improve the intestinal epithelial mucosal barrier injury and promote intestinal health in IUGR weaned piglets.4 Effect of diet supplemented with dihydroartemisinin on intestinal inflammatory response in IUGR weaned pigletsThe experimental design was the same as Experiment 2.The results showed that:(1)IUGR piglets significantly increased AI and the protein expression of caspase-3,and decreased PI and the protein expression of PCNA in the jejunum and ileum of piglets(P<0.05).Dietary DHA supplementation decreased AI and the protein expression of caspase-3,and increased PI and the expression levels of and PCNA protein in IUGR piglets(P<0.05).(2)The concentrations of intestinal LPS,jejunal IL-1β,IL-6 and TNF-αand ileal IL-1βand IL-6 of IUGR-CON group were significantly higher than those of NBW-CON group(P<0.05).DHA supplementation significantly decreased the concentrations of intestinal LPS,jejunal IL-1βand IL-6 and ileal IL-1β,IL-6 and TNF-αof IUGR piglets(P<0.05).(3)The m RNA expressions of jejunal TLR4,My D88,IRAK1,NOD1,RIPK2 and NF-κB p65 and ileal TLR4,NOD1,NOD2,RIPK2 and NF-κB p65 of IUGR piglets were significantly up-regulated when compared with NBW piglets(P<0.05).DHA supplementation down-regulated the m RNA expression of jejunal TLR4,IRAK1,NOD1,RIPK2 and NF-κB p65 and ileal TLR4,IRAK1,NOD1,NOD2,RIPK2 and NF-κB p65 of IUGR piglets(P<0.05).(4)The m RNA expression levels of Tollip,ERBB2IP and CENTB1 in the jejunum and SOCS1,ERBB2IP and CENTB1 in the ileum of piglets in IUGR-CON group were significantly lower than those in NBW-CON group(P<0.05).DHA supplementation significantly increased the m RNA expression of the above genes in IUGR piglets(P<0.05).(5)IUGR significantly increased the protein expressions of TLR4,p IκBαand nuclear p NF-κB,and decreased cytoplasmic p NF-κB in jejunum and ileum of piglets.Dietary DHA supplementation effectively improved the changes of the above protein expression levels in IUGR piglets(P<0.05).In conclusion,dietary DHA supplementation could regulate the apoptosis and proliferation intestinal epithelial cells,and alleviate the intestinal inflammatory injury by inhibiting the gene and protein expressions of TLR4/NODs/NF-κB signaling pathway and increasing the gene expressions of negative regulatory factors of TLR4/NODs pathway.5 Effect of diet supplemented with dihydroartemisinin on the diversity and composition of intestinal microflora in IUGR weaned pigletsThe experimental design was the same as Experiment 2.After slaughter,jejunum and ileum contents were collected for 16S r RNA sequencing.The results showed that:(1)IUGR significantly reduced the Shannon index and increased the Simpson index in the jejunum of piglets(P<0.05).Dietary DHA supplementation significantly increased the Shannon index and decreased the Simpson index of IUGR piglets(P<0.05).(2)In the jejunum,the dominant microflora at the phylum level were Firmicutes and Cyanobacteria,and those at the genus level were Lactobacillus,norank＿f＿＿norank＿o＿＿Chloroplast,Clostridium＿sensu＿stricto＿1,and Streptococcus.In the ileum,the dominant microflora in the phylum level of three groups were Firmicutes,while those in the IC group were Firmicutes and Proteobacteria.The dominant microflora at the genus level of NC group were Lactobacillus and Clostridium＿sensu＿stricto＿1,while those in IC group were lactobacillus,Clostridium＿sensu＿stricto＿1 and Escherichia-Shigella.(3)At the phylum level,IUGR significantly decreased the relative abundance of jejunal Actinobacteriota and increased ileal Proteobacteria of piglets(P<0.05).Dietary DHA supplementation significantly increased the relative abundance of Actinobacteriota in the jejunum of IUGR piglets(P<0.05).At the genus level,IUGR significantly reduced the relative abundance of Blautia,Ruminococcus＿gauvreauii＿group,Subdoligranulum,Collinsellaand unclassified＿f＿＿Ruminococcaceae in the jejunum of piglets(P<0.05).Dietary supplemented with DHA significantly increased the relative abundance of jejunal Streptococcus,Blautia,Ruminococcus＿gauvreauii＿group,Staphylococcus,Subdoligranulum,Collinsella and unclassified＿f＿＿Ruminococcaceae,and ileal Clostridium＿sensu＿stricto＿1 of IUGR piglets.(P<0.05).(4)The results of PCo A analysis showed that the distance between the samples in both jejunum and ileum of NC and ID group was close,and the distance between the samples of IC and NC group was far.(5)The m RNA expressions of tight junction protein of piglets were positively correlated with beneficial bacteria and negatively correlated with harmful bacteria in the small intestine(P<0.05).The m RNA expressions of inflammation related genes were negatively correlated with beneficial bacteria and positively correlated with harmful bacteria(P<0.05).Therefore,dietary DHA supplementation could significantly improve the intestinal microbial diversity and composition in IUGR weaned piglets,and the intestinal microflora correlated with expressions of tight junction protein and inflammation related genes.Therefore,this experiment concluded that 80 mg/kg DHA was the optimal dose in the diet of weaned piglets.IUGR reduced the growth performance,inhibited the intestinal development,and damaged the intestinal morphology and function of weaned piglets.Dietary DHA supplementation could improve intestinal mucosal barrier damage,reduce intestinal inflammatory injury and regulate intestinal microflora,which may be the potential mechanism of DHA in improving growth performance and digestive and absorptive function of IUGR weaned piglets.