| Early weaning has been widely used to improve reproductive efficiency in large-scale pig farming.However,the digestive and immune system of early-weaned piglets are insufficiently developed,easily lead to weaning stress reaction and diarrhea.Diarrhea is known closely related to the change of intestinal peristalsis rhythm.Weaning can potentially cause injury to enteric nervous system,resulting in abnormal intestinal peristalsis rhythm.Since intestinal peristalsis rhythm has been known to be regulated by the myenteric plexus,in this study,the underlying mechanism of longitudinal muscle-myenteric plexus(LM-MP)injury and regulation by nutrients calcium and phosphorus in weaned piglets were investigated.Experiment 1: Fourteen Duroc × Landrace × Yorkshire weaned piglets at 21 days of age were selected and randomly assigned into two groups,each group consists of seven replicates with one piglet of each.One-way experimental design was used in this study,and the main factor was weaning.The experiment lasted for 4 days.The results showed obvious symptoms of diarrhea and a significant increase in slow wave frequency of ileum and colon of piglets in the Day 24 group,compared with the Day 21 group(P<0.05).The distribution and number of interstitial cells of Cajal(ICC)were analyzed.The results showed that the expression of c-kit(ICC biomarker)in the ileum and colon of piglets in the Day 24 group was significantly lower than that of piglets in the Day 21 group(P<0.05),indicating the decrease of ICC cell or loss of c-kit protein in the intestine of weaned piglets.The results of oxidative damage of LM-MP in piglets showed significant up-regulation of 8-OHd G and down-regulation of Trx in the ileum and colon of piglets in the Day 24 group(P<0.05),indicating DNA and protein oxidative damage of intestinal LM-MP of weaned piglets.In addition,Atf3 was significantly upregulated in ileal LM-MP of piglets in the Day 24 group(P<0.05),indicating damage of ileal LM-MP.Experiment 2: In view of the observation that nerve damage occurred in the ileal LM-MP of weaned piglets in experiment 1,this study aimed to understand the underlying mechanism of ileal LM-MP injury using transcriptomics analysis.Notably,compared with the Day 21 group,a total of 243 differentially expressed genes were identified in the ileal LM-MP of piglets in the Day 24 group,including 209 down-regulated genes and 34 up-regulated genes.GO and KEGG enrichment analysis showed that differentially expressed genes were cell cycle regulation pathways related,and most of them were down-regulated in the ileal LM-MP of piglets in the Day 24 group,indicating changes of the cell cycle of ileal LM-MP of weaned piglets.RT-q PCR was used to confirm the expression levels of key genes involved in the cell cycle signaling pathway.The results showed that the m RNA levels of p21 were significantly up-regulated in the ileal LM-MP of the Day 24 group,and the m RNA levels of cyclin A2,cyclin B1 and PCNA were significantly down-regulated(P<0.05).Collectively,the results suggested the occurrence of G2/M phase arrest in the ileal LM-MP cells of weaned piglets,which affected the peristaltic rhythm of ileum.Experiment 3: Calcium and phosphorus were reported to regulate nerve signaling and cell cycle,thus,this experiment was conducted to investigate the effects of dietary supplementation of calcium and phosphorus on growth performance and regulatory mechanism of ileal LM-MP cell cycle of immune-stressed piglets.Eighteen Duroc × Landrace× Yorkshire weaned piglets at 22 days of age were selected and randomly assigned to three groups,each group consists of six replicates with one piglet of each replicate.The control and model group were fed with a basal diet,while the experimental group was fed with a diet containing a higher level of calcium and phosphorus [1.5 times NRC(2012)standard].From day 23,irinotecan(CPT)18 mg/kg BW was intraperitoneally injected to piglets in both model and experimental group for 4 days,to establish the immune-stressed piglet model,while piglets in the control group were injected with the same amount of saline.The experiment lasted for 28 days.The results showed that CPT can induce diarrhea,decrease the growth performance of piglets,and reduce the number of immune cells in the blood,indicating that the immune stress model of piglets was successfully established.Dietary supplementation of1.5 times of NRC standard calcium and phosphorus can alleviate the effects of CPT on average daily gain of piglets,reduce diarrhea,restore the number of blood monocytes and promote the recovery of immune function.In addition,calcium and phosphorus probably alleviate cell cycle arrest by down-regulating the levels of p21 and p53 in ileal LM-MP.In conclusion,oxidative stress and nerve injury occurred in intestine of weaned piglets.It probably caused diarrhea in piglets by affecting the cell cycle of intestinal LM-MP and interfering rhythm regulation function of LM-MP.Dietary calcium and phosphorus supplementation can relieve diarrhea,and reduce the effects of immune stress in the ileal LMMP cell cycle of piglets. |
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