Effect Of Glucagon-like Peptide-2 On Intestinal Development And Adaptation Of Weaned Piglet And The Study Of The Related Mechanism

Although most nutritionists acknowledge that Glucagon-like Peptide-2 affects intestinal mucosa development,regulates small intestinal adaptation and immune function by promoting proliferation and inhibiting apoptosis of intestinal enterocyte cells such as mice,rats,fetal pigs and neonatal piglets,it is not clear yet whether GLP-2 can regulate the intestinal adaptation of weaned piglets or not and what are the operative properties of GLP-2 function on the intestinal enterocyte cells of weaned piglets with the features of rapid renewal.To anwer this question,the study used 4 series experiments to explore the effects of GLP-2 on the intestinal growth of weaned piglets and the operative properties of GLP-2 function on proliferation,metabolism and apoptosis of weaned piglets' intestinal enterocyte cells,which will provide theoretical and experimental basis for further studying regulation and mechanism of GLP-2 on weanling stress by using weaned piglets in situ.Exp.1 The relationship between GLP-2's secretion and intestinal growth of different genotypes pigletsThe experiment preliminarily studied the relationship between GLP-2's secretion and intestinal growth of piglets with different growth rate by studying GLP-2 secretion rule and intestinal growth parameters of piglets with different genotypes during weaning.1-d old healthy piglets(20 MeiShan Pigs,20 Yorkshire×MeiShan Pigs and 20 Landraee), were assigned to three treatments with five replicates and four piglets per replicate according to Single-factor design principle.The piglets were fed in environment control laboratory and weaned on the 25^th day.Blood samples from 24-d,26-d and 30-d old piglets close to average weight in each replicate were collected through precaval vein for the determination of GLP-2 concentration.30-d old piglets were slaughtered for measuring intestinal growth parameters and investigating the relationship between the height of intestinal villis of 30-d piglets and serum GLP-2 concentrations of 24-d,26-d and 30-d old piglets.The results were as follows:at 30-d old,the length and the intestines length index of the Yorkshire×MeiShan Pig were longer than that of Landrace(P＜0.05) and the intestines weight index also outweighed that of Landraee(P＜0.05);the intestines weight index of the MeiShan Pig outweighed that of Landrace(P＜0.05) and the intestines length index was also longer than that of Landrace(P＜0.01).In addition,at 30-d old,the height of intestinal villis of Yorkshire×MeiShan Pigs was highter than that of Landrace and MeiShan Pig(P＞0.05) and the serum GLP-2 content was higher than Landrace and MeiShan Pig(P＜0.01);at 24-d old and at 26-d old,the serum GLP-2 content of Yorkshire×MeiShan Pigs was higher than MeiShan Pig(P＜0.01);at 26-d old,the serum GLP-2 content of Yorkshire×MeiShan Pigs was higher than Landrace(P＜0.05). Furthermore,at 30-d old,there was a significant correlation between the height of intestinal villis and serum GLP-2 content of different genotypes(P＜0.01).The study showed that GLP-2 activity of piglets with different genotypes increased gradually after weaning.Yorkshire×MeiShan Pigs had the largest change range and reached the highest activity at 30-d old,with intestinal growth superior to MeiShan Pigs and Landrace.Serum GLP-2 content of the three 30-d old genotypes was significantly correlated with the height of intestinal villis at 30-d old.The experimental results suggest that it is a feasible approach to select the Yorkshire×MeiShan Pig or its intestinal enterocyte cells as model to study GLP-2's possible mechanism and its regulation on the intestinal adaptation of piglets during weaning.After the confirmation that the secretion level and activity of GLP-2 in different genotypes piglets during weaning increased gradually,and that the height of intestinal villis was significantly correlated with serum GLP-2 concentration in circulation,the studying team further studied nutritional effects of GLP-2 on intestinal enterocyte cells of Yorkshire×MeiShan Pigs(Exp.2).The results indicated that GLP-2 in vitro was capable of promoting the proliferation of intestinal enterocyte cells of 28-d old weaned piglets, inhibiting cell apoptosis and maintaining the integrity of cell morphology and function.On this ground,the studying team made the following studies.Exp.3 Effects of the combined use of Glucagon-like peptide-2 and dipeptidyl peptidase-â…£Inhibitors on Intestinal Enterocyte Cells of Weaned Piglets in VitroThis study was conducted to investigate in vitro the effects of the combined use of Glucagon-like peptide-2(GLP-2) and dipeptidyl peptidase-â…£(DPP-â…£) inhibitors with different concentrations on cell proliferation,metabolism and enzyme activity of 28-d old weaned piglets' intestinal enterocyte cells.The experiment,taking in vitro 28-d old weaned piglets' intestinal enterocyte cells as model,studied effects of DPP-â…£(KR62436) inhibitors with different concentrations on the cell proliferation and metabolism according to the single-factor design principle.The method of 2×3 factorial design was adopted to study effects of the combined use of different GLP-2 concentrations(1×10^-10,1×10^-9 and 1×10^-8 mol/L) and different KR62436 concentrations(0 and 1×10^-11 mol/L) on cell proliferation,metabolism and apoptosis,with cell culture time 144h.The results were as follows:with the single use of DPP-â…£inhibitors,there was no significant change in cell under the observation of inverted microscope;although the cell quantity presented an increasing trend,MTT OD,protein retention and protein consumption firstly increased and then decreased(P＞0.05).LDH activity and CK activity presented a decreasing trend with low dosage of KR62436(P＞0.05).With higher concentration of KR62436,MTT OD value was signigicantly lower than control groups(P＜0.05),LDH activity and CK activity were signigicantly higher(P＜0.05),but Na⁺,K⁺-ATP enzyme activity changed insignificantly. When GLP-2 and KR62436 were both added into the cultura media,with the increasing of hGLP-2 concentrations,cell quantity increased signigicantly(P＜0.01),MTT OD value increased signigicantly(P＜0.01),total protein content and protein retention increased signigicantly(P＜0.01),and Na⁺,K⁺-ATP enzyme activity increased signigicantly(P＜0.01) but LDH activity and CK activity decreased signigicantly(P＜0.01).The results indicated that DPP-â…£inhibitors with a lower concentration had insignificant effects on cell growth. Although the DPP-â…£inhibitor with a higher concentration promoted the proliferation of intestinal enterocyte cells,it had negative effects on cell metabolism and cell integrality. The addition of DPP-â…£inhibitors in the culture media enhanced the effects of in vitro GLP-2 on cell proliferation,cell metabolism and cell integrality.GLP-2 and DPP-â…£inhibitors had significant synergistic effect.The experimental results suggested that KR62436 should be cautiously used to promote GLP-2 effects.Exp.4 Cytoprotection and repairing effects of GLP-2 on Intestinal Enterocyte Cells of 28-d Weaned Piglets following Injury byβ-conglycininThe experiment was conducted to study the effects of hGLP-2 on survival, proliferation,metabolism and apoptosis of intestinal enterocyte cell in 28-day-old weaned piglets after injury byβ-conglycinin and GLP-2's possible mechanism.According to Single-factor design principle,differentβ-conglycinin concentrations of 1.2mg/ml and 2.4mg/ml were used for conteracting toxic substances to primary culture enterocyte cells of weaned piglets.The cell damage model was established on the investigation of the cell survival,proliferation,metabolism and apoptosis.The experiment of 2×3 factorial design was adopted to study the impacts of the sensitized cultured cell with different hGLP-2 concentrations of 1×10^-9,1×10^-8 and 1×10^-7mol/l.The results were as follows:when β-conglycinin were used for counteracting toxic substances,the MTT OD significantly decreased(P＜0.05),especially protein retention and protein consumption extremely significantly decreased(P＜0.01),Na⁺,K⁺-ATP enzyme activity significantly decreased (P＜0.05),LDH activity and Caspase-3 significantly increased(P＜0.01).Whenβ-conglycinin were used for conteracting toxic substances after addition of hGLP-2,the MTT OD,protein retention and protein consumption and Na⁺,K⁺-ATP enzyme activity significantly(P＜0.05).LDH activity gradually decreased(P＜0.05) and Caspase-3 extremely significantly decreased(P＜0.01) along with the increase of hGLP-2 concentration.The results indicated thatβ-conglycinin had adverse effects on intestinal enterocyte cell proliferation and cell integrality in vitro.GLP-2 could relieve or avoid the adverse effects on intestinal enterocyte cell proliferation and cell integrality by regulating the Caspase-3 activity.To summarize,GLP-2 has effects on intestinal development of weaned piglets,and the height of intestinal villis is highly correlated with GLP-2 secretion levels.GLP-2 in vitro is capable of promoting the proliferation of the intestinal enterocyte cells and inhibiting their apoptosis in a dose-dependent manner.Although the single use of KR62436 may impair cell integrity,the addition of DPP-IV inhibitors enhances GLP-2 effects,which indicates that the combination of DPP-â…£inhibitors and GLP-2 has good synergistic effect,β-conglycinin extracts had adverse effects on proliferation and integrality of intestinal enterocyte cells.GLP-2 could relieve or avoid the adverse effects on intestinal enterocyte cell proliferation and cell apoptosis by protecting cells.The experimental results effectively support the further research and related work on in situ effects of GLP-2 on intestinal adaptation and acting mechanism of weaned piglets,and provide a fresh thinking on how to use GLP-2 to regulate weanling stress as well.