早期断奶仔猪肠上皮细胞氧化与损伤及二氢杨梅素对其调控作用
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摘要
本文以21日龄早期断奶仔猪为研究对象，以肠上皮细胞氧化—酶发育—促炎/抗炎性因子分泌—生长和损伤修复再生—应激营养调控为研究主线，从以下四个试验探讨了早期断奶仔猪肠上皮细胞氧化与损伤及二氢杨梅素(DMY)对其调控作用。1早期断奶对仔猪肠上皮细胞氧化和损伤影响的研究
试验一：选取21±1日龄断奶、平均体重6.25±0.37kg的健康仔猪80头，分成2组（断奶组与吮乳组），每组4重复，每个重复10头仔猪。分别于断奶当天、断奶后第1、3、5、7、10、14天采集样品，每次每组每个重复随机宰杀1头仔猪，取肠组织、刮取肠粘膜、分离肠上皮细胞，取肝脏和血液，测定氧化、损伤、细胞因子和酶等指标。结果如下：
（1）早期断奶第1-3天，仔猪肠上皮细胞XO活性急剧升高、尿酸含量增多，抗氧化酶（CAT、SOD、GSH-Px）活性下降、过氧化产物MDA含量升高，AKP、Na＋-K＋-ATP活性急剧下降，细胞因子IL-1上升、IL-10含量下降，EGF/EGFR的含量及mRNA表达量显著降低，肠上皮细胞发生水泡变性，肠绒毛中的毛细血管有充血和出血现象；第5-7天，上述指标变化达到峰顶或峰谷，而后开始恢复，10-14天恢复到正常水平。表明早期断奶导致肠上皮细胞过氧化、AKP及Na＋-K＋-ATP酶发育受阻、促炎性因子上升、抗炎性因子降低、EGF/EGFR生成减少，在形态结构上表现为肠绒毛萎缩、隐窝增生。
（2）早期断奶第1-3天，仔猪肝脏XO活性升高、尿酸含量增多，抗氧化酶活性下降、过氧化产物MDA含量升高，Na＋-K＋-ATP和GS活性急剧下降；第3-5天，上述指标变化达到峰顶或峰谷，而后开始恢复，7-10天恢复到正常水平。表明早期断奶导致肝脏过氧化、Na＋-K＋-ATP和GS活性下降；肝GS活性下降可能也是影响肠上皮细胞能量代谢、更新和损伤的原因之一。
（3）早期断奶第1-3天，仔猪血清抗氧化酶活性下降、过氧化产物MDA含量升高，IL-1升高、IL-10和slgA含量下降；第3-5天，抗氧化酶活性及MDA含量变化达到峰顶或峰谷，而后开始恢复，7-10天恢复到正常水平；IL-1、IL-10和slgA含量都在第7天达到最高值，且10-14天都维持在较高水平。
（4）仔猪肠上皮细胞、肝和血清中相同指标变化比较，肠上皮细胞指标变化幅度最大，持续时间最长。说明，早期断奶对仔猪肠上皮细胞影响较肝脏和机体更大。2体外HX/XO对肠上皮细胞氧化、损伤及DMY保护作用研究
试验二：常规培养IEC-6细胞系，选取贴壁良好的细胞建立次黄嘌呤/黄嘌呤氧化酶（HX/XO）体外肠上皮细胞的氧化与损伤模型，选择别嘌呤醇作为该系统的抑制剂。细胞分为６个处理组：Ⅰ组为空白对照组，其余五组分别依次加入HX/XO的终浓度为25/25、50/50、100/100、150/150、200/200μmol/L，应激培养24h后，测定细胞内各指标变化情况。得出：50/50μmol/L的HX/XO可显著降低IEC-6细胞CAT和SOD活性，增加过氧化产物MDA含量；显著降低IEC-6细胞AKP及DAO活性和EGF/EGFR含量及mRNA表达量；显著降低IEC-6细胞活力；其各项指标变化模式与早期断奶应激引起的仔猪肠上皮细胞相应指标的变化相一致。添加HX/XO体系中常用的抑制剂别嘌呤醇逆转由HX/XO体系引起的肠细胞损伤，上述结果表明，黄嘌呤氧化酶途径是肠上皮细胞过氧化产生的途径之一，肠上皮细胞的损伤与氧化应激直接相关。
试验三：向肠上皮细胞体外氧化应激模型中加入不同浓度的DMY，观察DMY对体外肠上皮细胞氧化损伤的保护作用。细胞分为6处理组，其中Ⅰ组为空白对照组，Ⅱ组为氧化组（50/50μmol/L），Ⅲ组为抑制剂组（50/50μmol/L+200μmol/L），其余三组为氧化+DMY组，依次分别加入DMY的终浓度为50、75、100μmol/L，检测IEC-6细胞活力及氧化和损伤修复相关指标。结果显示：75-100μmol/L的DMY能增强HX/XO氧化体系中IEC-6细胞抗氧化酶CAT及SOD活性，减少氧化产物MDA含量；提高IEC-6细胞中AKP活性，减少细胞培养液中DAO活性；提高IEC-6细胞EGF/EGFR含量及mRNA表达量，促进IEC-6细胞的增殖活力；添加别嘌呤醇浓度为200μmol/L时，各项指标与空白对照组（不加氧化剂）无差异显著（P>0.05）。
试验二和试验三从正、反两途径证明了HX/XO体系产生的氧化应激导致体外肠上皮细胞损伤。3DMY对早期断奶仔猪肠上皮细胞氧化与损伤修复作用的研究
试验四：选取21±1日龄断奶、平均体重6.33±0.34kg健康仔猪72头，随机分成2个处理组，分别为断奶组和断奶DMY添加组（添加量为500克/吨），每组4个重复，每个重复9头仔猪。分别于断奶当天、断奶后第1、3、7、14天每组每个重复宰杀1头仔猪，取肠组织、分离肠上皮细胞，取肝脏、血液。于第7天、14、28天称量体重、记录饲料消耗量。结果如下：
（1）添加0.05%DMY，第7天可显著降低仔猪肠上皮细胞XO活性、尿酸含量，升高CAT和SOD活性、降低MDA含量，增强AKP和Na＋-K＋-ATP活性，降低细胞因子IL-1含量，增加IL-10含量，增加EGF/EGFR的含量及mRNA表达量，肠绒毛-隐窝结构基本完整。表明添加DMY减轻早期断奶对仔猪肠上皮细胞的损伤，促进肠上皮细胞增殖分化，促进绒毛-隐窝结构的生长发育。
（2）添加0.05%DMY，第7天可显著降低仔猪肝脏XO活性、尿酸含量，升高CAT和SOD活性、降低MDA含量，增强Na＋-K＋-ATP活性，对GS活性影响不显著。
（3）添加0.05%DMY，第7天可显著升高仔猪血清CAT和SOD活性、降低MDA含量，降低IL-1含量、升高IL-10和slgA含量。
（4）添加0.05%DMY，提高断奶仔猪日增重和饲料转化率。
综合上述四个试验结果：早期断奶通过XO途径诱导仔猪肠上皮细胞过氧化，肠上皮细胞抗氧化酶活性降低、分化成熟相关酶发育受阻、IL-1分泌增加、IL-10及sIgA分泌减少，生长修复因子转录合成减少,从而导致肠绒毛—隐窝结构损伤和修复更新受阻，在形态结构上表现为绒毛萎缩，隐窝加深。早期断奶仔猪日粮添加DMY能够显著抑制XO产生的过氧化，减轻早期断奶对仔猪肠上皮细胞的损伤，增强修复功能，促进肠上皮细胞的增殖分化，促进肠绒毛-隐窝结构的发育，提高仔猪的生产性能。
The destination of this study was the age of21days’ piglets. The main line of the study was theintestinal epithelial cellular oxidation-development of enzyme-secret of promotinginflammation/anti-inflammation factors-growth and impairment recovery-stress and regulation ofnutrition. From the four experiments done, we evaluate the impairment and oxidation of piglets’intestinal epithelial cell caused by early-weaning and its regulation of DMY.
1The study of early weaning to piglets’ intestinal epithelial impairment and oxidationExperiment1:Eighty healthy piglets aged21±1days with average body weight6.25±0.37kg wereselected and divided into two groups which were suckling and weaning groups with four replicates ineach group, and ten piglets in each replicate. Samples were collected on the day of1、3、5、7、10、14after weaning. One piglet was scarified in each replicate in each group at once. The intestine usedto isolated intestinal epithelial cell, liver and blood used to inspect the indexes of oxidation,impairment, cellular factor and enzyme were harvested.
(1) The activity of XO rose acutely and the concentration of uric acid rose on the1-3days afterweaning while the activity of antioxidational enzymes (CAT、SOD、GSH-Px) reduced and the contentof MDA rose. The activity of AKP、Na＋-K＋-ATP reduced. The content of cytokine IL-1rose and IL-10reduced. The concentration of EGF/EGFR and transcription of its mRNA reduced significantly.Intestinal epithelial cell happened to baffle denaturation. Capillary vessel in intestinal villus happenedto hyper-tention and blood. On5-7days after weaning, the indexes reached the submit or lowest，thenbecome to recover. On10-14days after weaning, they recovered to normal. It was concluded thatintestinal epithelial cell happened to oxidation and the development of enzymes AKP and Na＋-K＋-ATPwas obstructed causing by the early weaning. The content of promoting inflammation factor rose andthe anti-inflammation factor reduced. The generation of EGF/EGFR reduced. Intestinal villus atrophiedand crypt proliferation in structure.
(2) On1-3days after early-weaning the activity of hepatic XO and the content of uric acid rose,while the activity of antioxidational enzymes reduced. The concentration of the oxidational productMDA rose. The activity of Na＋-K＋-ATP and GS reduced acutely. On3-5days after early-weaning,those indexes rose the submit or lowest, then become to recover which recover to normal on7-10days.It could be referred that liver happened to oxidation, activity of Na＋-K＋-ATP and GS reduced caused bythe early-weaning. That activity of GS reduced may be one of the reasons that influence the energeticmetabolism of intestinal epithelial cell, regeneration and impairment.
(3) On1-3days after early-weaning the activity of antioxidational enzyme in piglets’ serumreduced and the concentration of oxidaional product MDA rose. The concentration of IL-1rose, IL-10and slgA reduced. On the3-5days after weaning, the activity of antioxidational enzyme and thecontent of MDA reached submit or lowest, then become to recover which recover to normal on7-10days. The content of IL-1、IL-10and slgA reached submit on the7d after weaning, and maintained high level on10-14days.
(4) When compared the same indexes in intestinal epithelial cell, liver and serum, we canconcluded that range of intestinal epithelial cellular indexes was the biggest, which lasted the longest.It suggested that impairment caused by early-weaning to intestinal epithelial cell was bigger than thatin liver and mechanism.
2The oxidation and impairment of intestinal epithelial cell caused by HX/XO in vitro andprotection of supplemented DMY.
Experiment2: Normally cultured the IEC-6cell lines. Choose the cell stick the wall and growwell to establish the oxidational model of xanthine and xanthine oxidase (HX/XO) in vitro. Allopurinolwas the inhibitor of this model. The cells were divided into6treatments: group Ⅰwas control. Theother five groups were added into different concentration of HX/XO to their last concentration reached25/25,50/50,100/100,150/150,200/200μmol/L, which was cultured for24h. Then inspected theindexes in cells. Results indicated that the activity of CAT and SOD could be reduced and theconcentration of oxidational product MDA was increased with a concentration of50/50μmol/LHX/XO. The activities of AKP and DAO was reduced significantly also the content of EGF/EGFRand the transcription of mRNA. The cellular activity of IEC-6was reduced significantly. The changes ofother indexes were the same as those of intestinal epithelial cell causing by the stress of early weaning.Added inhibitor:allopurinol to this model could prohibit the impairment of intestinal cell. The resultsadvised that the impairement of intestinal cell is directly related with the oxidational stress.
Experiment3: To observe the protection of DMY to oxidation and impairment of intestinal cell.There were6treatment, which group Ⅰ was control. Ⅱ was oxidational group（50/50μmol/L）, Ⅲwasthe inhibitor group（50/50μmol/L+200μmol/L），the remained groups were oxidation and DMY groupwhich the concentration of DMY was50、75、100μmol/L. Inspect the activity of IEC-6and indexes ofits oxidation and impairment. The results indicated that the activities of cellular antioxidational enzymesCAT and SOD were improved in HX/XO oxidational model with a concentration of75-100μmol/LDMY supplemented. The content of oxidational product MDA reduced and the activity of cellular AKPimproved. The activity of DAO in cellular culturation reduced and the content of EGF/EGFR improvedas well as the mRNA transcription. The activity of cellular proliferation was promoted. Supplementedallopurinol with a dose of200μmol/L, there were no differences on the indexes with the control.
That the system of HX/XO could produce oxidation and stress to cells which caused impairmentto intestinal epithelial cell was proved by the experiment2and3in two different ways
3The study of supplemented DMY to early-weaning piglets on intestinal epithelial cell oxidation,impairment and recovery
Experiment4: Seventy-two healthy piglets aged21±1days with average body weight6.33±0.34kg were selected and divided into two groups which were weaning and DMY supplemented groupswith4replicates in each group, and9piglets in each replicate. Intestinal tissue used to isolatedintestinal epithelial cell, liver and blood were harvested from one piglet in one replicate in a group on0、1、3、7、14days after weaning. Body weight was recorded on the days of7,14and28and the consumption of feedstuff.
(1) Dietary supplemented DMY at a dose of0.05%could reduce the activity of piglets’ intestinalepithelial cellular XO and content of uric acid. Improved the activity of CAT and SOD. Reduced theconcentration of MDA. Improved the activity of AKP and Na＋-K＋-ATP. Reduced the content ofcytokine IL-1. Improved the content of IL-10and EGF/EGFR also their transcription of mRNA.Structure of intestinal villus and crypt were intact overall. It advised that dietary supplemented DMYcould reduce the impairment of early-weaning piglets’ intestinal epithelial cell and promote theproliferation and differentiation of intestinal epithelial cell. Promote growth and development ofintestinal villus and crypt.
(2) Dietary supplemented DMY with a dose of0.005%could reduce the activity of piglets’hepatic XO and the content of uric acid on the7days after weaning. Improved the activity of CATand SOD. Reduced the concentration of MDA, Improved the activity of Na＋-K＋-ATP while there wereno significant influences on activity of GS.
(3) Dietary supplemented DMY with a dose of0.005%could improve the activity of piglets’serum CAT and SOD and the content of IL-10and slgA. Reduced the concentration of MDA and IL-1.
(4) Dietary supplemented DMY with a dose of0.005%could improve the daily body weight andfeedstuff conversion rate.
In summary of the above four experiments: the oxidation of intestinal epithelial cell was inducedby early-weaning from the way of XO which made the activity of antioxidational enzymes reduce andthe development of secreted enzymes prohibit. The concentration of IL-1improved. Concentration ofIL-10and sIgA reduced. Growth and recovery factors transcription and synthesis reduced which maycontribute to structure of intestinal villus and crypt impaired and recovery inhibited. Intestinal villusshowed atrophy and crypt became deep in structure. Dietary supplemented DMY to piglet couldprohibit the oxidation made by XO and reduce the impairment of intestinal epithelial cell caused byearly-weaning. Improved the function of recovery. Promoted the proliferation and differentiation ofintestinal epithelial cell and development of intestinal villus and crypt. Improved the growthperformance of piglet.
试验一：选取21±1日龄断奶、平均体重6.25±0.37kg的健康仔猪80头，分成2组（断奶组与吮乳组），每组4重复，每个重复10头仔猪。分别于断奶当天、断奶后第1、3、5、7、10、14天采集样品，每次每组每个重复随机宰杀1头仔猪，取肠组织、刮取肠粘膜、分离肠上皮细胞，取肝脏和血液，测定氧化、损伤、细胞因子和酶等指标。结果如下：
（1）早期断奶第1-3天，仔猪肠上皮细胞XO活性急剧升高、尿酸含量增多，抗氧化酶（CAT、SOD、GSH-Px）活性下降、过氧化产物MDA含量升高，AKP、Na＋-K＋-ATP活性急剧下降，细胞因子IL-1上升、IL-10含量下降，EGF/EGFR的含量及mRNA表达量显著降低，肠上皮细胞发生水泡变性，肠绒毛中的毛细血管有充血和出血现象；第5-7天，上述指标变化达到峰顶或峰谷，而后开始恢复，10-14天恢复到正常水平。表明早期断奶导致肠上皮细胞过氧化、AKP及Na＋-K＋-ATP酶发育受阻、促炎性因子上升、抗炎性因子降低、EGF/EGFR生成减少，在形态结构上表现为肠绒毛萎缩、隐窝增生。
（2）早期断奶第1-3天，仔猪肝脏XO活性升高、尿酸含量增多，抗氧化酶活性下降、过氧化产物MDA含量升高，Na＋-K＋-ATP和GS活性急剧下降；第3-5天，上述指标变化达到峰顶或峰谷，而后开始恢复，7-10天恢复到正常水平。表明早期断奶导致肝脏过氧化、Na＋-K＋-ATP和GS活性下降；肝GS活性下降可能也是影响肠上皮细胞能量代谢、更新和损伤的原因之一。
（3）早期断奶第1-3天，仔猪血清抗氧化酶活性下降、过氧化产物MDA含量升高，IL-1升高、IL-10和slgA含量下降；第3-5天，抗氧化酶活性及MDA含量变化达到峰顶或峰谷，而后开始恢复，7-10天恢复到正常水平；IL-1、IL-10和slgA含量都在第7天达到最高值，且10-14天都维持在较高水平。
（4）仔猪肠上皮细胞、肝和血清中相同指标变化比较，肠上皮细胞指标变化幅度最大，持续时间最长。说明，早期断奶对仔猪肠上皮细胞影响较肝脏和机体更大。2体外HX/XO对肠上皮细胞氧化、损伤及DMY保护作用研究
试验二：常规培养IEC-6细胞系，选取贴壁良好的细胞建立次黄嘌呤/黄嘌呤氧化酶（HX/XO）体外肠上皮细胞的氧化与损伤模型，选择别嘌呤醇作为该系统的抑制剂。细胞分为６个处理组：Ⅰ组为空白对照组，其余五组分别依次加入HX/XO的终浓度为25/25、50/50、100/100、150/150、200/200μmol/L，应激培养24h后，测定细胞内各指标变化情况。得出：50/50μmol/L的HX/XO可显著降低IEC-6细胞CAT和SOD活性，增加过氧化产物MDA含量；显著降低IEC-6细胞AKP及DAO活性和EGF/EGFR含量及mRNA表达量；显著降低IEC-6细胞活力；其各项指标变化模式与早期断奶应激引起的仔猪肠上皮细胞相应指标的变化相一致。添加HX/XO体系中常用的抑制剂别嘌呤醇逆转由HX/XO体系引起的肠细胞损伤，上述结果表明，黄嘌呤氧化酶途径是肠上皮细胞过氧化产生的途径之一，肠上皮细胞的损伤与氧化应激直接相关。
试验三：向肠上皮细胞体外氧化应激模型中加入不同浓度的DMY，观察DMY对体外肠上皮细胞氧化损伤的保护作用。细胞分为6处理组，其中Ⅰ组为空白对照组，Ⅱ组为氧化组（50/50μmol/L），Ⅲ组为抑制剂组（50/50μmol/L+200μmol/L），其余三组为氧化+DMY组，依次分别加入DMY的终浓度为50、75、100μmol/L，检测IEC-6细胞活力及氧化和损伤修复相关指标。结果显示：75-100μmol/L的DMY能增强HX/XO氧化体系中IEC-6细胞抗氧化酶CAT及SOD活性，减少氧化产物MDA含量；提高IEC-6细胞中AKP活性，减少细胞培养液中DAO活性；提高IEC-6细胞EGF/EGFR含量及mRNA表达量，促进IEC-6细胞的增殖活力；添加别嘌呤醇浓度为200μmol/L时，各项指标与空白对照组（不加氧化剂）无差异显著（P>0.05）。
试验二和试验三从正、反两途径证明了HX/XO体系产生的氧化应激导致体外肠上皮细胞损伤。3DMY对早期断奶仔猪肠上皮细胞氧化与损伤修复作用的研究
试验四：选取21±1日龄断奶、平均体重6.33±0.34kg健康仔猪72头，随机分成2个处理组，分别为断奶组和断奶DMY添加组（添加量为500克/吨），每组4个重复，每个重复9头仔猪。分别于断奶当天、断奶后第1、3、7、14天每组每个重复宰杀1头仔猪，取肠组织、分离肠上皮细胞，取肝脏、血液。于第7天、14、28天称量体重、记录饲料消耗量。结果如下：
（1）添加0.05%DMY，第7天可显著降低仔猪肠上皮细胞XO活性、尿酸含量，升高CAT和SOD活性、降低MDA含量，增强AKP和Na＋-K＋-ATP活性，降低细胞因子IL-1含量，增加IL-10含量，增加EGF/EGFR的含量及mRNA表达量，肠绒毛-隐窝结构基本完整。表明添加DMY减轻早期断奶对仔猪肠上皮细胞的损伤，促进肠上皮细胞增殖分化，促进绒毛-隐窝结构的生长发育。
（2）添加0.05%DMY，第7天可显著降低仔猪肝脏XO活性、尿酸含量，升高CAT和SOD活性、降低MDA含量，增强Na＋-K＋-ATP活性，对GS活性影响不显著。
（3）添加0.05%DMY，第7天可显著升高仔猪血清CAT和SOD活性、降低MDA含量，降低IL-1含量、升高IL-10和slgA含量。
（4）添加0.05%DMY，提高断奶仔猪日增重和饲料转化率。
综合上述四个试验结果：早期断奶通过XO途径诱导仔猪肠上皮细胞过氧化，肠上皮细胞抗氧化酶活性降低、分化成熟相关酶发育受阻、IL-1分泌增加、IL-10及sIgA分泌减少，生长修复因子转录合成减少,从而导致肠绒毛—隐窝结构损伤和修复更新受阻，在形态结构上表现为绒毛萎缩，隐窝加深。早期断奶仔猪日粮添加DMY能够显著抑制XO产生的过氧化，减轻早期断奶对仔猪肠上皮细胞的损伤，增强修复功能，促进肠上皮细胞的增殖分化，促进肠绒毛-隐窝结构的发育，提高仔猪的生产性能。
The destination of this study was the age of21days’ piglets. The main line of the study was theintestinal epithelial cellular oxidation-development of enzyme-secret of promotinginflammation/anti-inflammation factors-growth and impairment recovery-stress and regulation ofnutrition. From the four experiments done, we evaluate the impairment and oxidation of piglets’intestinal epithelial cell caused by early-weaning and its regulation of DMY.
1The study of early weaning to piglets’ intestinal epithelial impairment and oxidationExperiment1:Eighty healthy piglets aged21±1days with average body weight6.25±0.37kg wereselected and divided into two groups which were suckling and weaning groups with four replicates ineach group, and ten piglets in each replicate. Samples were collected on the day of1、3、5、7、10、14after weaning. One piglet was scarified in each replicate in each group at once. The intestine usedto isolated intestinal epithelial cell, liver and blood used to inspect the indexes of oxidation,impairment, cellular factor and enzyme were harvested.
(1) The activity of XO rose acutely and the concentration of uric acid rose on the1-3days afterweaning while the activity of antioxidational enzymes (CAT、SOD、GSH-Px) reduced and the contentof MDA rose. The activity of AKP、Na＋-K＋-ATP reduced. The content of cytokine IL-1rose and IL-10reduced. The concentration of EGF/EGFR and transcription of its mRNA reduced significantly.Intestinal epithelial cell happened to baffle denaturation. Capillary vessel in intestinal villus happenedto hyper-tention and blood. On5-7days after weaning, the indexes reached the submit or lowest，thenbecome to recover. On10-14days after weaning, they recovered to normal. It was concluded thatintestinal epithelial cell happened to oxidation and the development of enzymes AKP and Na＋-K＋-ATPwas obstructed causing by the early weaning. The content of promoting inflammation factor rose andthe anti-inflammation factor reduced. The generation of EGF/EGFR reduced. Intestinal villus atrophiedand crypt proliferation in structure.
(2) On1-3days after early-weaning the activity of hepatic XO and the content of uric acid rose,while the activity of antioxidational enzymes reduced. The concentration of the oxidational productMDA rose. The activity of Na＋-K＋-ATP and GS reduced acutely. On3-5days after early-weaning,those indexes rose the submit or lowest, then become to recover which recover to normal on7-10days.It could be referred that liver happened to oxidation, activity of Na＋-K＋-ATP and GS reduced caused bythe early-weaning. That activity of GS reduced may be one of the reasons that influence the energeticmetabolism of intestinal epithelial cell, regeneration and impairment.
(3) On1-3days after early-weaning the activity of antioxidational enzyme in piglets’ serumreduced and the concentration of oxidaional product MDA rose. The concentration of IL-1rose, IL-10and slgA reduced. On the3-5days after weaning, the activity of antioxidational enzyme and thecontent of MDA reached submit or lowest, then become to recover which recover to normal on7-10days. The content of IL-1、IL-10and slgA reached submit on the7d after weaning, and maintained high level on10-14days.
(4) When compared the same indexes in intestinal epithelial cell, liver and serum, we canconcluded that range of intestinal epithelial cellular indexes was the biggest, which lasted the longest.It suggested that impairment caused by early-weaning to intestinal epithelial cell was bigger than thatin liver and mechanism.
2The oxidation and impairment of intestinal epithelial cell caused by HX/XO in vitro andprotection of supplemented DMY.
Experiment2: Normally cultured the IEC-6cell lines. Choose the cell stick the wall and growwell to establish the oxidational model of xanthine and xanthine oxidase (HX/XO) in vitro. Allopurinolwas the inhibitor of this model. The cells were divided into6treatments: group Ⅰwas control. Theother five groups were added into different concentration of HX/XO to their last concentration reached25/25,50/50,100/100,150/150,200/200μmol/L, which was cultured for24h. Then inspected theindexes in cells. Results indicated that the activity of CAT and SOD could be reduced and theconcentration of oxidational product MDA was increased with a concentration of50/50μmol/LHX/XO. The activities of AKP and DAO was reduced significantly also the content of EGF/EGFRand the transcription of mRNA. The cellular activity of IEC-6was reduced significantly. The changes ofother indexes were the same as those of intestinal epithelial cell causing by the stress of early weaning.Added inhibitor:allopurinol to this model could prohibit the impairment of intestinal cell. The resultsadvised that the impairement of intestinal cell is directly related with the oxidational stress.
Experiment3: To observe the protection of DMY to oxidation and impairment of intestinal cell.There were6treatment, which group Ⅰ was control. Ⅱ was oxidational group（50/50μmol/L）, Ⅲwasthe inhibitor group（50/50μmol/L+200μmol/L），the remained groups were oxidation and DMY groupwhich the concentration of DMY was50、75、100μmol/L. Inspect the activity of IEC-6and indexes ofits oxidation and impairment. The results indicated that the activities of cellular antioxidational enzymesCAT and SOD were improved in HX/XO oxidational model with a concentration of75-100μmol/LDMY supplemented. The content of oxidational product MDA reduced and the activity of cellular AKPimproved. The activity of DAO in cellular culturation reduced and the content of EGF/EGFR improvedas well as the mRNA transcription. The activity of cellular proliferation was promoted. Supplementedallopurinol with a dose of200μmol/L, there were no differences on the indexes with the control.
That the system of HX/XO could produce oxidation and stress to cells which caused impairmentto intestinal epithelial cell was proved by the experiment2and3in two different ways
3The study of supplemented DMY to early-weaning piglets on intestinal epithelial cell oxidation,impairment and recovery
Experiment4: Seventy-two healthy piglets aged21±1days with average body weight6.33±0.34kg were selected and divided into two groups which were weaning and DMY supplemented groupswith4replicates in each group, and9piglets in each replicate. Intestinal tissue used to isolatedintestinal epithelial cell, liver and blood were harvested from one piglet in one replicate in a group on0、1、3、7、14days after weaning. Body weight was recorded on the days of7,14and28and the consumption of feedstuff.
(1) Dietary supplemented DMY at a dose of0.05%could reduce the activity of piglets’ intestinalepithelial cellular XO and content of uric acid. Improved the activity of CAT and SOD. Reduced theconcentration of MDA. Improved the activity of AKP and Na＋-K＋-ATP. Reduced the content ofcytokine IL-1. Improved the content of IL-10and EGF/EGFR also their transcription of mRNA.Structure of intestinal villus and crypt were intact overall. It advised that dietary supplemented DMYcould reduce the impairment of early-weaning piglets’ intestinal epithelial cell and promote theproliferation and differentiation of intestinal epithelial cell. Promote growth and development ofintestinal villus and crypt.
(2) Dietary supplemented DMY with a dose of0.005%could reduce the activity of piglets’hepatic XO and the content of uric acid on the7days after weaning. Improved the activity of CATand SOD. Reduced the concentration of MDA, Improved the activity of Na＋-K＋-ATP while there wereno significant influences on activity of GS.
(3) Dietary supplemented DMY with a dose of0.005%could improve the activity of piglets’serum CAT and SOD and the content of IL-10and slgA. Reduced the concentration of MDA and IL-1.
(4) Dietary supplemented DMY with a dose of0.005%could improve the daily body weight andfeedstuff conversion rate.
In summary of the above four experiments: the oxidation of intestinal epithelial cell was inducedby early-weaning from the way of XO which made the activity of antioxidational enzymes reduce andthe development of secreted enzymes prohibit. The concentration of IL-1improved. Concentration ofIL-10and sIgA reduced. Growth and recovery factors transcription and synthesis reduced which maycontribute to structure of intestinal villus and crypt impaired and recovery inhibited. Intestinal villusshowed atrophy and crypt became deep in structure. Dietary supplemented DMY to piglet couldprohibit the oxidation made by XO and reduce the impairment of intestinal epithelial cell caused byearly-weaning. Improved the function of recovery. Promoted the proliferation and differentiation ofintestinal epithelial cell and development of intestinal villus and crypt. Improved the growthperformance of piglet.
引文
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