pBD-2对热应激致小鼠小肠形态结构损伤及紧密连接蛋白表达的修复作用

Repairing effects of pBD-2 on heat stress-induced morphological and structural damage and tight junction protein expression in mouse small intestine

为了研究猪β-防御素-2(porcinβ-defensin2,pBD-2)对热应激致小鼠小肠形态结构损伤及紧密连接蛋白表达的修复作用,试验将36只1月龄昆明小鼠随机分为空白对照组、热应激组和热应激+pBD-2组,每组12只。空白对照组和热应激组每天每只小鼠按体重10μL/g以灌胃的方式给予生理盐水,热应激+pBD-2组每天每只小鼠按体重0.01μg/g以灌胃的方式给予pBD-2。灌胃后将热应激组和热应激+pBD-2组放入40℃温箱热应激处理1 h,取出后与空白对照组一起放置于室温下进行饲养。按照相同的方式共处理28 d,并于第28天处理后断颈处死。每天灌胃前观察各组的行为,记录采食量并称重,试验结束后计算各组平均日采食量和平均日增重,并对小鼠进行剖检,分别取十二指肠、空肠、回肠组织进行石蜡切片制作和H.E.染色,比较各组肠绒毛高度与隐窝深度比值和单位面积内杯状细胞数目,并采用免疫组织化学技术对紧密连接蛋白ZO-1、Claudin-3和Occludin进行检测与相对表达量分析。结果表明:试验期间,空白对照组无异样行为;热应激组探索行为增加,焦虑好动;热应激+pBD-2组比热应激组安静。热应激组平均日采食量显著高于热应激+pBD-2组和空白对照组(P<0.05),而热应激+pBD-2组和空白对照组间差异不显著(P>0.05)。空白对照组和热应激+pBD-2组平均日增重显著高于热应激组(P<0.05),空白对照组和热应激+pBD-2组平均日增重差异不显著(P>0.05)。空白对照组肠道健康、无红肿,肠道内容物适中;热应激组肠道表面充血、红肿,肠道内容物较多;热应激+pBD-2组肠道无红肿现象,肠道内容物较少。热应激组十二指肠、空肠、回肠绒毛高度与隐窝深度比值均显著低于空白对照组和热应激+pBD-2组(P<0.05),空白对照组和热应激+pBD-2组间差异不显著(P>0.05)。热应激+pBD-2组十二指肠和空肠单位面积内杯状细胞数量显著高于空白对照组和热应激组(P<0.05),空白对照组显著高于热应激组(P<0.05);热应激+pBD-2组回肠单位面积内杯状细胞数量显著高于热应激组(P<0.05)且与空白对照组间差异不显著(P>0.05),热应激组与空白对照组间差异不显著(P>0.05)。空白对照组和热应激+pBD-2组十二指肠、空肠和回肠中ZO-1蛋白的相对表达量显著高于热应激组(P<0.05),空白对照组和热应激+pBD-2组间差异不显著(P>0.05)。空白对照组十二指肠和空肠中Claudin-3蛋白的相对表达量显著高于热应激组和热应激+pBD-2组(P<0.05),热应激+pBD-2组显著高于热应激组(P<0.05);各组回肠中Claudin-3蛋白的相对表达量差异不显著(P>0.05)。空白对照组十二指肠中Occludin蛋白的相对表达量显著高于热应激组,但热应激+pBD-2组与空白对照组、热应激组均差异不显著(P>0.05);空白对照组和热应激+pBD-2组空肠和回肠中Occludin蛋白的相对表达量显著高于热应激组(P<0.05),但空白对照组和热应激+pBD-2组间差异不显著(P>0.05)。说明热应激处理会影响小鼠的消化吸收功能和小肠形态结构,降低小鼠小肠中紧密连接蛋白的表达量,而pBD-2对热应激造成的上述影响具有较好的修复作用。

In order to investigate the repairing effects of porcinβ-defensin-2(pBD-2)on heat stress-induced morphological and structural damage and expression of tight junction proteins in mouse small intestine,in the experiment,thirty-six 1-month-old Kunming mice were randomly divided into a blank control group,a heat stress group,and a heat stress+pBD-2 group;with 12 mice in each group.The blank control group and heat stress group were given saline by gavage at 10μL/g of body weight per mouse per day;the heat stress+pBD-2 group was given pBD-2 by gavage at 0.01μg/g of body weight per mouse per day.After gavage,the heat stress group and the heat stress+pBD-2 group were placed in a 40℃incubator for 1 h.After removal,they were kept at room temperature together with the blank control group.The groups were treated in the same way for a total of 28 d,and were killed by breaking their necks on the 28th day of treatment.The behavior of each group was observed before gavage every day,and the feed intake was recorded and weighed;at the end of the experiment,the average daily feed intake and average daily weight gain of each group were calculated;and the abdominal cavity of the mice was dissected.Duodenum,jejunum and ileum tissues were taken for paraffin section making and H.E.staining respectively,to compare the ratio of height of intestinal villi to depth of crypts and the number of goblet cells per unit area in each group;and the tight junction proteins,ZO-1,Claudin-3 and Occludin,were detected by immunohistochemistry and analyzed for their relative expression.The results showed that during the trial,the blank control group had no unusual behavior;the heat stress group had increased exploratory behavior and was anxious and active;and the heat stress+pBD-2 group was quieter than the heat stress group.The average daily feed intake was significantly higher in the heat stress group than in the heat stress+pBD-2 group and the blank control group(P<0.05),while the difference between the heat stress+pBD-2 group and the blank control group was not significant(P>0.05).The average daily weight gain of the blank control group and the heat stress+pBD-2 group were significantly higher than that of the heat stress group(P<0.05);the average daily weight gain of the black control group and the heat stress+pBD-2 group had no significant differences(P>0.05).In the blank control group,the intestines were healthy,without redness and swelling,with moderate intestinal contents;in the heat stress group,the intestinal surface was congested with dark red color,redness and swelling,with more intestinal contents;and in the heat stress+pBD-2 group,the intestines were without redness and swelling,with less intestinal contents.The ratio of villus height to crypt depth in the duodenum,jejunum,and ileum was significantly lower in the heat stress group than in the blank control group and the heat stress+pBD-2 group(P<0.05);the difference between the blank control group and the heat stress+pBD-2 group was not significant(P>0.05).The number of goblet cells per unit area of duodenum and jejunum in heat stress+pBD-2 group was significantly higher than that in blank control group and heat stress group(P<0.05),and blank control group was significantly higher than that in heat stress group(P<0.05).The number of goblet cells per unit area of ileum in heat stress+pBD-2 group was significantly higher than that in heat stress group(P<0.05),and there was no significant differences between heat stress group and blank control group(P>0.05),and no significant differences between heat stress group and blank control group(P>0.05).The relative expression of ZO-1 protein in the duodenum,jejunum and ileum was significantly higher in the blank control group and the heat stress+pBD-2 group than in the heat stress group(P<0.05);the difference between the blank control group and the heat stress+pBD-2 group was not significant(P>0.05).The relative expression of Claudin-3 protein in the duodenum and jejunum was significantly higher in the blank control group than in the heat stress group and the heat stress+pBD-2 group(P<0.05);it was significantly higher in the heat stress+pBD-2 group than in the heat stress group(P<0.05);the relative expression of Claudin-3 protein in the ileum did not differ significantly among the groups(P>0.05).The relative expression of Occludin protein in the duodenum was significantly higher in the blank control group than in the heat stress group;however,the difference between the heat stress+pBD-2 group and both the blank control and heat stress groups was not significant(P>0.05).The relative expression of Occludin protein in the jejunum and ileum was significantly higher in the blank control group and the heat stress+pBD-2 group than in the heat stress group(P<0.05);however,the difference between the blank control group and the heat stress+pBD-2 group was not significant(P>0.05).The results suggested that heat stress affected the digestion and absorption function,the morphology and structure of the small intestine,and decreased the expression of tight junction proteins in the small intestine of mice,and that pBD-2 had a better repairing effect on the above effects caused by heat stress.