【作者】 王欢；

【导师】 杨淑华； 张淑枝；

【作者基本信息】 沈阳农业大学 ， 兽医硕士（专业学位）， 2020， 硕士

【摘要】 赭曲霉毒素A(Ochratoxin A,OTA)普遍存在于动物赖以生存的环境中,它之所以能够对养殖业造成巨大的经济损失,是因为有大量的动物饲料和食品被OTA污染。当OTA进入动物体内时,首先它会直接接触肠道,其对动物肠道屏障造成严重危害并影响动物机体健康。虾青素(Astaxanthin,AST)是类胡萝卜素合成的高级产物,是强的抗氧化剂,能够显著提高机体免疫功能。AST的抗氧化性有助于维持肠屏障的完整性,对机体免受真菌毒素的侵害具有很强的保护作用,服用AST可以维持肠道的正常功能。但是AST是否能够减轻OTA所致小鼠空肠毒性的作用尚不清楚。为了研究AST对OTA诱导的小鼠空肠屏障功能障碍的影响作用,我们将80只C57小鼠随机平均分为对照组(不含OTA)、OTA组(5 mg/kg body weight)、AST组(100 mg/kg body weight)和OTA+AST组(5 mg/kg body weight+100 mg/kg body weight)。试验周期为27 d(7 d给药,2 d自由采食)。通过检测小鼠血清生化指标(二胺氧化酶(DAO)、D-乳酸(D-lactate)、脂肪酸结合蛋白(IFABP))可以看出:与对照组相比OTA组中的血清生化指标含量极显著升高(P<0.01);与OTA组相比,OTA+AST组中的血清生化指标含量极显著下降(P<0.01)。接着通过观察组织病理切片和透射电镜发现:与对照组相比,OTA组中的小鼠空肠损伤严重;与OTA组相比,OTA+AST组中的小鼠空肠损伤远小于OTA组中小鼠的空肠损伤。我们又通过炎症指标(肿瘤坏死因子-α(TNF-α)、干扰素-γ(INF-γ)、白细胞介素-10(IL-10))和抗感染免疫的抗体:分泌性免疫球蛋白A(SIg A)发现:与对照组相比,OTA组中TNF-α和INF-γ的含量极显著高于对照组(P<0.01),IL-10和SIg A的含量极显著低于对照组(P<0.01);与OTA组相比,OTA+AST组中TNF-α和INF-γ的含量极显著低于OTA组(P<0.01),IL-10和SIg A的含量极显著高于OTA组(P<0.01)。最后,我们通过免疫组化(Claudin-1,Occludin,ZO-1)以及Western-blot试验验证各组空肠中各个蛋白的表达情况发现:OTA组与其他几组的紧密连接蛋白的定位并没有显著变化,但是各组的紧密连接蛋白的表达量发生了变化。与对照组相比,OTA组的Claudin-1和Occludin的表达量极显著减少(P<0.01),ZO-1的表达量显著减少(P<0.05);与OTA组相比,Claudin-1的表达量极显著增多(P<0.01),Occludin和ZO-1的表达量显著增多(P<0.05)。通过以上结果表明,AST对OTA诱导的小鼠空肠的损伤具有缓解作用,并对空肠屏障功能有保护作用。更多还原

【Abstract】 OTA is ubiquitous in the environment where animals depend.The reason why it can cause huge economic losses to the breeding industry is because a large amount of animal feed and food are contaminated by OTA.When OTA enters the animal’s body,it will directly contact the intestinal tract,which will seriously damage the animal’s intestinal barrier and affect the animal’s health.AST is an advanced product synthesized from carotenoids.It is a strong antioxidant and can significantly improve the body’s immunological function.The antioxidant properties of AST contribute to maintain the integrity of the intestinal barrier and protect the body against mycotoxins.Therefore,taking AST can maintain the normal function of the intestine.However,whether AST can alleviate the jejunal toxicity of mice caused by OTA is unclear.To investigate the effect of AST on OTA-induced jejunal barrier dysfunction in mice.we randomly divided 80 C57 mice into control group(without OTA),OTA group(5 mg/kg body weight),AST group(100 mg/kg body weight)and OTA+AST group(5 mg/kg body weight +100 mg/kg body weight).The test period is 27 days(7 days of administration,2 days of free feeding).By testing mouse serum biochemical indicators(DAO,D-lactate,IFABP),it can be seen that the serum biochemical indicators in the OTA group are significantly higher than those in the control group Increased(P<0.01);compared with the OTA group,the serum biochemical indicators in the OTA+AST group decreased significantly(P<0.01).Then through observation of histopathological sections and transmission electron microscopy,it was found that compared with the control group,the jejunum of the mice in the OTA group suffered severe damage;the jejunum injury of the mice in the OTA+AST group was much less than that in the OTA group.We also detected the inflammation indicators(TNF-α,INF-γ,IL-10)and antibodies against infectious immunity(SIg A)and found that the content of TNF-α and INF-γ in the OTA group was much higher than that of the control group(P<0.01),and the content of IL-10 and SIg A was much lower than the control group(P< 0.01);the content of TNF-α and INF-γ in the OTA+AST group was much lower than the OTA group(P<0.01),and the content of IL-10 and SIg A was much higher than the OTA group(P<0.01)).Finally,we used immunohistochemistry(Claudin-1,Occludin,ZO-1)and Western-blot test to verify the expression of each protein in the jejunum of each group and found that the localization of tight junction proteins in the OTA group and other groups did not significantly changes,but the expression of tight junction proteins in each group has changed.Compared with the control group,the expression of Claudin-1 and Occludin in the OTA group was significantly reduced(P<0.01),and the expression of ZO-1 was reduced(P<0.05);compared with the OTA group,the expression of Claudin-1 significantly increased(P<0.01),the expression of Occludin and ZO-1 increased(P<0.05).The above results show that AST has a relieving effect on OTA-induced damage to the mouse jejunum and has a protective effect on the jejunum functional barrier.更多还原