敌草快诱导肉鸡氧化损伤模型的建立及其分子机制研究

Establishment and Molecular Mechanism Study of Diquat Induced Oxidative Damage Model in Broilers Chickens

旨在建立敌草快诱导的肉鸡氧化损伤模型,并初步揭示其分子机制。将160只1日龄肉鸡随机分为4组,对照组(C)、低剂量组(T1)、中剂量组(T2)和高剂量组(T3)组,每组4个重复,每个重复10只。在第16日龄(第一阶段)时,每个重复抽取5只鸡,第37日龄(第二阶段)时抽取剩余5只鸡,称取肉鸡体重,计算每组平均体重,腹腔注射敌草快。T1、T2和T3组分别腹腔注射5、10、20 mg·kg^(-1)的敌草快,对照组注射相应剂量的生理盐水。两次注射后的当天和第5天,每个重复抽取2只,采血并取样。结果表明:两次注射敌草快后,与对照组相比,1)试验组肉鸡精神沉郁、饮食减少,T3组注射后6 h有2只鸡出现死亡,注射后72 h和96 h试验组肉鸡体重均极显著降低(P<0.01);2)T2和T3组血清中二胺氧化酶(DAO)、谷丙转氨酶(ALT)和谷草转氨酶(AST)活性均极显著(P<0.01)升高;3)T2和T3组血清中谷胱甘肽过氧化物酶(GSH-Px)活性均极显著降低(P<0.01),丙二醛(MDA)水平均极显著升高(P<0.01),第二阶段T3组血清中超氧化物歧化酶(SOD)活性显著降低(P<0.05);4)试验组各组血清中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量均极显著升高(P<0.01);5)试验组出现炎性细胞浸润,部分肝细胞空泡样变,细胞质疏松明显,且各肠段绒毛脱落较为严重;组织学评分显示,与对照组相比,T2组与T3组肝脏和小肠评分均极显著升高(P<0.01),除第二阶段十二指肠评分外,T1组与对照组相比无显著差异;6)各试验组肉鸡肝脏和空肠中ZO-1和OCLDN mRNA表达量均极显著降低(P<0.01);7)肝脏中T2组HO-1 mRNA表达量显著低于T1组(P<0.05);空肠中T2组和T3组Nrf2和HO-1 mRNA表达量较T1组均极显著降低(P<0.01)。综上所述,本研究成功建立敌草快诱导的肉鸡氧化损伤模型,且以10 mg·kg^(-1)的注射剂量为建立氧化损伤模型最为适宜,同时发现敌草快可能通过Nrf2/HO-1信号通路来诱导炎症相关因子生成且抑制抗氧化酶的表达,引起肉鸡肠道屏障损伤,从而引起肉鸡炎症反应。

The aim of this study was to establish a model of oxidative damage induced by diquat in broilers and to preliminarily reveal its molecular mechanism.A total of 1601-day-old broilers were randomly divided into four groups:control group(C),low-dose group(T1),medium-dose group(T2)and high-dose group(T3),4 replicates per group,10 replicates each.Five chickens were sampled in each replicate at the 16 th(the first stage),and the remaining five chickens were sampled at 37 th(the second stage)day of age,broilers were weighed,the average body weight of each group was calculated,and diquat was injected intraperitoneally.The T1,T2 and T3 groups were intraperitoneally injected with 5,10,and 20 mg·kg^(-1)of diquat,respectively,and the control group was injected with the corresponding dose of normal saline.The day and the fifth day after the two injections,two broilers were randomly taken from each replicate and blood collection and sampling were conducted.The results showed that after two injections of diquat,compared with the control group,1)The broilers in the experimental groups were depressed and drank less water and eaten less,2 broilers in the T3 group died at 6 h after each injection,and the body weight of broilers in the experimental groups were extremely significant reduced at 72 h and 96 h after each injection(P<0.01);2)The serum activity of diamine oxidase(DAO),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the T2 and T3 groups were extremely significant increased(P<0.01);3)The serum activity of glutathione peroxidase(GSH-Px)in the T2 and T3 groups were extremely significant decreased(P<0.01),and the serum levels of malondialdehyde(MDA)were extremely significant increased(P<0.01).The serum activity of superoxide dismutase(SOD)in the T3 group after the second infection was significantly decreased(P<0.05);4)The serum contents of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in each experimental group were extremely significant increased(P<0.01);5)In the experimental groups,inflammatory cell infiltration was present,some hepatocytes were vacuolated,cytoplasmic was obvious laxity,and the villi loss of each intestinal segment was serious;Histological scores showed that the liver and small intestine scores in both the T2 and T3 groups were extremely significant higher than control group(P<0.01),and except the histological score of duodenum at the second stage,there was no significant difference between the T1 group and the control group;6)The mRNA expressions of ZO-1 and OCLDN in the liver and jejunum of broilers in each experimental group were extremely significant decreased(P<0.01);7)The mRNA expression of HO-1 in the liver of T2 group was significantly lower than T1 group(P<0.05);The mRNA expressions of Nrf2 and HO-1 in jejunum of T2 and T3 groups were extremely significant lower than T1 group(P<0.01).In summary,this study successfully established a model of oxidative damage induced by diquat in broilers,and the injection dose of 10 mg·kg^(-1)was the most appropriate to establish the oxidative damage model,and it was found that diquat may induce the production of inflammation-related factors and inhibit the expression of antioxidant enzymes through the Nrf2/HO-1 signaling pathway,causing intestinal barrier damage in broilers,thereby causing inflammatory response in broilers.