猪β-防御素-2在黄曲霉毒素B1致仔猪空肠上皮细胞炎症反应中的作用  

Role of porcineβ-defensin-2 in inflammatory response of IPEC-J2 cells induced by aflatoxin B1

作　　者：李庆豪 张曼 孙娟 彭雅婷 刘勇[1] 金鑫 李奎[1] LI Qinghao;ZHANG Man;SUN Juan;PENG Yating;LIU Yong;JIN Xin;LI Kui(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;The Third Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]河南牧业经济学院动物医药学院,河南郑州450046 [3]郑州大学第三附属医院,河南郑州450052

出　　处：《中国兽医学报》2022年第3期558-565,共8页Chinese Journal of Veterinary Science

基　　金：河南省自然科学青年基金资助项目(212300410156);河南省博士后科研启动资助项目(202003063);河南农业大学博士科研启动项目(30602102)。

摘　　要：以仔猪空肠上皮细胞(IPEC-J2)为模型,探究猪β-防御素-2(porcineβ-defensin 2,pBD-2)在黄曲霉毒素B1(aflatoxin B1,AFB1)致细胞炎症反应中的作用。使用CCK-8法确认不同质量浓度(20,40,60,80,100 mg/L)AFB1及不同质量浓度(2,4,6,8,10 mg/L)pBD-2的细胞毒性,筛选两者的可用质量浓度,再通过qPCR确认不同质量浓度(10,20,30,40,50 mg/L)的AFB1对IPEC-J2细胞TNF-αmRNA及IL-1βmRNA表达的影响,建立AFB1致细胞损伤模型。然后分别以2,4,6,8 mg/L pBD-2处理细胞损伤模型作为试验组,同时分别以单独使用AFB1和pBD-2处理的细胞作为阳性对照组和阴性对照组,未经任何处理的细胞作为空白对照组,所有细胞处理均进行12 h。处理结束后提取所有组细胞总RNA和总蛋白,使用qPCR及Western blot检测TNF-α及IL-1β的表达变化。细胞存活率检测结果显示,与空白对照组相比,60 mg/L及更高剂量AFB1组细胞存活率显著降低(P<0.05);pBD-2在10 mg/L出现细胞毒性,细胞存活率显著低于空白对照组(P<0.05)。细胞损伤模型建立结果显示,与空白对照组相比,AFB1在30 mg/L时显著上调了TNF-αmRNA和IL-1βmRNA的表达(P<0.05)。使用pBD-2处理细胞损伤模型,qPCR结果显示,4,6 mg/LpBD-2可显著下调AFB1所致TNF-αmRNA及IL-1βmRNA的过量表达至空白对照组水平(P<0.05);Western blot结果显示,AFB1显著上调TNF-α及IL-1β的表达后(P<0.05),8 mg/L pBD-2可下调TNF-α的表达至显著低于空白对照组水平(P<0.05),6,8 mg/L pBD-2可显著下调IL-1β的表达至空白对照组水平(P<0.05)。结果表明,pBD-2可通过调控细胞炎性因子的表达来缓解AFB1对小肠上皮细胞造成的炎症反应。This experiment aims to explore the role of porcineβ-defensin-2(pBD-2)in inflammatory response induced by aflatoxin B1(AFB1)using IPEC-J2 cell as a model.The cytotoxicity of different concentrations(20,40,60,80,100 mg/L)of AFB1 and different concentrations(2,4,6,8,10 mg/L)of pBD-2 was confirmed by CCK-8 assay to screen the available dosage of both.Then the impact of different concentrations(10,20,30,40,50 mg/L)of AFB1 on TNF-αmRNA and IL-1βmRNA expression in IPEC-J2 cells were confirmed by qPCR to ensure the establishment of IPEC-J2 cells injury model induced by AFB1.The cell injury model was treated with different concentrations(2,4,6,8 mg/L)of pBD-2 as experimental groups,respectively.At the same time,the positive group treated with AFB1 and the negative group treated with pBD-2 only were set up,the cells without AFB1 and pBD-2 was the control group,all the treatment was carried out for 12 h.After treatment,the total RNA and total proteins of all groups were extracted to detect the changes of TNF-αand IL-1βby qPCR and Western blot.The results showed that the viability of cells treated with 60 mg/L and higher doses of AFB1 was significantly lower than the control group(P<0.05);the pBD-2 showed cytotoxicity at 10 mg/L,it caused the cell viability significantly lower than that of the control group(P<0.05).The results of qPCR and Western blot showed that30 mg/L AFB1 significantly up-regulated the expression of TNF-αmRNA and IL-1βmRNA(P<0.05).Using pBD-2 to treat the cell injury model,qPCR results showed that 4,6 mg/L pBD-2 could down-regulate the overexpression of TNF-αmRNA and IL-1βmRNA irritated by AFB1 to the same level of control group(P<0.05);after the expression of TNF-αand IL-1βwere significantly up-regulated by AFB1(P<0.05),Western blot results showed that 8 mg/L pBD-2 could downregulate the expression of TNF-αand made it significantly lower than that of control group(P<0.05),and 6,8 mg/L pBD-2 could down-regulate the expression of IL-1βto the same level of control group(P<0.05).Collectively,th

关 键 词：猪β-防御素-2 黄曲霉毒素B1 IPEC-J2细胞 炎症反应 

分 类 号：S859.8[农业科学—临床兽医学]