猪肺炎支原体感染猪肺泡巨噬细胞诱导氧化应激反应机制的初步研究  

A preliminary study on the mechanism of oxidative stress induced by porcine alveolar macrophages infected with Mycoplasma hyopneumoniae

作　　者：周颖 陈忠伟 段群棚 程珂 林昌华[2,4] 许艺兰 张胜斌 覃国喜 许心婷 杨讯业 秦毅斌 赵硕 全琛宇[1] 陈婷婷 徐悦 何颖 卢冰霞 ZHOU Ying;CHEN Zhongwei;DUAN Qunpeng;CHENG Ke;LIN Changhua;XU Yilan;ZHANG Shengbin;QIN Guoxi;XU Xinting;YANG Xunye;QIN Yibin;ZHAO Shuo;QUAN Chenyu;CHEN Tingting;XU Yue;HE Ying;LU Bingxia(Guangxi Veterinary Research Institute/Guangxi Zhuang Autonomous Region key Laboratory of Veterinary Biotechnology/Ministry of Agriculture and Rural Affairs Key Laboratory of China(Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control,Nanning 530001,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Jinguang Co.,Ltd.,of Guangxi Nongken Yongxin Animal Husbandry Group,Nanning 530042,China;Xijiang Co.,Ltd.,of Guangxi Nongken Yongxin Animal Husbandry Group,Guigang 537100,China)

机构地区：[1]广西壮族自治区兽医研究所/广西兽医生物技术重点实验室/农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,南宁530001 [2]广西大学动物科学技术学院,南宁530004 [3]广西农垦永新畜牧集团金光有限公司,南宁530042 [4]广西农垦永新畜牧集团西江有限公司,广西贵港537100

出　　处：《黑龙江畜牧兽医》2025年第4期1-7,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：广西现代农业产业技术体系生猪产业创新团队项目(nycytxgxcxtd-2023-15-02);南宁市科学研究与技术开发计划项目(20232039);广西基本科研业务费专项(桂科专项23-4);广西畜牧产业科技先锋队项目(桂农科盟202409-01-3);广西农业科技自筹经费项目(Z2023033,Z202222)。

摘　　要：为了明确猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)体外感染猪肺泡巨噬细胞是否会诱导细胞发生氧化应激反应,并初步揭示相关机制,试验分别用5×10^(6),1×10^(7),2×10^(7),5×10^(7)CCU/mL Mhp感染猪肺泡巨噬细胞,以未感染Mhp的细胞为对照,并分别于第6,12,18,24,36小时收集细胞,检测氧化应激相关指标[活性氧(reactive oxygen species,ROS)水平、一氧化氮(nitric oxide,NO)浓度、丙二醛(malondialdehyde,MDA)含量、还原型一氧化氮合成酶(induciblenitric oxide synthase,iNOS)和总超氧化物歧化酶(total superoxide dismutase,T-SOD)活性];根据氧化应激相关指标的测定结果,选择氧化应激水平最明显的感染浓度和时间点,采用荧光定量PCR方法检测氧化应激通路相关因子{核因子E2相关因子2(nuclear factor erythroid-2-related factor2,Nrf2)、Kelch样环氧氯丙烷相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)、磷酸酰胺腺嘌呤二核苷酸醌氧化还原酶-1[NAD(P)H:quinoneoxidoreductase-1,NQO1]、血红素加氧酶-1(heme oxygenase-1,HO-1)}的mRNA表达水平,并采用Western-blot方法检测上述因子的蛋白表达水平。结果表明:与正常细胞比较,4个浓度的Mhp感染细胞中ROS水平在第6,12,18,24,36小时均显著升高(P<0.05);5×10^(6)CCU/mL Mhp感染细胞的NO浓度仅在第24小时显著升高(P<0.05),1×10^(7)CCU/mL Mhp感染细胞的NO浓度在第6,12,18小时显著升高(P<0.05),2×10^(7)CCU/mL和5×10^(7)CCU/mL Mhp感染细胞的NO浓度在第6,12,18,24小时显著升高(P<0.05);5×10~(6)CCU/mL Mhp感染细胞的MDA含量仅在第36小时显著升高(P<0.05),1×10^(7)CCU/mL Mhp感染细胞的MDA含量在第24,36小时显著升高(P<0.05),2×10^(7)CCU/mL Mhp感染细胞的MDA含量在第6,12,18,36小时显著升高(P<0.05),5×10^(7)CCU/mL Mhp感染细胞的MDA含量在第6,18,24小时显著升高(P<0.05);5×10^(6)CCU/mL Mhp感染细胞的iNOS活性在第6,12,24,36小时显著升高(P<0.05),1×10^(7)CCU/mL Mhp感染细胞iNOS活性在第6,12,18In order to determine whether in vitro infection of 3D4/21 cells with Mycoplasma hyopneumoniae(Mhp)could induce oxidative stress in cells,and preliminarily reveal the relevant mechanism,in the test,porcine alveolar macrophages were infected with 5×10^(6),1×10^(7),2×10^(7)and 5×10^(7)CCU/mL of Mhp,respectively,and the cells not infected with Mhp were used as the control.Cells were collected at 6,12,18,24 and 36 h,respectively.Oxidative stress-related indexes(reactive oxygen species[ROS]levels,nitric oxide[NO]concentration,malondialdehyde[MDA],reduced nitric oxide synthase[iNOS]and total superoxide dismutase[T-SOD]activity)were detected.After determining the optimal infection conditions,the mRNA expression levels of oxidative stress-related factors(nuclear factor erythroid-2-related factor2[Nrf2],Kelch-like ECH-associated protein 1[Keap1],NAD(P)H:quinoneoxidoreductase-1[NQO1],heme oxygenase-1[HO-1])were detected by fluorescence quantitative PCR.Western-blot was used to detect the protein expression levels of the above factors.The results showed that compared with normal cells,the ROS levels of Mhp infected cells at 4 concentrations were significantly increased at 6,12,18,24 and 36 h(P<0.05).The NO concentration of Mhp-infected cells at 5×10^(6)CCU/mL was significantly increased only at 24 h(P<0.05);Mhp-infected cells with 1×10^(7)CCU/mL were significantly increased at 6,12 and 18 h(P<0.05);the NO concentrations of 2×10^(7)CCU/mL and 5×10^(7)CCU/mL Mhp infected cells were significantly increased at 6,12,18 and 24 h(P<0.05).The content of MDA in 5×10^(6)CCU/mL Mhp infected cells increased significantly at 36 h(P<0.05);the MDA content of 1×10^(7)CCU/mL Mhp infected cells was significantly increased at 24 and 36 h(P<0.05);the MDA contents of 2×10^(7)CCU/mL Mhp infected cells were significantly increased at 6,12,18 and 36 h(P<0.05);the MDA contents of 5×10^(7)CCU/mL Mhp infected cells were significantly increased at 6,18 and 24 h(P<0.05).The activity of iNOS of 5×10^(6)CCU/mL Mhp was significantly increased

关 键 词：猪肺炎支原体 氧化应激 核因子E2相关因子2 活性氧 Kelch样环氧氯丙烷相关蛋白1 血红素加氧酶-1 

分 类 号：S858.28[农业科学—临床兽医学]