黄连解毒散对热应激条件下巴马香猪PBMC TLRs mRNA转录及其介导的炎症因子表达的影响  

Effects of Coptis detoxification powder on transcription of PBMC TLRs mRNA and its mediated expression of inflammatory factors in Bama pigs under heat stress

作　　者：董林[1,2,3] 王艳萍 徐倩倩[1] 刘吉山[1,2] 王金良[1,2] 王化磊 沈志强[1,2] DONG Lin;WANG Yanping;XU Qianqian;LIU Jishan;WANG Jinliang;WANG Hualei;SHEN Zhiqiang(Binzhou Animal Science and Veterinary Medicine Institute,Binzhou,Shandong 256600,China;Shandong Lyudu Bio-Sciences&Technology Co.Ltd.,Binzhou,Shandong 256600,China;College of Animal Science and Veterinary Science,Jilin University,Changchun 130062,China)

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600 [3]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2024年第8期1813-1820,共8页Chinese Journal of Veterinary Science

基　　金：山东省自然科学基金资助项目(ZR2023MC212)。

摘　　要：为探索黄连解毒散对热应激导致的动物机体免疫抑制和炎症反应的影响。以巴马香猪为实验动物,建立热应激动物模型,研究黄连解毒散体内条件下对热应激导致的PBMC中TLRs mRNA转录激活及其介导的炎症因子表达的影响。采用人工气候温室建立热应激动物模型,制备中药“黄连解毒散”,通过预防给药和治疗给药,研究其对热应激及其炎症的抑制效应。利用细胞分离培养和间接免疫等技术,实时荧光定量PCR测定HSP70、TLRs和IL-17 mRNA转录变化,ELISA定量测定血清中IL-2、IL-6、TNF-α含量动态变化,Western blot与间接免疫荧光(IFA)测定NF-κβ炎症因子通路中p65磷酸化蛋白核易位与蛋白表达,分析介导热应激条件下巴马香猪PBMC TLRs mRNA转录及其介导的炎症因子表达与效应功能,研究中药“黄连解毒散”抗热应激效应及其作用通路。结果显示,成功建立了热应激巴马香猪实验动物模型,热应激显著上调了HSP70、TLR2、TLR4和炎症因子IL-17 mRNA转录水平,导致血清中炎症因子IL-2、IL-6、TNF-α的表达显著性增强,显著促进NF-κβ通路中p65核内磷酸化水平和胞质蛋白表达。中药“黄连解毒散”可显著降低热应激条件对HSP70和TLRs mRNA转录水平的上调效应,从而抑制效应炎症因子IL-17mRNA转录和血清中IL-2、IL-6和TNF-α的表达,显著性拮抗热应激导致的NF-κβ转录活性,显示良好的抗热应激效应。结果表明,研究了中兽药方剂“黄连解毒散”抗炎效应和其作用通路及其效应因子,分析了其抑制TLRs mRNA相关联的通路调控相关因子的表达与分泌水平,对其对临床上抗热应激、抗炎的功能效应和作用机理进行初步探索,为该方剂的推广应用和相关机理研究奠定了数据基础。In order to explore the effects of Coptis detoxification powder on immune suppression and inflammatory response caused by heat stress in animals,Bama pigs were used as experimental animals to establish an animal model of heat stress,and the effects of Coptis detoxification powder on transcriptional activation of TLRs mRNA in PBMC induced by heat stress and its mediated expression of inflammatory factors were studied in vivo.The animal model of heat stress was established by artificial climate greenhouse,Chinese medicine“Coptis detoxification powder”was prepared,and its inhibitory effect on heat stress and inflammation was studied by preventive and therapeutic administration.Real-time fluorescence quantitative PCR was used to determine the transcriptional changes of HSP70,TLRs and IL-17mRNA,and ELISA was used to determine the dynamic changes of serum IL-2,IL-6and TNF-α.Western blot and indirect immunofluorescence(IFA)were used to determine the nuclear translocation and protein expression of p65phosphorylated protein in NF-κβinflammatory factor pathway.The transcription of PBMC TLRs mRNA and its mediated inflammatory factor expression and function in Bama pigs under heat stress were systematically analyzed.The results showed that the experimental animal model of Bama pigs under heat stress was successfully established,and heat stress significantly up-regulated the mRNA transcription levels of HSP70,TLR2,TLR4and inflammatory factor IL-17,resulting in significantly enhanced expressions of serum inflammatory factors IL-2,IL-6and TNF-α.The NF-κβpathway significantly promoted the level of p65nuclear phosphorylation and the expression of cytoplasmic protein.The Chinese medicine“Coptis detoxification powder”could significantly reduce the upregulation effect of heat stress conditions on the transcription levels of HSP70and TLRs mRNA,thus inhibiting the mRNA transcription of inflammatory factor IL-17and the expression of IL-2,IL-6and TNF-αin serum,and significantly antagonizing the transcriptional activity of

关 键 词：黄连解毒散 热应激 TLRS 炎症因子 巴马香猪 

分 类 号：S852.2[农业科学—基础兽医学]