【摘要】 肌动蛋白相关蛋白2/3复合体亚基3(actin-related protein 2/3 complex subunit 3, ARPC3)和ARPC4作为调控肌动蛋白细胞骨架的重要信号分子，在炎症发展过程中对于维持上皮细胞屏障功能完整发挥重要作用。为探究ARPC3/4调控奶牛(Bos taurus)乳腺上皮细胞(mammary alveolar cells-large T antigen, MAC-T)屏障损伤与氧化应激的作用机制，本研究利用细胞计数试剂盒-8(cell counting kit-8,CCK-8)筛选脂多糖(lipopolysaccharide, LPS)和ARPC3/ARPC4抑制剂(CK666)的最适作用浓度，qPCR和Western blot技术检测核因子κB(nuclear factor kappa-B, NF-κB)信号通路、抗氧化和紧密连接相关蛋白及基因的表达。结果显示，LPS可显著提高活性氧(reactive oxygen species, ROS)含量，上调髓样分化因子88(myeloid differentiation primary response gene 88, MyD88)、Toll样受体4(Toll-like receptor 4, TLR4)、核因子κB抑制物激酶β(inhibitor of nuclear factor kappa-B kinase subunit beta, IKKβ)和NF-κB的表达，下调过氧化氢酶(catalase, CAT)、超氧化物歧化酶1(superoxide dismutase 1, SOD1)、谷胱甘肽过氧化物酶1(glutathione peroxidase 1, GPx1)、封闭蛋白1(claudin 1)、闭合蛋白(occludin)和闭锁小带蛋白-1(zonula occludens-1, ZO-1)的表达。CK666处理抑制ARPC3/ARPC4后，与LPS组相比，热休克蛋白70(heat shock protein 70, HSP70)的表达进一步上调，同时逆转了LPS导致的NF-κB、MyD88、TLR4和IKKβ表达上调，CAT、SOD1、GPx1、Claudin1、ZO-1和Occludin的表达下调，并降低了ROS含量。结果表明，奶牛乳腺上皮细胞炎症模型中ARPC3/ARPC4通过破坏紧密连接使上皮细胞屏障受损，进而激活NF-κB信号通路并抑制抗氧化基因的表达，促进炎症和氧化应激的发生，表明其具有作为乳房炎治疗靶点的潜力。本研究为奶牛乳房炎的防治提供了新的思路和理论依据。更多还原

【Abstract】 Actin-related protein 2/3 complex subunit 3(ARPC3) and ARPC4, as important signaling molecules regulating actin cytoskeleton, plays an important role in maintaining the integrity of epithelial barrier function during the development of inflammation. In order to explore the mechanism of ARPC3/4regulating barrier injury and oxidative stress in bovine(Bos taurus) mammary alveolar cells-large T antigen(MAC-T), cell counting kit-8(CCK-8) was used to screen the optimal concentration of lipopolysaccharide(LPS) and ARPC3/ARPC4 inhibitor(CK666) in this study. qPCR and Western blot were used to detect the expression of nuclear factor-κB(NF-κB) signaling pathway, antioxidant and tight junction related proteins and genes. The results showed that LPS significantly increased the content of reactive oxygen species(ROS) and up-regulated the expression of myeloid differentiation factor 88(MyD88), Toll-like receptor 4(TLR4), nuclear factor-κB inhibitor kinase β(IKKβ) and NF-κB. The expression of catalase(CAT), superoxide dismutase 1(SOD1), glutathione peroxidase 1(GPx1), Claudin1, Occludin and zonula occludens-1(ZO-1) were downregulated. After inhibiting ARPC3/ARPC4 by CK666 treatment, the expression of heat shock protein 70(HSP70) was further up-regulated compared with LPS group, and the up-regulation of NF-κB, MyD88, TLR4and IKKβ induced by LPS was reversed. The expression of CAT, SOD1, GPx1, Claudin1, ZO-1 and Occludin was down-regulated, and the content of ROS was reduced. The results showed that in the inflammatory model of bovine mammary epithelial cells, ARPC3/ARPC4 impaired the epithelial cell barrier by destroying the tight junction, thereby activating the NF-κB signaling pathway and inhibiting the expression of antioxidant genes to promote inflammation and oxidative stress, indicating its potential as a therapeutic target for mastitis. This study provides new ideas and theoretical basis for the prevention and treatment of dairy cow mastitis.更多还原