奎宁酸通过NF-κB与NLRP3炎性小体对牛乳腺细胞、小鼠乳腺炎性反应和细胞焦亡的抑制效应  

Inhibitory effect of quinic acid on inflammatory response and pyroptosis of bovine mammary gland cells and mouse mammary gland tissue through NF-κB and NLRP3 inflammasome

作　　者：吕祥 刘梦燕 康钰晨 龙泉 张以诺 林涛 蒋曹德[1] LYU Xiang;LIU Mengyan;KANG Yuchen;LONG Quan;ZHANG Yinuo;LIN Tao;JIANG Caode(College of Animal Science and Technology,Southwest University,Beibei,Chongqing 400715,China;Leshan Normal College,Leshan,Sichuan 641000,China;College of Grassland Agriculture,Northwest A&F University,Yangling,Shannzi 712100,China)

机构地区：[1]西南大学动物科学技术学院,重庆北碚400715 [2]乐山师范学院,四川乐山641000 [3]西北农林科技大学草业与草原学院,陕西杨凌712100

出　　处：《中国兽医学报》2024年第9期1982-1991,共10页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32171673)。

摘　　要：奎宁酸(QA)具有抗氧化、抗癌和抗炎的作用,但其对牛乳腺炎的保护作用还有待进一步研究。旨在探究QA对脂多糖(lipopolysaccharide,LPS)诱导的牛乳腺上皮细胞(MAC-T)、小鼠乳腺组织炎症与细胞焦亡的抑制作用及其机制。采用CCK-8法筛选QA处理MAC-T细胞的最佳质量浓度,ELISA法检测炎性因子、氧化应激因子以及细胞焦亡指示因子的表达水平;免疫组化法检测小鼠乳腺组织中CD3含量;Western blot检测NF-κB、NOD样受体3(NLRP3)炎症小体、Caspase-11和GSDMD(gasdermin D)的表达水平;免疫荧光检测核因子κB(NF-κB)关键蛋白p65核转移情况。结果表明,QA(20、40、60、80 mg/L)显著提高了MAC-T细胞活性(P<0.05)。QA处理显著降低了MAC-T细胞和小鼠乳腺中LPS诱导的炎性因子(TNF-α、IL-1β、IL-6)、氧化应激因子(COX-2、iNOS)以及细胞焦亡指示因子(ROS、LDH、IL-18)的表达水平,并具有剂量依赖性(P<0.05);小鼠腹腔注射QA(5、10、20 mg/kg)后LPS诱导的CD3的表达显著下调(P<0.05),小鼠乳腺组织T淋巴细胞浸润减少。QA显著抑制了MAC-T细胞和小鼠乳腺组织中LPS诱导的NF-κB(IκBα、p65、p-IκBα和p-p65)的表达水平、MAC-T细胞NF-κB核转移、NLRP3炎症小体(NLRP3、ASC、Caspase-1)、Caspase-11和GSDMD的蛋白表达水平(P<0.05)。上述结果表明,QA通过NF-κB和NLRP3炎症小体抑制MAC-T、小鼠乳腺炎性反应与细胞焦亡。本研究为植物活性成分防治乳腺炎提供了新的数据。Quinic acid(QA)has antioxidant,anticancer and anti-inflammatory effects,but its protective effect against bovine mastitis remains to be further investigated.The aim of this study was to investigate the inhibitory effects and mechanisms of quinic acid(QA)on lipopolysaccharide(LPS)-induced inflammation and pyroptosis in bovine mammary epithelial cells(MAC-T)and mouse mammary tissues.The CCK-8 method was applied to screen the treatment concentration of QA in MAC-T cells.The ELISA method was used to detect the expression levels of inflammatory factors,oxidative stress factors and pyroptosis indicators.The distribution of CD3 in mouse mammary tissues was detected by the immunohistochemical method.p65 nuclear translocation was measured by immunofluorescence.Western blot was performed to test the protein and phosphorylation levels of NF-κB,the inflammasome of NOD-like receptor(NLRP3),Caspase-11 and gasdermin D(GSDMD).The results showed that QA(20,40 and 60 mg/L)significantly increased the activity of MAC-T cells(P<0.05).QA treatment significantly reduced LPS-induced expression of inflammatory factors(TNF-α,IL-1βand IL-6),oxidative stress indicators(COX-2 and iNOS)and pyroptosis indicators(ROS,LDH and IL-18)in both MAC-T cells and mouse mammary tissues in a manner of dose-dependence(P<0.05).Moreover,after intraperitoneal injection of QA at 5,10and 20 mg/kg,respectively,the expression of T-lymphocyte marker CD3 induced by LPS was significantly downregulated in mouse breast tissues(P<0.05).In addition,QA significantly decreased the LPS-induced expression of NF-κB(IκBα,p65,p-IκBαand p-p65),inflammasome(NLRP3,ASC and Caspase-1),Caspase-11 and DSDMD in both MAC-T cells and mouse mammary tissues,and inhibited p65 nuclear transfer in MAC-T cells(P<0.05).Overall,the above results indicate that QA inhibits inflammatory response and pyroptosis through NF-κB and NLRP3 inflammasome in both MAC-T cells and mouse mammary tissues.The results of this study provide novel data on the prevention and treatment of mastitis by plant

关 键 词：奎宁酸 乳腺炎症 NF-ΚB NLRP3炎性小体 细胞焦亡 

分 类 号：S858.23[农业科学—临床兽医学]