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.
Abstract
Quinic acid(QA)has antioxidant,anticancer and anti-inflammatory effects,but its pro-tective 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 mam-mary tissues was detected by the immunohistochemical method.p65 nuclear translocation was measured by immunofluorescence.Western blot was performed to test the protein and phosphoryl-ation levels of NF-κB,the inflammasome of NOD-like receptor(NLRP3),Caspase-11 and gasder-min 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-1β and 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 5,10 and 20 mg/kg,respectively,the expression of T-lymphocyte marker CD3 induced by LPS was sig-nificantly downregulated in mouse breast tissues(P＜0.05).In addition,QA significantly de-creased the LPS-induced expression of NF-κB(IκBα,p65,p-IκBα and p-p65),inflammasome(NL-RP3,ASC and Caspase-1),Caspase-11 and DSDMD in both MAC-T cells and mouse mammary tis-sues,and inhibited p65 nuclear transfer in MAC-T cells(P＜0.05).Overall,the above results indi-cate that QA inhibits inflammatory response and pyroptosis through NF-κB and NLRP3 inflamma-some in both MAC-T cells and mouse mammary tissues.The results of this study provide novel da-ta on the prevention and treatment of mastitis by plant active ingredients.
维普
万方数据