甘草查尔酮A对副猪嗜血杆菌感染猪肺泡巨噬细胞氧化应激的影响

【目的】 探究甘草查尔酮A对副猪嗜血杆菌感染猪肺泡巨噬细胞引发的氧化应激的影响, 为开发治疗副猪嗜血杆菌感染的新型药物提供参考。【方法】 利用支气管肺泡灌洗法分离猪肺泡巨噬细胞, 分为6组: 阴性对照组, 未感染副猪嗜血杆菌的猪肺泡巨噬细胞; 阳性对照组, 感染副猪嗜血杆菌的猪肺泡巨噬细胞; DMSO组, 猪肺泡巨噬细胞感染副猪嗜血杆菌后给予0.1% DMSO; 5、10和20 μg/mL甘草查尔酮A组, 猪肺泡巨噬细胞感染副猪嗜血杆菌后给予相应浓度的甘草查尔酮A。各组细胞培养24 h后, 用CCK-8法检测细胞活力, 用酶标仪或可见光分光光度计检测各组细胞上清中乳酸脱氢酶(lactate dehydrogenase, LDH)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)和超氧化物歧化酶(superoxide dismutase, SOD)活性及丙二醛(malondialdehyde, MDA)、一氧化氮(nitric oxide, NO)和总抗氧化能力(total antioxidant capacity, T-AOC)水平, 用活性氧(reactive oxygen species, ROS)荧光探针(DCFH-DA)检测ROS水平。【结果】 与阴性对照组相比, 副猪嗜血杆菌感染组猪肺泡巨噬细胞活力均显著降低(P<0.05), 上清液中LDH活性均显著提高(P<0.05), MDA、NO、ROS水平及SOD活性均显著增加(P<0.05);与阳性对照组相比, 5、10和20 μg/mL甘草查尔酮A组猪肺泡巨噬细胞活力均显著提高(P<0.05), 上清液LDH活性均显著降低(P<0.05), 10和20 μg/mL甘草查尔酮A组MDA、NO和ROS水平均显著降低(P<0.05);5、10和20 μg/mL甘草查尔酮A处理组GSH-Px、SOD和T-AOC活性均呈剂量依赖性显著升高(P<0.05)。【结论】 副猪嗜血杆菌感染猪肺泡巨噬细胞导致氧化和抗氧化平衡失调, 5~20 μg/mL甘草查尔酮A可以降低细胞氧化应激反应。

Effects of Licochalcone A on Oxidative Stress Induced by Haemophilus parasuis Infection in Porcine Alveolar Macrophages

【Objective】 This study was to explore the effects of licochalcone A (Lico A) on the oxidative stress of porcine alveolar macrophages (PAMs) infected with Haemophilus parasuis, so as to provide reference for the development of new drugs for the treatment of Haemophilus parasuis infection.【Method】 PAMs were obtained from the lungs by bronchoalveolar lavage and divided into 6 groups. Negative control group, PAMs were not infected with Haemophilus parasuis; Positive control group, PAMs were infected with Haemophilus parasuis only; DMSO group, PAMs were treated with 0.1% DMSO after infection with Haemophilus parasuis; 5, 10 and 20 μg/mL Lico A group, PAMs were treated with 5, 10 and 20 μg/mL Lico A after infection with Haemophilus parasuis.PAMs were treated according to the above groups and cultured for 24 h.Cell viability were measured by CCK-8 method, the activities of lactate dehydrogenase (LDH), glutathione peroxidase(GSH-Px)and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA), nitric oxide (NO) and total antioxidant capacity (T-AOC) in the supernatant were measured by enzyme labeling or visible light photometer, respectively.The intracellular reactive oxygen species (ROS) level was observed using a fluorescence probe, 2', 7'-dichlorofluorescin diacetate (DCFH-DA).【Result】 Compared with negative control group, after infection with Haemophilus parasuis, the activity of PAMs was decreased significantly (P < 0.05), the activity of LDH in supernatant was significantly increased (P < 0.05), the levels of MDA, NO and ROS and the activity of SOD were significantly increased (P < 0.05).Compared with positive group, the cell viability in 5, 10 and 20 μg/mL Lico A groups was significantly increased (P < 0.05), the activity of LDH in supernatant was significantly decreased (P < 0.05), the levels of MDA, NO and ROS in 10 and 20 μg/mL Lico A groups were significantly decreased (P < 0.05).The activities of GSH-Px, SOD and T-AOC in 5, 10 and 20 μg/mL Lico A groups were significantly increased in a dose dependent manner (P < 0.05).【Conclusion】 Haemophilus parasuis infection results in an imbalance between oxidant and antioxidant in PAMs, and 5-20 μg/mL Lico A could decrease the cellular response to oxidative stress.