Center for Food Quality Supervision and Testing (Zhanjiang)Ministry of Agriculture and Rural Affairs PR China, Agricultural Products Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, 52400, China; Institute for Fisheries Sciences, Guangxi University, Nanning, 53000, China.
Center for Food Quality Supervision and Testing (Zhanjiang)Ministry of Agriculture and Rural Affairs PR China, Agricultural Products Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, 52400, China.
Fish Shellfish Immunol. 2019 Nov;94:479-484. doi: 10.1016/j.fsi.2019.08.073. Epub 2019 Aug 28.
The present study was conducted to evaluate the anti-inflammatory activity of florfenicol (FFC) against lipopolysaccharide (LPS)-induced inflammatory responses in Ctenopharyngodon idella in vivo and in vitro. Head-kidney (HK) macrophages were pre-treated with 10 μg/mL LPS and then exposed to different concentrations of FFC to determine its in vitro anti-inflammatory activity. Inhibitory effect of FFC on inflammatory mediators TNF-α, IL-6 and IL-1β, as well as LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production were assayed by ELISA. The expression level of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. Expression level of TLR-related genes (TLR1, TLR2, TLR4, TLR7, TLR8) expression, tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor-b-activated kinase 1 (TAK1), Myeloid differentiation factor 88 (MyD88), nucleus p65, NF-κBα (IκBα) were measured by RT-PCR after grass carp were treated with 50, 100 and 200 mg FFC/kg body weight for 3 days. Results from in vitro tests demonstrated that FFC dose-dependently inhibited LPS-induced inflammatory cytokines TNF-α, IL-6 and IL-1β, inflammatory factors NO and PGE 2 production in macrophages. In addition, iNOS and COX-2 expression levels decreased significantly as compared with LPS treated group. In vivo test demonstrated that treatment with FFC prevented the LPS-induced upregulation of TNF-α, IL-6, IL-1β, NO and PGE 2. The expression level of iNOS, and COX-2 in FFC-treated grass carp were also downregulated as compared with LPS treated fish. Besides, FFC blocked the expression of Toll-like receptor 2 (TLR2) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of IκBα. Furthermore, administration of FFC inhibited the up-regulation of IRAK4, TRAF6 and TAK1 induced by LPS. These results suggest that the anti-inflammatory properties of FFC might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expressions through the down-regulation of Toll/NF-κB signaling pathways.
本研究旨在评估氟苯尼考（FFC）在体内和体外对鲤鱼头肾（HK）巨噬细胞脂多糖（LPS）诱导的炎症反应的抗炎活性。先用 10μg/mL LPS 预处理 HK 巨噬细胞，然后用不同浓度的 FFC 处理，以确定其体外抗炎活性。通过 ELISA 测定 FFC 对炎性介质 TNF-α、IL-6 和 IL-1β以及 LPS 诱导的一氧化氮（NO）和前列腺素 E2（PGE2）产生的抑制作用。通过 RT-PCR 检测诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的表达水平。用 RT-PCR 检测 TLR 相关基因（TLR1、TLR2、TLR4、TLR7、TLR8）、肿瘤坏死因子受体相关因子 6（TRAF6）、转化生长因子-β激活激酶 1（TAK1）、髓样分化因子 88（MyD88）、核 p65、NF-κBα（IκBα）的表达水平。鲤鱼用 50、100 和 200mg FFC/kg 体重处理 3 天后进行检测。体外试验结果表明，FFC 呈剂量依赖性地抑制 LPS 诱导的 HK 巨噬细胞中炎性细胞因子 TNF-α、IL-6 和 IL-1β、炎性因子 NO 和 PGE2 的产生。此外，与 LPS 处理组相比，iNOS 和 COX-2 的表达水平显著降低。体内试验表明，FFC 可防止 LPS 诱导的 TNF-α、IL-6、IL-1β、NO 和 PGE2 的上调。与 LPS 处理的鱼类相比，FFC 处理的鲤鱼中 iNOS 和 COX-2 的表达水平也下调。此外，FFC 阻断了 Toll 样受体 2（TLR2）的表达，从而抑制了核转录因子-kappa B（NF-κB）p65 和 IκBα 抑制剂的磷酸化。此外，FFC 抑制了 LPS 诱导的 IRAK4、TRAF6 和 TAK1 的上调。这些结果表明，FFC 的抗炎特性可能是通过抑制 iNOS、COX-2、IL-6、IL-1β 和 TNF-α 的表达，从而抑制 Toll/NF-κB 信号通路来实现的。
