龙眼蛋白对C57BL/6小鼠及RAW264.7巨噬细胞的炎症因子的影响研究

Effects of Dimocarpus longan Lour. protein on inflammatory factors in C57BL/6 mice and RAW264.7 macrophages

中文关键词:  龙眼  巨噬细胞  炎症因子  腺苷酸活化蛋白激酶（AMP-activatedprotein kinase，AMPK）/核因子κB（nuclear factor kappa-B，NF-κB） 信号通路

英文关键词:Dimocarpus longan Lour. protein  macrophages  inflammatory cytokines  AMP-activatedprotein kinase/nuclear factor kappa-B signaling pathway

中文摘要:

      目的 探讨龙眼蛋白对C57BL/6小鼠及RAW264.7巨噬细胞的炎症因子影响及作用机制。方法 采用反向色谱-质谱法(reverse-phase liquid chromatography-mass spectrometry, RPLC-MS)鉴定龙眼蛋白的组成。采用100 mg/(kg·d)和200 mg/(kg·d)龙眼蛋白腹腔注射C57BL/6小鼠, 酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)测定小鼠血液炎症因子的表达, 苏木精-伊红(hematoxylin-eosin, HE)染色法染色分析肺部炎症病理反应; 用0.2、0.4、0.6 mg/mL龙眼蛋白处理小鼠RAW264.7巨噬细胞, 噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, MTT]法测定细胞活力, 实时荧光定量聚合酶链式反应法(real-time quantitative polymerase chain reaction, Q-PCR)和ELISA检测炎症因子表达, 蛋白质免疫印迹法(western blot, WB)分析腺苷酸活化蛋白激酶(AMP-activatedprotein kinase, AMPK)/核因子κB (nuclear factor kappa-B, NF-κB)信号通路相关蛋白表达情况。结果 通过RPLC-MS结合数据库检索, 共鉴定到肽段覆盖率在30%以上的蛋白质25种。腹腔注射100 mg/(kg·d)龙眼蛋白使小鼠肺部产生炎症病理反应, 小鼠血清中的炎症因子[血清淀粉样蛋白A (serum amyloid A, SSA)、超敏C反应蛋白(hypersensitive C-reactive protein, hs-CRP)、降钙素原(procalcitonin, PCT)、白细胞介素-6 (interleukin-6, IL-6)]均显著升高。0.2 mg/mL龙眼蛋白处理使RAW264.7细胞IL-6、白细胞介素-8 (interleukin-8, IL-8)、肿瘤坏死因子-α (tumor necrosis factor α, TNF-α)的mRNA和蛋白表达均呈剂量依赖式上升, AMPK磷酸化水平表达下调, p65磷酸化水平表达上调。结论 龙眼蛋白能够促进小鼠和RAW264.7巨噬细胞的炎症反应, 其作用机制可能是龙眼蛋白促进了AMPK/NF-κB信号通路相关炎症因子的表达。

英文摘要:

      Objective To investigate the effects of Dimocarpus longan Lour. protein on inflammatory factors in C57BL/6 mice and RAW264.7 macrophages and its mechanism. Methods The composition of Dimocarpus longan Lour. protein was analyzed by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS). C57BL/6 mice were injected with 100 mg/(kg·d) and 200 mg/(kg·d) Dimocarpus longan Lour. protein. The expression of inflammatory factors in the blood of mice was determined by enzyme linked immunosorbent assay (ELISA), and the pathological reaction of lung inflammation was analyzed by hematoxylin-eosin (HE) staining method. RAW264.7 cells were treated with 0.2, 0.4 and 0.6 mg/mL Dimocarpus longan Lour. protein. The cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), the expression of inflammatory factors was detected by real-time quantitative polymerase chain reaction (Q-PCR) and ELISA, and the expression of related proteins in AMP-activatedprotein kinase (AMPK)/nuclear factor kappa-B (NF-κB) signaling pathway was analyzed by western blot (WB). Results The 25 proteins with more than 30% peptide coverage were identified through RPLC-MS combined with database retrieval. The intraperitoneal injection of 100 mg/(kg·d) Dimocarpus longan Lour. protein produced inflammatory and pathological reactions in the lungs of mice. The serum inflammatory factors [serum amyloid A (SSA), hypersensitive C-reactive protein (hs-CRP), procalcitonin (PCT) and interleukin-6 (IL-6)] were significantly increased. By treated with 0.2 mg/mL Dimocarpus longan Lour. protein with RAW264.7 cells, mRNA and protein expressions of IL-6, interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) were increased in a dose-dependent manner, the phosphorylation level of AMPK was down-regulated and the phosphorylation level of p65 was up-regulated. Conclusion Dimocarpus longan Lour. protein can promote the inflammatory response of mice and RAW264.7 macrophages, which may be related to promoting the expression of inflammatory factors through AMPK/NF-κB signaling pathway.