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

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 factorα,TNF-α)的m RNA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。

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/(kgꞏd)and 200 mg/(kgꞏd)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/(kgꞏd)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 factorα(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.