甘草黄酮对脂多糖诱导小胶质细胞i NOS表达和NF-кB活化影响
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摘要
目的研究甘草黄酮对脂多糖诱导的BV-2小胶质细胞诱导型一氧化氮合酶(inducible nitricoxide,iNOS)表达影响和对核因子-kB(nuclear factor-kappa B,NF-kB)活化的影响。方法将BV-2细胞随机分为四个组:Control组、脂多糖组、3μg/mL和5μg/mL甘草黄酮处理组。采用免疫荧光染色法定性分析iNOS在BV-2细胞的表达;采用免疫蛋白印迹法定量分析甘草黄酮处理前后胞浆iNOS蛋白表达量和胞核NF-кB p65蛋白含量变化。结果各组i NOS阳性BV-2细胞数量(F=8.09,P<0.01),胞浆iNOS蛋白表达量(F=7.89,P<0.01)和胞核NF-кB p65蛋白含量(F=6.12,P<0.01)差异均有统计学意义。被脂多糖刺激后,大量BV-2细胞呈iNOS阳性(P<0.01),部分细胞胞体变圆钝,突起变粗短;胞浆iNOS蛋白表达量较对照组急剧增多(P<0.01),同时伴有胞核NF-кB p65蛋白含量的显著增加(P<0.01)。而经两种浓度脂多糖处理过的BV-2细胞,在脂多糖刺激后,iNOS阳性细胞数量则均较脂多糖组有明显减少(P<0.01),同时细胞胞体缩小,突起增多;胞浆iNOS蛋白表达量也均较脂多糖组有明显降低(P<0.01),同时伴有胞核NF-кB p65蛋白含量的降低(P<0.01)。结论甘草黄酮可抑制脂多糖诱导的小胶质细胞iNOS表达,其机制可能与调节NF-кB活化有关。
Objective To investigate effects of Glabridin on iNOS expression and NF-кB activation in the BV-2 microglial cells induced by LPS. Methods BV-2 cells were randomly divided into four groups: control group, LPS group, 3 μg/mL and 5 μg/mL Glabridin treatment group. Immunofluorescence staining was used to quantify iNOS expression in BV-2 cells. The cytoplasmic i NOS protein and nuclear NF-кB p65 protein were detected by Western blot.Results There were significant differences in the number of iNOS positive BV-2 cells(F =8.09, P <0.01) and the expression levels of iNOS protein(F=7.89, P<0.01) and NF-кB p65 protein(F=6.12, P<0.01). After being stimulated by LPS, a large number of BV-2 cells expressed iNOS in LPS group(P <0.01) and the shapes of some BV-2 cells were round and blunt with their processes thick and short. Compared with control group, both the cytoplasmic expression of iNOS protein and nuclear NF-кB p65 protein increased significantly(P <0.01). In Glabridin-pretreatment groups, the numbers of LPS-induced i NOS positive cells were significantly lower(P<0.01) and the body of BV-2 cells shrunk and their protrusions increased. Compared with LPS group, the cytoplasmic i NOS and nuclear NF-кBp65 significantly decreased in two Glabridin-treatment groups(P <0.01). Conclusion Glabridin can inhibit i NOS expression in LPS-induced microglia, which is related to the regulation of NF-кB activation.
Objective To investigate effects of Glabridin on iNOS expression and NF-кB activation in the BV-2 microglial cells induced by LPS. Methods BV-2 cells were randomly divided into four groups: control group, LPS group, 3 μg/mL and 5 μg/mL Glabridin treatment group. Immunofluorescence staining was used to quantify iNOS expression in BV-2 cells. The cytoplasmic i NOS protein and nuclear NF-кB p65 protein were detected by Western blot.Results There were significant differences in the number of iNOS positive BV-2 cells(F =8.09, P <0.01) and the expression levels of iNOS protein(F=7.89, P<0.01) and NF-кB p65 protein(F=6.12, P<0.01). After being stimulated by LPS, a large number of BV-2 cells expressed iNOS in LPS group(P <0.01) and the shapes of some BV-2 cells were round and blunt with their processes thick and short. Compared with control group, both the cytoplasmic expression of iNOS protein and nuclear NF-кB p65 protein increased significantly(P <0.01). In Glabridin-pretreatment groups, the numbers of LPS-induced i NOS positive cells were significantly lower(P<0.01) and the body of BV-2 cells shrunk and their protrusions increased. Compared with LPS group, the cytoplasmic i NOS and nuclear NF-кBp65 significantly decreased in two Glabridin-treatment groups(P <0.01). Conclusion Glabridin can inhibit i NOS expression in LPS-induced microglia, which is related to the regulation of NF-кB activation.
引文
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[12]KWON HM, CHOI YJ, CHOI JS, et al. Blockade of cytokineinduced endothelial cell adhesion molecule expression by licorice isoliquiritigenin through NF-kappaB signal disruption[J]. ExpBiol Med(Maywood), 2007, 232(2):235-245.
[2]王玲玲,陈思,马俊,等.帕金森病患者外周血单核细胞CD14和TLR4中mRNA的表达水平[J].中国神经精神疾病杂志, 2016, 42(2):104-108.
[3]王茜,张辉,刘名,等. P38信号通路调控帕金森病小鼠黑质NF-κB和iNOS的表达[J].南方医科大学学报, 2014, 34(8):1176-1180.
[4]赵焱,卢祖能.光甘草定对MPTP致帕金森病小鼠海马区ERK信号通路的影响[J].中华医学杂志, 2017, 97(26):2050-2054.
[5]Block ML, Hong JS. Microglia and inflammation-mediated neurodegeneration:multiple triggers with a common mechanism[J].ProgNeurobiol, 2005, 76(2):77-98.
[6]ARIMOTO T, BING G. Up-regulation of inducible nitric oxide synthase in the substantia nigra by lipopolysaccharide causes microglial activation and neurodegeneration[J]. Neurobiol Dis,2003, 12(1):35-45
[7]GANSTER RW, TAYLOR BS, SHAO L, et al. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B[J]. Proc Natl Acad Sci USA, 2001,98(15):8638-8643
[8]陈浩,师亮,王燕宏,等.甘草黄酮对MPTP帕金森病小鼠的实验性治疗研究[J].中风与神经疾病杂志, 2013, 30(12):1112-1113.
[9]KIM KR, JEONG CK, PARK KK, et al. Anti-inflammatory effects of licorice and roasted licorice extracts on TPA-induced acute inflammation and collagen-induced arthritis in mice[J]. J Biomed Biotechnol, 2010, 2010:709378.
[10]肖辉.甘草黄酮对UVB诱导HaC aT细胞ERS中IRE1信号通路的影响[D].兰州:兰州大学, 2016.
[11]THIYAGARAJAN P, CHANDRASEKARAN CV, DEEPAK HB,et al. Modulation of lipopolysaccharide-induced pro-inflammatory mediators by an extract of Glycyrrhizaglabra and its phytoconstituents[J]. Inflammopharmacology, 2011, 19(4):235-241.
[12]KWON HM, CHOI YJ, CHOI JS, et al. Blockade of cytokineinduced endothelial cell adhesion molecule expression by licorice isoliquiritigenin through NF-kappaB signal disruption[J]. ExpBiol Med(Maywood), 2007, 232(2):235-245.