摘要
目的:观察肺纤维化中诱导免疫损伤的高迁移率族蛋白Box-1(high mobility group Box-1 protein,HMGB1)刺激人肺成纤维细胞(HFL1)后细胞外调节蛋白激酶1/2(extracellular regulated protein kinase1/2,ERK1/2),核转录因子-κB(nuclear factor kappa B,NF-κB)信号通路的活化情况,以及补阳还五汤含药血清对HMGB1刺激HFL1后以上信号通路的干预作用。方法:采用含药血清药理学的实验方法,以5%,10%,15%,20%的补阳还五汤含药血清和空白血清来干预HMGB1诱导刺激的HFL1细胞。采用酶联免疫吸附测定(ELISA)法检测白细胞介素-1β(IL-1β)的含量;采用蛋白质免疫印迹法(Western blot)检测细胞总蛋白中ERK1/2,胞浆胞核中NF-κB p65蛋白的表达。结果:与空白组比较,HMGB1刺激组诱导后ERK1/2蛋白,胞浆和胞核中NF-κB p65蛋白及IL-1β含量的表达均增高(P<0.05,P<0.01);与20%空白血清预培养加HMGB1组比较,HMGB1刺激组诱导后ERK1/2蛋白,胞浆和胞核中NF-κB p65蛋白及IL-1β含量的表达均增高(P<0.05);与HMGB1刺激组比较,补阳还五汤含药血清各组能够降低ERK1/2蛋白,胞浆和胞核中NF-κB p65蛋白及IL-1β含量的表达,其中15%和20%补阳还五汤含药血清组降低ERK1/2的表达(P<0.05);胞浆10%补阳还五汤含药血清组和胞核20%补阳还五汤含药血清组降低NF-κB p65蛋白的表达(P<0.05);15%补阳还五汤含药血清组降低IL-1β的含量(P<0.05)。结论:补阳还五汤含药血清在体外实验中,可抑制HMGB1引起的人肺成纤维细胞中ERK1/2/NF-κB信号通路的活化,减少IL-1β等炎症因子分泌。提示补阳还五汤可能通过调控人肺成纤维细胞中ERK1/2/NF-κB信号通路,防治肺纤维化的发生发展。
Objective： To observe the activation of extracellular regulated protein kinase 1/2 （ERK1/2） and nuclear factor kappa B （NF-KB） signaling pathway after stimulating normal lung fibroblasts （HFL1） by high mobility group Box-1 protein （HMGB1） , which can induce immune damage in pulmonary fibrosis, as well as the intervening effect of Buyang Huanwu Tang-containing serum on above signal pathways. Method： The drugcontaining serum pharmacological methodology was adopted. Buyang Huanwu Tang-containing serum with concentrations of 5% , 10% , 15% , 20% , and blank serum were adopted to intervene normal lung fibroblasts （HFL1） stimulated by HMGB1. The enzyme-linked immunosorbent assay （ELISA） method was used to detect the expression of interleukin-1β （IL-1β）, and the immune protein imprinting method （Western blot method） was applied to detect total protein expression of ERK1/2 and cell cytoplasm and nucleus protein expression of NF-κB p65. Result： Compared with blank control group, HMGB1 can promote the expressions of IL-1β, ERK1/2, NF-KB p65 in HFL1 cell （P 〈0.05, P 〈0.01）. Compared with the 20% serum control added with HMGB1 group, the expressions of IL-1β, ERK1/2, NF-κB p65 were increased in HMGB1 stimulation group. Compared with HMGB1 stimulation group, all of Buyang Huanwu Tang-containing serum groups could reduce the expressions of IL-1β, ERK1/2, NF-κB p65. Among them, 15% and 20% Buyang Huanwu Tang-containing serum could obviously reduce the expression of ERK1/2 （P 〈 0.05 ） ; 10% Buyang Huanwu Tang-containing serum in cell cytoplasm, and 20% Buyang Huanwu Tang containing-serum in cell nucleus could clearly decrease the expression of NF-κB p65 （P 〈0.05）; And 15% Buyang Huanwu Tang-containing serum could visibly reduce the expression of IL-1/3 （P 〈 0.05）. Conclusion： In the in vitro experiment, Buyang Huanwu Tang-containing serum can inhibit the activation of ERK1/2-NF-κB signaling pathways caused by HMGB1 in HFL-1, and reduce the secretion of inflammatory factor IL-1β. The results suggest that Buyang Huanwu Tang may prevent and treat pulmonary fibrosis by regulating ERK1/2-NF-κB signaling pathways.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2017年第13期153-157,共5页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金面上项目(81373597)
