HMGB1对人肺成纤维细胞ERK1/2-NF-κB信号通路的影响及补阳还五汤含药血清的干预作用

Effect of HMGB1 on ERK1/2-NFκB Signaling Pathway and Intervening Effect of Buyang Huanwu Tang-Containing Serum

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。

Abstract:

Objective: To observe the activation of extracellular regulated protein kinase 1/2(ERK1/2) and nuclear factor kappa B(NF-κB) 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 drug-containing 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-κB 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-1β (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.