基于MAPK/ERK信号通路探究参七虫草方对特发性肺纤维化炎症反应的影响

Exploring effects of Shenqi Chongcao Recipe on the inflammatory reaction of idiopathic pulmonary fibrosis based on MAPK/ERK signal pathway

关键词:  特发性肺纤维化  参七虫草方  炎症反应  胶原沉积

Key words:  idiopathic pulmonary fibrosis  Shenqi Chongcao Recipe  inflammatory reaction  collagen deposition

摘要:

[目的] 探究参七虫草方对丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）信号通路的影响，探讨其干预特发性肺纤维化（IPF）炎症反应、延缓肺纤维化的机制。[方法] 将140只雄性SD大鼠随机分成5组：空白组、模型组、参七虫草方组、吡非尼酮组、精氨酸脱亚胺酶组，每组28只。除空白组外，其余4组大鼠均采用气管内滴注博来霉素（BLM）的方法建立IPF大鼠模型。空白组及模型组大鼠予0.9%氯化钠溶液灌胃，其余3组分别予参七虫草方、吡非尼酮灌胃及精氨酸脱亚胺酶腹腔注射，于药物干预第7、14、21、28天分4批处死大鼠。苏木精-伊红（HE）、Masson染色法观察肺组织病理变化；流式细胞术分别检测各组大鼠肺组织单细胞悬液中调节性T淋巴细胞（Th）1/Th2、自然杀伤T细胞（NKT细胞）阳性率；酶联免疫吸附（ELISA）法检测各组大鼠血清中转化生长因子-β1（TGF-β1）及白细胞介素-10（IL-10）的相对表达水平；蛋白免疫印迹（Western Blot）法检测各组大鼠肺组织中磷酸化丝裂原活化蛋白激酶（P-MEK）、磷酸化ERK（P-ERK）、核转录因子-κB p65（NF-κB p65）及活化蛋白酶-1（AP-1）的相对表达水平。[结果] 与模型组比较，各治疗组大鼠肺组织中炎症细胞浸润及蓝色胶原沉积均有不同程度改善，但随时间推移，肺纤维化仍逐渐进展。与空白组比较，模型组大鼠相同时间点肺组织单细胞悬液中Th1/Th2降低（P＜0.05），NKT细胞阳性率升高（P＜0.01），血清中TGF-β1表达水平上升（P＜0.01），IL-10表达水平下降（P＜0.01），肺组织中P-MEK、P-ERK、NF-κB p65及AP-1的表达水平增加（P＜0.01）。与模型组比较，各治疗组大鼠相同时间点肺组织单细胞悬液中Th1/Th2无明显差异（P＞0.05），NKT细胞阳性率降低（P＜0.01），血清中TGF-β1表达水平下降（P＜0.01），IL-10表达水平上升（P＜0.01），肺组织中P-MEK、P-ERK、NF-κB p65及AP-1的表达水平降低（P＜0.01）。[结论] 参七虫草方可能通过抑制MAPK/ERK信号通路，提高IPF模型大鼠血清中IL-10的表达水平，降低TGF-β1的表达水平，同时下调肺组织中NF-κB p65、AP-1的表达水平，从而减轻炎症细胞浸润及胶原沉积，改善IPF病情进展。

Abstract:

[Objective] To study the effect of Shenqi Chongcao Recipe on MAPK/ERK signal pathway，and to explore the mechanism of its intervention in idiopathic pulmonary fibrosis（IPF） inflammatory response and delaying pulmonary fibrosis. [Methods] The 140 male SD rats were randomly divided into 5 groups：Blank group，Model group，SCF group，PFD group and ADI group，with 28 rats in each group. Except the Blank group，the other four groups of rats were used to construct IPF models by intratracheal instillation of BLM. The Blank group and Model group were given 0.9% sodium chloride solution intragastric administration，while the remaining three groups were given Shenqi Chongcao Recipe，pirfenidone intragastric administration and ADI by intraperitoneal injection respectively. And the rats were put to death in four batches on the 7th，14th，21st and 28th days of the drug intervention. HE and Masson staining were used to observe the histopathological changes in the lungs. Flow cytometry was used to detect Th1/Th2，and NKT cells positivity rate in single-cell suspensions of rat lung tissues in each group respectively. Detection the relative expression levels of TGF-β1 and IL-10 in the serum of each group of rats by ELISA. Detection the relative expression levels of P-MEK，P-ERK，NF-κB p65 and AP-1 in the lung tissue of each group of rats by Western Blot. [Results] Compared with Model group，inflammatory cell infiltration and blue collagen deposition in the lung tissues of rats in all treatment groups were improved to different degrees，but lung fibrosis still gradually progressed with time. Compared with blank group，at the same time point，Th1/Th2 was significantly reduced（P＜0.05），and the positive rate of NKT cells was significantly higher in single-cell suspensions of lung tissues（P＜0.01） in rats of Model group. The expression levels of TGF-β1 were improved significantly（P＜0.01） and the expression levels of IL-10 was decreased significantly（P＜0.01） in the serum. The expression levels of P-MEK，P-ERK，NF-κB p65 and AP-1 in the lung tissue was increased significantly（P＜0.01）. Compared with Model group，at the same time point，there was no significant difference in Th1/Th2（P＞0.05） and the positive rate of NKT cells was significantly lower（P＜0.01） in single-cell suspensions of lung tissues in rats of all treatment groups. The expression levels of TGF-β1 were reduced significantly（P＜0.01） and the expression levels of IL-10 was increased significantly（P＜0.01） in the serum. The expression levels of P-MEK，P-ERK，NF-κB p65 and AP-1 in the lung tissue was decreased significantly（P＜0.01）. [Conclusion] Shenqi Chongcao Recipe can reduce the expression levels of TGF-β1 and improve the expression levels of IL-10 in the serum，while down-regulated the expression levels of P-MEK，P-ERK，NF-κB p65 and AP-1 in the lung tissue of IPF model rats by inhibiting MAPK/ERK signal pathway，thus reducing inflammatory cells infiltration，decreasing collagen deposition and delaying the progression of IPF.