基于MAPK/ERK/Nrf2信号通路的参七虫草方对肺纤维化大鼠免疫串化的动态实验研究

Dynamic experimental study on the effect of Shenqi Chongcao Formula on immune tandem reaction in IPF model rats based on the MAPK/ERK/Nrf2 signal pathway

关键词:  参七虫草方  特发性肺纤维化  免疫反应  巨噬细胞  韩明向

Key words:  Shenqi Chongcao Formula  idiopathic pulmonary fibrosis  immune response  macrophage  HAN Mingxiang

摘要:

[目的] 基于MAPK/ERK/Nrf2信号通路,观察国医大师韩明向所创参七虫草方对特发性肺纤维化(IPF)模型大鼠免疫串化的影响。[方法] SPF级SD大鼠150只随机分为五组,空白组、模型组、参七虫草组、吡非尼酮组及Nrf2抑制剂组,每组各30只大鼠。除空白组外均构建IPF大鼠模型,于造模后的第7、14、21、28天称取大鼠体质量并处死各组中的6~8只大鼠,HE、Masson染色观察大鼠肺组织病理变化;ELISA法检测大鼠血清核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)的含量;RT-PCR法检测大鼠肺组织丝裂原活化蛋白激酶1(MEK1)、丝裂原活化蛋白激酶2(MEK2)、细胞外调节蛋白激酶1(ERK1)、细胞外调节蛋白激酶2(ERK2)、衔接蛋白复合物-1(AP-1)mRNA的表达水平;流式细胞术检测各组大鼠肺组织M1及M2型巨噬细胞的阳性率后计算M1/M2,评价各组大鼠的免疫反应程度。[结果] 同时间点下相较于空白组,其余四组大鼠于造模后第7、14、21、28天的肺组织均有不同程度的炎性细胞浸润和胶原纤维沉积,第14、21、28天的体质量均较轻(P<0.05),第7、14、21、28天的血清Nrf2、HO-1含量及肺组织MEK1、MEK2、ERK1、ERK2、AP-1 mRNA的相对表达量均明显升高(P<0.05);相较于模型组,药物干预的三组大鼠第14、21、28天的体质量较重,第7、14、21、28天的肺组织病变程度较轻、血清Nrf2、HO-1含量及肺组织MEK1、MEK2、ERK1、ERK2、AP-1 mRNA的相对表达量较低(P<0.05)。相较于空白组,模型组大鼠早期肺组织M1/M2明显升高(P<0.05),后期M1/M2明显降低(P<0.05);相较于模型组,药物干预的三组大鼠早期肺组织M1/M2明显降低(P<0.05),后期M1/M2明显升高(P<0.05)。[结论] 参七虫草方同吡非尼酮和Nrf2抑制剂,能够降低大鼠肺纤维化程度,其机制可能为通过抑制MAPK/ERK/Nrf2信号通路,降低AP-1、HO-1的表达水平,从而干预IPF大鼠肺部的免疫反应,减轻炎症损伤,减少胶原沉积,延缓肺纤维化进展。

Abstract:

[Objective] To observe the effect of Chinese medical master HAN Ming-xiang' Shenqi Chongcao Formula on the idiopathic pulmonary fibrosis(IPF) rats based on the mitogen activated protein kinase(MAPK)/extracellular regulated protein kinases(ERK)/nuclear factor-erythroid 2-related factor 2(Nrf2) signal pathway. [Methods] 150 Specific Pathogen-Free(SPF)-grade Sprague-Dawley(SD) rats were randomly and evenly divided into 5 groups:the blank group,the model group,Shenqi Chongcao group,Pirfenidone group and the inhibitor of nuclear factor erythroid 2-related factor 2(Nrf2) group. IPF rat models were established except the blank group,then 6~8 rats in each group were weighed and euthanized on the 7th,14th,21st,and 28th days of the experiment. Hematoxylin-eosin(HE) and Masson staining were used to observe the pathological changes in the lung tissues;ELISA method was used to detect the contents of nuclear Nrf2 and heme oxygenase-1(HO-1) in the rats,serum;Reverse Transcription-Polymerase Chain Reaction(RT-PCR) was used to detect the expression levels of mitogen-activated protein kinase 1(MEK1),mitogen-activated protein kinase 2(MEK2),extracellular regulated protein kinases 1(ERK1),extracellular regulated protein kinases 2(ERK2) and adaptor protein complex-1(AP-1) mRNA in the rats,lung tissues;Flow cytometry was used to detect the positive rates of M1 and M2 macrophages,and then M1/M2 was calculated to evaluate the immune response level. [Results] Compared with the blank group and at the same time,on the 14th,21st,and 28th days,the other four groups of rats showed lower weight;on the 7th,14th,21st,and 28th days,the four groups of rats showed varying degrees of inflammatory cell infiltration,collagen fiber deposition in the lung tissue,and higher contents of Nrf2 and HO-1 in the serum,higher expression levels of MEK1,MEK2,ERK1,ERK2 and AP-1 mRNA in the lung tissues. Compared with the model group,on the 14th,21st,and 28th days,the other three groups showed higher weight;on the 7th,14th,21st,and 28th days,the three groups showed milder lesion severity,lower contents of Nrf2 and HO-1,and lower expression levels of MEK1,MEK2,ERK1,ERK2,AP-1 mRNA(P<0.05). Compared with the blank group,the lung tissue M1/M2 of the model group rats was significantly higher in the early stage(P<0.05),and in the late stage it was significantly lower(P<0.05). Compared with the model group,in the early stage the other three groups had significantly lower M1/M2(P<0.05),and in the late stage had significantly higher M1/M2(P<0.05). [Conclusion] Shenqi Chongcao Formula can reduce the degree of pulmonary fibrosis in rats like Pirfenidone and the inhibitor of Nrf2. The mechanism maybe that Shenqi Chongcao Formula can reduce the expression levels of AP-1 and HO-1 by inhibiting MAPK/ERK/Nrf2 signaling pathway,thereby interving the immune reaction in the lungs of IPF rats,reducing inflammatory response and collagen deposition,and delaying the progress of pulmonary fibrosis.