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目的探讨补肾益肺消癥方干预肺纤维化大鼠内质网应激和JNK细胞凋亡信号通路的作用机制。方法将SD大鼠随机分为空白组、模型组、阳性药物组、中药预防组、中药治疗组、中药防治组,除空白组外,其他组均以博莱霉素致大鼠肺纤维化模型。中药预防组及中药防治组造模1 d后开始给予补肾益肺消癥方12.68 g/(kg·d)灌胃,空白组和模型组予等量生理盐水灌胃。造模28 d后确定造模是否成功,并处死中药预防组大鼠,观察预防性给药对IPF的影响;造模成功后,阳性药物组给予吡非尼酮53.57 mg/(kg·d)灌胃,中药治疗组及中药防治组给予补肾益肺消癥方12.68 g/(kg·d)灌胃,空白组和模型组仍给予等量生理盐水灌胃,持续28 d。HE染色观察各组大鼠肺组织病理变化,real-time PCR及Westernblot检测肺组织中JNK信号通路相关因子GRP78、IRE1α、p-JNK、TRAF2基因表达及蛋白含量的差异。结果肺组织切片HE染色显示,阳性药物组、中药预防组及中药治疗组肺泡轻度破坏,成纤维细胞有一定程度增生;中药防治组肺泡结构完整,有少量成纤维细胞增生,较模型组纤维化程度明显改善;模型组肺组织中GRP78、IRE1α、p-JNK、TRAF2基因表达拷贝数比值及蛋白含量均明显高于空白组(P均<0.05);中药治疗组和中药防治组GRP78基因表达拷贝数比值及蛋白含量和TRAF2基因表达拷贝数比值均明显低于模型组(P均<0.05),阳性药物组和中药防治组IRE1α基因表达拷贝数比值均明显低于模型组(P均<0.05),阳性药物组、中药预防组、中药治疗组和中药防治组IRE1α蛋白含量均明显低于模型组(P均<0.05),中药防治组p-JNK蛋白含量明显低于模型组(P<0.05)。结论通过下调GRP78、IRE1α、p-JNK、TRAF2的水平,调控JNK信号通路,减轻内质网应激,减少细胞凋亡是补肾益肺消癥方延缓肺纤维化的作用机制之一。
Objective To investigate the mechanism of Bushen Yifei Xiaozheng Recipe in intervening endoplasmic reticulum stress and JNK apoptosis signal pathway in rats with pulmonary fibrosis. Methods SD rats were randomly divided into blank group, model group, positive drug group, traditional Chinese medicine prophylaxis group, traditional Chinese medicine treatment group, traditional Chinese medicine prevention and treatment group, except the blank group, all other groups were treated with bleomycin-induced pulmonary fibrosis model . The traditional Chinese medicine prevention group and the traditional Chinese medicine prevention and treatment group were given intragastric administration of 12.68 g / (kg · d) of Bushen Yifei Xiaozheng Decoction 1 d after modeling, and the rats in the blank group and model group were given the same amount of normal saline. After modeling for 28 days, the model was established and the rats in TCM preventive group were sacrificed to observe the effect of prophylactic administration on IPF. After successful modeling, the positive drug group received pirafenidone 53.57 mg / (kg · d) Gavage, Chinese medicine treatment group and traditional Chinese medicine prevention and treatment group were given Bushen Yifei Xiaojian Prescription 12.68 g / (kg · d) by gavage, the blank group and model group were still given equal volume of normal saline for 28 days. HE staining was used to observe the pathological changes of lung tissue in each group. Real-time PCR and Western blot were used to detect the expression of GRP78, IRE1α, p-JNK and TRAF2 in lung tissue and protein content. Results HE staining showed that the alveoli in the positive drug group, the traditional Chinese medicine prophylaxis group and the traditional Chinese medicine treatment group were mildly damaged and the fibroblasts proliferated to a certain degree. The alveolar structure of the TCM prevention and cure group was intact with a small amount of fibroblast proliferation, The expression of GRP78, IRE1α, p-JNK and TRAF2 in the model group were significantly higher than that in the blank group (P <0.05). The expressions of GRP78, Copy number ratio, protein content and TRAF2 gene copy number ratio were significantly lower than the model group (P <0.05), the positive drug group and the Chinese medicine control group IRE1α gene copy number ratio were significantly lower than the model group (P <0.05 (P <0.05). The protein content of p-JNK in the Chinese medicine prevention and treatment group was significantly lower than that in the model group (P <0.05). The protein content of IRE1α in the positive drug group, the traditional Chinese medicine prevention group, the traditional Chinese medicine treatment group and the traditional Chinese medicine prevention group were significantly lower than those in the model group ). Conclusion The mechanism of Bushen Yifei Xiaozhang Decoction delays lung fibrosis by down-regulating the levels of GRP78, IRE1α, p-JNK and TRAF2, regulating JNK signaling pathway, reducing endoplasmic reticulum stress and decreasing apoptosis.