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目的：通过博来霉素气管滴入法构建肺纤维化动物模型，探讨益肺通络方对模型大鼠IL-17RA/TRAF6/ACT1信号通路及肝肾功能的影响。方法：采用博来霉素气管滴入法构建SD大鼠肺纤维化模型，随机分为5组：空白对照组、模型组、强的松组、益肺通络方低剂量组和益肺通络方高剂量组，分别给予对应的干预，共干预14 d，取大鼠血清和肺组织做检测。通过聚合酶链式反应（polymerase chain reaction, PCR）检测IL-17RA、TRAF6、ACT1、IL-1β、TNF-α、IL-6 mRNA相对表达，通过蛋白质印迹（Western blot, WB）检测IL-17RA、TRAF6、ACT1蛋白表达，通过免疫荧光检测信号分子TRAF6和ACT1表达，通过酶联免疫吸附测定法（enzyme-linked immunosorbent assay, ELISA）检测大鼠血清细胞因子IL-1β和TNF-α含量，采用全自动生化分析仪检测大鼠的肝肾功能指标。结果：免疫荧光图像显示与空白对照组相比，模型组大鼠肺组织信号分子TRAF6、ACT1的荧光强度较高，在益肺通络方干预后荧光强度显著降低。PCR结果显示与空白对照组相比，模型组大鼠显著上调IL-17RA、TRAF6、ACT1、IL-1β、TNF-α、IL-6 mRNA相对表达（P<0.05）；与模型组相比，益肺通络方组大鼠上述基因相对表达均明显减少（P<0.05）。WB检测结果显示与空白对照组相比，模型组大鼠肺组织IL-17RA、TRAF6、ACT1蛋白表达明显升高（P<0.05）；与模型组相比，益肺通络方可显著抑制上述蛋白表达（P<0.05）。ELISA显示与空白对照组相比，模型组大鼠细胞因子IL-1β、TNF-α含量明显升高（P<0.05）；与模型组相比，益肺通络方组大鼠上述细胞因子含量均明显降低（P<0.05）。全自动生化分析仪结果显示大鼠肝肾功能相关指标均在正常范围内，各组间无统计学差异（P>0.05）。结论：益肺通络方可能通过调控IL-17RA/TRAF6/ACT1信号通路发挥抗肺纤维化作用。益肺通络方通过下调IL-17A相关细胞因子分泌发挥抗炎作用。益肺通络方对肝肾功能无毒副作用。
Abstract：Objective: To investigate the effects of Yifei Tongluo Formula on the IL-17RA/TRAF6/ACT1 signaling pathway and liver and kidney function in an animal model of pulmonary fibrosis established by using bleomycin tracheal instillation method. Methods: The SD rat with pulmonary fibrosis was constructed using bleomycin tracheal instillation method, and randomly divided into 5 groups: the blank control group, the model group, the prednisone group, the low-dose Yifei Tongluo Formula group, and the high-dose Yifei Tongluo Formula group. Corresponding interventions were given for 14 days, and rat serum and lung tissue were taken for testing. The relative expression of IL-17RA, TRAF6, ACT1, IL-1β, TNF-α, and IL-6 m RNA was detected by polymerase chain reaction(PCR), and the protein expression of IL-17RA, TRAF6, and ACT1 was detected by Western blot(WB). The expression of signal molecules TRAF6 and ACT1 was detected by immunofluorescence. The levels of serum cytokines IL-1β and TNF-α in rats were assessed using an enzyme-linked immunosorbent assay(ELISA), and liver and kidney function indicators were detected by a fully automated biochemical analyzer. Results: The immunofluorescence images indicated that the fluorescence intensity of the signal molecules TRAF6 and ACT1 in the lung tissue of the model group rats was elevated when compared to the blank control group. Furthermore, a significant decrease in fluorescence intensity was observed following the intervention with Yifei Tongluo Formula. The results from the PCR analysis indicated that, in comparison to the blank control group, the model group rats exhibited a notable increase in the relative expression levels of IL-17RA, TRAF6, ACT1, IL-1β, TNF-α, and IL-6 m RNA(P<0.05). Additionally, when compared to the model group, the Yifei Tongluo Formula group rats demonstrated a significant decrease in the relative expression of the aforementioned genes(P<0.05). The results of the WB test indicated that the expression levels of IL-17RA, TRAF6, and ACT1 proteins in the lung tissues of the rats in the model group were significantly higher compared to those in the blank control group(P<0.05). Additionally, when compared to the model group, the Yifei Tongluo Formula demonstrated a significant inhibitory effect on the expression of these proteins(P<0.05). The results from the ELISA indicated that the model group rats exhibited a marked increase in the cytokine levels of IL-1β and TNF-α compared to the blank control group(P<0.05). Furthermore, when compared to the model group, there was a significant reduction in the levels of these cytokines in the group of rats treated with the Yifei Tongluo Formula(P<0.05). The results of the fully automated biochemical analyzer showed that the liver and kidney function-related indicators of rats were within the normal range, and there was no statistical difference between the groups(P>0.05). Conclusion: Yifei Tongluo Formula may exert anti pulmonary fibrosis effects by regulating the IL-17RA/TRAF6/ACT1 signaling pathway. Yifei Tongluo Formula exerts anti-inflammatory effects by downregulating the secretion of IL-17A related cytokines and has no toxic side effects on liver and kidney function.
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基本信息:
DOI：10.13210/j.cnki.jhmu.20250115.004
中图分类号:R285.5
引用信息:
[1]马先,冯家腾,周博文,等.益肺通络方对肺纤维化模型大鼠IL-17RA/TRAF6/ACT1信号通路及肝肾功能影响的研究[J].海南医科大学学报,2025,31(20):1573-1579+1590.DOI:10.13210/j.cnki.jhmu.20250115.004.
基金信息:
2022年青年岐黄学者培养项目[国中医药人教函（2022）256号]; 山东省中医药高层次人才培育项目[鲁卫函（2023）143号]~~
2025-01-16
2025-01-16
2025-01-16