摘要
目的:探讨毒素清保护肺泡上皮屏障改善衰老小鼠肺炎模型的作用机制。方法:72只SPF级C57 BL/6J小鼠随机取36只颈背部皮下注射D-半乳糖200 mg/kg为衰老组,其余注射生理盐水为青年组,1次/d,连续8 w,建立衰老小鼠模型。将30只衰老小鼠和30只青年小鼠分别随机分为正常对照组、模型对照组、毒素清7.8、15.6 g/kg组、头孢曲松钠0.2 g/kg组,一次性气管内滴注肺炎克雷伯杆菌建立肺炎模型,6 h后分别灌胃给予生理盐水或相应药物,第3 d处死小鼠。计算脏器湿质量、体质量,衰老小鼠脏器指数;试剂盒检测氧化应激水平;HE染色观察肺组织病理变化;ELISA法检测炎症因子含量;蛋白质免疫印迹、免疫荧光检测闭锁小带蛋白1(ZO-1)、闭合蛋白(OCC)表达;免疫组化法观察细胞周期依赖性激酶抑制蛋白1A(P21)、髓过氧化物酶(MPO)、中性粒细胞弹性蛋白酶(NE)、磷酸化表皮生长因子受体(p-EGFR)、磷酸化细胞外信号调节激酶(p-ERK)蛋白阳性表达。结果:与青年对照组比较,衰老对照组小鼠脾指数、胸腺指数、超氧化物歧化酶(SOD)活力明显降低(P<0.05或P<0.01),丙二醛(MDA)含量、P21蛋白阳性表达显著升高(P<0.01);与正常对照组比较,模型对照组肺组织见大量炎性细胞浸润,肺间质和肺泡内水肿,肺间隔增厚,肺损伤及肺泡炎评分显著升高(P<0.01);与模型对照组比较,毒素清7.8、15.6 g/kg组、头孢曲松钠0.2 g/kg组肺泡结构明显,炎性细胞浸润减轻,肺损伤及肺泡炎评分明显降低(P<0.05或P<0.01)。与青年模型对照组比较,衰老模型对照组炎性细胞浸润较重;与衰老正常对照组比较,衰老模型对照组肺泡灌洗液总细胞数和白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)含量、MPO、NE蛋白阳性表达水平升高(P<0.05或P<0.01),ZO-1、OCC蛋白表达明显降低(P<0.05或P<0.01);与衰老模型对照组比较,衰老毒素清15.6 g/kg组肺泡炎评分、肺损伤评分、肺泡灌洗液总细胞数、IL-6、TNF-α含量、MPO、NE蛋白阳性表达水平明显降低(P<0.05或P<0.01),ZO-1、OCC蛋白表达明显升高(P<0.05或P<0.01)。网络药理学富集分析毒素清的333个核心靶点显示,毒素清改善老年人肺炎可能与EGFR、ERK、MAPK等信号通路有关。其中EGFR与毒素清的清风藤碱、异甘草素均可较好结合,且毒素清15.6 g/kg可以显著降低衰老小鼠肺炎模型肺组织p-EGFR和p-ERK表达(P<0.05或P<0.01)。结论:毒素清可以保护肺泡上皮屏障,改善衰老小鼠肺炎模型,其机制可能与抑制EGFR/ERK信号有关。
Objective:To investigate the mechanism of Dusuqing(毒素清)in protecting the alveolar epithelial barrier and ameliorating pneumonia in aging mice.Methods:Seventy-two SPF-grade C57BL/6J mice were used in this study,from which 36 mice were randomly selected and injected subcutaneously with D-galactose(200 mg/kg)at the back of the neck for the modeling of aging,and the remaining mice(youth group)were injected with normal saline once daily for 8 weeks.Thirty aging mice and 30 youth mice were respectively randomized into blank control,model control,Dusuqing(15.6 g/kg and 7.8 g/kg),and ceftriaxone sodium groups.One-time intratracheal dripping of Klebsiella pneumoniae was performed for the modeling of pneumonia.After 6 h,mice were administrated with corresponding drugs or normal saline by gavage and then were euthanized on day 3.The organ wet mass/body weight index was determined to calculate the organ index.Oxidative stress was measured by biochemical test kits.Pathological changes in the lung tissue were observed through hematoxylin-eosin staining.The levels of inflammatory factors were determined by enzyme-linked immunosorbent assay.The expression of zona occludens-1(ZO-1)and occludin(OCC)was detected by Western blotting and immunofluorescence assay.Additionally,the expression levels of cyclin-dependent kinase inhibitor 1A(P21),myeloperoxidase(MPO),neutrophil elastase(NE),phosphorylated epidermal growth factor receptor(p-EGFR),and phosphorylated extracellular signal-regulated kinase(p-ERK)were analyzed by immunohistochemistry.Results:Compared with the youth group,the aging group showed decreases in spleen index,thymus index,and superoxide dismutase(SOD)content(P<0.05 or P<0.01)and increases the malondialdehyde(MDA)and P21 levels(P<0.01).Compared with the blank control group,the model control group presented inflammatory cell infiltration,lung interstitial and intra-alveolar edema,thickened lung septa,and increased lung injury and alveolitis scores(P<0.01).Compared with the model control group,Dusuqing(7.8 g/kg and 15.6 g/kg)and ceftriaxone sodium groups showed clear alveolar structures,reduced inflammatory cell infiltration,and decreased lung injury and alveolitis scores(P<0.05 or P<0.01).Compared with the youth model control group,the aging model control group exhibited severe inflammatory cell infiltration.Compared with the youth Dusuqing(15.6 g/kg)group,the aging Dusuqing(15.6 g/kg)group showed reduced inflammatory cell infiltration and declined lung injury and alveolitis scores.Compared with the aging blank control group,the aging model control group showed increases in total cell count and expression levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),MPO,and NE but decreases in the expression levels of ZO-1 and OCC in the bronchoalveolar lavage fluid(P<0.05 or P<0.01).Compared with the aging model control group,the aging Dusuqing(15.6 g/kg)group presented declines in alveolitis score,lung injury score,total cell count,and expression levels of IL-6,TNF-α,MPO,and NE(P<0.05 or P<0.01)but upregulation in the expression levels of ZO-1 and OCC(P<0.05 or P<0.01).Network pharmacology enrichment of 333 core targets of Dusuqing indicated that the amelioration of pneumonia in aging mice by Dusuqing may be associated with EGFR,ERK,and MAPK signaling pathways.Specifically,EGFR showed good binding affinity with both sinoacutine and isoliquiritigenin.Dusuqing at 15.6 g/kg down-regulated the expression levels of p-EGFR and p-ERK in the lung tissue of the mouse model of pneumonia-aging(P<0.05 or P<0.01).Conclusion:Dusuqing protects the alveolar epithelial barrier and ameliorates pneumonia in aging mice by inhibiting EGFR/ERK signaling.
作者
万冉
程俞梦
程思远
江宇航
赵帅军
赵鹏
刘学芳
WAN Ran;CHENG Yumeng;CHENG Siyuan;JIANG Yuhang;ZHAO Shuaijun;ZHAO Peng;LIU Xuefang(Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province&Education Ministry of P.R.China,Henan Key Laboratory of Chinese Medicine for Respiratory Disease,Henan University of Chinese Medicine,Zhengzhou 450046;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450046;The First Clinical Medical School of Henan University of Chinese Medicine,Zhengzhou 450046;Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046)
出处
《中药药理与临床》
北大核心
2025年第10期37-45,共9页
Pharmacology and Clinics of Chinese Materia Medica
基金
河南省高等学校重点科研项目(编号:24A360013)
河南省本科高校青年骨干教师培养计划(编号:2023GGJS084)
河南省高校科技创新人才支持计划(编号:24HASTIT073)。
关键词
毒素清
衰老小鼠肺炎模型
肺泡上皮屏障
表皮生长因子受体/细胞外信号调节激酶
Dusuqing
Mouse model of pneumonia-aging
Alveolar epithelial barrier
Epidermal growth factor receptor(EGFR)/extracellular signal-regulated kinase(ERK)
