基于MEK/ERK通路清热解毒扶正方调控中性粒细胞胞外诱捕网治疗肺炎机制
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1.河南中医药大学医学院;2.河南中医药大学呼吸疾病中医药防治省部共建协同创新中心

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河南省自然科学基金资助项目(242300421294)


Mechanism of Heat-Clearing, Detoxifying, and Supporting Formula in Treating Pneumonia by Regulating Neutrophil Extracellular Traps via the MEK/ERK Pathway
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1.School of Medicine,Henan University of Chinese Medicine;2.Henan Provincial Education Office and Henan University of Traditional Chinese Medicine to Establish Joint Innovation Center for Respiratory Diseases in Traditional Chinese Medicine,Key Experimental Laboratory of Chinese Medicine for Respiratory Diseases in Henan Province

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    摘要:

    目的:探讨清热解毒扶正方——毒素清通过MEK/ERK通路调控中性粒细胞胞外诱捕网治疗肺炎的机制。方法:采用气管滴注肺炎克雷伯杆菌,建立肺炎小鼠模型。将小鼠分为空白组、模型组、头孢曲松组、毒素清低剂量组、毒素清高剂量组。从一般情况、肺泡灌洗液细胞计数、肺病理等方面评价毒素清对肺炎小鼠的药效作用;试剂盒检测肺组织髓过氧化物酶(MPO)活力;ELISA法检测肺组织肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)含量变化;qRT-PCR法检测肺组织TNF-α、IL-6、白细胞介素-1β(IL-1β)、淋巴细胞抗原6复合体位点G(Ly6G)、MPO、中性粒细胞弹性蛋白酶(NE)、瓜氨酸化组蛋白H3(Cit-H3)、肽基精氨酸脱亚氨酶4(PAD4)mRNA的表达;IHC观察Ly6G、MPO、NE、Cit-H3在肺组织切片中的表达;IF观察NETs在肺组织切片中的表达;Western blot法检测肺组织MEK、P-MEK、ERK、P-ERK、PAD4、Cit-H3蛋白的表达。结果:与空白组相比,模型组小鼠肺泡灌洗液细胞计数显著升高(P<0.01),肺组织大量炎细胞浸润、肺泡水肿及肺泡壁增厚;肺组织MPO活力明显升高(P<0.01);肺组织TNF-α、IL-6表达升高(P<0.01);肺组织TNF-α、IL-6、IL-1β、Ly6G、MPO、NE、Cit-H3、PAD4 mRNA表达升高(P<0.01);肺组织切片中Ly6G、MPO、NE、Cit-H3表达升高(P<0.01);肺组织切片中NETs表达升高(P<0.01);肺组织p-MEK/MEK和p-ERK/ERK比值、PAD4、Cit-H3蛋白表达显著升高(P<0.05或P<0.01)毒素清干预3天可有效改善肺炎小鼠呼吸急促、活动度降低、体重下降等症状,减轻肺组织炎细胞浸润与肺组织损伤;毒素清高剂量可减少肺泡灌洗液细胞数目(P<0.05);毒素清各组可降低肺组织MPO的活力(P<0.05或P<0.01);降低肺组织TNF-α、IL-6的表达(P<0.05或P<0.01),减轻炎症反应;毒素清低剂量组降低肺组织TNF-α、IL-1β mRNA的表达(P<0.01);毒素清高剂量组降低IL-6 mRNA的表达(P<0.01);毒素清各组显著降低Ly6G、MPO、NE、Cit-H3、PAD4 mRNA的表达(P<0.01);降低Ly6G、MPO、NE、Cit-H3在肺组织切片中的表达(P<0.05或P<0.01);降低NETs在肺组织切片中表达(P<0.01);毒素清高剂量组降低肺组织p-MEK/MEK蛋白表达(P<0.01);毒素清各组降低肺组织p-ERK/ERK比值、PAD4、Cit-H3蛋白表达(P<0.05或P<0.01)。结论:MEK/ERK通路促进NETs形成参与肺炎发生发展。毒素清通过调节MEK/ERK抑制NETs形成继而减轻肺部炎症反应。

    Abstract:

    OBJECTIVE: To explore the mechanism of the Qinghe detoxification and Fuzheng formula, Toxin Clear, in the treatment of pneumonia by regulating the neutrophil extracellular trapping network through the MEK/ERK pathway. METHODS: Tracheal drip injection of Klebsiella pneumoniae was used to establish a mouse model of pneumonia. The mice were divided into blank group, model group, ceftriaxone group, toxin clear low dose group, and toxin clear high dose group. The pharmacodynamic effects of toxocarb on mice with pneumonia were evaluated in terms of general condition, alveolar lavage fluid cell count, and lung pathology; the kit detected the MPO viability in lung tissues; ELISA detected the changes of TNF-α and IL-6 content in lung tissues; and qRT-PCR detected the TNF-α, IL-6, IL-1β, Ly6G, MPO, NE, and Cit-H3 in lung tissues, PAD4 mRNA expressio;IHC to observe the expression of Ly6G, MPO, NE, and Cit-H3 in lung tissue sections;IF observation of NETs expression in lung tissue sections;Western blot method to detect the expression of MEK, P-MEK, ERK, P-ERK, PAD4, Cit-H3 proteins in lung tissues; RESULTS: Compared with the blank group, the cell counts of alveolar lavage fluid of mice in the model group were significantly elevated (P < 0.01), with a large number of inflammatory cell infiltration, alveolar edema and alveolar wall thickening in the lung tissues; the vitality of MPO in the lung tissues was significantly elevated (P < 0.01); the expression of TNF-α and IL-6 in the lung tissues was elevated (P < 0.01); the expression of TNF-α, IL-6, IL-1β, Ly6G , MPO, NE, Cit-H3, and PAD4 mRNA expression was elevated (P < 0.01); Ly6G, MPO, NE, and Cit-H3 expression was elevated in lung tissue slices (P < 0.01);NETs expression was elevated in lung tissue sections (P < 0.01); and p-MEK/MEK and p-ERK/ERK ratios, PAD4, and Cit-H3 protein expression were significantly elevated in lung tissue (P< 0.05 or P< 0.01)Toxocin intervention for 3 days could effectively improve the symptoms of shortness of breath, reduced activity and weight loss in mice with pneumonia, and reduce the infiltration of inflammatory cells in lung tissues and lung tissue damage; the high dose of toxocin reduced the number of cells in alveolar lavage fluid (P < 0.05); toxocin in all groups reduced the viability of MPO in lung tissues (P<0.05 or P<0.01); and reduced the expression of TNF-α, IL -6 expression in lung tissue (P<0.05 or P<0.01), and reduced inflammatory response; the low-dose group of toxocarbamol reduced the expression of TNF-α and IL-1β mRNA in lung tissue (P<0.01); the high-dose group of toxocarbamol reduced the expression of IL-6 mRNA (P<0.01); and the various groups of toxocarbamol significantly reduced the expression of Ly6G, MPO, NE, Cit-H3, and PAD4 mRNA expression (P< 0.01); decreased Ly6G, MPO, NE, and Cit-H3 expression in lung tissue sections (P< 0.05 or P< 0.01); decreased expression of NETs in lung tissue sections(P< 0.01); toxocarbophilus high dose group decreased lung tissue p-MEK/MEK protein expression (P< 0.01); toxocarbophilus groups decreased lung tissue p-ERK/ERK ratio, PAD4, Cit-H3 protein expression (P< 0.05 or P<0.01). CONCLUSION: MEK/ERK promote the formation of NETs involved in the development of pneumonia. Toxin clear inhibits the formation of NETs through the regulation of MEK/ERK and then attenuates the inflammatory response in the lung.

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  • 收稿日期:2025-09-28
  • 最后修改日期:2026-01-20
  • 录用日期:2026-04-01
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