摘要
目的:探讨鹿鹅鼻炎方含药血清通过miR-149-5p靶向调控Notch信号通路调节小鼠鼻黏膜内皮细胞炎症反应干预变应性鼻炎(AR)的机制。方法:通过细胞增殖实验筛选出鹿鹅鼻炎方含药血清最佳浓度作为后续实验的处理因素;将细胞分为空白组、模型组、鹿鹅鼻炎方含药血清组、miR-149-5p模拟组(miR-149-5p agomir组)、miR-149-5p对照组(agomir-NC组)、miR-149-5p抑制剂组(miR-149-5p inhibitor组)、miR-149-5p抑制剂对照组(inhibitor-NC组)。采用50 ng/mL IL-13诱导小鼠鼻黏膜内皮细胞炎症反应,构建AR细胞模型。采用ELISA法检测细胞培养基上清液中IL-17、IL-10、TGF-β的含量;Western blot法检测细胞中Foxp3、RORγt、Notch1、Hes-1蛋白水平;qPCR法检测细胞中Foxp3、RORγt、Notch1、Hes-1mRNA表达。结果:与模型组比较,鹿鹅鼻炎方含药血清组IL-10、TNF-β1水平及Foxp3蛋白表达显著上升,Foxp3 mRNA表达无明显差异,但有升高趋势,IL-17,RORγt、Notch1、Hes-1蛋白及mRNA水平显著下降(P<0.01);与模型组比较,miR149-5p agomir组的IL-10、TNF-β1,Foxp3蛋白及mRNA表达显著上升,IL-17,RORγt、Notch1、Hes-1蛋白及mRNA水平显著下降(P<0.01)。鹿鹅鼻炎方可上调miR-149-5p表达,抑制Notch通路相关蛋白Notch1、Hes-1表达,下调Th17细胞主转录因子RORγt的表达水平,减少Th17标志性细胞因子IL-17生成,抑制Th17细胞分化,上调Treg细胞主转录因子Foxp3的表达水平,增加Treg细胞因子IL-10、TNF-β1的生成,促进Treg细胞分化,恢复Th17/Treg免疫平衡。结论:鹿鹅鼻炎方可通过上调miR-149-5p表达从而抑制Notch通路进而恢复Th17/Treg免疫失衡发挥治疗AR的作用。
Objective:To explore the mechanism of Lu'e Rhinitis Formula drug-containing serum intervene on nasal mucosal endothelial cells of mice with allergic rhinitis(AR)by miR-149-5p targering Notch signaling pathway.Methods:Through cell proliferation experiment,the optimal concentration of medicated serum of Lu'e Rhinitis Formula was selected as the treatment factor for the subsequent experiment.The cells were divided into blank group,model group,Lu'e Rhinitis Formula medicated serum group,miR-149-5p simulated group(mmu-miR-149-5p agomir)and miR-149-5p control group(agomir-NC),miR-149-5p inhibitor group(mmu-miR-149-5p inhibitor),miR-149-5p inhibitor control group(invitor-NC).50 ng/mL IL-13 was used to induce the inflammatory response of mice nasal mucosal endothelial cells,and the cell model of allergic rhinitis was established.The contents of IL-17,IL-10 and TGF-βin the supernatant of cell culture medium were detected by ELISA.The protein levels of Foxp3,RORyt,Notch1 and Hes-1 were detected by Western blot.The mRNA expressions of Foxp3,RORyt,Notch1 and Hes-1 were detected by qPCR.Results:Compared with the model group,treatment with Lu'e Rhinitis Formula containing serum resulted in a significant upregulation of levels of IL-10,TGF-β1,and Foxp3 protein expression.Although Foxp3 mRNA expression did not reach statistical significance,it showed a clear upward trend.Additionally,this treatment led to a significant downregulation of IL-17,RORγt,Notch1 and Hes-1 proteins and mRNA(P<0.01).Similarly,the miR-149-5p agomir group demonstrated significantly increased expression of IL-10,TGF-β1,and Foxp3 at both protein and mRNA levels,along with a significant reduction in IL-17,RORγt,Notch1 and Hes-1 proteins and mRNA,relative to the model group(P<0.01).Lu'e Rhinitis Formula can up-regulate the expression of miR-149-5p,inhibit the expression of Notch pathway related proteins Notchl and Hes-1,down-regulate the expression level of Thl7 cell main transcription factor RORγt,reduce the production of Th17 signature cytokine IL-17,and inhibit Thl7 cell differentiation.Up-regulated the expression level of main transcription factor Foxp3 in Treg cells,increased the production of Treg cytokine IL-10,promoted the differentiation of Treg cells,and restored Thl7/Treg immune balance Conclusion:The mechanism of Lu'e Rhinitis Formula intervention in AR may be achieved by up-regulating the expression of miR-149-5p,inhibiting the expression of Notch pathway and regulating Th17/Treg immune homeostasis.
作者
贾明月
张美怡
肖锶瑶
于洋
邵翔
韩桂玲
JIA Mingyue;ZHANG Meiyi;XIAO Siyao;YU Yang;SHAO Xiang;HAN Guiling(Department of Integrated Traditional Chinese and Western Medicine for Pulmonary Diseases Part 1,National Center for Integrative Medicine,National Center for Respiratory Medicine,State Key Laboratory of Respiratory Health and Multimorbidity,National Clinical Research Center for Respiratory Diseases,Institute of Respiratory Medicine,Chinese Academy of Medical Sciences,Center of Respiratory Medicine,China-Japan Friendship Hospital,Beijing 100029,China;Graduate School,Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中华中医药杂志》
北大核心
2025年第11期5379-5384,共6页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金青年科学基金项目(No.82104760)
中华中医药学会青年求实项目(No.2022-QNQSDEP-09)
中日友好医院第二批菁英计划骨干人才项目(No.ZRJY2023-GG05)
中央高水平医院临床科研业务费资助项目(No.2022-NHLHCRFLX-01-0308)。
