康复新液通过miR-449a/Notch1轴改善脂多糖诱导的口腔黏膜上皮细胞炎症损伤*

doi:10.3870/j.issn.1672-0741.25.10.008

摘要/Abstract

摘要： 目的探究康复新液(KFX)对脂多糖(LPS)诱导的口腔黏膜上皮细胞(OMECs)炎症损伤的影响及其机制。方法体外分离、鉴定、培养小鼠口腔黏膜上皮细胞(mOMECs),以不同稀释倍数(0、0.078%、0.156%、0.313%、0.625%、1.25%、2.5%、5%、10%)KFX及不同浓度(0、0.1、1、10 μg/mL)LPS分别处理mOMECs 48 h,采用CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡水平,确定KFX和LPS干预的最佳浓度。将mOMECs分为对照组(Control)、LPS组(1 μg/mL LPS)、KFX组(0.625% KFX)、KFX+抑制剂对照组(KFX+inhibitor-NC)、KFX+miR-449a抑制剂组(KFX+miR-449a inhibitor)。采用CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡水平,ELISA检测细胞培养上清中TNF-α和IL-1β水平,qRT-PCR检测细胞中miR-449a和Notch1 mRNA表达水平,Western blot检测细胞中Notch1、cleaved-Caspase-3、Bcl-2和Bax蛋白表达水平。通过生物信息学方法预测miR-449a与Notch1的靶向结合位点,双荧光素酶报告基因实验、qRT-PCR和Western blot验证miR-449a与Notch1的靶向调控关系。结果与Control组比较,LPS组细胞增殖活性、miR-449a和Bcl-2蛋白表达水平降低(均P<0.05),细胞凋亡率,TNF-α和IL-1β水平、Notch1 mRNA及Notch1、cleaved-Caspase-3、Bax蛋白表达水平升高(均P<0.05);与LPS组比较,KFX组细胞增殖活性、miR-449a表达水平和Bcl-2蛋白表达水平升高(P<0.05),细胞凋亡率,TNF-α和IL-1β水平,Notch1 mRNA及Notch1、cleaved-Caspase-3、Bax蛋白表达水平降低(均P<0.05);与KFX组比较,KFX+miR-449a inhibitor组上述指标变化趋势与之相反(均P<0.05),而KFX+inhibitor-NC组无显著变化(P>0.05)。双荧光素酶报告基因实验、qRT-PCR和Western blot结果表明,miR-449a靶向抑制Notch1表达。结论 KFX可能通过miR-449a/Notch1轴改善LPS诱导的mOMECs炎症损伤。

关键词: 康复新液, miR-449a/Notch1轴, 脂多糖, 口腔黏膜上皮细胞, 炎症损伤

Abstract: Objective To investigate the effect of Kangfuxin liquid(KFX)on lipopolysaccharide(LPS)-induced inflammatory injury in oral mucosal epithelial cells(OMECs)and its underlying mechanism. Methods Mouse OMECs(mOMECs)were isolated,identified,and cultured ex vivo,then treated with various concentrations of KFX(0,0.078%,0.156%,0.313%,0.625%,1.25%,2.5%,5%,10%)or LPS(0,0.1,1,10 μg/mL)for 48 h.Cell proliferation and apoptosis were assessed by CCK-8 assay and flow cytometry,respectively,to determine the optimal concentrations of KFX and LPS for intervention.The mOMECs were then divided into the following groups:Control,LPS(1 μg/mL LPS),KFX(0.625% KFX),KFX+negative control inhibitor(KFX+inhibitor-NC),and KFX+miR-449a inhibitor.Cell proliferation was assessed by CCK-8 assay,and apoptosis by flow cytometry.The levels of TNF-α and IL-1β in the supernatant were measured by ELISA.The expression of miR-449a and Notch1 mRNA was determined by qRT-PCR.The protein expression levels of Notch1,cleaved-Caspase-3,Bcl-2,and Bax were detected by Western blotting.Potential binding sites between miR-449a and Notch1 were predicted using bioinformatics tools.The targeting relationship between miR-449a and Notch1 was verified by dual-luciferase reporter assay,qRT-PCR,and Western blotting. Results Compared with the Control group,the LPS group showed significantly decreased cell proliferation,miR-449a expression,and Bcl-2 protein levels(all P<0.05),along with increased apoptosis,TNF-α and IL-1β levels,Notch1 mRNA expression,and Notch1,cleaved-Caspase-3,and Bax protein levels(all P<0.05).Compared with the LPS group,the KFX group exhibited increased cell proliferation,miR-449a expression,and Bcl-2 protein levels(all P<0.05),and decreased apoptosis,TNF-α and IL-1β levels,Notch1 mRNA expression,and Notch1,cleaved-Caspase-3,and Bax protein levels(all P<0.05).Compared with the KFX group,the KFX+miR-449a inhibitor group showed reversed trends in all aforementioned indicators(all P<0.05),whereas no significant changes were observed in the KFX+inhibitor-NC group(P>0.05).Dual-luciferase reporter assay,qRT-PCR,and Western blotting confirmed that miR-449a targeted and inhibited Notch1 expression. Conclusion KFX may ameliorate LPS-induced inflammatory injury in mOMECs via the miR-449a/Notch1 axis.

Key words: Kangfuxin liquid, miR-449a/Notch1 axis, lipopolysaccharide, oral mucosal epithelial cells, inflammatory injury

中图分类号:

R781.05

王钊鑫, 向国林. 康复新液通过miR-449a/Notch1轴改善脂多糖诱导的口腔黏膜上皮细胞炎症损伤^*[J]. 华中科技大学学报（医学版）, 2026, 55(2): 226-234.

Wang Zhaoxin, Xiang Guolin. Kangfuxin Liquid Ameliorates LPS-induced Inflammatory Injury in Oral Mucosal Epithelial Cells via the miR-449a/Notch1 Axis[J]. Acta Medicinae Universitatis Scientiae et Technologiae Huazhong, 2026, 55(2): 226-234.

参考文献

[1] Randall D A,Wilson Westmark N L,Neville B W.Common oral lesions[J].Am Fam Physician,2022,105(4):369-376.
[2] Philipone E M,Peters S M.Ulcerative and inflammatory lesions of the oral mucosa[J].Oral Maxillofac Surg Clin North Am,2023,35(2):219-226.
[3] 龙厚宁,杨璟,沈巧琳.康复新液药理作用与临床应用及安全性研究进展[J].临床合理用药,2024,17(13):173-178.
Long H N,Yang J,Shen Q L.Research progress on pharmacological action,clinical application and safety of Kangfuxin liquid[J].Chinese Journal of Clinical Rational Drug Use,2024,17(13):173-178.
[4] Shen L,Chen X,Zhang T.Assessment of the anti-inflammatory efficacy and symptom resolution time of kangfuxin liquid combined with metronidazole in recurrent oral ulcers[J].J Inflamm Res,2025,18:11925-11934.
[5] Shang R,Lee S,Senavirathne G,et al.microRNAs in action:Biogenesis,function and regulation[J].Nat Rev Genet,2023,24(12):816-833.
[6] Barati T,Mirzaei Z,Ebrahimi A,et al.miR-449a:A promising biomarker and therapeutic target in cancer and other diseases[J].Cell Biochem Biophys,2024,82(3):1629-1650.
[7] 卢静,李建红,彭巍.过表达miR-449a对溃疡性结肠炎大鼠肠黏膜的保护作用及机制研究[J].局解手术学杂志,2021,30(10):835-841.
Lu J,Li J H,Peng W.Protective effect and mechanism of overexpression of miR-449a on intestinal mucosa of rats with ulcerative colitis[J].Journal of Regional Anatomy and Operative Surgery,2021,30(10):835-841.
[8] Cai Y,Jiang C,Zhu J,et al.miR-449a inhibits cell proliferation,migration,and inflammation by regulating high-mobility group box protein 1 and forms a mutual inhibition loop with Yin Yang 1 in rheumatoid arthritis fibroblast-like synoviocytes[J].Arthritis Res Ther,2019,21(1):134.
[9] 李蓉,富国文,朱红林,等.山羊原代口腔上皮细胞的分离培养及鉴定[J].动物医学进展,2025,46(4):72-77.
Li R,Fu G W,Zhu H L,et al.Isolation,culture and identification of primary oral epithelial cells of goats[J].Progress in Veterinary Medicine,2025,46(4):72-77.
[10] Jiang S,Li H,Zhang L,et al.Generic diagramming platform(GDP):A comprehensive database of high-quality biomedical graphics[J].Nucleic Acids Res,2025,53(D1):D1670-D1676.
[11] Zou L F,Dong Y,Xu N S,et al.Clinical observation and experimental study on Kangfuxin fluid in treating indwelling needle-related phlebitis[J].TMR Mod Herb Med,2022,5(4):21.
[12] 王钦,刘克娜,孔彩华,等.美洲大蠊有效成分的提取及药理活性研究进展[J].中华中医药学刊,2021,39(8):108-111.
Wang Q,Liu K N,Kong C H,et al.Research progress on extraction and pharmacological activities of effective constituents from Meizhou Dalian(periplaneta Americana)[J].Chinese Archives of Traditional Chinese Medicine,2021,39(8):108-111.
[13] Zeng C,Liao Q,Hu Y,et al.The role of periplaneta Americana(blattodea:Blattidae)in modern versus traditional Chinese medicine[J].J Med Entomol,2019,56(6):1522-1526.
[14] Sun H,Yang C,Liu X,et al.Effectiveness of negative pressure wound therapy of diabetic foot ulcers using periplaneta Americana(kangfuxin liquid)irrigation[J].Int J Low Extrem Wounds,2025,24(4):1165-1172.
[15] 秦爱丽,郑蕾,蒋海晓,等.康复新液治疗复发性口腔溃疡患儿的临床效果研究[J].中华全科医学,2020,18(9):1516-1518,1522.
Qin A L,Zheng L,Jiang H X,et al.Clinical effect of kangfuxin liquid on children with recurrent oral ulcer[J].Chinese Journal of General Practice,2020,18(9):1516-1518,1522.
[16] Jiang L,Zhu J.Review of MiRNA-disease association prediction[J].Curr Protein Pept Sci,2020,21(11):1044-1053.
[17] Chen Q,Yang Z,Pan G,et al.Tumor suppressor miR-449a inhibits the development of gastric cancer via down-regulation of SGPL1[J].RSC Adv,2018,8(46):26020-26028.
[18] Zhang J,Lu W W,Meng L P.miR-449a induces phenotypic transformation of vascular smooth muscle cells by targeting KLF4[J].Chin J Pathol,2020,49(2):180-182.
[19] Wang H,Wang F,Wang Y,et al.Study on the mechanism of BMSCs in regulating NF-κB signal pathway by targeting miR-449a to improve the inflammatory response to peripheral nerve injury[J].J Musculoskelet Neuronal Interact,2022,22(4):546-561.
[20] 牛春雪,马蕾,薛海波,等.Notch1信号通路参与自身免疫性疾病发病机制的研究进展[J].现代免疫学,2020,40(1):67-71.
Niu C X,Ma L,Xue H B,et al.Research progress of Notch1 signaling pathway involved in the pathogenesis of autoimmune diseases[J].Current Immunology,2020,40(1):67-71.
[21] Liu Z,Lei Y,Zuo J,et al.Activated Notch1 promotes macrophage polarization and exacerbates sepsis-induced acute lung injury via β-catenin/NF-κB signaling[J].Biochem Pharmacol,2025,236:116892.
[22] Ni X Q,Zhang Y R,Jia L X,et al.Inhibition of Notch1-mediated inflammation by intermedin protects against abdominal aortic aneurysm via PI3K/Akt signaling pathway[J].Aging,2021,13(4):5164-5184.
[23] Zhang Y,Liu J,Zhang D,et al.LianChuang ZhiXue liquid enema modulates the macrophage polarization of ulcerative colitis via inhibiting the jagged-1/Notch1 signaling pathway[J].Drug Des Devel Ther,2025,19:3253-3268.

康复新液通过miR-449a/Notch1轴改善脂多糖诱导的口腔黏膜上皮细胞炎症损伤^*

Kangfuxin Liquid Ameliorates LPS-induced Inflammatory Injury in Oral Mucosal Epithelial Cells via the miR-449a/Notch1 Axis

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 4

编辑推荐

Metrics

本文评价

[1]	陈匀, 庄晓磊, 肖玲玲, 骆菲, 汪吉梅. LncRNASNHG15通过靶向miR-942-5p调控脂多糖诱导的肺泡上皮细胞损伤[J]. 华中科技大学学报（医学版）, 2024, 53(4): 433-437.
[2]	谭媛, 廖勇杰, 岳嗣凤. 胆囊收缩素通过胆囊收缩素受体 A 减弱炎症反应降低脂多糖诱导的急性肺损伤[J]. 华中科技大学学报（医学版）, 2022, 51(5): 625-630.
[3]	张志发, 朱梦娇, 杨少兵, 陶怡芝, 王学仁△ . 右美托咪定对高迁移率族蛋白1诱导人脐静脉内皮细胞炎性反应的影响[J]. 华中科技大学学报（医学版）, 2022, 51(1): 38-43.
[4]	何琼, 包树臻, 王玉巧, 阚天燕, 张跃斌, 李艳△. 白藜芦醇通过调节 NADPH 氧化酶表达缓解脂多糖诱导的急性肺损伤[J]. 华中科技大学学报（医学版）, 2022, 51(1): 68-71.