南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (7): 1370-1381.doi: 10.12122/j.issn.1673-4254.2024.07.17

• • 上一篇    

尿石素A通过活化miR-136介导的Sirt1信号通路减轻呼吸道合胞病毒诱导的新生小鼠肺部感染

王洪哲1(), 谢海棠1(), 徐乌兰1, 李明2   

  1. 1.内蒙古民族大学附属医院儿科,内蒙古 通辽 028000
    2.通辽市科尔沁区第四人民医院儿科,内蒙古 通辽 028000
  • 收稿日期:2024-04-16 出版日期:2024-07-20 发布日期:2024-07-25
  • 通讯作者: 谢海棠 E-mail:wanghongzhe66@163.com;15104758818@163.com
  • 作者简介:王洪哲,主治医师,E-mail: wanghongzhe66@163.com
  • 基金资助:
    内蒙古自治区高等学校科学研究项目(NJZY19151)

Urolithin A alleviates respiratory syncytial virus-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling

Hongzhe WANG1(), Haitang XIE1(), Wulan XU1, Ming Li2   

  1. 1.Department of Pediatrics, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000, China
    2.Department of Pediatrics, Fourth People's Hospital of Horqin District, Tongliao 028000, China
  • Received:2024-04-16 Online:2024-07-20 Published:2024-07-25
  • Contact: Haitang XIE E-mail:wanghongzhe66@163.com;15104758818@163.com

摘要:

目的 探讨尿石素A(UA)治疗呼吸道合胞病毒(RSV)引起的新生小鼠肺部感染的疗效及作用机制。 方法 将Babl/c小鼠(5~7 d)随机分为5组(n=10):正常对照(Control)组、RSV感染(RSV)组、低剂量UA(UA-L)组、中剂量UA(UA-M)组、高剂量UA(UA-H)组。BEAS-2B细胞同样分为Control组、RSV组、UA-L组、UA-M组和UA-H组。除正常对照组外,其余4组小鼠或细胞均给予RSV感染。RSV感染2 h后,UA-L组、UA-M组和UA-H组小鼠分别腹腔注射2.5、5和10 mg/kg的UA,1次/d,连续注射2周;UA-L组、UA-M组和UA-H组细胞分别用2.5、5和10 µmol/L的UA处理48 h。HE染色评价肺组织病理学改变。收集支气管肺泡灌洗液用于炎症细胞计数和炎症因子检测。炎症、细胞活力、凋亡和自噬分别采用酶联免疫吸附实验、CCK-8实验、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记实验、流式细胞术、Western blotting和免疫荧光染色检测。qRT-PCR检测miR-136和Sirt1 mRNA的表达,双荧光素酶报告基因系统验证miR-136和Sirt1的相互关系。 结果 与Control组相比,RSV感染组小鼠肺组织病理评分、病毒荷载量、TUNEL阳性细胞数、LC3-II/I、Beclin-1和miR-136表达及BALF中总细胞数、炎症细胞数和炎症因子含量显著升高(P<0.0001),而p62和Sirt1表达显著减少(P<0.0001);与RSV组相比,UA处理则呈剂量依赖性逆转上述检测指标值(P<0.05)。与Control组相比,RSV组BEAS-2B凋亡细胞数、LC3B阳性细胞数及miR-136表达明显增多,Sirt1表达减少(P<0.01);与RSV组相比,UA处理剂量依赖性地减弱上述指标的改变(P<0.01)。与miR-NC组相比,miR-136组Sirt1-WT的荧光素酶活性显著降低,Sirt1表达减少(P<0.001),Sirt1-MUT的荧光素酶活性不变(P>0.05)。与RSV+UA组相比,RSV+UA+miR-136组和RSV+UA+Ex527组炎症因子含量和凋亡细胞数明显增加(P<0.05),LC3B表达显著减少(P<0.0001);miR-136与Ex527共处理进一步改变上述指标值(P<0.05)。 结论 UA通过活化miR-136介导的Sirt1信号通路减轻RSV诱导的新生小鼠肺部感染。

关键词: 尿石素A, 呼吸道合胞病毒, miR-136, Sirt1

Abstract:

Objective To observe the therapeutic effects of urolithin A (UA) on respiratory syncytial virus (RSV)-induced lung infection in neonatal mice and explore the underlying mechanisms. Methods Babl/c mice (5-7 days old) were subjected to nasal instillation of RSV and received intraperitoneal injection of saline or 2.5, 5 and 10 mg/kg UA 2 h after the infection and then once daily for 2 weeks. Bronchoalveolar lavage fluid (BALF) was then collected for detection of inflammatory cells and mediators, and lung pathology was evaluated with HE staining. RSV-infected BEAS-2B cells were treated with 2.5, 5 or 10 µmol/L UA. Inflammatory factors, cell viability, apoptosis and autophagy were analyzed using ELISA, CCK-8 assay, TUNEL staining, flow cytometry, Western blotting and immunofluorescence staining. The cellular expressions of miR-136 and Sirt1 mRNAs were detected using qRT-PCR. A dual-luciferase reporter system was used to verify the binding between miR-136 and Sirt1. Results In neonatal Babl/c mice, RSV infection caused obvious lung pathologies, promoted pulmonary cell apoptosis and LC3-II/I, Beclin-1 and miR-136 expressions, and increased the total cell number, inflammatory cells and factors in the BALF and decreased p62 and Sirt1 expressions. All these changes were alleviated dose-dependently by UA. In BEAS-2B cells, RSV infection significantly increased cell apoptosis, LC3B-positive cells and miR-136 expression and reduced Sirt1 expression (P<0.01), which were dose-dependently attenuated by UA. Dual-luciferase reporter assay confirmed the binding between miR-136 and Sirt1. In RSV-infected BEAS-2B cells with UA treatment, overexpression of miR-136 and Ex527 treatment both significantly increased the inflammatory factors and cell apoptosis but decreased LC3B expression, and these changes were further enhanced by their combined treatment. Conclusion UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway.

Key words: urolithin A, respiratory syncytial virus, miR-136, Sirt1