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

UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway. 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。

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.

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.

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.