目的:观察尿石素A(UA)对呼吸道合胞病毒(RSV)诱导的新生小鼠肺部感染的治疗作用并探讨其机制。
方法:Babl/c小鼠(5-7日龄)经鼻腔滴注RSV,并在感染2小时后腹膜内注射盐水或2.5、5和10mg/kgUA,然后每天一次,持续2周。然后收集支气管肺泡灌洗液(BALF)用于检测炎症细胞和介质,HE染色评价肺部病理。用2.5、5或10μmol/LUA处理RSV感染的BEAS-2B细胞。炎症因子,细胞活力,使用ELISA分析细胞凋亡和自噬,CCK-8测定,TUNEL染色,流式细胞术,Western印迹和免疫荧光染色。使用qRT-PCR检测miR-136和Sirt1mRNA的细胞表达。使用双荧光素酶报告系统来验证miR-136和Sirt1之间的结合。
结果:在新生Babl/c小鼠中,RSV感染引起明显的肺部病变,促进肺细胞凋亡与LC3-Ⅱ/Ⅰ,Beclin-1和miR-136表达,并增加了细胞总数,BALF中的炎症细胞和因子和p62和Sirt1表达降低。所有这些变化都被UA剂量依赖性地缓解。在BEAS-2B细胞中,RSV感染显著增加细胞凋亡,LC3B阳性细胞和miR-136表达及Sirt1表达降低(P<0.01),其被UA剂量依赖性地减毒。双荧光素酶报告基因测定证实了miR-136和Sirt1之间的结合。在用UA处理的RSV感染的BEAS-2B细胞中,过表达miR-136和Ex527处理均显著增加炎症因子和细胞凋亡,但降低LC3B表达,这些变化通过联合治疗得到了进一步增强。
结论:UA通过激活miR-136介导的Sirt1信号通路改善RSV诱导的新生小鼠肺部感染。
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.
CONCLUSIONS: UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway.