炙甘草来源的sRNA可能通过靶向TNF-α缓解轻度ADRS模型小鼠的肺损伤

Baked licorice-derived sRNA alleviates lung injury mild ARDS mouse models possibly through targeting TNF-α

目的探索靶向TNF-α的小RNA(sRNA)在急性呼吸窘迫综合征(ARDS)中的影响和作用机制。方法以TNF-α为靶点,通过生物信息学方法从四逆汤水煎液sRNA数据库中筛选sRNAs;用双荧光素酶报告基因系统验证sRNAs靶向TNF-α的效果;将sRNA转染进细胞中,通过ELISA检测脂多糖(LPS)或poly(I:C)刺激的人肺腺癌细胞系A549的炎性因子IL-1β、IL-6和TNF-α;通过RT-qPCR检测LPS或poly(I:C)刺激的A549细胞炎性因子IL-1β、IL-6和TNF-α的mRNA水平;用LPS或poly(I:C)气管滴注构建小鼠轻度ARDS模型,将小鼠分为native组、模型组、NC组和sRNA治疗组,通过ELISA检测小鼠支气管肺泡灌洗液(BALF)和血清中炎性因子IL-1β、IL-6和TNF-α的浓度;观察轻度ARDS模型小鼠存活情况,统计分析小鼠的存活率。结果生物信息学分析筛选出50条可能与TNF-αmRNA结合的sRNA;双荧光素酶报告基因系统和人单核细胞白血病细胞系(THP1)炎性细胞模型筛选出1条能靶向TNF-α的TNF-α-sRNA-9,这条sRNA来源于四逆汤中的炙甘草;与NC组相比,TNF-α-sRNA-9降低了LPS或poly(I:C)刺激的A549细胞炎性因子IL-1β、IL-6和TNF-α水平(P<0.05),降低了mRNA水平(P<0.05);与NC组相比,TNF-α-sRNA-9的治疗降低了轻度ARDS模型小鼠的支气管肺泡灌洗液和血清中的IL-1β、IL-6和TNF-α水平(P<0.05),增加了小鼠的生存率。结论TNF-α-sRNA-9可能通过靶向TNF-α缓解轻度ARDS模型小鼠的肺损伤。

Objective To explore the effect and mechanism of small RNA(sRNA)targeting at TNF-α in acute respiratory distress syndrome(ARDS).Methods Taking TNF-α as the target,screen sRNA from the sRNA database of Sini decoction by bioinformatics methods.The effect of sRNA targeting at TNF-α was verified by dual-luciferase reportergene system;the sRNA was transfected into the cells,then IL-1β,IL-6 and TNF-α of LPS or poly(I:C)induced A549 cells were detected by ELISA;the mRNA level of IL-1β,IL-6 and TNF-α in lipopolysaccharide(LPS)or poly(I:C)induced A549 cells was detected by RT-qPCR;Mild ARDS mouse models was developed by intratracheal instillation of LPS or poly(I:C)in mice.The mice were divided into native group,model group,NC group and sRNA-treated group.The concentration of IL-1β,IL-6 and TNF-α in BALF and serum was detected by ELISA.The survival of mild ARDS mouse models was observed.The survival rate of mice was counted.Results Bioinformatic analysis identified TNF-α mRNA binding 50 sRNAs and the dual-luciferase reporter gene system and THP1 inflammatory cell model identified the TNF-α-sRNA-9 that targeted at TNF-α,which was derived from Baked licorice in Sini decoction;Compared with NC group,TNF-α-sRNA-9 decreased the level of IL-1β,IL-6 and TNF-α in LPS or poly(I:C)induced A549 cells(P<0.05),and decreased the mRNA level(P<0.05).Compared with NC group,TNF-α-sRNA-9 treatment decreased the level of IL-1β,IL-6,and TNF-α in BALF and serum of mild ARDS mouse models induced by LPS or poly(I:C)(P<0.05)and increased the survival rate of mice.Conclusions TNFα-sRNA-9 alleviates lung injury in mild ARDS mouse models with potential mechanism of targeting at TNF-α.