NO诱导富含miR-126的胎盘间充质干细胞外泌体治疗大鼠脓毒症

NO-induced miR-126-rich placental mesenchymal stem cell exosomes to treat sepsis in rats

目的探索NO诱导的胎盘间充质干细胞(PMSCs)外泌体对脓毒症大鼠的治疗效果及药理机制。方法分别利用NO、miR-126抑制剂、无效对照抑制剂对PMSCs进行诱导培养,超速离心获取3种外泌体(N-Exo、I-Exo和Exo)。利用透射电镜观察外泌体形态,qPCR检测miR-126含量。取清洁级SD大鼠160只,等分成4组。对照组(假手术+PBS)、脓毒症组(CLP+PBS)、N-Exo组(CLP+N-Exo)、I-Exo组(CLP+I-Exo)。利用盲肠结扎穿孔术(CLP)构建大鼠脓毒症模型。检测各组大鼠术后48h血生化指标(血清ALT、AST、BUN、IL-6、IL-10、TNF-α、IFN-γ、IL-1β含量);取肺和肾,HE染色进行病理评分、测量血管微渗漏指数和干/湿重之比;Westernblot检测肾脏内高迁移率族蛋白B1(HMGB1)、晚期糖基化终产物受体(RAGE)、Toll样受体2(TRL-2)、TRL-4、p-p65蛋白含量、qPCR测量miR-126-3p和miR-126-5p的含量;最终观察各组大鼠7d存活率。结果①miR-126含量:N-Exo>Exo>I-Exo(P<0.05);②7d存活率:脓毒症组和I-Exo组显著低于N-Exo组和对照组(P<0.05);③肝肾功能及炎症因子含量、肺和肾病理评分、血管微渗漏及干/湿重之比:脓毒症组和I-Exo组显著高于N-Exo组和对照组(P<0.05);④肾脏内miR-126-3p、miR-126-5p及HMGB1、RAGE、TRL-2、TRL-4、p-p65蛋白含量:脓毒症组和I-Exo组显著低于N-Exo组和对照组(P<0.05)。结论N-Exo可能是通过miR-126-HMGB1-NF-κB来抑制炎症反应,保护血管内皮细胞,最终提高脓毒症大鼠的生存率。

Objective To investigate the therapeutic effect of NO-induced placental mesenchymal stem cell (PMSC) exosomes in septic rats and its pharmacological mechanism. Methods PMSCs were induced by NO,miR-126 inhibitor and null control inhibitor,and three exosomes (N-Exo,I-Exo and Exo) were obtained by ultracentrifugation.The morphology of exosomes was observed by transmission electron microscope,and the content of miR-126 was detected by qPCR.160 clean grade SD rats were taken and divided into 4 groups.Control group (sham operation+PBS),sepsis group (CLP+PBS),N-Exo group (CLP+N-Exo),I-Exo group (CLP+I-Exo).A rat sepsis model was constructed using cecal ligation and puncture(CLP).Blood biochemical parameters (serum ALT,AST,BUN,IL-6,IL-10,TNF-α,IFN-γ,IL-1β content) were measured at 48 h after operation in each group.Taking lung and kidney,HE staining for pathological score,measuring the ratio of vascular microleakage index and dry/wet weight;Western blot was used to analyze high mobility group box-1 protein (HMGB1),advanced glycation end product receptor (RAGE),Toll-like receptor 2 (TRL-2),TRL-4,p-p65 protein content,qPCR measurement the content of miR-126-3p and miR-126-5p;Finally,the survival rate of rats in each group was observed at 7 days. Results ① miR-126 content: N-Exo>Exo>I-Exo ( P <0.05);② 7 d survival rate: the sepsis group and the I-Exo group were significantly lower than the N-Exo group and the control group ( P <0.05);③ The ratio of liver and kidney function and inflammatory factors,lung and renal pathological score,vascular microleakage and dry/wet weight: the sepsis group and the I-Exo group were significantly higher than the N-Exo group and the control group ( P <0.05);④ Contents of miR-126-3p,miR-126-5p and HMGB1,RAGE,TRL-2,TRL-4,and p-p65 in the kidney: the sepsis group and the I-Exo group were significantly lower than the N-Exo group and the control group ( P <0.05). Conclusion N-Exo may inhibit inflammatory response through miR-126-HMGB1-NF-κB,protect vascular endothelial cells,and ultimately improve the survival rate of septic rats.