骨髓间充质干细胞源外泌体对急性肺损伤大鼠肺泡巨噬细胞M1/M2极化的影响及其机制

Effect of bone marrow mesenchymal stem cell-derived exosomes on M1/M2 polarization of alveolar macrophages in rats with acute lung injury and its mechanism

目的:探讨大鼠骨髓间充质干细胞来源的外泌体(bone marrow mesenchymal stem cells derived exosomes,BMSCs-Exos)对脂多糖(LPS)诱导的急性肺损伤大鼠肺泡巨噬细胞NR8383 M1/M2极化的影响及其潜在的机制。方法:分离SD大鼠骨髓间充质干细胞并制备BMSCs-Exos。分别用0.1、1 mg/mL BMSCs-Exos预处理NR8383细胞1 h,再用1μg/mL LPS诱导48 h,采用ELISA和蛋白免疫印迹分别检测细胞上清液中炎性因子含量以及细胞内极化标志物蛋白表达。将NR8383细胞单独培养或与BMSCs共培养,经20μmol/L外泌体抑制剂GW4869处理并予以1μg/mL LPS诱导48 h,采用实时荧光定量PCR和蛋白免疫印迹分别检测细胞内miR-212-5p以及IL-4Rα和p-STAT6蛋白相对表达。生物信息学预测miR-212-5p与IL-4RαmRNA结合位点并通过荧光素酶实验验证。结果:成功制备BMSCs-Exos。ELISA和免疫印迹结果表明,1μg/mL LPS诱导48 h可促进NR8383细胞分泌炎性因子(TNF-α,IL-1β和IL-10)及M1极化,BMSCs-Exos预处理可明显降低LPS诱导的NR8383细胞培养上清液中炎性因子TNF-α和IL-1β含量,增加抗炎因子IL-10含量,同时抑制细胞M1极化并促进其M2极化,且1 mg/mL BMSCs-Exos明显强于0.1 mg/mL BMSCs-Exos。细胞共培养实验结果表明,1μg/mL LPS诱导48 h可增加NR8383细胞中miR-212-5p相对表达量以及IL-4Rα和p-STAT6蛋白表达,与BMSCs共培养可有效抑制LPS对上述指标的影响,但BMSCs的作用可被GW4869阻断。荧光素酶实验结果显示,miR-212-5p可与IL-4RαmRNA 3′UTR结合并促进其蛋白表达。结论:BMSCs-Exos可能通过miR-212-5p/IL-4Rα/STAT6通路抑制LPS诱导的急性肺损伤大鼠肺泡巨噬细胞NR8383的M1极化并促进其M2极化。

Objective:To investigate the effect of bone marrow mesenchymal stem cell-derived exosomes(BMSCs-Exos)on the M1/M2 polarization of alveolar macrophages NR8383 in rats with lipopolysaccharide(LPS)-induced acute lung injury and its potential mechanism.Methods:Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley(SD)rats,and BMSCs-Exos were prepared.NR8383 cells were pretreated with 0.1 or 1 mg/mL BMSCs-Exos for 1 h,followed by induction with 1μg/mL LPS for 48 h,respectively.ELISA and Western blotting were used to detect the inflammatory factor levels in the cell supernatant and the expression of intracellular polarization marker proteins,respectively.NR8383 cells were cultured alone or co-cultured with BMSCs,treated with 20μmol/L exosome inhibitor GW4869,and induced with 1μg/mL LPS for 48 h.Quantitative real-time PCR(qRT-PCR)and Western blotting were used to detect the intracellular miR-212-5p expression and the relative expression of IL-4Rαand p-STAT6 proteins,respectively.The binding site of miR-212-5p and IL-4RαmRNA were predicted by bioinformatics and verified by luciferase assay.Results:BMSCs-Exos were successfully prepared.ELISA and Western blotting results showed that 1μg/mL LPS induced for 48 h promoted the secretion of inflammatory factors(TNF-α,IL-1β,and IL-10)and M1 polarization of NR8383 cells.Pretreatment with BMSCs-Exos significantly reduced the contents of LPS-induced inflammatory factors TNF-αand IL-1β,upregulated the content of anti-inflammatory factor IL-10,inhibited M1 polarization and promoted M2 polarization of NR8383 cells,and the effect of 1 mg/mL BMSCs-Exos was significantly stronger than that of 0.1 mg/mL BMSCs-Exos.The cell co-culture experiment results showed that 1μg/mL LPS induced for 48 h increased miR-212-5p levels and IL-4Rαand p-STAT6 protein expression in NR8383 cells.Co-culture with BMSCs greatly inhibited the effects of LPS on the above indicators,however the effect of BMSCs could be blocked by GW4869.Luciferase assay results indicated that miR-212-5p could bind to the 3′UTR of IL-4RαmRNA and promote its protein expression.Conclusion:BMSCs-Exos could inhibit M1 polarization and promote M2 polarization of LPS-induced rat alveolar macrophage NR8383 through the miR-212-5p/IL-4Rα/STAT6 pathway.