间充质干细胞来源的外泌体通过抑制核因子κB信号通路减轻放射性肺纤维化的机制研究

Mesenchymal stem cells-derived exosomes attenuate radiation-induced pulmonary fibrosis by inhibiting the NF-κB signaling pathway

目的探讨间充质干细胞来源的外泌体(MSCs-EXO)对放射性肺纤维化(RIPF)的作用机制。方法将15只雄性SD大鼠随机分为对照组、放射(irradiation,IR)组、IR+MSCs-EXO(IR+EXO)组,每组5只。IR组及IR+EXO组大鼠右肺给予单次照射,剂量为30 Gy。IR+EXO组分别于照射后1 h、14 d两个时间点,采用尾静脉推注方式给予每千克1×109个的外泌体;对照组和IR组大鼠同步尾静脉推注等体积的生理盐水。于照射后12周处死各组大鼠,收集大鼠右侧新鲜肺组织。采用HE和Masson染色检测肺组织病理损伤和胶原纤维沉积情况,采用免疫组织化学染色和RT-qPCR检测肺组织中纤维化相关分子α平滑肌肌动蛋白(α-SMA)和I型胶原蛋白alpha 1链(COL1A1)的表达,采用Western blot检测肺组织中核因子κB(NF-κB)信号通路相关分子表达。结果照射后12周,与IR组相比,IR+EXO组大鼠肺组织病理受损明显减轻,Ashcroft score评分明显降低(P<0.001);IR+EXO组肺组织中胶原纤维沉积明显减少,胶原容积分数显著低于IR组,差异有统计学意义(P<0.001);IR+EXO组中α-SMA和COL1A1蛋白阳性表达水平明显低于IR组,α-SMA和COL1A1 mRNA表达水平也均明显低于IR组,差异有统计学意义(P<0.01);与IR组相比,IR+EXO组p-NF-κB p65(Ser536)蛋白表达明显下调,差异有统计学意义(P<0.01),α-SMA和COL1A1蛋白表达也明显下调,差异有统计学意义(P<0.01)。结论MSCs-EXO可能通过抑制NF-κB信号通路减轻放射性肺纤维化。

Objective To explore the effect and potential mechanism of mesenchymal stem cells-derived exosomes(MSCs-EXO)on radiation-induced pulmonary fibrosis(RIPF).Methods Human umbilical cord derived MSCs were isolated and cultured,and their exosomes(EXO)were collected.Exosomes were identified by western blot,nanoparticle tracking analysis and transmission electron microscopy.Fifteen SD rats were randomly divided into control group,irradiation(IR)group and IR+MSCs-EXO(IR+EXO)group,with five rats in each group.The rats in IR group and IR+EXO group were given a single dose of 30 Gy to the right lung.At 1 h and 14 d after irradiation,1×109 exosomes/kg were injected via tail vein in IR+EXO group.Rats in the control group and IR group were simultaneously injected with equal volume of normal saline via tail vein.The rats in each group were sacrificed at 12th week after irradiation,and the fresh lung tissues on the right side of the rats were collected.He and Masson staining were used to detect the pathological damage and collagen fiber deposition of lung tissue.Immunohistochemical staining and RT-qPCR were used to detect the expression of fibrosis related moleculesα-SMA and COL1A1 in lung tissue.Western blot was used to detect the expression of NF-κB signaling pathway related molecules in lung tissue.Results At 12th week after irradiation,compared with IR group,the pathological damage of lung tissue in IR+EXO group was significantly reduced,and the Ashcroft score was significantly lower than that in IR group(P<0.001).The deposition of collagen fibers in lung tissue of IR+EXO was significantly reduced,and the collagen volume fraction was significantly lower than that of IR group(P<0.001).The positive expression levels ofα-SMA and COL1A1 proteins in the IR+EXO group were significantly lower than those in the IR group,and the mRNA expression levels ofα-SMA and COL1A1 were also significantly lower than those in the IR group(P<0.01).Compared with the IR group,the p-NF-κB p65(Ser536)protein expression in the IR+EXO group was significantly downregulated(P<0.01),the expression ofα-SMA and COL1A1 proteins was also significantly downregulated(P<0.01).Conclusion MSCs-EXO may alleviate radiation-induced pulmonary fibrosis by inhibiting NF-κB signaling pathway.