外泌体介导的辐射旁效应对心肌成纤维细胞的干预及黄芪甲苷的防护作用

Intervention of Exosome-Mediated Radiation Bystander Effect on Cardiac Fibroblasts and Protection of Astragaloside IV

为探讨放射性外泌体介导的辐射旁效应对心肌成纤维细胞(cardiac fibroblasts,CFs)的影响并观察黄芪甲苷(astragaloside IV,AST)的防护效应,该研究以2 Gy X线辐照大鼠CFs,48 h后超速离心提取放射性外泌体(X-exo),进行外泌体形态、浓度和表面标志蛋白鉴定。将CFs分为对照组(Control)、照射组(X-CFs)、CFs与放射性外泌体共培养组(X-exo+CFs)和黄芪甲苷干预组(X-exo+AST-CFs),采用流式细胞术检测各组细胞周期;划痕实验检测细胞迁移能力;Western blot检测纤维化相关分子TGF-β1、Col-I的表达情况。结果显示,X-CFs组和X-exo+CFs组在干预后24 h、48 h处于G/G期的细胞比例均低于Control组(P<0.01),S期和G/M期的细胞比例均高于Control组(P<0.01);X-CFs和X-exo+CFs组细胞迁移率在12 h、24 h、48 h时均显著高于Control组(P<0.05);在48 h时X-CFs组和X-exo+CFs组TGF-β1、Col-I蛋白表达量均高于Control组(P<0.01)。X-exo+AST-CFs组在干预后24 h、48 h时处于G/G期的细胞比例高于X-exo+CFs组(P<0.01),处于S期、G/M期的细胞比例均低于X-exo+CFs组(P<0.01);对比X-exo+CFs组,X-exo+AST-CFs组干预后,12 h、24 h、48 h细胞迁移率分别降低了62.6%、40.6%、41.2%(P<0.01),48 h时TGF-β1、Col-I蛋白表达量分别降低了15%、21.9%(P<0.01)。这说明X线诱导的外泌体(放射性外泌体)能促进共培养的CFs增殖、迁移、TGF-β1和Col-I高表达,且AST对这种放射性外泌体通过辐射旁效应引起的促纤维化有一定的抑制效应。

To investigate the effects of exosome-mediated radiation bystander on CFs (cardiac fibroblasts) and the intervention of AST (astragaloside IV),X-exo (radioactive exosomes) were extracted by overspeed centrifugation from rat CFs after 2 Gy X-ray irradiation.Then,the identification of exosome morphology,concentration and surface marker proteins was performed.CFs were divided into control group (Control),irradiation group (X-CFs),co-culture group of CFs and radioactive exosomes (X-exo+CFs),and AST intervention group (X-exo+AST-CFs).Flow cytometry was used to detect cell cycle;Scrape assay was used to detect CFs migration ability;Western blot was used to detect the expression of fibrosis-related molecules TGF-β1 and Col-I.The results showed that,compared with the Control group,the proportion of CFs in G/Gphase decreased in X-CFs and X-exo+CFs groups (P<0.01) at 24 h and 48 h after intervention,the proportion of cells in S and G/M phases increased (P<0.01);the cell mobility of X-CFs and X-exo+CFs groups increased at 12 h,24 h,48 h,respectively (P<0.05);the expression of TGF-β1 and Col-I protein in X-CFs groups and X-exo+CFs groups increased at 48 h (P<0.01).Compared with X-exo+CFs group,the proportion of cells in G/Gphase increased at 24 h and 48 h in X-exo+AST-CFs group (P<0.01),while S stage and G/M stage ratio decreased (P<0.01);the cell mobility of X-exo+AST-CFs group decreased by 62.6%,40.6%,and 41.2% at 12 h,24 h,and 48 h,respectively (P<0.01);TGF-β1 and Col-I protein expression at 48 h decreased by 15% and 21.9%,respectively (P<0.01).These aforementioned findings indicate that X-ray induced exosomes (radioactive exosomes) can promote the proliferation of CFs,enhance the migration ability of CFs and promote the high expression of fibrosis factors,and AST has some inhibitory effect on the profibrotic effect caused by this exosome-mediated radiation bystander effect.