Protective effect and mechanism of mesenchymal stem cell-derived extracellular vesicle on radiation-induced liver injury
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摘要:
目的 探讨间充质干细胞来源的细胞外囊泡（MSC-EV）对照射诱导的小鼠放射性肝损伤和肝细胞系损伤的保护作用及相关机制。 方法 分别将C57BL/6小鼠随机分为空白组、造模组和MSC-EV治疗组（治疗组），每组9只；将AML12细胞随机分为对照组、照射组和MSC-EV干预组（干预组）。分别通过一次性15 Gy和6 Gy X线照射建立动物和细胞放射性损伤模型，照射后48 h取小鼠的肝组织与血清进行相关实验，照射后15 h对细胞进行相关检测。检测血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）水平，肝组织和细胞丙二醛（MDA）含量，实时荧光定量逆转录聚合酶链反应（RT-qPCR）检测白细胞介素（IL）-1β、IL-6、转化生长因子（TGF）-β、CXC趋化因子配体（CXCL）10信使RNA（mRNA）相对表达量，肝组织苏木素-伊红（HE）染色进行肝组织病理损伤评分，脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记（TUNEL）染色法、碘化丙啶（PI）染色法分别检测肝组织、细胞凋亡情况，蛋白质印迹法测定谷胱甘肽过氧化酶（GPX）4、铁死亡抑制蛋白（FSP）1的蛋白表达情况，二氢乙啶（DHE）染色检测活性氧簇（ROS）产生水平，并检测线粒体通透性转换孔（mPTP）荧光强度。 结果 与空白组比较，造模组小鼠血清AST、ALT水平均增高，小鼠肝组织IL-1β、TGF-β和CXCL10 mRNA相对表达量均增加，MDA含量增加，肝损伤评分升高，细胞凋亡率增加，细胞内ROS水平升高，小鼠肝组织GPX4、FSP1蛋白相对表达量下降，差异均有统计学意义（均为P<0.05）；与造模组比较，治疗组小鼠血清AST、ALT水平均降低，小鼠肝组织IL-1β、TGF-β和CXCL10 mRNA相对表达量均下降，MDA含量减少，肝损伤评分降低，细胞凋亡率下降，细胞内ROS水平降低，小鼠肝组织GPX4、FSP1蛋白相对表达量增加，差异均有统计学意义（均为P<0.05）。与对照组比较，照射组细胞凋亡率增加，细胞内ROS水平升高，mPTP荧光强度减弱，IL-1β、TGF-β、IL-6的mRNA相对表达量均增加，MDA含量增加，GPX4、FSP1的蛋白相对表达量均下降，差异均有统计学意义（均为P<0.05）；与照射组比较，干预组细胞凋亡率降低，细胞内ROS水平减少，mPTP荧光强度增强，IL-1β、TGF-β、IL-6的mRNA相对表达量均降低，MDA含量减少，GPX4、FSP1的蛋白相对表达量均增加，差异均有统计学意义（均为P<0.05）。 结论 MSC-EV可通过减少肝细胞铁死亡、提高抗氧化水平、减少脂质过氧化物的产生，从而有效缓解照射导致的放射性肝损伤。 Abstract:Objective To evaluate the protective effect and the underlying mechanism of mesenchymal stem cell-derived extracellular vesicle (MSC-EV) on radiation-induced liver injury and liver cell line injury in mouse models. Methods C57BL/6 mice were randomly divided into the blank group, model group and MSC-EV treatment group (treatment group), with 9 mice in each group. AML12 cells were randomly divided into the control group, irradiation group and MSC-EV intervention group (intervention group). Animal and cell models with radiation-induced injury were established by one-time 15 Gy and 6 Gy X-ray irradiation, respectively. At 48 h after irradiation, liver tissues and serum samples of mice were collected and prepared for subsequent experiments. At 15 h post-irradiation, cell experiment was carried out. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and content of malondialdehyde (MDA) in liver tissues and cells were measured. The relative expression levels of interleukin (IL)-1β, IL-6, transforming growth factor (TGF)-β and CXC chemokine ligand (CXCL)10 messenger RNA (mRNA) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). Liver tissues were prepared for hematoxylin-eosin (HE) staining to calculate liver pathological injury score. The apoptosis of liver tissues and cells was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and propidiumiodide (PI) staining, respectively. The expression levels of glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) proteins were detected by Western blot. The production level of reactive oxygen species (ROS) was detected by dihydroethidine (DHE) staining. The fluorescence intensity of mitochondrial permeability transition pore (mPTP) was determined. Results Compared with the blank group, serum levels of AST and ALT were up-regulated, and the relative expression levels of IL-1β, TGF-β and CXCL10 mRNA in the mouse liver tissues were up-regulated, and MDA content was increased, liver injury score was elevated, cell apoptosis rate was increased, intracellular ROS level was elevated, and the relative expression levels of GPX4 and FSP1 proteins in the mouse liver tissues were down-regulated in the model group, and the differences were statistically significant (all P<0.05). Compared with the model group, serum levels of AST and ALT were decreased, and the relative expression levels of IL-1β, TGF-β and CXCL10 mRNA in the liver tissues of mice were down-regulated, MDA content was declined, liver injury score was declined, cell apoptosis rate was decreased, intracellular ROS level was decreased, and the relative expression levels of GPX4 and FSP1 proteins in the liver tissues of mice were up-regulated in the treatment group, and the differences were statistically significant (all P<0.05). Compared with the control group, cell apoptosis rate was increased, intracellular ROS level was elevated, the fluorescence intensity of mPTP was weakened, the relative expression levels of IL-1β, TGF-β and IL-6 mRNA were up-regulated, MDA content was increased, and the relative expression levels of GPX4 and FSP1 proteins were down-regulated in the irradiation group, and the differences were statistically significant (all P<0.05). Compared with the irradiation group, cell apoptosis rate was declined, intracellular ROS level was decreased, the fluorescence intensity of mPTP was strengthened, the relative expression levels of IL-1β, TGF-β and IL-6 mRNA were down-regulated, MDA content was decreased and the relative expression levels of GPX4 and FSP1 proteins were up-regulated in the intervention group, and the differences were statistically significant (all P<0.05). Conclusions MSC-EV may effectively alleviate radiation-induced liver injury by reducing ferroptosis of liver cells, enhancing antioxidant level and decreasing the production of lipid peroxide, thereby effectively alleviating radiation-induced liver injury. -
图 1 MSC-EV对小鼠肝脏放射性损伤的保护作用
注：A图为各组小鼠血清AST、ALT水平；B图为各组小鼠肝组织的IL-1β、CXCL10、TGF-β mRNA相对表达量；C图为各组小鼠肝组织MDA含量；D、E图为各组小鼠肝组织HE染色结果（×400）和肝损伤评分；F、H图为各组小鼠肝组织TUNEL染色结果（×100）及细胞凋亡情况；G、I图为各组小鼠肝脏DHE染色结果（×200）及ROS水平；J图为蛋白质印迹法测定各组小鼠肝组织GPX4和FSP1蛋白表达。与空白组比较，aP<0.05；与造模组比较，bP<0.05。
Figure 1. Protective effect of MSC-EV on liver radiation injury in mice
图 2 MSC-EV对AML12细胞放射性损伤的保护作用
注：A、C图为各组AML12细胞PI染色结果（×200）及细胞凋亡情况；B、D图为各组AML12细胞DHE染色结果（×200）及ROS水平；E、F图为各组AML12细胞mPTP染色结果（×200）；G图为各组AML12细胞IL-1β、TGF-β和IL-6的mRNA相对表达量；H图为各组AML12细胞MDA含量；I图为蛋白质印迹法检测各组AML12细胞GPX4、FSP的蛋白表达。与对照组比较，aP<0.05；与照射组比较，bP<0.05。
Figure 2. Protective effect of MSC-EV on liver radiation injury in AML12 cell
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