Mechanism of human umbilical cord mesenchymal stem cell-derived exosomes against mouse renal ischemia/reperfusion injury

doi:10.12307/2025.048

Abstract

Abstract: BACKGROUND: Human umbilical cord mesenchymal stem cell-derived exosomes are involved in multiple injury repair processes, and the effects and the specific mechanisms of renal ischemia/reperfusion injury have not been fully elucidated.
OBJECTIVE: To investigate the molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes in the treatment of renal ischemia/reperfusion injury.
METHODS: (1) Human umbilical cord mesenchymal stem cells were cultured and exosomes were obtained and identified using an exosome extraction kit. (2) The distribution of exosomes in the kidney of mice with renal ischemia/reperfusion injury was examined by intravital fluorescence imaging. (3) Thirty C57/BL6 male mice were divided into five groups according to the random number table method: sham operation group, renal ischemia/reperfusion group, sham operation group+Compound C group, renal ischemia/reperfusion+exosome group (exosome group), and renal ischemia/reperfusion+exosome+Compound C group (exosome+Compound C group), with 6 mice in each group. Except the sham operation group, bilateral renal pedicles were clamped for 45 minutes and a mouse model of renal ischemia/reperfusion injury was established after 24 hours of reperfusion. In sham operation+Compound C group and exosome+Compound C group, AMPK inhibitor Compound C was intraperitoneally injected 30 minutes before model establishment. In the exosome group and exosome+Copmpound C group, exosomes were injected through the tail vein 15 minutes before renal pedicle clipping. The levels of serum creatinine and urea nitrogen, interleukin 6, and tumor necrosis factor α in renal tissue, and the expression of apoptosis-related factors in renal tissue were detected after 24 hours of reperfusion in each group.
RESULTS AND CONCLUSION: (1) Human umbilical cord mesenchymal stem cell exosomes had the typical tea tray morphology, with the diameter distribution in the range of 40-160 nm, and expressed the specific marker membrane protein of exosome surface. (2) Murine kidneys after renal ischemia/reperfusion injury were more likely to gather human umbilical cord mesenchymal stem cell exosomes compared with the sham operation group. (3) Exosome pretreatment reduced renal injury and the level of renal cell apoptosis in mice treated with renal ischemia/reperfusion injury. Moreover, this protective effect could be reversed by AMPK inhibitors. These findings verify that human umbilical cord mesenchymal stem cell-derived exosomes exerting a protective effect on renal ischemia/reperfusion injury may be related to the activation of the AMPK/YAP1 pathway to antiapoptosis.

Key words: human umbilical cord mesenchymal stem cell, exosome, AMPK-YAP1 pathway, apoptosis, kidney, ischemia/reperfusion injury

CLC Number:

Li Lingyu, Wei Huafeng, Luo Hao, Wang Hao, He Jiahui, Yao Yawei, Lyu Xinghua. Mechanism of human umbilical cord mesenchymal stem cell-derived exosomes against mouse renal ischemia/reperfusion injury[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(13): 2706-2712.

Figures/Tables 2

2.1 HucMSCs-exo的鉴定结果透射电子显微镜下，HucMSCs-exos为外泌体典型茶托样结构，见图1A，纳米颗粒跟踪分析结果表明，HucMSCs-exos直径在40-160 nm之间，见图1B。Western blot检测表面蛋白CD9、CD63和TSG101表达呈阳性，见图1C。以上结果表明成功分离得到hucMSCs-exos。 2.2 HucMSCs-exo在小鼠双肾缺血再灌注损伤部位富集在造模后30 min及6，12，24，48 h肾脏荧光强度见图2A。与假手术组相比，可见在造模后30 min及6，12，24 h肾缺血再灌注组小鼠肾脏荧光强度更强，48 h荧光强度差异无显著性意义，证明外泌体在30 min及6，12，24 h时优先聚集到损伤的肾脏，48 h时外泌体聚集无显著差异，揭示了外泌体聚集的时间特征，见图2B。 2.3 HucMSCs-exo通过AMPK/YAP通路抗小鼠肾缺血再灌注损伤 2.3.1 小鼠肾组织苏木精-伊红染色与假手术组对比，假手术+CC组小鼠肾组织结构正常，二者之间无明显差异，表明Compound C对小鼠肾无额外损伤；肾缺血再灌注组肾组织结构损伤严重，出现肾小管坏死、扩张，大量炎症细胞浸润和刷状缘消失，病变呈弥漫性改变。给予外泌体后病理改变有所减轻，肾小管管腔部分扩张，肾小管上皮细胞部分刷状缘消失，病变呈局灶性改变；外泌体+CC组小鼠肾组织病理损伤较外泌体组有加重趋势，见图3。 2.3.2 小鼠血清肾功能指标、肾组织炎性因子改变与假手术组比较，假手术+CC组血清肌酐和尿素氮水平未见明显变化，表明Compound C对小鼠肾功能未造成显著差异损伤；肾缺血再灌注组血清肌酐和尿素氮水平明显升高；与肾缺血再灌注组比较，外泌体组血清肌酐和尿素氮水平明显降低；与外泌体组相比，外泌体+CC组血清肌酐和尿素氮水平增高，见图4A，B。 ELISA检测小鼠肾组织中炎性因子白细胞介素6 和肿瘤坏死因子α水平，由于苏木精-伊红染色与肾功能检测结果表明Compound C对小鼠肾功能未造成显著差异损伤，故后续实验不再检测假手术+CC组。与假手术组比较，肾缺血再灌注组肾组织白细胞介素6和肿瘤坏死因子α水平明显升高；与肾缺血再灌注组比较，外泌体组肾组织白细胞介素6 和肿瘤坏死因子α水平明显降低，与外泌体组相比，外泌体+CC组肾组织白细胞介素6 和肿瘤坏死因子α水平增高，表明HucMSCs-exos对小鼠肾缺血再灌注损伤治疗有效，见图4C，D。 2.3.3 小鼠肾组织AMPK/YAP1通路蛋白表达变化 Western blot 检测显示：与假手术组比较，肾缺血再灌注组肾组织AMPK、p-AMPK、YAP1蛋白表达降低；与肾缺血再灌注组比较，外泌体组肾组织AMPK、p-AMPK、YAP1蛋白表达升高；与外泌体组比，外泌体+CC组肾组织AMPK、p-AMPK、YAP1蛋白表达降低，提示HucMSCs-exos可通过激活AMPK/YAP1通路减轻小鼠缺血再灌注损伤，见图5。"

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