郭文文, 袁圆, 王浩, 等. 人脐带间充质干细胞来源外泌体在肾缺血-再灌注损伤中的保护作用及其机制研究[J]. 器官移植, 2023, 14(3): 371-378. DOI: 10.3969/j.issn.1674-7445.2023.03.008
引用本文: 郭文文, 袁圆, 王浩, 等. 人脐带间充质干细胞来源外泌体在肾缺血-再灌注损伤中的保护作用及其机制研究[J]. 器官移植, 2023, 14(3): 371-378. DOI: 10.3969/j.issn.1674-7445.2023.03.008
Guo Wenwen, Yuan Yuan, Wang Hao, et al. Protective role and mechanism of human umbilical cord mesenchymal stem cell-derived exosome in renal ischemia-reperfusion injury[J]. ORGAN TRANSPLANTATION, 2023, 14(3): 371-378. DOI: 10.3969/j.issn.1674-7445.2023.03.008
Citation: Guo Wenwen, Yuan Yuan, Wang Hao, et al. Protective role and mechanism of human umbilical cord mesenchymal stem cell-derived exosome in renal ischemia-reperfusion injury[J]. ORGAN TRANSPLANTATION, 2023, 14(3): 371-378. DOI: 10.3969/j.issn.1674-7445.2023.03.008

人脐带间充质干细胞来源外泌体在肾缺血-再灌注损伤中的保护作用及其机制研究

Protective role and mechanism of human umbilical cord mesenchymal stem cell-derived exosome in renal ischemia-reperfusion injury

  • 摘要:
      目的  探究人脐带间充质干细胞来源外泌体(hucMSC-Exo)在肾缺血-再灌注损伤(IRI)中的保护作用,明确瞬时受体电位阳离子通道蛋白(TRPC)6/聚腺苷二磷酸核糖聚合酶(PARP)1信号通路在该过程中的重要作用及其调控机制。
      方法  采用超速离心法提取hucMSC-Exo,通过透射电子显微镜(透射电镜)、纳米颗粒追踪分析和蛋白质印迹法对其进行鉴定。将SD大鼠随机分成假手术组(S组)、假手术+TRPC6抑制剂SKF96365组(SS组)、肾IRI组(IRI组)、外泌体处理组(EXO组)、外泌体+TRPC6抑制剂SKF96365组(ES组),每组6只。检测血清肌酐和血尿素氮水平,苏木素-伊红(HE)染色观察肾组织病理学改变并行Paller评分,蛋白质印迹法检测大鼠肾组织中坏死性凋亡关键分子,包括受体相互作用蛋白激酶(RIPK)1、RIPK3和混合谱系激酶结构域样蛋白(MLKL)、TRPC6和PARP1的表达水平。
      结果  透射电镜下观察到典型茶托样结构,纳米颗粒追踪分析结果显示所提取物质的平均直径为125.9 nm,蛋白质印迹法检测其表面标志CD9、CD63、CD81表达阳性,证实提取物为外泌体。与S组比较,IRI组血清肌酐、血尿素氮水平增加,肾组织病理损伤加重,Paller评分增加,TRPC6、PARP1蛋白相对表达量下降,RIPK1、RIPK3、MLKL蛋白相对表达量增加(均为P < 0.05);与IRI组比较,EXO组血清肌酐、血尿素氮水平降低,肾组织病理损伤减轻,Paller评分降低,TRPC6、PARP1蛋白相对表达量增加,RIPK1、RIPK3、MLKL蛋白相对表达量下降(均为P < 0.05);与EXO组比较,ES组血清肌酐、血尿素氮水平增加,肾组织病理损伤加重,Paller评分增加,TRPC6、PARP1蛋白相对表达量下降,RIPK1、RIPK3、MLKL蛋白相对表达量增加(均为P < 0.05)。
      结论  hucMSC-Exo可减轻大鼠肾IRI所致的坏死性凋亡,其保护机制与TRPC6/PARP1通路激活有关。

     

    Abstract:
      Objective  To investigate the protective effect of human umbilical cord mesenchymal stem cell-derived exosome (hucMSC-Exo) on renal ischemia-reperfusion injury (IRI), and to clarify the critical role and regulating mechanism of transient receptor potential canonical (TRPC) 6/poly adenosine-diphosphate-ribose polymerase (PARP) 1 signaling pathway during this process.
      Methods  The hucMSC-Exo was extracted by ultracentrifugation, and identified by transmission electron microscope (TEM), nanoparticle tracing analysis and Western blot. SD rats were randomly divided into the sham operation group (group S), sham operation+TRPC6 inhibitor SKF96365 group (group SS), renal IRI group (group IRI), exosome treatment group (group EXO) and exosome +TRPC6 inhibitor SKF96365 group (group ES), with 6 rats in each group. Serum creatinine and blood urea nitrogen levels were detected. Pathological changes of renal tissues were observed by hematoxylin-eosin (HE) staining and Paller score was calculated. The expression levels of key molecules of necroptosis in rat renal tissues, including receptor-interacting protein kinase (RIPK)1, RIPK3 and mixed-lineage kinase domain-like protein (MLKL), TRPC6 and PARP1, were detected by Western blot.
      Results  Typical saucer-like structure was observed under TEM. Nanoparticle tracing analysis showed that the average diameter of the extracted substance was 125.9 nm. Western blot revealed that the surface markers of CD9, CD63 and CD81 were positively expressed, confirmed that the extracted substance was exosome. Compared with group S, the serum creatinine and blood urea nitrogen levels were up-regulated, the pathological damage of renal tissues was worsened, Paller score was elevated, the relative expression levels of TRPC6 and PARP1 proteins were down-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were up-regulated in group IRI (all P < 0.05). Compared with group IRI, the serum creatinine and blood urea nitrogen levels were down-regulated, the pathological damage of renal tissues was mitigated, Paller score was decreased, the relative expression levels of TRPC6 and PARP1 proteins were up-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were down-regulated in group EXO (all P < 0.05). Compared with group EXO, the serum creatinine and blood urea nitrogen levels were up-regulated, the pathological damage of renal tissues was aggravated, Paller score was increased, the relative expression levels of TRPC6 and PARP1 proteins were down-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were up-regulated in group ES (all P < 0.05).
      Conclusions  hucMSC-Exo may alleviate the necroptosis induced by renal IRI in rat models, which is related to the activation of TRPC6/PARP1 signaling pathway.

     

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