Protective role and mechanism of human umbilical cord mesenchymal stem cell-derived exosome in renal ischemia-reperfusion injury
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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。 -
关键词:
- 间充质干细胞来源外泌体 /
- 瞬时受体电位阳离子通道蛋白（TRPC） /
- 聚腺苷二磷酸核糖聚合酶（PARP） /
- 坏死性凋亡 /
- 缺血-再灌注损伤 /
- Paller评分 /
- 血清肌酐 /
- 血尿素氮
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. -
图 1 hucMSC-Exo的鉴定
注：A图为透射电镜检测hucMSC-Exo的形态（×200 000）；B图为纳米颗粒追踪分析测定hucMSC-Exo粒径大小（平均直径125.9 nm）；C图为蛋白质印迹法检测hucMSC-Exo中CD9、CD63和CD81的表达。
Figure 1. Characterization of hucMSC-Exo
图 2 各组肾组织HE染色及Paller评分
注：A~E图分别为S组、SS组、IRI组、EXO组、ES组大鼠肾组织HE染色结果（×400）；F图为各组大鼠肾组织Paller评分。与S组比较，aP < 0.05；与IRI组比较，bP < 0.05；与EXO组比较，cP < 0.05。
Figure 2. HE staining and Paller score of renal tissues in each group
图 3 各组血清肌酐和血尿素氮水平
注：A图为各组大鼠血清肌酐水平；B图为各组大鼠血尿素氮水平。与S组比较，aP < 0.05；与IRI组比较，bP < 0.05；与EXO组比较，cP < 0.05。
Figure 3. Serum creatinine and blood urea nitrogen levels in each group
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[1] THAPA K, SINGH TG, KAUR A. Cyclic nucleotide phosphodiesterase inhibition as a potential therapeutic target in renal ischemia reperfusion injury[J]. Life Sci, 2021, 282: 119843. DOI: 10.1016/j.lfs.2021.119843. [2] G BARDALLO R, PANISELLO-ROSELLÓ A, SANCHEZ-NUNO S, et al. Nrf2 and oxidative stress in liver ischemia/reperfusion injury[J]. FEBS J, 2022, 289(18): 5463-5479. DOI: 10.1111/febs.16336. [3] WU MY, YIANG GT, LIAO WT, et al. Current mechanistic concepts in ischemia and reperfusion injury[J]. Cell Physiol Biochem, 2018, 46(4): 1650-1667. DOI: 10.1159/000489241. [4] MARTIN JL, GRUSZCZYK AV, BEACH TE, et al. Mitochondrial mechanisms and therapeutics in ischaemia reperfusion injury[J]. Pediatr Nephrol, 2019, 34(7): 1167-1174. DOI: 10.1007/s00467-018-3984-5. [5] JUN W, BENJANUWATTRA J, CHATTIPAKORN SC, et al. Necroptosis in renal ischemia/reperfusion injury: a major mode of cell death?[J]. Arch Biochem Biophys, 2020, 689: 108433. DOI: 10.1016/j.abb.2020.108433. [6] FENG Y, IMAM ALIAGAN A, TOMBO N, et al. RIP3 translocation into mitochondria promotes mitofilin degradation to increase inflammation and kidney injury after renal ischemia-reperfusion[J]. Cells, 2022, 11(12): 1894. DOI: 10.3390/cells11121894. [7] SHEN B, HE Y, ZHOU S, et al. TRPC6 may protect renal ischemia-reperfusion injury through inhibiting necroptosis of renal tubular epithelial cells[J]. Med Sci Monit, 2016, 22: 633-641. DOI: 10.12659/msm.897353. [8] HUANG J, CAO H, CUI B, et al. Mesenchymal stem cells-derived exosomes ameliorate ischemia/reperfusion induced acute kidney injury in a porcine model[J]. Front Cell Dev Biol, 2022, 10: 899869. DOI: 10.3389/fcell.2022.899869. [9] DING C, ZHENG J, WANG B, et al. Exosomal microRNA-374b-5p from tubular epithelial cells promoted M1 macrophages activation and worsened renal ischemia/reperfusion injury[J]. Front Cell Dev Biol, 2020, 8: 587693. DOI: 10.3389/fcell.2020.587693. [10] ZHANG Y, WANG J, YANG B, et al. Transfer of microRNA-216a-5p from exosomes secreted by human urine-derived stem cells reduces renal ischemia/reperfusion injury[J]. Front Cell Dev Biol, 2020, 8: 610587. DOI: 10.3389/fcell.2020.610587. [11] SHEN B, MEI M, PU Y, et al. Necrostatin-1 attenuates renal ischemia and reperfusion injury via meditation of HIF-1α/miR-26a/TRPC6/PARP1 signaling[J]. Mol Ther Nucleic Acids, 2019, 17: 701-713. DOI: 10.1016/j.omtn.2019.06.025. [12] ZHANG Y, CHANG Y, HAN Z, et al. Estrogen protects against renal ischemia-reperfusion injury by regulating Th17/Treg cell immune balance[J]. Dis Markers, 2022: 7812099. DOI: 10.1155/2022/7812099. [13] ZHOU Z, YOU B, JI C, et al. Implications of crosstalk between exosome-mediated ferroptosis and diseases for pathogenesis and treatment[J]. Cells, 2023, 12(2): 311. DOI: 10.3390/cells12020311. [14] LIN Z, WU Y, XU Y, et al. Mesenchymal stem cell-derived exosomes in cancer therapy resistance: recent advances and therapeutic potential[J]. Mol Cancer, 2022, 21(1): 179. DOI: 10.1186/s12943-022-01650-5. [15] LAI JJ, CHAU ZL, CHEN SY, et al. Exosome processing and characterization approaches for research and technology development[J]. Adv Sci (Weinh), 2022, 9(15): e2103222. DOI: 10.1002/advs.202103222. [16] KEERTHIKUMAR S, CHISANGA D, ARIYARATNE D, et al. ExoCarta: a web-based compendium of exosomal cargo[J]. J Mol Biol, 2016, 428(4): 688-692. DOI: 10.1016/j.jmb.2015.09.019. [17] HU Q, SU H, LI J, et al. Clinical applications of exosome membrane proteins[J]. Precis Clin Med, 2020, 3(1): 54-66. DOI: 10.1093/pcmedi/pbaa007. [18] SOHRABI B, DAYERI B, ZAHEDI E, et al. Mesenchymal stem cell (MSC)-derived exosomes as novel vehicles for delivery of miRNAs in cancer therapy[J]. Cancer Gene Ther, 2022, 29(8/9): 1105-1116. DOI: 10.1038/s41417-022-00427-8. [19] CHEN S, SUN F, QIAN H, et al. Preconditioning and engineering strategies for improving the efficacy of mesenchymal stem cell-derived exosomes in cell-free therapy[J]. Stem Cells Int, 2022: 1779346. DOI: 10.1155/2022/1779346. [20] KIMIZ-GEBOLOGLU I, ONCEL SS. Exosomes: large-scale production, isolation, drug loading efficiency, and biodistribution and uptake[J]. J Control Release, 2022, 347: 533-543. DOI: 10.1016/j.jconrel.2022.05.027. [21] FERREIRA D, MOREIRA JN, RODRIGUES LR. New advances in exosome-based targeted drug delivery systems[J]. Crit Rev Oncol Hematol, 2022, 172: 103628. DOI: 10.1016/j.critrevonc.2022.103628. [22] 王光华, 王桂斌, 滕晓华. 间充质干细胞来源外泌体治疗缺血再灌注损伤分子机制及研究进展[J]. 生物医学工程与临床, 2021, 25(5): 639-643. DOI: 10.13339/j.cnki.sglc.20210820.019.WANG GH, WANG GB, TENG XH. Review of exosomes derived from mesenchymal stem cells in protecting ischemia-reperfusion injury[J]. Biomed Eng Clin Med, 2021, 25(5): 639-643. DOI: 10.13339/j.cnki.sglc.20210820.019. [23] LIAO YJ, MA YX, HUANG LL, et al. Augmenter of liver regeneration protects the kidney against ischemia-reperfusion injury by inhibiting necroptosis[J]. Bioengineered, 2022, 13(3): 5152-5167. DOI: 10.1080/21655979.2022.2037248. [24] 刘昕蓓, 刘文静. 调控性细胞死亡在肾脏缺血再灌注损伤中的研究进展[J]. 生理学报, 2022, 74(2): 320-332. DOI: 10.13294/j.aps.2022.0025.LIU XB, LIU WJ. The role of regulated cell death in renal ischemia-reperfusion injury[J]. Acta Physiol Sin, 2022, 74(2): 320-332. DOI: 10.13294/j.aps.2022.0025. [25] DEGTEREV A, HUANG Z, BOYCE M, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury[J]. Nat Chem Biol, 2005, 1(2): 112-119. DOI: 10.1038/nchembio711. [26] DELLA TORRE L, NEBBIOSO A, STUNNENBERG HG, et al. The role of necroptosis: biological relevance and its involvement in cancer[J]. Cancers (Basel), 2021, 13(4): 684. DOI: 10.3390/cancers13040684. [27] ASHOUR H, HASHEM HA, KHOWAILED AA, et al. Necrostatin-1 mitigates renal ischaemia-reperfusion injury - time dependent - via aborting the interacting protein kinase (RIPK-1)-induced inflammatory immune response[J]. Clin Exp Pharmacol Physiol, 2022, 49(4): 501-514. DOI: 10.1111/1440-1681.13625. [28] LIU Z, LI C, LI Y, et al. Propofol reduces renal ischemia reperfusion-mediated necroptosis by up-regulation of SIRT1 in rats[J]. Inflammation, 2022, 45(5): 2038-2051. DOI: 10.1007/s10753-022-01673-6. [29] YUAN X, LI D, CHEN X, et al. Extracellular vesicles from human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs) protect against renal ischemia/reperfusion injury via delivering specificity protein (SP1) and transcriptional activating of sphingosine kinase 1 and inhibiting necroptosis[J]. Cell Death Dis, 2017, 8(12): 3200. DOI: 10.1038/s41419-017-0041-4. [30] WANG H, CHENG X, TIAN J, et al. TRPC channels: structure, function, regulation and recent advances in small molecular probes[J]. Pharmacol Ther, 2020, 209: 107497. DOI: 10.1016/j.pharmthera.2020.107497. [31] ENGLISCH CN, PAULSEN F, TSCHERNIG T. TRPC channels in the physiology and pathophysiology of the renal tubular system: what do we know?[J]. Int J Mol Sci, 2022, 24(1): 181. DOI: 10.3390/ijms24010181. [32] DRYER SE, KIM EY. The effects of TRPC6 knockout in animal models of kidney disease[J]. Biomolecules, 2022, 12(11): 1710. DOI: 10.3390/biom12111710. [33] 郭文文, 袁圆, 魏华锋, 等. 瞬时受体电位阳离子通道蛋白6在缺血-再灌注损伤中的作用研究进展[J]. 器官移植, 2022, 13(5): 659-665. DOI: 10.3969/j.issn.1674-7445.2022.05.017.GUO WW, YUAN Y, WEI HF, et al. Research progress on the role of transient receptor potential canonical 6 in ischemia-reperfusion injury[J]. Organ Transplant, 2022, 13(5): 659-665. DOI: 10.3969/j.issn.1674-7445.2022.05.017. [34] ZHENG Z, TSVETKOV D, BARTOLOMAEUS TUP, et al. Role of TRPC6 in kidney damage after acute ischemic kidney injury[J]. Sci Rep, 2022, 12(1): 3038. DOI: 10.1038/s41598-022-06703-9. [35] SHEN B, MEI M, AI S, et al. TRPC6 inhibits renal tubular epithelial cell pyroptosis through regulating zinc influx and alleviates renal ischemia-reperfusion injury[J]. FASEB J, 2022, 36(10): e22527. DOI: 10.1096/fj.202200109RR. [36] PU Y, ZHAO H, SHEN B, et al. TRPC6 ameliorates renal ischemic reperfusion injury by inducing Zn2+ influx and activating autophagy to resist necrosis[J]. Ann Transl Med, 2022, 10(5): 249. DOI: 10.21037/atm-21-5837. -
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